Cancer Cell Structures, Carcinogens and Genomic Instability (Leon P. Bignold 2006)
Cancer Cell Structures, Carcinogens and Genomic Instability (Leon P. Bignold 2006)
Cancer Cell Structures, Carcinogens and Genomic Instability (Leon P. Bignold 2006)
Carcinogens and
Genomic Instability
Edited by Leon P. Bignold
Birkhuser Verlag
Basel Boston Berlin
Editor
Leon P. Bignold
Division of Tissue Pathology
Institute of Medical and Veterinary Science
PO Box 14
Rundle Mall, SA 5001
Australia
e-ISBN: 3-7643-7378-4
www. birkhauser.ch
Contents
List of Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preface
VII
...............................................
XI
25
49
Andreas Luch
The mode of action of organic carcinogens on cellular structures
...
65
97
. . . . . . . . . . . . . . . . 131
. . . . . 159
. . . . . 201
VI
Contents
. . . . . . . . . . . . . . . . . . . . . . . . . 303
VII
List of Contributors
Vinay Arora, Apoptosis Research Centre, Childrens Hospital of Eastern
Ontario, Research Institute, 401 Smyth Road, Ottawa, Ontario K1H 8L1,
Canada; e-mail: [email protected]
Benjamin J. Arthurs, Chemical Structure & Dynamics, Pacific Northwest
National Laboratory, MS K8-88, 3335 Q Ave, Richland, WA 99354, USA;
e-mail: [email protected]
Bharati Bapat, Samuel Lunenfeld Research Institute, Department of Pathology
and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue,
9th Floor, Room 992B, Toronto, Ontario M5G 1X5, Canada; and
Department of Laboratory Medicine and Pathobiology, University of
Toronto, Toronto, ON, M5S 1A1, Canada; e-mail: [email protected]
Leon P. Bignold, Division of Tissue Pathology, Institute of Medical and
Veterinary Science, PO Box 14, Rundle Mall, SA 5001, Australia; e-mail:
[email protected]
Herman H. Cheung, Apoptosis Research Centre, Childrens Hospital of
Eastern Ontario, Research Institute, 401 Smyth Road, Ottawa, Ontario K1H
8L1, Canada; e-mail: [email protected]
B. L. D. Coghlan, Centre for European Studies and General Linguistics,
University of Adelaide, Adelaide, SA 5005, Australia
William B. Coleman, Department of Pathology and Laboratory Medicine, UNC
Lineberger Comprehensive Cancer Center, University of North Carolina
School of Medicine, Campus Box 7525, Room 515, Brinkhous-Bullitt
Building, Chapel Hill, NC 27599, USA; e-mail: william.coleman@
pathology.unc.edu
James R. Davie, Manitoba Institute of Cell Biology, 675 McDermot Avenue,
Winnipeg, Manitoba, R3E 0V9 Canada; e-mail: [email protected]
Michael J. Davies, The Heart Research Institute, 145 Missenden Road,
Camperdown, Sydney, NSW 2050, Australia; e-mail: [email protected]
Bojan Drobic, Manitoba Institute of Cell Biology, 675 McDermot Avenue,
Winnipeg, Manitoba, R3E 0V9 Canada; e-mail: [email protected]
Anette Duensing, Molecular Virology Program, University of Pittsburgh
Cancer Institute, Hillman Cancer Center, Research Pavillion, Suite 1.8, 5117
Centre Avenue, Pittsburgh, PA 15213, USA; e-mail: [email protected]
Stefan Duensing, Molecular Virology Program, University of Pittsburgh
Cancer Institute, Hillman Cancer Center, Research Pavillion, Suite 1.8, 5117
Centre Avenue, Pittsburgh, PA 15213, USA; e-mail: [email protected]
Katherine L. Dunn, Manitoba Institute of Cell Biology, 675 McDermot Avenue,
Winnipeg, Manitoba, R3E 0V9 Canada; e-mail: [email protected]
VIII
List of contributors
List of contributors
IX
[email protected]
Karl Mnger, The Channing Laboratory, Room 861, Brigham and Womens
Hospital, 181 Longwood Avenue, Boston, MA 02115, USA; e-mail:
[email protected]
Asha S. Multani, Department of Molecular Genetics, Unit # 011, The
University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd.,
Houston, TX 77030, USA
Christine L. Nguyen, The Channing Laboratory, Room 861, Brigham and
Womens Hospital, 181 Longwood Avenue, Boston, MA 02115, USA;
e-mail: [email protected]
Kazuo Okimoto, Toxicology Group, Safety Research Laboratories, Dainippon,
Pharmaceutical Co., Ltd., 33-94, Enoki-cho, Suita, Osaka 564-0053, Japan;
e-mail: [email protected]
Sen Pathak, Department of Molecular Genetics, Unit # 011, The University of
Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX
77030, USA; e-mail: [email protected]
David I. Pattison, The Heart Research Institute, 145 Missenden Road,
Camperdown, Sydney, NSW 2050, Australia; e-mail: [email protected]
Stavroula Raptis, Samuel Lunenfeld Research Institute, Department of
Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University
Avenue, 9th Floor, Room 992B, Toronto, Ontario M5G 1X5, Canada; and
Department of Laboratory Medicine and Pathobiology, University of
Toronto, Toronto, ON, M5S 1A1, Canada; e-mail: [email protected]
Marianne Sowa, Chemical Structure & Dynamics, Pacific Northwest National
Laboratory, MS K8-88, 3335 Q Ave, Richland, WA 99354, USA; e-mail:
[email protected]
C. B. Seymour, Medical Physics and Applied Radiation Sciences Unit,
McMaster University, Hamilton, Ontario L8S 4K1, Canada
Elizabeth T. Snow, Centre for Cellular and Molecular Biology, School of
Biological and Chemical Sciences, Deakin University, 221 Burwood
Highway, Burwood, Victoria 3125, Australia; e-mail: [email protected]
Gregory J. Tsongalis, Laboratory of Molecular Pathology, Department of
Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756,
USA
XI
Preface
This volume began with an invitation from the publishers to edit a volume of
EXS on Cancer. This invitation undoubtedly derived from my articles in
Cellular and Molecular Life Sciences in 2002 and 2003 on the relationships
between the morphology, aetiology and pathogenesis of tumours, especially in
relation to genetic instability. After many years of teaching the theories of cancer in undergraduate medical school courses, it seemed to me that the variably
chaotic histopathologic features of tumours parallel in some way, the variably
unstable genomes of tumour cells, which were being discovered in the 1990s.
Thus the title of the volume has come to include morphology, carcinogenesis
and genetic instability.
The invitation came while I was working with Herrn Dr. med. Hubertus
Jersmann (MD Dsseldorf, PhD, now Senior Lecturer in Medicine of the
University of Adelaide) and Professor Brian Coghlan (Emeritus Professor of
German, the University of Adelaide), on the work of the nineteenth century
cancer pathologists, especially David Paul von Hansemann (18581920). With
the delivery of the manuscripts from the authors of the chapters, it became
obvious that a background chapter for the volume could include some of the
material which we had uncovered together. Because of this, chapter 1 is
authored by the three of us, and the new material figures prominently.
I am extremely grateful to the contributors of the chapters, without whom,
of course there could be no volume at all, and to Dr. Beatrice Menz, the supervising editor at Birkhuser, who has been unfailingly helpful and patient.
The staff of the Barr-Smith Library at the University of Adelaide were very
helpful throughout and Mr. Peter Dent of the Photography Department of the
Institute of Medical and Veterinary Science, Adelaide, assisted with the design
of the cover.
Leon P. Bignold
March 2005
Division of Tissue Pathology, Institute of Medical and Veterinary Science, PO Box 14, Rundle Mall,
SA 5001, Australia
2
Centre for European Studies and General Linguistics, University of Adelaide, Adelaide, SA 5005,
Australia
3
Department of Medicine, University of Adelaide, Adelaide, SA 5005, Australia
Summary. Morphological abnormalities of both the nuclei and the cell bodies of tumour cells were
described by Mller in the late 1830s. Abnormalities of mitoses and chromosomes in tumour cells
were described in the late 1880s. Von Hansemann, in the 1890s, suggested that tumour cells develop
from normal cells because of a tendency to mal-distribution and other changes of chromosomes
occurring during mitosis. In the first decades of the 20th century, Mendelian genetics and gene mapping of chromosomes were established, and the dominant or recessive bases of the familial predispositions to certain tumour types were recognised. In the same period, the carcinogenic effects of ionising radiations, of certain chemicals and of particular viruses were described. A well-developed
somatic gene-mutational theory of tumours was postulated by Bauer in 1928. In support of this, in
the next three decades, many environmental agents were found to cause mitotic and chromosomal
abnormalities in normal cells as well as mutations in germ-line cells of experimental animals.
Nevertheless, mitotic, chromosomal, and other mutational theories were not popular explanations of
tumour pathogenesis in the first half of the 20th century. Only in the 1960s did somatic mutational
mechanisms come to dominate theories of tumour formation, especially as a result of the discoveries
of the reactivity of carcinogens with DNA, and that the mutation responsible for xeroderma pigmentosum causes loss of function of a gene involved in the repair of DNA after damage by ultraviolet
light (Cleaver in 1968). To explain the complexity of tumourous phenomena, multi-hit models
gained popularity over single-hit models of somatic mutation, and epigenetic mechanisms of
gene regulation began to be studied in tumour cells. More recently, the documentation of much larger-than-expected numbers of genomic events in tumour cells (by Stoler and co-workers, in 1999) has
raised the issue of somatic genetic instability in tumour cells, a field which was pioneered in the
1970s mainly by Loeb. Here these discoveries are traced, beginning with nuclear instability though
mitotic-and-chromosomal theories, single somatic mutation theories, multi-hit somatic theories,
somatic, non-chromosomal, genetic instability and epigenetic mechanisms in tumour cells as a
background to the chapters which follow.
Key words: Cancer, carcinogenesis, chromosomes, genetic instability, historical, nuclei.
Introduction
There are several excellent histories of the study of cell biology and of
tumours [114], which give coverage of most of the various aspects of cells
and this disease process. However, no perfectly satisfactory account of the history of investigations of the relationships between the morphological features
and aetiological factors of tumours is available, especially in terms of the
genetic theories of the pathogenesis of tumour formation. Essentially, the history of the investigation of mutations in tumour cells is characterised by an
early attempt by von Hansemann to provide a theory that embraced morphological abnormalities and chromosomal changes. This theory was rejected and
largely forgotten, but was followed, over a period of approximately 70 years,
by slow recognition, first of single mutations in tumours, then of multiple
mutations in tumours. Recently, using molecular methods based on the polymerase chain reaction, so many mutations in tumour cells have been demonstrated that only acquired somatic non-mitotic, non-chromosomal genetic
instability, together with alterations of gene expression, appears to provide a
likely explanation. This chapter sketches the major milestones of tumour
genetics, with detail given mainly when it has not previously been published
in English.
The nature of the essential abnormality of cancerous tissues has been discussed since the disease was recognised as a process separate from inflammatory disorders, by the Hippocratic School in the 6th century BC. However,
studies of tumours according only to clinical features and macroscopic appearances, by Greek, Roman, Arab, and medieval and Renaissance Europeans led
to concepts and schemes of classification of tumours that were largely arbitrary and unhelpful [2, 8, 9, 12].
Beginning in the 16th century, various forms of microscopes were developed in Europe. The compound form is said to have been invented by the
Janssen brothers in about 1590 [15, 16]. Early compound microscopes suffered especially from poorly made glass, as well as chromatic and spherical
aberration, and were not necessarily more useful than the simple microscopes
of the period, for example, those of Leewenhoek (16321723) [15, 16]. The
progress of microscopic discoveries in the 17th and 18th centuries was
achieved mainly through gradual improvements in the making of the glass for
lenses. Thus, early in the period, lacteal and lymphatic vessels were described
and only later were red cells in blood and globules in lymph, as well as fibres
in many tissues discovered [4]. One of the last, but important discovery using
a simple microscope with a non-achromatic lens (see below) was that of the
nucleus by Robert Brown in the early 1830s [17]. Regarding tumours, one
view in this period was that tumours derive from particular (plastic) types of
lymph, perhaps through a process of coagulation [8, 12, 18].
(17691869, father of Lord Lister [13]) in 1830, were useful achromatic lenses manufactured [16]. Microscopes with these lenses could magnify 500 times,
and a wealth of scientific discovery followed. Within a few years, it was appreciated that cells are the basic living units of the body, and which, by multiplying and secreting extracellular materials, form all the tissues, i.e. the Cell theory. Wolff [2] gives priority to Raspail (a French histochemist, 17941878
[13]), both for this idea, and for being first to use the word cell for the microscopic structures now recognised as such. The same idea, however, was propounded in detail by Schleiden (18041881) (for plant cells) in 1838 [19] and
by his friend Schwann (18101882) (who gave generous credit to Schleiden)
for animal cells in 1839 [20].
Schwann did this work while he was an assistant to Johannes Mller
(18011858), Professor of Anatomy and Physiology at the University of
Berlin, who was perhaps the most remarkable scientific teacher in modern history. This is because his students included not only Schwann, His
(18111887), Henle (18091885) and Klliker (18171905) [13], but also
Virchow (18211902), who was the major proponent of cellular pathology
(see below), Helmholtz (18211894), who propounded the law of conservation of energy as well as contributing to optics and acoustics, and Wilhelm
Wundt (18321920), who founded experimental psychology. Another of
Mllers students was Brcke (18191892), who in turn was a major influence
on Sigmund Freud (18561939) [21].
Although Mllers research interests were mainly in neurophysiology, in
1838 he published [22] studies of the cells and their nuclei in tumours using a
Schiek microscope of the latest type. Mller [22] documented the variable
sizes and shapes of cells and nuclei, not only between tumour types, but also
between cases of the same tumour type, and also among the cells of individual
tumours. In so far as nuclei were understood to probably contain at least some
of the hereditary material of cells [23], the finding of variability of nuclear
morphology in cells might be considered the original observation of a form of
hereditary/genetic instability in tumour cells, even if it was not appreciated as
such at the time.
1. How do cells arise? Schwann [20], Mller [22] and Klliker [24] thought
that cells could arise spontaneously in some specific (but invisible) type of
interstitial fluid, which they referred to as blastema. Mller in particular
thought that the process involved first crystallisation of nuclear material in the
blastema and, second, the aggregation of cytoplasm around the crystals [22].
Mller thought cancer cells arose from particular cancerous invisible particles,
which he called seminum morbii [22]. The alternative view, being that cells
must always develop from pre-existing cells was espoused by Raspail, Remak
(18151865) and, most famously, Virchow. For accounts of this well-documented controversy, which lasted into the late 19th century, see especially [5,
812] and references therein.
2. How do cells and nuclei divide to generally provide for daughter cells,
which are the same as each other and the mother cell? Even with the best
achromatic lenses, it was not possible in histological preparations to see any
more detail of nuclear division than a division of nuclear material into two
parts, followed by division of the cell, and this matter was not resolved in this
period (see below).
3. To what degree do the embryological origins of the cells determine their ultimate morphology? Histological studies of embryos had led to suggestions, particularly by Remak and His [1, 3, 4, 812], that there are two or three basic
embryological layers, from which adult cell types derive. Tracing the developmental pedigrees of cell types in adults was a major activity in embryology
until the 20th century [1, 3]. A part of the stimulus for these studies was the urge
to further investigate earlier observations of Caspar Wolff (17331794), von
Baer (17921876) and Oken (17791851) [3, 4, 25, 26], who had shown that
the phases of embryonic development which occur in one species resemble,
albeit temporarily, the adult forms of simpler species that are lower or earlier in the evolutionary tree. The results of these histological studies generally
supported the earlier observations, and were popularised as the saying
Ontogeny recapitulates Phylogeny, especially by Haeckel (18341919) [4,
2628]. These studies formed part of the well-documented struggle (broadly
from 18581940) to establish scientific evidence for and against Darwins theory of evolution [2933].
4. Can cells undergo changes of mature morphological type in adults? Or are
they always faithful to their original lineage? Virchow pointed out that chondrocytes and osteocytes associated with the callus of healing of fractures of
bone arise from basic connective or supportive tissue [34] (Bindegewebe: for
a useful note on the English translation of this word, see Translators Notes in
[25]). For these changes of cell type, Virchow used the phrase histological
substitution in the second edition of his Cellular pathology (1858) [34], but
used metaplasia for the same process in the fourth edition of Cellular
pathology in 1871 [35] and in a later article in 1884 [32] (see also [36]).
However, the same authors who supported fixed continuities of embryological
layer to adult cell type also tended to support a view of fixity of cell type in
adult tissues (see above).
5. As an extension of the fourth issue above, can cells of tumours come from
a single tumour precursor cell in connective tissue or from the more specialised cells of each type? Again, Virchow insisted that interchanges of cell
types are common phenomena and that cancers do not come from epithelial
cells, but rather from particular cells of the Bindegewebe [34, 35]. Eventually,
the opposite view, especially in regards to the derivation of carcinomas from
epithelial cells only, came to be most widely held, due especially to the work
of Waldeyer (18361921) and Thiersch (18221895) [812]. The present
view is that some, but not all, cell types or their local stem cells retain some
ability to adopt different directions of histological differentiation under particular circumstances [37].
6. What is the stimulus to the excessive growth of tumours? By the mid-19th
century, ideas of blastemas and plastic lymph had been abandoned, and
Virchows [34] suggestion that a chronic local irritation must be the first step
of tumour formation was popular. Thiersch, in 1865 suggested that a local
over-nutrition of tissues might cause excessive growth [2], and Bol, in 1876
proposed that tumour cells derive their growth in some way from some influence of embryonal-like mesenchymal cells at the site of tumour formation [2].
Cohnheims idea [38], published in 1882, was that the cells of tumours are
essentially embryonal in nature, being left-over embryonal cells. In this way,
carcinomas are epithelial because they arise from left-over embryonal
epithelial cells. To account for the sudden activation of these dormant
embryonal cells, Cohnheim suggested a mechanism of local hyper-nutrition
[39]. Ribbert (18551920) initially invoked embryonal rests as the source of
tumour cells [2], but later thought that normal adult cells might be stimulated
to grow entirely because of a loss of normal local inhibitory tissue tension
[39] (reviewed in English [2, 40, 41]).
7. What is the mechanism of the nuclear pleomorphism to tumours? None of
the various mechanisms of tumour formation (above), however, provide an
explanation for nuclear pleomorphism in tumour cells. Perhaps stimulated in
part by this consideration, much effort was expended in the late 19th century
on the (intranuclear-) parasitic theories, for which the best evidence obtained
was that the intranuclear irregularities of tumour cells resemble either the bodies of, or the effects of, parasites. (Wolff [2] devotes 150 pages to these theories. Shimkin [8] gives a useful table.) These were very popular up to the early
20th century and were mentioned by Ewing as late as 1940 [42], although the
evidence in their favour was entirely morphological, and conversion of normal
cells to cancer cells by the intranuclear cancer parasites was not achieved.
8. In the most general sense, what is the relationship of disease processes to
normal physiological processes? The Ancient Greeks and Romans, especially
Galen, held that all diseases, excepting trauma and parasitic disorders, are due
to imbalances of (normal physiological) humours and hence are endogenous
in origin [2, 5, 812]. Mller [23] held that diseases are abnormal physiologies and Virchow repeatedly and strongly stated the same view [9, 43, 44]. To
give just two quotations, in 1855 [45], Virchow stated All pathological for-
tumour formation. The general outline of his concepts appeared in his first
paper in 1890 [47], while later articles and two books [49, 50] contained extensions and modifications of his ideas, as well as responses to the frequently negative comments published by other authors.
The first paper (1890) [47] is difficult to understand for two reasons. First,
von Hansemann probably felt that, before he could elaborate a notion of cancer, he had to describe a normal biological process which, when mildly abnormal, would produce appearances resembling those of cancer (perhaps under
the influence of Virchow, see above). Thus, it was probably not enough, in
1890, for von Hansemann to observe that, if chromosomes carry the genetic
material of the cell, and are abnormal in cancer cells, then the abnormal chromosomes are probably the cause of the abnormalities of cancer cells. Put
another way, von Hansemann possibly had to satisfy Virchows somewhat
abstract notions of disease pathogenesis (see above) and find a whole analogous system of biological process, which in some way resembled many if not
most of the tumourous phenomena. Second, at the time von Hansemann wrote
the paper, the differences between the chromosomal replications and divisions
in meiosis and mitosis were not recognised, nor were the numbers of chromosomes in human adult cells or gametes known. Furthermore, the individuality
of chromosomes was only one theory among many at the time, and genes
and gene maps lay in the future.
At the beginning of this paper [47], von Hansemann discussed ideas of the
variability of amounts of chromatin in cells generally. He then noted that injection of the chromatin of a sperm is an important aspect of fertilisation of the
egg, and that the amount of chromatin increases and decreases in cells associated with spermatogenesis in testicular tissue. Von Hansemann then observed
that in tumour tissue, increases and decreases of chromatin in tumour cells
occurs, and that the smallest nuclei appear to become degenerate. This last
process that von Hansemann observed, seems to be in some way analogous to
the expulsion of the polar bodies (referring to them as did Hertwig [25] as
Richtungskrperchen, which later came to be used for centrioles) from the
developing egg in the ovary. Next, von Hansemann discussed asymmetric distribution of chromosomes in mitosis as the main mechanism of the formation
of small nuclei in some detail. In the next section of the paper, he reviewed theories of cell heredity as they were known in 1890, mentioning especially the
ideas of Weismann and Naegeli, in relation to quantitative and qualitative
asymmetries of cell division (not nuclear division) during formation of the
blastula. This discussion led to consideration of the progress of differentiation
of the cells in the early embryo, with two pivotal statements being made, to
try to link differentiation, autonomy and growth. They were:
1. With every further qualitative work division, the cells lose the capability to
exist autonomously.
2. With every new generational phase, a changed growth energy [nutritional, formative, and functional activity (Virchow)] takes place which often
10
tion of tumours prior to 1890, was of homologous (like the adjacent normal
tissue) and heterologous (unlike the adjacent normal tissue). This classification, which dated from Laennec in 1804 [2] and was used by Virchow [34]
and others, did not permit any intermediary types, while von Hansemanns
concept (dedifferentiation/anaplasia) was of a process which could occur in
grades and degrees [2]. Von Hansemanns views were based on actual
histopathological phenomena, which were being more and more widely documented in diagnostic histopathology throughout the world from the 1890s
onwards, using the new techniques and microscope lenses (see above).
Dedifferentiation and anaplasia entered the medical lexicon, where they
remain firmly to this day.
Reappraisal
Perhaps correctly, von Hansemanns notion of anaplasia (with its component
of either the correct dedifferentiation or the incorrect undifferentiation
concept of the cell) has been discounted. However, from the perspective of the
21st century, we can see that his basic idea of chromosomal disorder as the
basis of tumour formation may well be valid, and current aspects of chromatin
and chromosomes in cancer are the subjects of chapters 2 and 3 in this volume.
On the basis of all of this, it would appear that von Hansemann may deserve
more recognition as a contributor to genetic theories of tumours and oncology
generally, than he is currently awarded.
11
mental animals were conducted, especially in the UK and the USA [8], and it
was found that, indeed, the inbred offspring of animals with certain tumour
types were more liable to the same tumour, but not to tumours generally. Maud
Slye (18791954) thought that the results of such studies showed that human
tumours are of a recessive type, but this was not supported by other workers, notably Little (18881971) [8, 64].
Another line of investigation was the transplantability of experimental
tumours between members of the same species, and across species [8, 6569].
Initially, transplantation experiments were conducted in the investigation of
infectious theories of cancer according to the Kochs postulates used for
infectious diseases. No transfers of disease to normal recipient cells by tumour
tissue occurred. Subsequently, transplantation of tumours was used to study
the hereditary factors associated with the susceptibility of the recipient animals
to tumour take, and claims were made that this was dominantly inherited
[66]. Later, the effects of immunological reactions to these transplants were
recognised, and it transpired that most reactions appeared to be due to the
recipients reactions to the species-related antigens of the donor, rather than
any reactions to tumour-specific antigens [67]. Other studies were directed at
factors associated with metastasis, which was similar to von Hansemanns feature of capacity of the tumour cells for independent existence, or autonomy (see above). Leo Loeb [68] in 1937 considered that the major determinants of growth of transplanted tumours include immune reactions of the host,
but also that the growth energy or growth momentum of the transplanted
tumour tissue is important. Because growth rate of tumours and degree of dedifferentiation are often related, and rapidly growing tumours may access host
blood vessels faster than the host tissues can react with fibrosis, this may be an
adequate explanation of tumourous greater capacity for independent existence (see above).
Despite this unsatisfactory situation concerning the actual significance of
heterotypic survival, these transplants of tumours provided useful models of
cancer for the study of various aspects of cancer, not the least of which was
anti-cancer therapies. The distinction between degree of autonomy and
susceptible to immunological rejection by the recipient animal could not be
made easily until the advent of the nude mouse in the 1980s [70].
Chemical agents
Although workers in certain occupations had been known to be susceptible to
cancers in the 18th century, the chemical or physical basis of these were not
widely considered. This may have been due in part to the fact that these diseases were still considered to be due to some generalised imbalances of
humours, and thus direct action of these agents on cells were perhaps not
understood to be relevant. Snuff cancer was described in 1761 by John Hill,
chimney-sweeps cancer 1775 by Percival Pott and pipe smokers cancer (of
12
Physical agents
Ultraviolet light was discovered in 1801 by Rittner, who noted the ability of a
component of sunlight beyond violet light to darken silver chloride [76].
Sunlight was suggested to be the cause of sailors cancers by Unna in 1894 and
ultraviolet light was shown to be able to cause skin cancers in white mice in
1928 [8]. Chapter 6 of this volume deals with current issues in ultraviolet carcinogenesis.
X-rays were discovered in 1895 by Roentgen (18451923) [8, 12], and uranium salts were shown to emit gamma rays in 1896 by Becquerel (18521908)
[8, 12]. The former were understood to cause skin cancers as early as 1902 by
Freiben [8, 12] and isotopes taken internally were reported to cause bone cancers in 1925 [8]. Experimental induction of germ-line mutation in Drosophila
by X-rays was demonstrated in 1928 by H.J. Muller [6], and it was established
in the 1930s that irradiation causes chromosomal lesions in cell cultures in
vitro [77]. Chapters 7 and 11 deal with current aspects of radiation-induced
carcinogenesis.
13
Infectious agents
Numerous parasitic theories of neoplasia were proposed from the 19th century on the basis of structures suggested to be these parasites in the cytoplasm
and nuclei of tumour cells (see above). The association of bilharzia and bladder cancer was suggested as early as 1889 [8].
Peyton Roux, in 1911, reported that a tumour of fowls could be due to a
transmissible, filterable agent [8, 12], and subsequently Shope reported that a
filterable agent could transmit papillomata of the skin of rabbits [78].
Subsequent developments in the field showed that oncogenic viruses may be
of either RNA or DNA type [79]. Current aspects of viral oncogenesis in relation to the host genome are discussed in chapter 8 of this volume.
14
15
16
In relation to the mutations and the development of tumours, some chemical carcinogens were found to be germ-line mutagens by many authors [6,
105]. Nevertheless, the idea of somatic mutation as an important direct effect
of carcinogens was resisted especially by Berenblum [106, 107], Foulds [82]
Willis [84] and Burdette [108]. Another example was Earle who, in 1943
[109], reported that normal cells cultured in vitro can change into cells that can
form tumours when they are injected back into the animal of origin. He did so
without mention of mutation, just as later reviewers of this topic into the 1960s
omitted discussion of mutation [110, 111].
Only the discovery of viral oncogenes by Huebner and Todaro in 1968
[112], followed by later documentation of endogenous cellular
oncogenes/growth factors, established mutation as a widely held basis of
tumour formation (see reviews [113115]).
17
sue cells of a particular organ unstable, so that mutation takes place, resulting
in excessive mitosis of that particular cell (resulting in the adenoma).
However, Lockhart-Mummerys view of the number of mutations required
to cause a malignancy is somewhat unclear, because of his statement
Malignancy arises because of a second accident associated with excessive
proliferation. If accident here refers to a mutation, Lockhart-Mummerys
theory is of three hits for malignancy.
Also on the basis of clinical studies, Nichols [122] in 1969, suggested that
the tumours of neurofibromatosis arise by a second (somatic) mutation of the
already mutant locus (the n locus) of predisposed cells. Comings [123] elaborated a general theory of carcinogenesis, and made the same suggestion
that tumours arise by mutation of both copies of diploid pairs of regulatory
genes (i.e. both alleles of one gene Comings used the term gene for allele
in his paper). Applying concepts of recessive oncogenesis (see above),
Knudson ([124, 125] reviewed [126]) has proposed that perhaps only two
mutations are required for carcinogenesis generally.
However, single or even two or three mutations do not explain all the phenomena of more complex tumours, such as carcinoma of the colon. For this
tumour, activating mutations of multiple genes have been proposed. For
example, Vogelstein and co-workers [127, 128] proposed a five-step model
involving a series of oncogenes. Another issue is the importance of sequential
timing of these mutations. Fearon and Vogelstein (1990) [127] were of the definite opinion that Accumulation, rather than order, is most important in carcinogenesis.
More complex models, which go beyond a simple chains of activations,
have been proposed recently [129, 130].
In experimental studies, once pure carcinogens were prepared in the 1930s
(see above), it became possible to study possible synergistic effects of two or
more carcinogens. The latter studies, undertaken especially by Berenblum and,
independently, Mottram [73] showed that for many chemicals, tumour formation required the application of one particular type of chemical (the initiator), before the application of another particular type of chemical (the promoter). Neither chemical alone produced tumours, and no tumours were
caused by the chemicals in the reverse sequence. It was recognised, however,
that some chemicals (complete carcinogens) could have both effects, and in
some cases, the sequence did not matter (co-carcinogenesis). These data led
to the popular two-stage concept of carcinogenesis with initiation and
promotion being necessary phases of tumour formation [73, 74]. At the time,
the mechanism of each of these processes was unclear, but later it was proposed that initiation represented a primary mutation of some particular cancer critical gene [131134], and promotion was probably related to epigenetic phenomena [135].
In the context of these clinical and experimental findings, epidemiological
investigations were carried out in the 1950s and 1960s, to try to confirm the
18
19
Conclusions
The history of the relationships of the morphology of tumour cells and their
cellular genetics has involved numerous contributions from many apparently
separate fields of biology. The broad cellular morphological observations, in
terms of the variability of form, function and behaviour, were established in
the 19th century due to the work of Mller, Virchow, von Hansemann and others. In particular, mitotic and chromosomal lesions noted by von Hansemann,
although virtually ignored at the time, may find support in more recent
karyokinetic studies of chronic myelocytic leukaemia, and some sarcomatous
conditions. The genetic observations of most recent times, however, have
revealed so much previously unsuspected genomic disturbance in tumour cells
that some form of non-mitotic, non-chromosomal instability appears to be
involved. Because all of these processes involve nuclear structures, and appear
to be provokable by carcinogens, this volume has been designed to bring
together chapters which deal with many aspects of these studies, and illuminate the latest aspects of these contemporary issues in cancer research.
Acknowledgements
We are indebted to the National Library of Congress (Washington D.C.) for the gift of a copy of reference [84] and to the staffs of the Barr-Smith Library, University of Adelaide, and the Library of the
University of Leipzig for assistance. Ms Elizabeth Goodwin typed many of the translations, and help
was rendered also by S. Coghlan, A. Coglan and M.-L. Krone.
20
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25
Summary. Nuclear morphometric descriptors such as nuclear size, shape, DNA content and chromatin
organization are used by pathologists as diagnostic markers for cancer [1]. Tumorigenesis involves a
series of poorly understood morphological changes that lead to the development of hyperplasia, dysplasia, in situ carcinoma, invasive carcinoma, and in many instances finally metastatic carcinoma.
Nuclei from different stages of disease progression exhibit changes in shape and the reorganization of
chromatin, which appears to correlate with malignancy [2]. Multistep tumorigenesis is a process that
results from alterations in the function of DNA. These alterations result from stable genetic changes,
including those of tumor suppressor genes, oncogenes and DNA stability genes, and potentially
reversible epigenetic changes, which are modifications in gene function without a change in the DNA
sequence [35]. DNA methylation and histone modifications are two epigenetic mechanisms that are
altered in cancer cells. The impact of genetic (e.g., mutations in Rb and ras family) and epigenetic
alterations with a focus on histone modifications on chromatin structure and function in cancer cells
are reviewed here.
Key words: Chromatin, histones, MAPK, nucleus, ras gene, tumors.
Introduction
Histones are basic proteins that have a vital role in the organization of DNA in
the human cell nucleus. In addition to establishing a hierarchy of chromatin
structures, resulting in compaction of the nuclear DNA about 10,000-fold, the
histones have critical roles in differential packaging of decondensed euchromatin and condensed heterochromatin regions of the genome. Both euchromatin and heterochromatin are organized by the basic repeating structural unit
in chromatin, the nucleosome [6]. The nucleosome core particle consists of a
histone octamer core around which 146 base pairs of DNA are wrapped. The
core histones are arranged as a (H3H4)2 tetramer and two H2A-H2B dimers
positioned on both sides of the tetramer. The core histones have a similar structure with a basic N-terminal domain, a globular domain organized by the histone fold, and a C-terminal tail (Fig. 1). The histone-fold domains of the four
core histones mediate histone-histone and histone-DNA interactions [6].
The nucleosomes are joined by linker DNA, which is of varying length. A
fifth class of histone, the H1 histones or linker histones, binds to the linker
DNA and to core histones. H1 has a tripartite structure consisting of a central
globular core and lysine-rich N- and C-terminal domains (see Fig. 2). The
globular domain binds to one linker DNA strand as it exits or enters the
nucleosome and to nucleosomal DNA near the dyad axis of symmetry of the
26
B. Drobic et al.
K79 M
M P
H3
ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPH
5
10
15
MM
20
Ac
30
25
Ac
35
Ac
Ac
Ac
Ac
Ac
K77 K79
H4
SGRGKGGKGLGKGGAKRHRKVLRDNI
10
Ac Ac
15
Ac
20
25
Ac
K115 Ac
K122 Ac
IRGERA
Ac
K91
R92 M
K59 M
P
SGRGKQGGKARAKAKTRSSRAGLQ
5
Ac
15
10
Ac
20
H2A
Ac
15
Ac Ac
125 127
PEPAKSAPAPKKGSKKAVTKAQKKDGKK
10
119
Ac Ac
K43 M
mm
AVLLPKKTESHHKAKGK
20
Ac
25
K85 Ac
R99 M
H2B
K AVSEGTKAVTKYTSSK
108
116
Ac
Ac
120
Figure 1. Core histone modifications. The N-terminal and in some cases C-terminal amino acid
sequences of human histone are shown. The modifications include methylation (M), acetylation (Ac),
phosphorylation (P), ubiquitination (U), and biotinylation (B). Methylation sites that are uncertain are
denoted as (m).
nucleosome [7]. H1 and the histone tails stabilize the higher order compaction
of chromatin.
The core histones undergo a wide range of post-synthetic modifications,
most of which are reversible. These modifications include acetylation, phosphorylation, methylation, ubiquitination, poly ADP ribosylation, and biotinylation [8, 9] (Fig. 1). The majority of modified amino acids reside in the tail
domains, but there is an increasing awareness of modified residues occurring
in the histone-fold domains [10]. The amino-terminal tails of the four core histones play an important role in chromatin compaction. These tails protrude
from the nucleosome, with that of H3 protruding the farthest. The tails of H3,
being the longest and positioned in such a way as to allow several contacts
UV-B
27
EGF
RTKs
GDP
GTP
Y-P
Grb2
MEKK3
MEKK6
Sos
Ras
Raf
RasGRP
PKC
DAG
TPA
MEK
p38/SAPK2
Mos
ERK
ERK
p38/SAPK2
NUCLEUS
Msk1
CBP
SWI/SNF
Complex
cyclin E- Cdk2
Histone H1
PIC
Transcription
Factor
P
N E E
E S
2
7 15 17
HDAC
Complex
P P
P P
S
T T 169 S
T H1-3 E
151
134
35
114
185
222
Ac
P Ac K Ac Ac
Ac
P
K 18 K
S
N K 14
23 K S
10
9
2728
H3
Figure 2. MAPK signal transduction pathways and the modification of chromatin. The Ras-MAPK
pathway is activated by EGF and TPA. TPA acts through PKC and/or RasGRP. UV-B activates both
the Ras-MAPK and the p38 kinase pathways. Inserts: Left panel, The sites of phosphorylation of H1
subtype H1.3 are located on the N- and C-terminal tails. Right panel, The H3 phosphorylation sites
are nestled in a region of acetylation (P, phosphorylation, Ac acetylation). CBP, CREB binding protein (histone acetyltransferase); Cdk2, cyclin-dependent kinase 2; DAG, diacylglycerol; GDP, guanosine diphosphate; GTP, guanosine 5'-triphosphate; HDAC complex, histone deacetylase complex;
PIC, preinitiation complex; RasGRP, Ras guanyl nucleotide-releasing protein; RTKs, receptor tyrosine kinases; SOS, Son of Sevenless; TPA, 12-O tetradecanoylphorbol-13-acetate.
with linker DNA, are crucial for the formation of higher order chromatin fibers
[11]. Acetylation and phosphorylation of the N-terminal tail of H3 promotes
28
B. Drobic et al.
29
through direct or indirect interaction with various promoter-binding transcription factors including CREB, nuclear hormone receptors and oncoprotein activators such as c-Fos, c-Jun and c-Myb. The broad spectrum of p300/CBP interacting proteins provides a general mechanism for integration of several signaling and transcription-response pathways that p300 and CBP modulate [18].
Defects in the expression and function of HATs have been reported in cancer cells. The mis-targeting, ill-timed activation or irregular increase in activity of HATs can lead to expression of genes that allow tumorigenesis [19].
Chromosomal translocations, deletions and mutations that affect genes encoding potent HATs have been associated to the genesis of several malignant conditions, particularly hematological disorders. The following section discusses
malignancies that result from aberrant HATs and subsequent consequences to
chromatin structure and function.
30
B. Drobic et al.
Rubenstein-Taybi
syndrome
CBP
mutated
CBP
Somatic
translocation
MLL-CBP
MORF-CBP
CBP-MORF
MOZ-CBP ; MOZ-TIF2
CBP-MOZ
Hematological malignancies
PLZF-RARalpha
PML-RARalpha
N-CoR (SMRT)
RbAP48
AML1/ETO
SAP30
mSin3
HDAC1, 2
LAZ3/ BCL6
non-Hodgkin
Lymphomas
Figure 3. Somatic translocations and mis-targeting of HATs and HDACs. Somatic translocations and
mutations involving the CBP gene can interfere with the normal function and targeting of CBP, a
potent histone acetyltransferase and coactivator, resulting in a variety of hematological malignancies.
Mis-targeting of HDAC complexes, which deacetylate histones and transcription factors and are corepressors, by fusion proteins arising from somatic translocations, can interfere with genetic programs,
resulting in a variety of hematological malignancies.
generating a chimeric protein. This MOZ-TIF2 protein can bind CBP and
mimic the MOZ-CBP fusion function that has been demonstrated to be necessary for transformation and leukemogenesis in vitro and in vivo [26].
In many cases, the development of leukemias can arise during recession
after primary treatment. These disorders are not only limited to AML, they are
also found in chronic myeloid leukemia (CLL) and myelodysplastic syndrome
31
32
B. Drobic et al.
to promoter coactivators and factors as well as stimulating chromatin remodeling. Furthermore, they are integral in the positive or negative coordination of
cell signaling pathways, growth, apoptosis, differentiation and embryogenesis.
Acetylation and deacetylation of histones and proteins not only affect local
areas of chromatin but also bulk conformations, dictating activity and threedimensional interactions of transcription factors involved [31]. The functional
availability of HATs alongside histone deacetylases (HDACs) have an effect
on which subsets of genes, a particular developmental stage or a cellular
process, are expressed at a given time, permitting the active on and inactive
off states. Formation of HAT fusion proteins from translocations and nonfunctional HATs from mutations generate aberrant acetyltransferases that
influence the balance of gene activation and repression, and alter transcriptional regulation, leading to genesis of cancers. Different HATs may function
differently according to their chromosomal contexts. Depending on the
domains retained or left intact after translocations and mutations (i.e., bromodomains, acetyltransferase domains), their DNA binding capabilities, factor binding domains and modifying potential are compromised allowing shifts
in the equilibrium of gene expression [32]. In the leukemic disorders mentioned above, the HAT fusion proteins involved potentially recruit anomalous
transcription factors that elevate HDAC complexes at transcription sites and
prompt the block in hematopoietic differentiation characteristic of the malignancies [33]. In certain scenarios, some investigations point to the potential
role of HATs as tumor suppressors, and this claim has been verified in mice
models but continues to be studied in humans [23].
33
HDACs in cancer
HDACs are chromatin-modifying enzymes that remove acetyl groups from the
N-terminal tails of histones. Deacetylation of histones is associated with
repression of transcriptional activity, thus HDACs are co-repressors of transcription. In addition to the deacetylation of histones, HDACs are responsible
for the deacetylation of non-histone proteins including E2F, MyoD, p53,
Hsp90, GATA-1 and tubulin [3740].
Mammalian HDACs belong to one of three families. The first class, consisting of HDACs 1, 2, 3, and 8, is defined by its relationship to the yeast deacetylase Rpd3. Class II HDACs are larger proteins related to yeast Hda1 and include
HDACs 47, 9 and 10. HDAC 11 shares properties with both class I and II
HDACs, and thus tends not to be classified. The third class of HDACs is often
referred to as the Sir2 family, and encompasses those HDACs with homology
to yeast Sir2. These enzymes require nicotinamide adenine dinucleotide to
function [37]. HDACs do not bind DNA directly, and instead are tethered to target sites by mediating factors present in various protein complexes [41].
HDACs are members of large multi-protein complexes. Class I HDACs are
found in a variety of protein complexes including Sin3, NuRD, and Co-REST
and interact with factors such as Sp1, YY1, and retinoblastoma (Rb) binding
protein-1 [38]. Additionally, they are found in complexes with nuclear receptor co-repressor (N-CoR) and silencing mediator for retinoic acid receptor and
thyroid hormone receptor (SMRT), which can contain other HDACs such as
HDACs 4, 5 and 7 [38].
Expression of HDACs does not appear to be altered in cancer [38].
However, HDACs are found in complexes with well-known tumor suppressors
and oncogenes, such as Rb and Mad; in a diseased state inclusion of HDACs
in these complexes could lead to abnormal recruitment of HDACs and aberrant
gene expression [42, 43].
Translocation and point mutation events in non-Hodgkins lymphoma often
result in overexpression of the BCL-6 oncogene (Fig. 3). The product of this
oncogene has been linked to the regulation of B cell proliferation and is able
to recruit HDAC activity through interactions with N-CoR and SMRT, thus
aberrant repression activities may be involved in this cancer [44, 45].
The mis-targeting of HDACs mediated by recruitment by fusion proteins is
evident in many hematological cancers (Fig. 3). Patients with acute promyelocytic leukemia often have translocations in which RAR is fused to PML or
PLZF [38, 45, 46]. This results in an oncoprotein able to recruit HDAC activity through N-CoR and SMRT, and is thought to lead to selective transcriptional repression [38, 45]. This prevents differentiation and results in the disproportionate proliferation of cells seen in these patients [38]. Although acute
34
B. Drobic et al.
35
36
B. Drobic et al.
37
38
B. Drobic et al.
39
[98]. Recently, a potent and selective inhibitor of all three human Aurora
kinases, VX-680 has been shown to decrease H3 phosphorylation at S10 in
the MCF-7 cell line as well as suppress tumor growth in vivo [104]. This finding implicates Aurora kinases in the processes leading to malignant transformation and carcinogenesis, and shows promise for a new approach for anticancer therapy since VX-680 was able to induce regression of a range of
human tumor types. The VX-680 inhibitor is progressing into clinical development [104].
40
B. Drobic et al.
The transcription factor E2F has a pivotal role in regulating the expression
of S phase-specific genes. Repression of these genes is through the Rb protein,
which binds to E2F. Rb recruits histone methyltransferases and histone
deacetylases to repress gene expression [111]. Rb bound to E2F recruits
SUV39H1 to the S phase-specific gene promoter (e.g., cyclin E), which in turn
recruits HP1. Rb phosphorylation abolishes its association with histone
deacetylase and histone H3 K9 methyltransferase. Thus, inactivation of this
tumor suppressor gene will result in the deregulation of Rb-guided epigenetic
pathways.
EZH2 is an H3 K27 histone methyltransferase that is a component of the
EED (embryonic ectoderm development)-EZH2 complex. Sequence-specific
DNA binding proteins Pho and Pho1 bind to the Polycomb response element
and recruit the EED-EZH2 complex, resulting in the methylation of H3 K27.
Methylated H3 K27 recruits Polycomb group (PcG) proteins and the
Polycomb repressive complex 1 to silence specific genes. PcG proteins maintain the silenced state of homeotic genes. EZH2 is overexpressed in prostate
and breast cancer cells, and this deregulation of EZH2 may result in alteration
of chromatin structure and deregulation of the downstream targets of the EEDEZH2 complex [112]. SMYD3 (SET- and MYND-domain containing protein
3) is an H3 K4 histone methyltransferase and sequence-specific DNA binding
protein that is overexpressed in colorectal carcinomas and hepatocellular carcinomas. Suppression of SMYD3 expression inhibited the growth of colorectal carcinoma and hepatocellular carcinoma cells. SMYD3 is involved in the
activation of oncogenes and genes associated with cell-cycle regulation [113].
41
Concluding comments
A great deal has been learned about the interplay between genetic and epigenetic processes in cancer. Pharmacological approaches using DNA methylation inhibitors, HDAC inhibitors or combinations of both to alter epigenetic
42
B. Drobic et al.
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49
Summary. Telomeres which protect the individual chromosomes from disintegration, end-to-end
fusion and maintain the genomic integrity during the somatic cell divisions play an important role in
cellular aging. Aging and cancer development are linked with each other because cancer is considered
a group of complex genetic diseases that develop in old cells and, in both, telomere attrition is
involved. Numeric chromosome imbalance also known as aneuploidy is the hallmark of most solid
tumors, whether spontaneous or induced by carcinogens. We provide evidence in support of the
hypothesis that telomere attrition is the earliest genetic alteration responsible for the induction of aneuploidy. Dysfunctional telomeres are highly recombinogenic leading to the formation of dicentric
chromosomes. During cell divisions, such complex chromosome alterations undergo breakage fusion
bridge cycles and may lead to loss of heterozygosity (LOH) and gene amplification. Furthermore, we
have provided evidence in support of the hypothesis that all types of cancer originate in the organ- or
tissue-specific stem cells present in a particular organ. Cancer cells and stem cells share many characteristics, such as, self-renewal, migration, and differentiation. Metaphases with abnormal genetic
constitution present in the lymphocytes of cancer patients and in some of their asymptomatic family
members may have been derived from the organ-specific stem cells. In addition, evidence and discussion has been presented for the existence of cancer-specific stem cells. Successful treatment of cancer, therefore, should be directed towards these cancer stem cells.
Key words: Aneuploidy, dysfunctional telomeres, fluorescence in situ hybridization, genetic instability, stem cell.
Introduction
Cancer is not a single disease. It comprises a group of complex genetic diseases of uncontrolled cell division and is also one of the characteristics of aged
cells. Aging and cancer development, therefore, are linked with each other.
Most cancers are caused by chromosome and gene mutations that accumulate
in a specific tissue or organ during the cellular aging. Genetic instability is
exhibited by aging cells, both in vitro and in vivo, in the form of numerical
(aneuploidy) and structural chromosomal alterations (translocation, deletion,
amplification and inversion) [1]. More than 90% cancers are caused by exposure to environmental carcinogens. The insult inflicted by carcinogens are first
faced by the termini of chromosomes, called telomeres, which are attached to
the inner nuclear wall. Among their many functions, telomeres determine the
domain and stability of individual chromosomes within the nucleus and serve
as guardian of the genome [2]. Functional telomeres are essential for the normal segregation and maintenance of chromosomes during mitotic and meiotic
50
divisions [3]. More recent information has shown that the maintenance of the
telomere depends on interactions with an enzyme, telomerase, with telomeric
proteins, and with some still undiscovered factors regulating the telomeric
functions. Dysfunctional telomeres support the survival of aneuploid cells, a
characteristic of many human and murine cancers.
The single unifying cellular mechanism that influences both aging and cancer development is the telomere dynamics [4, 5]. Cancer cells stabilize their
telomere repeats either by a telomerase-dependent pathway or by the telomereindependent or alternate lengthening of telomere (ALT) pathway [6]. Unlike
the murine somatic cells, human somatic cells lack or have diminished telomerase activity. This major difference in human and murine cells can easily
explain why it is difficult to transform normal human cells, but easy to transform mouse cells, in vitro. Mouse and human somatic cells differ in many
other respects, for example, in their responses to oxidative stress [7].
Most hematological neoplasms are known to arise from stem cells, whereas epithelial malignancies are generally considered to originate in differentiated organ- or tissue-specific somatic cells. Recently, a hypothesis was proposed
that not only the hematological malignancies but also all solid tumors originate
in organ- or tissue-specific stem cells or their immediate progeny (progenitor)
cells [5, 8, 9]. This hypothesis is based mainly on two recent observations: the
presence of stem cells in each and every human organ or tissue [1012], and
the presence of poorly differentiated cancer cells signaling a poor prognosis
for patients. Since stem cells, especially embryonic stem (ES) cells, have the
potential to differentiate into all three major tissue lineages, ectoderm, mesoderm and endoderm and their derivative organs [1315], it is not unreasonable
to propose that organ- or tissue-specific cancers originate in the organ- or tissue-specific stem cells [2, 8, 9]. Human ES cells have four unique characteristics: (1) self renewal, (2) differentiation into other cell types, (3) migration in
vivo, and (4) cell death under unfavorable conditions [16, 17].
The purpose of this chapter is to discuss, in brief, the relationships between
aneuploidy, stem cells and cancer development. That stem cells and aneuploidy play crucial roles in cancer development and metastasis will also be discussed in some detail under separate subtitles.
51
alterations, including translocations, amplifications or deletions of certain segments. Because of these inherent characteristics, most cancer cells have
genomic heterogeneity. In other words, each cell of a given tumor has its own
chromosomal features except for certain specific common marker chromosomes. Inherent chromosomal instability, which can be due to the telomere
erosion, plays a major role in causation of most cancers [2224].
As early as 1890, von Hansemann [25] first suggested that cancer originates
in an alteration in the genetic content of a cell. Later, Theodore Boveri [18,
26], while working on chromosomes of Ascaris and Paracentrotus sea urchin
eggs, proposed his famous theory of malignancy. According to Boveris theory, the neoplastic properties of a cancer are the consequence of chromosomal
aberrations, and a malignant transformation results from the clonal expansion
of a single genetically altered somatic cell. In our earlier publications we asked
[9], among several questions, Could this somatic cell undergoing neoplastic
transformation be an organ- or tissue-specific stem cell?. Boveri, who first
introduced the term centrosome [27], postulated that cancer cells are formed
due to abnormal chromosome distribution originating as the consequence of
multipolar mitosis, caused by the formation of multiple centrosomes. Recent
molecular studies have provided strong evidence in support of Boveris theory
of malignancy [2837]. The centrosome is an important actor of the cell division machinery. Its malfunction may cause abnormal chromosome segregation
or no segregation at all, resulting in aneuploidy, the hallmark of cancers
[3746].
The original definition of aneuploidy was deviation of one or more chromosomes from the haploid (1n) state [47], which in the human is 23 chromosomes, in mouse 20 chromosomes, in rat 21 chromosomes, in cat 19 chromosomes, in Syrian hamster 22 chromosomes and in Chinese hamster 11 chromosomes. Presence of an extra copy (trisomy) or the absence of a chromosome
(monosomy) is generally considered an example of aneuploidy. However,
recently, the term aneuploidy has been used even for the presence of an extra
segment or the deletion of a segment from a chromosome without a gain or
loss in the total chromosome/centromere numbers. Currently, this term is used
ambiguously to encompass all kinds of structural and numerical chromosome
instabilities. Is aneuploidy that is caused by a dysfunctional centrosome, an
early genetic change that initiates cancer formation? Some researchers, including the present authors, favor the opinion that aneuploidy indeed is the first
causal step in tumor development. According to the strict classical definition
of aneuploidy, primary leukemia and lymphomas, which do not show numerical anomalies but are characterized by their specific structural alterations,
including reciprocal translocations and inversions, should not be considered
aneuploid [47]. Presence of t(9;22) in chronic myelogenous leukemia, and
t(8;14) and t(14;18) in different lymphomas are typical examples of human
cancers. Only in the blast phase or at advance stage of the disease have numerical (aneuploidy) and additional structural anomalies been reported.
Practically all cancer types, hematological and solid, contain structural anom-
52
alies in their genomes [2, 5, 45, 46, 48, 49]. That structural chromosome anomalies precede aneuploidy has even been reported in immortal fibroblast cultures of Li-Fraumani syndromes [50]. The important question is: which comes
first, the numerical alterations (aneuploidy) or the structural modifications in
cancer initiation? The obvious reply is: structural alteration due to telomere
erosion comes first in the multistage carcinogenic process (Fig. 1).
Figure 1. Pathways for apoptosis, aneuploidy and cancer initiation. Reprinted with permission and
modified after [9].
53
54
first suggested that diploid human fibroblasts have a limited number of replication in culture, after which they stop dividing [63], and reach mortality stage
1 (M1) [64]. However, viral oncogenes, e.g., AgTsv40, may transform such
cells even with critically shortened telomeres. Such cells may enter another
mortality stage, stage 2 (M2), and in the senescent stage may remain metabolically active but without undergoing DNA synthesis. These cells with drastically changed morphology become large and express -galactosidase activity
[65]. Replicative senescence is also a genetically dominant phenotype [66].
The question here is: What factor(s) are responsible for driving primary
somatic cells to enter replicative senescence? One of the answers lies in telomere erosion and the absence of telomerase. In mTERC/ mutant mice, telomere attrition has been shown to cause genomic instability, progressive infertility and even the induction of epithelial malignancies in late generation animals
[6769]. Primary murine embryonic fibroblasts (MEFs) from the mTERC/
mouse were used to study the mechanism of dysfunctional telomeres and a
number of telomere-associated proteins (Tab. 1). These mice are telomerase
null because they lack the gene that encodes for telomerase RNA. MEFs
derived from such mutant mice, have reduced ability to immortalize sponta-
55
Interaction
Functions
at telomeres
Chromosome
localization
References
Telomerase
hTERT
hTERC
With T2AG3
overhang,
Telomerase
Telomere elongation
RNA subunit
5p15
[70]
3q26
[71]
Length maintenance
Negative length
regulator (dependent
on telomerase)
Negative length
regulator (independent
of telomerase)
Positive length regulator
Positive length regulator
Length regulator
Telomerase inhibitor
7
8q13
[72]
[73]
16q22
[74]
8p23/10q23
14q11
16
8p23
[75, 76]
[77, 78]
[79]
[80]
2q35/22q11
[81, 82]
Capping of telomere
T-loop sterilization
8q11
5q31
[83]
[84]
Recombination in
ALT cells
Telomere structure
maintenance
Telomere structure
15q15
[85]
8p12/15q26
[86, 87]
17p13
[88, 89]
Telomere chromatin
structure
11q22
[90, 91]
Specific proteins
Pot1
With G-rich strand
TRF1
T loops
TRF2
T loops
TANK1/2
TIN2
RAP1
PINX1
With telomere
With TRF1
With TRF2
With TRF1/Pin2
Nonspecific proteins
Ku70/Ku86
With telomeric
repeats
DNA-PKCA With telomeric DNA
Rad50 NSB/ With TRF2
MRE11
Rad51
In ALT cells
WRN/BLM
p53
With single-strand
T-loop
With TRF1
ATM
*
neously in culture. These findings indicate that telomere attrition limits the
replicative potential of MEF in vitro. A typical metaphase spread from a fifth
generation (G5) mTERC / mouse fibroblast revealing a chromosome fusion
product and 41 chromosomal arms is shown in Figure 3.
56
Figure 3. A typical metaphase spread from a fifth generation (G5) mTERC/ mouse fibroblast showing a fusion product (arrow) and 41 chromosomal arms, an example of aneuploidy.
Proliferate indefinitely
Self renewal by similar signals
Are heterogeneous, with different phenotypes
Migrate
Express telomerase
Have extended telomere repeats
Differentiate
Can be tissue-specific
Undergo organogenesis
Undergo apoptosis
Cancer cells
Proliferate indefinitely
Self-renewal by similar signals
Are heterogeneous, with different phenotypes
May metastasize (migrate)
Express telomerase
Metastatic cells have extended telomeric
repeats
Differentiate
Can be tissue-specific
Undergo limited organogenesis
Undergo apoptosis
57
58
further support [102, 103]. Recently, Reya and associates [104] and Kondo et
al. [105] have brought this hypothesis into the limelight by presenting molecular evidence in support of this concept. The later group has isolated cancer
stem cells, as a small side population (SP), even from the long-term established tumor cell lines including C6 glioma, MCF-7 breast cancer, B104 neuroblastoma and HeLa cell lines [105].
Evidence that cancer develops from stem cells, not differentiated somatic
cells
There is plenty of evidence to support the statement that dividing (cycling)
cells are more susceptible than the quiescent (non-dividing) cells to accumulate mutations when challenged by the environmental mutagens. Most differentiated somatic cells perform their functions in an organ- and tissue-specific
environment and are then replaced by proliferation of specialized stem or progenitor cells [106]. During wound healing and in disease conditions, organ- or
tissue-specific stem cells or their progenitors divide, migrate and help in repair.
The stem or progenitor cells form one cell that remains a stem cell and another cell that differentiates into the specialized function-oriented mature cell.
Fully differentiated somatic cells perform their functions and then undergo
apoptosis (Fig. 2). They are replaced by the newly differentiated somatic cells
in the organ. Since cancer cells originate by the interactions of environmental
carcinogens and the genetic make-up of the person, it is worth considering that
stem cells, being poorly differentiated somatic cells with the phenotypes of
their progenitors, also having telomerase activity, are the target of neoplastic
transformation [104, 105, 107109]. In a recent book chapter, Sell [110] speculated that most carcinomas and adenocarcinomas originate in the organ-specific stem cells. Of course, most hematological malignancies have their origin
in stem cells. In fact, cancer originates from an imbalance between the rate of
cell division and the rate of differentiation and cell death. Also, maturation
arrest of stem cell differentiation has been considered a common pathway for
the origin of teratocarcinoma and epithelial malignancies [111]. Inherently, the
stem cells are characterized by substantial longevity, replication potential and
telomerase activity. In addition, they are also known to have much longer mean
telomere length, which is a survival factor for the cells [2].
There is mounting evidence to suggest that practically every organ has its
own stem cell reservoir. Although these specialized cells are present in a small
pocket in the organ-specific environment, their potential to replace damaged
cells is controlled by the cell requirement. This is analogous to the Seed and
Soil hypothesis proposed by Paget [112] for metastatic breast cancer cells.
The presence of organ-specific stem cells has been reported in lung, mammary gland, liver, pancreas, kidney, heart, retina, muscle, skin and brain of mammalian species including human. Even during angiogenesis, angioblasts,
59
which are the precursors of endothelial cells, act as progenitor cells with several stem cell characteristics.
Are circulating abnormal metaphases derived from organ- or tissuespecific stem cells of cancer patients?
The initial (primary) genetic (chromosomal) alterations associated with cancer
development do not necessarily occur in every somatic cell [113]. It has been
proposed that predisposed individuals inherit susceptibility traits that makes
their specific chromosomes prone to breaking at a particular loci [49]. The
chromosomes of a cancer-predisposed individual may undergo specific alterations at relatively low frequency in all tissues, including peripheral blood
lymphocytes (PBL). Could this trait be the attrition of telomere in those chromosomes? Clastogens that are able to induce chromosome-specific aberrations
have been described previously in a separate report [2].
Specific cytogenetic alterations were first identified in PBL as being associated with chromosome 13 in retinoblastoma, with chromosome 11 in Wilms
tumor, with chromosome 3 in renal cell carcinoma, with chromosomes 2, 5
and 11 in colorectal cancer, with chromosomes 1, 6 and 9 in melanoma, with
chromosome 1 in endometrial cancer, and chromosomes 5, 7, 8, 10, and 16 in
prostate cancer (see reviews [2, 49]). Subsequent reports have shown specific
chromosomal changes in a small percentage (13%) of phytohemagglutininstimulated lymphocytic metaphases of various epithelial malignancies, including breast, lung, prostate and other adenocarcinomas [114, 115]. These circulating aberrant metaphases are not cancer cells because: (1) they have mainly
chromatid breaks, simple translocations or deletions; (2) the patients whose
tumor cells and PBL were both analyzed cytogenetically had mainly chromatid breaks in the PBL but stable marker chromosomes involving the same
chromosomes in their tumor cells [116]; and (3) such types of chromosomal
alterations are present even in the PBLs of some asymptomatic family members [117]. Could these rare abnormal metaphases be coming from the tissueor organ-specific stem cells? Undoubtedly, more research in future is required
to substantiate this hypothesis.
Conclusion
In conclusion, we provide evidence for the proposal that telomere attrition
(dysfunction) is the earliest genetic alteration in the organ- or tissue-specific
stem cells, which then is responsible for aneuploidy and is the cause of all
types of cancer. Metaphases with abnormal genetic constitutions present in the
PBL of cancer patients and some of their asymptomatic family members might
be derived from the organ-specific stem cells. Successful treatment of cancer
by different modalities and chemoprevention strategies should be directed
60
towards these cancer stem cells. It would be most rewarding to develop isolation procedures for these organ-specific stem cells for their further biological
characterization and to elucidate the mechanism of proliferation, progression
and metastasis of cancer.
Acknowledgements
We would like to thank Amanda Weppler and Gunjan P. Multani for preparing the figures and Burr
and Cynthia Furlong for their editorial comments. This work was supported in part by the institutional T.C. Hsu Molecular Cytogenetic Core established in the Department of Molecular Genetics. Our
apology to all those investigators whose work could not be cited here due to constrain of space.
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65
Abstract. Most genotoxic organic carcinogens require metabolic activation to exert their detrimental
effects. The present review summarizes the mechanisms of how organic carcinogens are bioactivated
into DNA-reactive descendants. Beginning with the history of discovery of some important human
organic carcinogens, the text guides through the development of the knowledge on their molecular
mode of action that has grown over the past decades. Some of the most important molecular mechanisms in chemical carcinogenesis, the role of the enzymes involved in bioactivation, the target gene
structures of some ultimate carcinogenic metabolites, and implications for human cancer risk assessment are discussed.
Key words: Aromatic amines, carcinogen-DNA adducts, cytochrome P450, metabolism, mutation
profiles, phase II enzyme activation, polycyclic aromatic hydrocarbons.
Introduction
Epidemiological evidence based on geographic and temporal variations in cancer incidences and studies of migrant populations suggest that environmental
exposures have a substantial impact on the causation of human cancer [1].
These studies led to the conclusion that the majority of cancer deaths in
Western industrial countries are attributable to exogenous factors such as
tobacco, diet, infections, and occupational exposures. The notion that the environment has the principal role in the causation of sporadic cancer is also supported by analyses of cancer cases in cohorts of twins [2] and by analyses of
family-cancer databases [3]. In either case there is strong evidence that the
influence of nonshared environmental factors predominates.
There is no doubt that a wide range of organic chemicals (as pure compounds or present in mixtures) are carcinogenic in humans [4]. Examples from
the list of known or suspected human chemical carcinogens are aromatic and
heterocyclic amines or amides (e.g., 2-naphthylamine, 2-NA; 2-acetylaminofluorene, 2-AAF; 2-amino-3-methylimidazo[4,5-f]quinoline, IQ), halogenated
and unsubstituted olefines (e.g., tetra- or perchloroethylene, PER), halogenated paraffins (e.g., 1,2-dibromoethane, 1,2-DBE), N-nitroso compounds (e.g.,
N-nitrosodimethylamine, NDMA; 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone, NNK), benzene and polycyclic aromatic compounds (e.g.,
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benzo[a]pyrene, B[a]P; 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD), estrogen-receptor agonists or antagonists with residual agonistic effects (e.g.,
diethylstilbestrol, DES; tamoxifen), wood dust (e.g., from oak and beech), natural compounds (e.g., aflatoxins, ochratoxin A, OTA; pyrrolizidine alkaloids),
and so forth. Some selected structures are shown in Figure 1.
Figure 1. Some examples of organic chemicals that are known or reasonably anticipated to be carcinogenic in humans.
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that this particular occupation was associated with increased rates of cancer in
the urinary bladder, subsequently referred to as aniline cancers. In 1898, the
German internist Otto Michael Ludwig Leichtenstern (18451900) considered
2-NA most likely to be involved in human bladder tumorigenesis [10].
Until the dawn of the 20th century, physicians were only able to collect the
unexpected outcome of an undesigned and undesirable grand scale (occupational exposure) experiment based on the rise of industrialization. By the year
1907 it was officially recognized by the Workmens Compensation Act of
Great Britain that epidermal cancer can be caused by pitch, tar or tarry compounds [11]. The imperative next step was that of experimental reproduction
of cancer. After many failures to reproduce the known human outcome in laboratory animals, the Japanese pathologist Katsusaburo Yamagiwa
(18631930) and his assistant Ichikawa successfully produced malignant
tumors through application of a chemical mixture (coal tar) to the ear of rabbits [12]. While epithelial proliferation could be chemically induced already
some years earlier [13, 14], this experiment produced undoubted malignant
epithelial cancer for the first time. It was then experimentally and epidemiologically confirmed that tar and soot is carcinogenic in the skin of mice and in
humans exposed at work place, respectively [1517]. After chemical synthesis
routes for pure higher molecular polycyclic aromatic hydrocarbons (PAHs)
had been first described, Sir Ernest Laurence Kennaway (18811958) and his
colleagues at the Royal Cancer Hospital in London successfully proved that
single PAHs such as 1,2:5,6-dibenzanthracene (dibenz[a,h]anthracene,
DB[a,h]A) and others are tumorigenic [18, 19]. As an indicator assay, they
applied the mouse skin bioassay, which had been introduced to chemical cancer research some years earlier [20]. In 1933, Cook, Hewett and Hieger from
the Cancer Hospital were successful in isolating the carcinogenic principle
out of coal tar pitch [21]. It turned out to be another PAH, the pentacyclic
3,4-benzpyrene (B[a]P), which nowadays is one of the most investigated carcinogens ever and a standard compound used in many cancer experiments as a
positive control (Fig. 1).
Studies with aromatic amines, aminoazo dyes and related compounds supplemented the large volume of experimental data on the carcinogenicity of
industrial chemicals that had been released in high amounts during this time.
The incriminated aniline itself failed to produce tumors in the urinary bladder
of rabbits, as did other aromatic amines such as 2-NA [22]. Later, bladder
papillomas and carcinomas were successfully induced in dogs by gavage or
dermal application of 2-NA [23] an experiment that supported the early prediction from Leichtenstern. In the meantime aminoazo dyes, such as
o-aminoazotoluene (reduction product of Scarlet Red) [24] and 4-dimethylaminoazobenzene (DAB, butter yellow; Fig. 1), were shown to be tumorigenic in rat liver [25]. Wilson et al. [26] reported on the tumorigenicity of
2-AAF in bladder, liver, and various other organs of rats. 2-AAF is an arylamide which was intended to be used as a pesticide, but has been later introduced as a model compound in experimental liver cancer research (Fig. 1).
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With the beginning of the 1940s, a huge amount of experimental data on the
bioactivity of pure and structurally defined organic compounds present in the
industrial environment had been collected.
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Figure 3. Bioactivation of aromatic amines or amides (AA) towards ultimate DNA-reactive sulfate or
acetoxy esters. The ultimate electrophiles and major DNA binding products are exemplified in the
case of 2-AAF. 2-AAF, 2-acetylaminofluorene; CYP, cytochrome P450-dependent monooxygenase;
dG, 2'-deoxyguanosine; SULT, sulfotransferase; NAT, N-acetyltransferase; The arylnitrenium ion is a
putative reactive intermediate. The grey arrows point to the position of a nucleophile attack of DNA,
protein, or GSH. See text and Figure 2 for further explanations.
detoxification of activated metabolites such as epoxides or dihydrodiol epoxides (e.g., from PAHs) has been widely acknowledged since then, more recent
evidence points to an additional but detrimental role of GST enzymes in the
activation of certain industrial chemicals from the classes of haloalkanes (e.g.,
1,2-DBE) and haloalkenes (e.g., PER; see Fig. 1). These compounds are likely to be human carcinogens due to similarities between susceptible animals
and humans in bioactivation [4]. In the case of PER, there is also some limited evidence from cohort studies of laundry and dry-cleaning workers, among
whom a higher than normal occurrence of non-Hodgkins lymphoma,
esophageal and cervical cancer was found [4]. Both groups of organic carcinogens are bioactivated into genotoxic GSH conjugates. While dihaloalkanes
may undergo GST-catalyzed conversion into DNA-binding GSH half mustard and GSH episulfonium electrophiles (Fig. 4) [57], haloalkene-GSH conjugates can be further converted via a kidney-specific cysteine conjugate
-lyase-dependent pathway. After release of the terminal amino acids -Glu
and Gly, lysis of the -bond in the remaining Cys adduct may eventually lead
to the generation of electrophilic and toxic thioketenes, which are capable of
binding to macromolecules in this tissue [58].
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Figure 5. Genotoxic and nongenotoxic modes of action of organic carcinogens. Chemical carcinogens
directly or indirectly affect the regulation and expression of genes involved in cell cycle control, DNA
repair, cell differentiation, or cell death. DNA damage- or receptor-induced alterations in cellular signal transduction processes may lead to the loss of growth control and to genome instability. AHR,
arylhydrocarbon receptor (agonists: TCDD, PAHs, PCBs); ER, estrogen hormone receptor (agonists:
estrogen, diethylstilbestrol; ER antagonist tamoxifen with residual agonistic effects). See text for further explanations.
lung, adrenal cortex, skin, lymph nodes etc. [60]. In addition, this compound
is also known as a potent tumor promotor in liver and skin in the two-stage initiation-promotion models for tumorigenesis (see below). TCDD is the
strongest agonist of the arylhydrocarbon receptor (AHR) [61], an ubiquitous
cytosolic protein originally discovered in connection to the inducibility of the
microsomal enzyme activity designated as aryl hydrocarbon hydroxylase
(AHH). The AHH activity (now identified as being identical to certain alleles of CYP1A1 and CYP1B1) could be induced in vitro and in vivo by planar
PAHs such as B[a]P and others [62, 63]. This cellular response is mediated by
AHR [64], a member protein of the bHLH-PAS family of transcription factors
characterized by an N-terminal basic helix-loop-helix DNA binding domain
and a homology region originally described in the transcription factors PER,
ARNT, and SIM (PAS domain) [65]. Upon binding to one of its ligands, the
complex translocates into the nuclear compartment, heterodimerizes with the
AHR nuclear translocator (ARNT), and then binds to specific arylhydrocarbon- or xenobiotic-responsive elements (XRE = 5'-TNGCGTG-3'). XRE
sequences are enhancer elements upstream of genomic target genes encoding
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In the two-stage mouse skin bioassay, which had been established in 1947
[84], strong carcinogenic PAHs act as complete carcinogens when repeatedly applied over time [85]. Such compounds are capable of inducing both
somatic mutations in critical target genes through DNA binding (initiation
phase) and subsequent outgrowth of cells that are irreversibly transformed
(promotion phase). This two-stage concept goes back to Friedewald and
Rous [86] who were the first to distinguish between the initiating and promoting effects in chemical carcinogenesis. In the early 1980s, induction of activating mutations in cellular H-Ras upon single application of carcinogenic
PAHs was proven to be an early event in tumor initiation [87]. In this experiment, genomic DNA from skin carcinomas of mice, induced by single application of DMBA and subsequent treatment with a chemical promotor of carcinogenesis (12-O-tetradecanoylphorbol-13-acetate, TPA), carried an activated H-Ras oncogene. Transfection experiments with this DNA led to morphological transformation of fibroblasts in culture. As known today, oncogenic
RAS increases cellular proliferation through multiple pathways, e.g., elevated
cyclin D1 expression [88] or mitogen-activated protein kinase (MAPK) pathways (e.g., JNK, ERKs) [89]. Nevertheless, repeated application of carcinogenic PAHs is mandatory to obtain maximal tumor yield in mouse skin. This
finding along with the requirement of a functional AHR protein supports the
notion that both the initiating and promotional activity of carcinogenic PAHs
in skin depends on AHR-mediated gene expression. This would be in agreement with the tumor promoting activity of TCDD in this organ.
Apoptotic resistance
Carcinogenic aromatic amines or amides such as 4-aminobiphenyl (4-ABP) or
2-AAF primarily induce bladder tumors in dogs, and tumors in the liver, lung
or mammary gland of rodents [90] (Figs 1 and 3). Despite some new insights
on the main CYP isoform involved in activation in vivo [91], N-hydroxylation
and subsequent reactive ester formation have been well characterized and sufficiently explain the genotoxicity of these compounds [90, 92] (Fig. 3).
However, additional tumor-promoting activities have been observed and investigated, particularly in the case of the model compound 2-AAF, a complete
carcinogen in rodent liver [93]. Chronic exposure of rats to 2-AAF was found
to trigger adaptive responses in mitochondria permeability transition pores and
BCL-2 expression levels of hepatocytes that resulted in an increased resistance
to apoptosis [94]. There is evidence that this effect is an early tissue response
to the presence of reactive oxygen species (e.g., hydroxy or superoxide anion
radicals) generated via redox-cycling of 2-AAF metabolites (i.e., 2-nitrosofluorene, cf. Fig. 3). Since mitochondrial resistance is established in the tissue
before the clonal outgrowth of preneoplastic cells, this nongenotoxic effect
contributes to the selection of resistant cells and hence to the tumor-promoting
activity of 2-AAF in its target organ liver.
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XMEs usually operate with high regio- and stereoselectivity. For instance,
CYP-mediated toxification of aflatoxin B1 (AFB1), a natural carcinogen produced by Aspergillus mould and linked to human liver carcinogenesis [111],
occurs at its 8,9-position (Fig. 7). Based on the isolation of the main DNA
adduct (at N7 in guanine bases), formation of an 8,9-epoxide intermediate had
been proposed and subsequently confirmed [112]. CYP3A4, the most important enzyme involved in AFB1 activation in human liver, exclusively forms the
exo isomer (Fig. 7). In contrast, another CYP form, CYP1A2, may add some
small amounts of the diastereomeric endo epoxide [113]. However, the predominant AFB1 exo-8,9-oxide is about 1000-fold more genotoxic than its endo
diastereomer due to the spatial configuration of the epoxide moiety within the
AFB1 exo-8,9-oxide-DNA intercalation complex. Intercalation of the furanocoumarine residue between DNA bases directs the exo epoxide ring in a
favorable position for an SN2 attack by the N7 atom of guanine [114, 115].
Follow-up products of the main N7-DNA adduct of AFB1 then result from
depurination or ring opening of the purine base. These products, along with the
detoxification products produced by GST- or mEH-mediated conversions, are
depicted in Figure 7.
Similarly, metabolic activation of PAHs is highly selective [80]. As demonstrated for a wide range of carcinogenic PAHs the initial epoxidation
hydrolysis sequence produces a dihydrodiol with R,R-configuration in high
enantiomeric excess (cf. above). Subsequent (diastereo)selective epoxidation
at the vicinal double bond then predominantly generates the R,S-dihydrodiol
S,R-epoxide with the epoxide moiety trans to the benzylic hydroxy group. In
the case of B[a]P, all four possible stereoisomeric 7,8-dihydrodiol 9,10-epoxides are depicted in Figure 6, with (+)-anti-B[a]PDE as the major species
formed during bioactivation. Depending on the activation system, some small
amounts of the other isomers, the ()-syn-(R,S,R,S)-, (+)-syn-(S,R,S,R) and ()anti-(S,R,R,S) dihydrodiol epoxides, may also be generated the latter two
through monooxygenation of the S,S-dihydrodiol (Fig. 6). However,
B[a]P-induced DNA damage in vitro or in vivo predominantly results from
covalent interaction of (+)-anti-B[a]PDE, most of which is trapped by 2'deoxyguanosine (dG) residues via trans opening of the epoxide moiety [80,
116] (Fig. 7). If not repaired properly, the product of this reaction is likely to
cause nucleotide misincorporation at the opposite DNA strand during the next
round of DNA replication, and, therefore, has the potential of inducing mutations (i.e., dG base substitutions or frame shifts). DNA lesions such as the (+)trans-anti-B[a]PDE-N2-dG adduct are subject to nucleotide excision repair
(NER). Induction of NER activity requires both the disruption of normal base
pairing and the presence of a chemical modification (bipartite damage recognition) [117]. It could be demonstrated that (+)-anti-B[a]PDE induces a considerably different degree of NER activity in a certain DNA base context
depending on the way of epoxide ring opening during adduct formation [118].
While the cis-opened adduct, i.e., the (+)-cis-anti-B[a]PDE-N2-dG, adopts an
intercalative, internal adduct conformation with the benzo[a]pyrenyl moiety
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Figure 7. Stereoselective activation of aflatoxin B1 (AFB1) and benzo[a]pyrene (B[a]P) and their main
reaction products with DNA. CYP3A4, the main CYP enzyme involved in AFB1 activation, stereoselectively produces AFB1 exo-8,9-epoxide. The endo-diastereomer is not formed by CYP3A4, yet it
may be formed in small amounts by CYP1A2. B[a]P is stereoselectively activated to (+)anti-B[a]PDE possessing R,S,S,R-configuration (cf. Fig. 6). The grey arrows point to the position of
the nucleophile (DNA, protein, GSH) attack. GSH conjugates of AFB1 exo-8,9-oxide and (+)anti-B[a]PDE are detoxification products. AFB1 exo-8,9-oxide forms primarily DNA adducts at position N7 in purine bases. The N7 adduct is then subject to further modification. Due to the positive
charge in the purine ring system, the purine adduct may be released from DNA to produce an apurinic
site (cf. Fig. 4). Ring opening leads to another secondary product, the formamidopyrimidine (FAPY)
adduct. The major DNA binding product of B[a]P, the (+)-trans-anti-B[a]PDE-N2-dG, derives from
trans opening of the epoxide moiety. See text for further explanations. GST, gluthathione S-transferase; mEH, microsomal epoxide hydrolase.
inserted into the double helix and concomitant displacement of the modified
base, the (+)-trans-anti-B[a]PDE-N2-dG displays an external conformation
with the aromatic ring system accommodated in the minor DNA groove [119].
Hence, the local DNA distortion induced by the cis product is much more
severe as compared to the trans product, thereby resulting in a 10-fold faster
removal through NER. Moreover, the poor enzymatic repair of the predominant (+)-trans-anti-B[a]PDE-N2-dG adduct is preceded by an insufficiently
activated DNA damage checkpoint [120]. At non-toxic doses of anti-B[a]PDE,
a significant number of synchronized cells in vitro were found to enter S phase,
with little increase of those in G1. The failure to induce a proper DNA damage
arrest in G1 (so-called stealth property) along with insufficient enzymatic
repair increases the likelihood of transforming mutations because DNA replication continues on a damaged template via engagement of error-prone Y-family polymerases (pol , , , , Rev1) during translesional synthesis [121]. Data
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from analyses of the cell cycle and the expression profiles of human mammary epithelial cells or tumor cells of epithelial origin in vitro revealed that the
TP53 CDKN1A (p21WAF1)-mediated G1 checkpoint response was improper
even at a DNA damage level of about 180000 anti-B[a]PDE-DNA adducts/cell
[122]. Although TP53 levels were rapidly increasing due to protein stabilization via Ser15 phosphorylation, the cells exposed to anti-B[a]PDE lacked a
timely induction of CDKN1A. Whereas the transactivation activity of TP53
was not impaired in the case of several other downstream targets (e.g.,
GADD45, WIP1, p53R2) [122], a similar anti-B[a]PDE-induced and TP53mediated transcriptional repression has been observed at the BRCA1 locus
[123]. The BRCA1 tumor suppressor is involved in DNA damage response,
DNA double-strand break and transcription-coupled repair, thereby contributing to the inhibition of genomic instability during the course of malignant cellular transformation [124]. At present, however, the reasons for the differential
activity of TP53 at various target gene promotors remain obscure.
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in vivo against DNA adduct formation and toxicity of B[a]P [140] or 4-ABP
[91]. Both enzymes are involved in activation of B[a]P (CYP1A1) or
4-ABP (CYP1A2; see Figs 2, 3 and 6), yet animals that lack the corresponding genes have been found to suffer from higher DNA adduct levels in
internal organs and increased toxicity. It seems likely that this effect is due
to the well-balanced expression levels of CYP1A1 and 1A2 along with
detoxifying phase-II enzymes in wild-type animals, but also a result of the
overcompensatory induction of other oxidative enzymes, such as CYP1B1
or flavin-dependent monooxygenases, which would substitute for the
absence of CYP1A1 and 1A2 [141].
2. Humans are mostly exposed to complex mixtures of compounds rather than
to single carcinogens. For instance, due to the manner of their generation
(incomplete combustion), more than 100 different PAHs can be detected in
airborne particulates [142]. Cigarette smoke entails the risk of being
exposed to about 60 known carcinogens from a variety of chemical classes
including 4-ABP, B[a]P, and traces of metal ions [143]. The interactions of
those individual compounds may result in synergistic effects within the biological system. At the level of DNA repair, for example, co-exposure of
cells to Ni ions [144], or As and its methylated metabolites [145], was found
to enhance B[a]P-mediated mutagenesis through inhibition of NER-catalyzed removal of anti-B[a]PDE-N2-dG adducts. Even the presence of
structurally and stereochemically different DNA lesions in the same
genome may cause repair inhibition through sequestration of critical NER
subunits by those modifications that are more repair resistant (so-called
decoy adducts) [146]. Despite being refractory to excision, decoy
adducts immobilize NER factors, and may therefore contribute to synergistic interactions between multiple genotoxic agents present in complex
environmental mixtures. Thus, the results obtained from single compound
experiments would not allow direct extrapolation to the corresponding
effects expected to be exerted from mixtures.
3. In experimental tumor models, animals or cells are treated with single compounds in very high doses as compared to the levels of human background
exposures. Therefore, regulatory toxicologists have to extrapolate the doseresponse relationships found in animals into the low-dose exposure ranges
of humans in order to assess the accompanying risk. Depending on the kind
of approach applied, the results may differ tremendously, and are often subject to believe or disbelieve [147, 148].
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CYP1A1*2A (MspI, 3' untranslated region) and *2B (Ile462Val, exon 7) are
associated with increased catalytic activity, higher levels of PAH-DNA adducts
and TP53 mutations in smokers. CYP1A1 MspI combined with GSTM1 0/0 leads
to an at risk genotype for tobacco-accociated DNA damage and lung cancer.
CYP1A2
Limited evidence for higher risk of bladder cancer in smokers with CYP1A2*1F
(163CA: SNP intron 1) entailing increased inducibility. Combination
effect with slow NAT2 acetylator phenotype.
CYP1B1
CYP2A6
CYP2E1
CYP2E1*6 (7632 TA: DraI RFLP intron 6): N7-alkyl levels in lung samples
elevated. Inadequate evidence for increased lung and breast cancer risk.
Substrates: arene oxides (e.g., AFB1, B[a]P). Limited evidence for higher AFB1
protein adduct levels and liver cancer susceptibility in subjects with mEH
Tyr113His (exon 3 polymorphism). Limited evidence for reduced lung cancer risk in
smokers [167].
SULT1A2
SULT2A1
Main form in liver. Several rare alleles with moderate effects on enzyme activity
(<2).
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Table 1. (Continued)
Genetic polymorphisms Biological effects/tumor susceptibility (selection)
N-Acetyltransferases (NAT, EC 2.3.1.5)
NAT1
Predominantly extrahepatic. Substrates: N-acetylation (detoxication), and O-acetylation or N,O-transacetylation (toxication) of arylamines/amides and some HCAs.
NAT1*10 allele (elevated activity) entails higher risk for colon and bladder cancer
(high rate of O-acetylation of HCAs in colon and arylamines in bladder).
NAT2
Predominantly hepatic. Substrates and reactions as NAT1, main form for HCAs,
but no N,O-transacetylation. Several NAT2 alleles (e.g. NAT2*5B) are associated
with a slow acetylator phenotype. Slow acetylators have higher levels of 4-ABPhemoglobin adducts and an increased risk of bladder cancer (impaired hepatic
N-acetylation of arylamine); yet they are at lower risk for colon cancer.
Glutathione S-transferases (GST, EC 2.5.1.18)
GSTM1
Substrates: epoxides of PAHs and olefines (AFB1, 1,3-butadiene, etc.), arylamine
esters. GSTM1 0/0 genotype (loss of activity): although mainly expressed in
liver, higher PAH-DNA adduct levels, cytogenetic damage, and TP53 mutations in
lungs of smokers; effect pronounced with combined CYP1A1*2A genotype.
Higher 4-ABP-hemoglobin levels in smokers and non-smokers; effect
pronounced with combined slow NAT2 acetylator genotype. GSTM1 0/0 alone is
only a weak modifier of lung (OR 1.41) and bladder cancer (OR 1.44). For lung
cancer, the effect significantly increases in the presence of an active CYP1A1
MspI genotype (OR 3-10).
GSTT1
Substrates: epoxides of PAHs, 1,3-butadiene, ethylene oxide (detoxication),
dihaloalkanes/-methanes (toxication). Accordingly, GSTT1 0/0 polymorphism
entails a higher or lower risk for genomic damage in vitro, depending on the
substrate. At present, no evidence for modulation of human cancer risk.
GSTP1
Equivocal results in vitro. Limited evidence for association between GSTP1
Ile105Val polymorphism and increased bladder cancer susceptibility. Inadequate
evidence for a role in human lung cancer.
Arylhydrocarbon receptor (AHR) pathway
AHR
Several polymorphisms in humans reported. Combination of Lys554Leu and
Val570Ile impairs TCDD-mediated CYP1A1 induction in vitro. Strong evidence for
a correlation of human lung, laryngeal, and oral cavity cancer with AHR
phenotype.
ARNT
Polymorphisms known, but functional roles as yet unknown [169].
DNA repair proteins
XRCC1/3 Involved in base excision and DNA double strand repair. XRCC1 Arg399Gln (exon
10) and XRCC3 Thr241Met (exon 7) are associated with higher bulky DNA adduct
levels in non-smokers and both non- and ex-smokers, respectively. Combined
effects of multiple gene variants on DNA damage levels observed. As yet only
insufficient evidence for an important role in human (lung) cancer susceptibility.
XPD
Helicase involved in NER. XPD Asp312Asn (exon 10) and Lys751Gln (exon 23)
correlate to significant elevated DNA adduct levels in non-smokers. Combination
effects observed. Limited evidence for role in cancer susceptibility: OR (lung
cancer) of 1.06-3.2.
a
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Prospects
Background levels of carcinogen-DNA adducts in human tissue samples from
unexposed individuals were found to be in the range of 1 per 105 (oxidative
lesions/human placenta), 1 per 107 (B[a]P/human placenta), 0.33.9 per 108
(4-ABP/human bladder), or 0.2 per 108 nucleotides (tobacco-specific
N-nitrosamines/peripheral lung) [177]. The improvement of our knowledge on
the efficiency of the enzymatic repair of such lesions and on the various biological effects (modes of action) exerted by genotoxic carcinogens (Fig. 5) is
necessary to determine molecularly defined no-adverse-effect levels as the
basis for setting practicable thresholds in the human environment [178].
Further, any risk assessment that would not consider the interindividual variability within the human population would be prone to severely underestimate
the risks to those subjects who are most vulnerable to carcinogen-induced
DNA damage. It therefore becomes important to identify individuals at risk
by means of toxicogenetics and molecular epidemiology [179, 180]. This
implies that we learn more about the role and interplay of additional, as yet
unknown, susceptibility and resistance genes targeted by human carcinogens
or involved in modulating human responses to carcinogenic compounds.
Beyond the range of known polymorphic enzymes in carcinogen metabolism
and repair (Tab. 1), additional genetic variants are likely to contribute to the
development of sporadic cancer [181, 182]. The discovery of these variants
and the characterization of their interactions with environmental exposures are
clearly among the major topics in chemical-related cancer research in the years
to come.
Acknowledgments
I am very grateful to my colleague and friend Dr. Gregory P. Tochtrop for his critical reading of the
manuscript. The work of the author was supported by the German Research Foundation (DFG: LU
841/2-1).
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Abstract. Metals are essential for the normal functioning of living organisms. Their uses in biological
systems are varied, but are frequently associated with sites of critical protein function, such as zinc
finger motifs and electron or oxygen carriers. These functions only require essential metals in minute
amounts, hence they are termed trace metals. Other metals are, however, less beneficial, owing to their
ability to promote a wide variety of deleterious health effects, including cancer. Metals such as arsenic,
for example, can produce a variety of diseases ranging from keratosis of the palms and feet to cancers
in multiple target organs. The nature and type of metal-induced pathologies appear to be dependent on
the concentration, speciation, and length of exposure. Unfortunately, human contact with metals is an
inescapable consequence of human life, with exposures occurring from both occupational and environmental sources. A uniform mechanism of action for all harmful metals is unlikely, if not implausible, given the diverse chemical properties of each metal. In this chapter we will review the mechanisms of carcinogenesis of arsenic, cadmium, chromium, and nickel, the four known carcinogenic
metals that are best understood. The key areas of speciation, bioavailability, and mechanisms of action
are discussed with particular reference to the role of metals in alteration of gene expression and maintenance of genomic integrity.
Key words: Arsenic, cadmium, carcinogenesis, chromium, nickel, oxidative stress.
Introduction
The association of metal exposure with cancer is a well-documented phenomenon. Metals such as arsenic (As), cadmium (Cd), chromium (Cr), and nickel
(Ni) are part of an ever growing list of environmental agents that have been
formally classified by the International Agency for Cancer Research (IARC)
as being known carcinogens [14]. For other metals such as iron, copper,
beryllium, lead, and mercury there exists an ever increasing body of evidence
to support their inclusion in the IARC listings [58]. Iron [8] and copper [7],
in particular, are carcinogenic in excess, but are highly regulated and generally only produce cancer in animal models or in people with genetic diseases
that prevent appropriate metabolic regulation. There is even less information
on beryllium carcinogenesis, and no definitive studies that indicate the species,
conditions or length of exposure by which lead and mercury metals cause cancer in humans. For these reasons, this review will focus on the known carcinogenic metals: As, Cd, Cr and Ni.
Despite increasing numbers of researchers in the field and the expanding
role of metals in environmental health issues, the nature of cancer induction by
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uals who have become resistant to normal therapies [32]. Occupational exposure to arsenic is greatest in mining and metal smelting industries; however, it
can also occur through glass manufacturing and as the result of coal burning
for power production [33]. But the greatest extent of exposure is from arseniccontaminated water sources.
Arsenic is normally found in close association with heavy metals such as
gold, copper and silver. Mining of these heavy metals brings arsenic to the surface where it is concentrated through the refining processes [34]. Arsenic can
also be brought to the surface when it is leached from the rock surrounding
underground aquifers. It is in this circumstance that arsenic has had its most
profound effects on human health. Since the 1980s countries such as
Bangladesh, India, and China, where surface water is frequently contaminated
with microbial pathogens, have invested heavily in alternate water sources that
are now known to be heavily contaminated with arsenic [34, 35].
Concentrations of arsenic in these water sources vary wildly; however, in
many regions they exceed by 10 to 15 times the current World Health
Organizations (WHO) recommended level of 10 ppm [36]. Even at these levels, arsenic is not acutely toxic. However, as early as 1968, similar high levels
of arsenic in the artesian well water in regions of southern Taiwan were recognized as a likely cause of carcinogenesis [37]. Increased cancer rates associated with arsenic-contaminated drinking water have now been recorded in
many countries, including Taiwan, Argentina, Chile, and Mexico. The arsenicassociated cancer incidence in Bangladesh and West Bengal, India is expected
to reach catastrophic levels over the next several decades [38, 39].
Arsenic in the environment can take a range of forms, both organic and inorganic. Inorganic arsenic has two possible valencies, arsenite, or As(III) and
arsenate, As(V). Arsenite is the more toxic of the two species with cell viability assays indicating that concentrations anywhere from 1 to 10 M and
upwards are able to promote toxicity [5]. Arsenate is approximately three to
fivefold less toxic than arsenite, presumably because As(V) requires reduction
to As(III) to exert its toxicity. Organoarsenic species can also be formed by
biometabolism. Many organoarsenic species are significantly less toxic than
inorganic As(III). However, methyl As(III) species can be significantly more
toxic than inorganic As(III) [40] and may contribute to arsenic carcinogenesis.
The relative toxicity of the different forms of arsenic is predominantly the
result of their different chemical properties, but may also relate to the relative
efficiency of their uptake [41, 42], the duration of the exposure, and the time
when the toxicity assay is performed [42, 43]. Arsenic excretion rates vary, but
it is generally accepted that arsenic, unlike other carcinogenic metals, is rapidly excreted by the body, to the extent that more than 50% is removed within
2 days in acute poisoning cases [33].
Organic metabolites of arsenic that are of most interest are the monomethyl
and dimethyl species of both arsenite and arsenate. These species are generated through biomethylation of inorganic arsenic, followed by reduction and
subsequent methylation of monomethyl As(III) to produce dimethyl As(V)
101
(DMA). The intermediate methylated As(III) species are thought to be considerably more toxic than either methyl As(V) species or even inorganic
As(III) species [40, 44]. However, the levels of available organic As(III) species in human tissues relative to other arsenic species are still largely undetermined.
Other organoarsenic species such as arsenobetaine and arsenosugars are
commonly found in marine species. Although arsenobetaine is often found in
high concentration in marine animals, it is largely excreted unmetabolized, and
has very low toxicity [45]. Arsenosugars are frequently found in seaweed and
in crustaceans [46]. Recent evidence suggests that these compounds may be
metabolized to DMA, which can then be further metabolized to more toxic
species [46]. This raises the possibility that consumption of seafoods can be a
source of considerable arsenic intake, some of which may result in the retention of some relatively toxic arsenic species. This may have implications for
Asian populations, particularly those which live on high fish diets, such as the
Japanese [46].
Arsenic pathology is complex. When ingested at very high doses, in excess
of 200 mg, it produces acute toxicity characterized by nausea, vomiting,
sloughing off of epithelial tissues, internal bleeding, changes to blood pressure, and atrial fibrillation [33]. This can lead to heart attack, coma, and
death. At sublethal doses arsenic ingestion can be treated with a range of
metal chelators, which reduce its effects; however, few if any other treatments
for arsenic ingestion exist. At very low doses, arsenic appears to have minimal short-term effect however, over longer periods a range of pathologies are
seen [2]. Chronic low-dose exposure initially produces blotching of the skin,
followed by hyperkeratosis of the palms and soles of the feet. If exposure continues, alterations to peripheral vasculature are seen along with the formation
of skin lesions, which left untreated, can become cancerous [47, 48]. Arsenic
is also associated with an increased risk for cancer of the lungs, liver and
bladder [47].
Induction of cancer by arsenic is not thought to originate from a single
exposure, but rather is the result of gradual changes to a variety of processes
within the cell. Different arsenic species enter cells by different mechanisms.
Arsenate is able to mimic phosphate, and hence is able to enter cells using
phosphate transport proteins. Arsenite, however, is thought to enter through
aquaglyceroporins [49]. Once in the blood stream, arsenic is taken to the liver
where biometabolism occurs. This process involves the progressive methylation of arsenic, with As(III) converted to the less toxic methyl As(V) species.
The ingested arsenic is excreted predominantly in the urine as inorganic
As(III) and As(V), methyl As(V), and dimethyl As(V), with the proportions of
these being variable and related to arsenic dose [5052]. Some intermediate
trivalent arsenic metabolites are also produced, and can be found in the urine
[53]. Despite their greater toxicity, relative to either inorganic As or organic
As(V) species, it is yet to be determined whether these methyl As(III) and
dimethyl As(III) species play a significant role in carcinogenesis.
102
Cadmium
Unlike many other metals, cadmium is found in only one valence state, that of
Cd(II). Exposure to cadmium has also been far less common than other carcinogenic metals. Of greatest note was the historical use of cadmium as a paint
additive giving rise to the bright yellows seen in many paintings, such as those
of Claude Monet [54]. Industrial use of cadmium is only a recent phenomenon,
beginning in the 1940s. Cadmium is now most commonly encountered in cadmium-nickel battery production [10], although it continues to be used in
paints, as well as in plastic production where it is an effective stabilizing agent.
Like arsenic, occupational exposure to cadmium can occur through metal
refining processes, where cadmium is often associated with copper and can be
released into the atmosphere during heating [55]. The greatest exposure to
cadmium, however, comes from cigarette smoke [10]. Particulate cadmium in
cigarette smoke collects in the lungs where it can be transported into the bloodstream across the alveoli. Unlike arsenic, cadmium has a long biological half
life, considered to be somewhere between 15 and 25 years [4, 56]. This means
cadmium can accumulate to levels many times greater than an individual
would be subjected to in a single exposure. Cadmium is only a weak mutagen,
but is a strong co-mutagen [4, 57, 58]. This is of particular concern for cigarette smokers who simultaneously inhale cadmium and benzo[a]pyrene, as
well as a range of other chemicals, including arsenic and other metals.
Health effects of cadmium are quite dissimilar to other metals. Non-toxic
doses of cadmium produce a wide variety of effects, many of which are related to bone development and maintenance. Individuals exposed to cadmium
can develop osteoporosis, anemia, eosinophilia, emphysema, and renal tubular
damage [59]. Long-term cadmium toxicity can produce Itai-Itai disease, in
which individuals suffer from bone fractures, severe pain, proteinuria and
severe osteomalacia [59]. Acute high-level exposure to cadmium is also able
to produce severe lung damage. However, like other metals, prolonged repeated exposures are required to induce carcinogenesis. Target organs for cadmium are varied however, lung cancers predominate [4]. Other tissues subject to
malignant transformation by cadmium include the prostate, pancreas and kidney. The testes are also thought to be a site of cadmium carcinogenesis; however, this has only been shown in animal models. Like arsenic, cadmium is
only a weak mutagen. This suggests that tumors result from either epigenetic
or co-carcinogenic effects, particularly in cases of smoking-induced lung cancer [10].
Chromium
Chromium is widely available, complex in action, and used industrially in a
myriad of applications including, pigment production, chrome plating, welding, production of ferrochrome metals, leather tanning and as a dietary sup-
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Inhalation of particulate Cr(VI) can cause irritation to the nasal tissue, leading
to nose bleeds, ulceration and formation of lesions in the nasal passage [60].
Damage to lung tissue is also not uncommon [70, 71]. Ingestion of Cr(VI) can
cause nausea, vomiting, ulceration of the stomach, damage to the liver and kidney, and finally death [60]. Both species of chromium can cause contact hypersensitivity, leading to rashes, swelling and ulcerations. Cr(VI) is the most carcinogenic form of chromium, with insoluble particulate chromium compounds
being the most persistent [66] and the most hazardous [72].
Nickel
Nickel has many common industrial uses, thanks largely to its unique chemical properties. Industrially, it is used in electroplating, electroforming, in circuitry, and in nickel-cadmium batteries. Nickel alloys, including stainless
steel, are used in a wide variety of objects, from kitchen knives to building
tools [73]. Nickel is also used in jewelry and medical implements. Metallic
nickel is non-carcinogenic to humans; however, all other nickel compounds,
such as nickel sulfides, oxides, and silicates, and other soluble salts, are known
carcinogens [12]. Carcinogenic nickel exposure is greatest through the inhalation of nickel-containing particulates. The burning of fossil fuels, as well as the
refining of metals such as copper, introduces considerable amounts of nickel
into the atmosphere [12]. Like arsenic, nickel can also be leached from soils
and rock, thereby contaminating water supplies. In lower organisms such as
bacteria, nickel is an essential trace element found in up to seven different
enzymes [74]. Higher organisms, however, have failed to show any definitive
role for nickel in normal cellular function. That said, studies in the 1970s and
80s showed that the removal of nickel from the diet of rats had significant
effects both physically and mentally, which, with continued exclusion of nickel from the diet, were more profound in the subsequent generations [75]. It
may be that nickel is not required for normal cellular function in humans, but
rather is essential for our intestinal microflora. Like both arsenic and chromium, nickel occurs in different oxidation states, ranging from I to IV, with Ni(II)
being most common in biological systems.
As with chromium, particulate nickel is most harmful to humans, especially in the lung where crystalline nickel becomes lodged in the mucous prior to
being phagocytized by both epithelial cells and macrophages [76]. Once inside
the cells, the nickel compounds are gradually broken down releasing reactive
nickel ions. The phagocytic nature of nickel uptake means considerable
amounts of nickel are able to accumulate over time, damaging lung tissue and
frequently causing latent effects in individuals who may have been exposed to
nickel many years earlier [76].
Nickel is not overly toxic to individuals at low doses; however, nickel-containing jewelry can produce contact hypersensitivity in many people [73]. This
normally results in rashes and inflammation of the region of contact. However,
105
106
(SOD), thereby producing the less reactive, but more mobile, H2O2 [92]. Like
O2 , H2O2, is also tightly regulated by a multiplicity of catalase and peroxidase
enzymes.
107
103, 109, 112, 113]. In myeloid leukemia (NB4) and epithelial cells, arsenic
treatment at low doses has been shown to induce NADPH oxidase [85, 90].
Recent data show that arsenic can also activate NADPH oxidase in endothelial
cells [85, 89].
In addition to ROS, nitrogen-based radicals, such as nitric oxide and peroxynitrite, have also been implicated in oxidative damage by arsenic. The formation of micronuclei and induction of poly(ADP-ribosylation) in Chinese
hamster ovary (CHO) cells and bovine endothelial cells and the formation of
oxidative DNA damage [measured by cleavage with formamidopyrimidineDNA glycosylase (Fpg) enzyme] have all been shown to be effectively blocked
by the addition of inhibitors of nitric oxide synthase, suggesting that these radicals may account for some of the damage seen in cells [114, 115]. In all, the
formation of radical species by arsenic appears to be an important mechanism
by which arsenic may promote its carcinogenic effects.
Chromium
Chromium, like arsenic, has been shown to produce oxidative stress in cells by
multiple mechanisms; however, the extent to which these are able to produce
cancer is still subject to debate. As mentioned above, Cr(VI) can undergo a
series of reductions leading to the formation of Cr(III). Chromium(VI) is a
strong oxidizing agent and, like copper and iron, can produce ROS directly
through Fenton type chemistry, whereby Cr(VI), or one of its metabolites, is
able to interact with H2O2 in the presence of a reductant to produce both superoxide and hydroxyl radicals [116119]. However, it is not only the ROS produced by the reduction of chromium species that can produce oxidative damage in cells, there is a growing body of evidence to suggest that the genotoxicity of chromium can be caused in part by the reactive chromium species
themselves, such as Cr(V) [120]. OBrien et al. [13] have raised the possibility that these species may in fact be the direct cause of the oxidative stress
response measured by DCFH and rhodamine 123. Even the use of ROS scavengers is not sufficient to rule out this possibility, since these scavanges can
also react directly with Cr(V) to prevent DNA damage [121, 122]. It must be
noted, however, that the formation of radicals by this mechanism has only been
shown to occur when both chromium and H2O2 were present at concentrations
that are unlikely to be physiologically achievable within cells.
Like most metals that have the capacity to undergo redox reactions,
chromium has been shown to deplete intracellular GSH and alter the regulation of the redox enzymes such as catalase and SOD [123125]. Glutathione
has shown to be a critical factor in the reduction of Cr(VI) to Cr(III). The relationship between chromium-induced oxidative stress, DNA damage and
repair processes, and apoptotic cell death are complex [13, 22]. Moreover, the
relationship between these processes and the induction of cancer is far from
well understood.
108
Cadmium
In contrast to chromium, cadmium has been shown not to have any capacity to
produce free radical species by Fenton type chemistry [10, 126]. However,
cadmium is able to promote oxidative stress in a variety of model systems via
the formation of superoxide and H2O2 radicals [127129]. Indirect evidence in
support of free radical generation in cells is also abundant. Studies of cell culture, rat and mouse models all show a general downregulation of GSH and
thioredoxin reductase, as well as expression changes in radical converting
enzymes such as SOD [10, 130, 131]. This suggests that cadmium may not
produce significant free radical species by itself, but rather prevents the normal regulation of radicals produced by other agents and metabolic processes
of the cell [132]. Similarly, it appears that cadmium may be able to induce the
release of iron from its bound state in proteins and biological membranes [133,
134]. The release of iron would then provide a catalyst for ROS production
through Fenton/Haber Weiss chemistry.
Nickel
Unlike either arsenic or chromium, nickel is not readily metabolized by cells
and, therefore, does not have the capacity to produce radicals by this mechanism. However, nickel is able to produce ROS by redox cycling and other less
direct mechanisms. Soluble nickel particles exist in cells in two states, either
as Ni(II) or Ni(III). Nickel has the capacity to bind to amino acid residues and
can subsequently undergo redox cycling reactions between these two states
in the presence of molecular oxygen and H2O2. These processes produce a
variety of radicals including OH, carbon- and sulfur-centered radicals, as
well as nickel-based radicals [6, 12, 135, 136]. Direct evidence for the formation of radical species by nickel in CHO, lymphoblast and A549 cells has
been shown by a number of groups [24, 137139]. Likewise, fumes from
nickel welding processes have been shown to promote the formation of both
radical species and lipid peroxidation of cell membranes [140]. Similarly,
8-oxo-dG and other oxidative base modifications have been generated in
DNA through interaction of nickel and H2O2, suggesting a capacity for nickel to generate damage by Fenton type reactions [12, 141]. Thus, phagocytosis of particulate nickel compounds such as nickel sulfide and nickel subsulfide and subsequent release of Ni(II) can produce oxidative stress in the lungs
and other tissues [12, 24, 142]. Moreover, dissolution of nickel by these
processes can occur over extended periods of time, leading to continuous
production of radicals within the cell [12], thereby initiating and actively promoting the development of tumors [143]. Nickel has indirectly been shown
to effect GSH levels and the levels of key enzymes such as SOD and glutathione peroxidase in both cell and animal models [140, 144146]. The
potential for nickel to generate radical species and oxidative stress by these
109
mechanisms, forms a likely means to both induce and promote alteration and
disregulation in cells.
Mismatch repair
Spontaneous alteration of DNA bases and mistakes by DNA polymerases are
commonly recognized and repaired by the MMR system [148]. The principle
role of MMR is to remove nucleotides that have been inadvertently incorporated opposite non-pairing partner bases and to correct the insertion/deletion
of bases. These errors normally occur as a byproduct of DNA replication and,
if not corrected, can result in either base substitution or frameshift errors [148,
149]. In E. coli, the MMR system consists of a number of key proteins, including: MutS, MutL, MutH, DNA polymerases, single-stranded binding proteins,
and DNA ligase [150]. Eukaryotes, however, have evolved a more complex
system whereby many of these proteins have been duplicated, and now have
specific roles in certain parts of the cell, or work only under certain circumstances. The specificity and efficiency of MMR means that defects in these
proteins can lead to an accumulation of errors in the genome, producing cancers such as hereditary nonpolyposis colon cancer (HNPCC) [151].
Although MMR plays a significant role in the repair of oxidative DNA damage [152], interactions between carcinogenic metals and the MMR pathway
appear to be limited. Currently, cadmium is the only carcinogenic metal shown
to interfere with MMR [153]. Physiologically relevant concentrations of cad-
110
mium, on the order of 5 M, can inhibit MMR in yeast and extracts from
human cells by between 20% and 50% [54, 153]. Inhibition of MMR to this
extent can have significant implications for the accumulation of errors in the
genome generated by endogenous processes [154].
111
NER proficient human cells in which nickel treatment reduced repair and
increased mutagenesis of benzo[a]pyrene adducts [171].
112
Direct repair
In contrast to other pathways mentioned previously, direct repair is by far the
most simple, generally consisting of a single protein which produces chemical
reversion of nucleotide damage. The best known of these reactions in mammalian cells is O6-methylguanine-DNA methyltransferase (MGMT) [150,
156]. Left unrepaired, O6-methylguanine lesions in DNA can produce large
numbers of GC AT transition mutations [156]. Importantly, arsenic can alter
methylation of the promoter region of this gene, downregulating protein
expression [101, 191]. Cadmium and nickel have also been shown to alter the
113
Arsenic
Trivalent arsenic species are well known to bind to protein thiols [195], particularly when the cysteine residues are in close proximity within the protein.
Binding of As(III) to critical cysteine residues has been demonstrated to inactivate both the glucocorticoid receptor [196, 197] and the glucose transporter,
GLUT4 [195, 198], as well as prevent the activation of NF-B [199].
Phenylarsine oxide has also been shown to bind a range of proteins including
NADPH oxidase, both stimulating and inhibiting ROS production dependant
on dose [90, 200].
Cadmium
Beyond the more obvious mechanisms of carcinogenesis, such as increased
ROS and altered gene expression, cadmium can also facilitate malignant transformation by altering cell-cell adhesion. Both vascular endothelial cells and
transport epithelia rely on cell adhesion complexes to control intercellular
transport. A number of key proteins have been identified in these adhesion complexes, including the catenins, connexins, cadherins, and integrins [201203].
Of particular interest are the cadherins, which appear to be most affected by
cadmium [10, 204, 205]. Cadherins are unique cell-cell adhesion proteins that
require calcium to facilitate binding. They are coupled to catenins, which in
turn link them to actin polymers within cells [201, 206]. It is the E-cadherins,
which link epithelial cells that are thought to be the most susceptible to cadmium [205]. E-cadherin is important to cell development and has also been shown
114
to suppress tumor formation in a range of tissues [204, 206]. The effect of cadmium on cell adhesion was first characterized by a significant loss of tissue
integrity that was not initially due to apoptosis [207209]. Later studies, especially those of Prozialeck et al. [210] showed that cell adhesion and, in particular, the integrity of E-cadherin was an early target of cadmium toxicity. It was
also shown that cadmium was able to exert its greatest effects on E-cadherins
when calcium levels were low, suggesting that cadmium competes for calcium
binding sites [211, 212] The loss of E-cadherins are thought to enhance tumor
metastasis, promote toxicity, and promote changes to gene expression profiles
through altered -catenin signaling [204, 206].
Chromium
Complexes formed by chromium are considerably more varied than those of
other metals discussed here. The binding of chromium to DNA does not occur
with Cr(VI); however, the reduced metabolites of chromium, Cr(III, IV, and V)
have all been shown to be reactive towards DNA [213, 214]. Although the structure and efficiency of formation of these chromium-DNA complexes is strongly affected by the reductant involved, such as GSH, ascorbate, or cysteine, most
of the resulting adducts seem to be both genotoxic and mutagenic [22]. Binding
of chromium species to DNA appears to be preferential for guanine nucleotides,
and occurs largely with phosphates in the backbone [22, 215]. The formation of
chromium-DNA adducts has a twofold effect, they both inhibit DNA replication and prevent DNA repair, thereby promoting mutagenesis.
Nickel
Nickel shows a strong affinity for histidines and, to a lesser extent, cysteines,
and is able to form complexes with a wide variety of proteins [216, 217]. As a
result, nickel is frequently used to extract and purify proteins that have been
histidine tagged [218, 219]. Proteins that have been shown to bind nickel
include: serum albumins, the neuroblastoma-associated tumor suppressor
(DAN), and histones [220222]. Like other metals that form protein complexes, it is thought that nickel interacts with proteins, altering their conformation
in such a way that they are no longer able to perform normal cellular functions.
Nickel has also been shown to crosslink DNA as the result of oxidation of
DNA-associated proteins [12, 223].
115
Arsenic
Arsenic can both induce and suppress gene expression, depending on its concentration and the length of exposure. Microarray analyses of gene expression
in arsenic-treated cells have identified hundreds of genes, most of which fall
within several categories: cellular stress response, cell cycle control, redox regulation, and DNA repair [25, 99, 229, 230]. Different cell types and different
treatment conditions can produce different effects on gene expression. In some
cases, for example, such as arsenic-induced Bowens disease, p53 expression is
upregulated compared to non-arsenic-related disease controls [231]. Low-dose
arsenic also promotes upregulation of p53 in cultured fibroblasts after both
acute and longer treatments [232, 233]. In contrast, microarray analysis of normal human keratinocytes exposed to between 0.005 and 5 M As(III), showed
a generalized downregulation of p53 [234]. Transcription factors such as AP-1
and NF-B are also regulated by arsenic, presumably the result of the activation
of signal transduction pathways and the formation of ROS [235237]. Hu et al.
[237] showed that acute low-dose treatments of human fibroblasts with arsenite
produced upregulation of both AP-1 and NF-B expression, while chronic
exposures lead to a downregulation of AP-1 and NF-B. The AP-1 transcription
factor is important for the regulation of DNA repair, inflammatory responses
and cell growth [238, 239]. The activation of NF-B can increase the expression
of cytokines and growth factors, which may be responsible for tumor promotion
[240, 241]. In aortic endothelial cells, it has been shown that acute low-dose
arsenic treatments promote nuclear accumulation of NF-B, similar to results
seen in rat lung slices [242, 243]. These changes in transcription factor expression and activation are likely to lead to the observed changes in gene expression,
and, more importantly perhaps, the observed inhibition of BER by arsenic.
116
It has only recently been discovered that arsenic can also modify DNA
methylation patterns [244]. Dose-dependent hypermethylation of gene promoters was first noticed in regions of the p53 gene following exposure of cultured cells to either As(III) or As(V) [245]. Similarly, the p53 promoter region
was shown to be hypermethylated in basal cell carcinomas (BCCs) from
arsenic-exposed individuals relative to BCCs from non exposed patients [246].
In contrast, Zhao et al. [244] showed that chronic treatment of rat liver cells
with arsenic caused global hypomethylation of promoters and malignant transformation. This hypomethylation was thought to occur as the result of depletion of S-adenosyl-methionine [244]. Changes to methylation patterns induced
by arsenic are persistent, destabilizing [247], and have the potential to promote
aberrant expression of genes involved in cell development and regulation,
leading to cancer induction [25, 97, 226].
Cadmium
Like most metals, cadmium is responsible for alterations in the expression of
many genes, including the immediate early response genes, c-fos, c-jun and
c-myc; stress response proteins, such as metallothionein and heat shock proteins; and transcription factors, such as NF-B [248251]. Zheng et al. [252]
have also shown that the livers of mice treated with 10 mol/kg CdCl2 exhibit
increased expression of c-jun and p53. All of these proteins are believed to be
involved in tumor promotion. Immediate early response genes (IEGs) induce
mitogenic growth signals causing increased proliferation, particularly of cells
that already possess mutations in critical regulatory genes.
Activation of stress response genes in response to changes in the extracellular environment enables cells to both protect themselves against oxidative
stress and maintain normal cellular function. Cadmium activates a variety of
these genes, the most notable of which is metallothionein. Metallothionein is
a cysteine-rich low molecular weight protein, which binds excess heavy metal
ions preventing their toxic effects [253]. Differential tissue expression of metallothioneins is thought to be a major reason for the tissue specificity of cadmium carcinogenesis [10, 254]. Cadmium is readily able to induce metallothionein expression in the liver and kidneys, but not in the testes or prostate [253,
255, 256]. Reduced expression of metallothionein in the testes and prostate
relative to the liver of rats, correlates with increased levels of tumors and toxicity in these tissues [253]. Similarly, the use of transgenic mice has demonstrated that metallothionein reduces cadmium-induced ROS formation and
activation of other genes that protect against oxidative stress [257]. Several key
antioxidant genes in cells, most notably SOD and catalase, show reduced levels of expression in response to cadmium treatment [10, 130, 131, 258].
Depression of these enzymes can facilitate an increased build up of ROS,
which can cause significant damage to cells.
117
Chromium
Chromium can also induce changes in gene expression due to its ability to produce radical species and oxidative stress. For example, as with arsenic and cadmium, both NF-B and AP-1 are modulated by chromium exposure, with
NF-B being up regulated, which in turn activates c-myc [16, 262, 263].
Microarray studies in various cell cultures and in vitro models exposed to low
to medium doses of chromium show an increase in a variety of genes, including those of the oxidative stress response, particularly those involved in redox
regulation [70, 262, 264, 265]. Chromium species, like nickel species, have
also been shown to affect the expression of hypoxia-inducible factor-1 (HIF-1)
proteins [266]. Unlike the other metals described here, there is very little evidence to suggest that chromium also produces epigenetic changes, with the
exception of a report by Cheng et al. [267] showing transgenerational changes
in hormonal control in mice fed a diet supplemented with high levels of
Cr(III).
Nickel
Nickel, like the other metals is able to alter the regulation of a variety of genes,
including NF-B [12]. Nickel has also been shown to promote the induction
of hypoxia through activation of the transcription factor HIF-1 [268].
Increased levels of HIF-1 correlate with angiogenesis of new vasculature in
tumors [269]. Other microarray studies have shown that nickel acetate exposure induces large-scale alterations of gene expression in human lung epithelial cells [270]. Some of the genes most strikingly affected include metallothionein and the heat shock proteins. Similarly, nickel sulfate-induced lung
injury in mice showed gene expression patterns representative of both hypoxic and oxidative stress responses [271].
118
In contrast to the other metals presented in this section, the principle carcinogenic mechanism of nickel appears to be epigenetic in nature (reviewed in
[12, 227]). The effects of nickel on DNA methylation were first suggested
when it was noted that nickel-immortalized cells could be induced to senesce
by demethylation with 5-azacytidine [272]. Since then it has been shown that
nickel treatment alters methylation-dependent chromatin condensation [224],
causes gene silencing [273], and modifies the activity of DNA methyltransferases [274]. Additionally, when mice are injected with nickel sulfide, the
resultant tumors all exhibit hypermethylation of the p16 gene, an important
regulator of cell cycle control [275].
More recently, it has been shown that nickel can induce epigenetic changes
by both hypoacetylation and localized hypermethylation [276, 277] and that
chemical demethylation and deacetylation can reverse gene silencing [278,
279].
Summary
Agents responsible for human carcinogenesis are grossly varied in their properties, and metals are no exception. However, it seems likely that metals share
several common means by which to induce cancer. Critically, the most important of these appears to be the generation of oxidative stress and deregulation
of key maintenance genes within cells. That said, the nature of the dose of each
of these metals, as well as confounding variables required to produce a carcinogenesis, remain at best an unresolved issue. However, with time, and as
research progresses, it is likely that a more complete picture will emerge on
metal-induced carcinogenesis.
Acknowledgements
This work was supported in part by the Electric Power Research Institute contract no. WOEPP4898/C2396, the U.S. Environmental Protection Agencys Science to Achieve Results (STAR) program, and the Centre for Cellular and Molecular Biology, School of Biological and Chemical
Sciences, Deakin University, Australia. These supporting agencies were not involved in any way in the
study design, data collection, or interpretation of the results presented here.
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Summary. Solar radiation is the primary source of human exposure to ultraviolet (UV) radiation.
Overexposure without suitable protection (i.e., sunscreen and clothing) has been implicated in mutagenesis and the onset of skin cancer. These effects are believed to be initiated by UV-mediated cellular damage, with proteins and DNA as primary targets due to a combination of their UV absorption characteristics and their abundance in cells. UV radiation can mediate damage via two different
mechanisms: (a) direct absorption of the incident light by the cellular components, resulting in excited state formation and subsequent chemical reaction, and (b) photosensitization mechanisms, where
the light is absorbed by endogenous (or exogenous) sensitizers that are excited to their triplet states.
The excited photosensitizers can induce cellular damage by two mechanisms: (a) electron transfer
and hydrogen abstraction processes to yield free radicals (Type I); or (b) energy transfer with O2 to
yield the reactive excited state, singlet oxygen (Type II). Direct UV absorption by DNA leads to
dimers of nucleic acid bases including cyclobutane pyrimidine species and pyrimidine (6-4) pyrimidone compounds, together with their Dewar isomers. These three classes of dimers are implicated in
the mutagenicity of UV radiation, which is typified by a high level of CCTT and CT transversions. Single base modifications can also occur via sensitized reactions including Type 1 and Type II
processes. The main DNA product generated by 1O2 is 8-oxo-Gua; this is a common lesion in DNA
and is formed by a range of other oxidants in addition to UV. The majority of UV-induced protein
damage appears to be mediated by 1O2, which reacts preferentially with Trp, His, Tyr, Met, Cys and
cystine side chains. Direct photo-oxidation reactions (particularly with short-wavelength UV) and
radicals can also be formed via triplet excited states of some of these side chains. The initial products of 1O2-mediated reactions are endoperoxides with the aromatic residues, and zwitterions with
the sulfur-containing residues. These intermediates undergo a variety of further reactions, which can
result in radical formation and ring-opening reactions; these result in significant yields of protein
cross-links and aggregates, but little protein fragmentation. This review discusses the formation of
these UV-induced modifications and their downstream consequences with particular reference to
mutagenesis and alterations in protein structure and function.
Key words: DNA, free radicals, photoproducts, protein, singlet oxygen, ultraviolet.
Introduction
Nature of UV and solar radiation
UV light is defined as the region of the electromagnetic spectrum with wavelengths from 200 to 400 nm. This light is broken down into three distinct wavelength bands, known as UVC (ca. 200280 nm), UVB (ca. 280320 nm) and
UVA (ca. 320400 nm). As with all electromagnetic radiation, the shortest
wavelength radiation (UVC) is the most energetic, and has the greatest potential for biological damage.
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The major source of human exposure to UV light is via the sun. Solar radiation contains all three forms of UV radiation, but UV radiation with wavelengths below 295 nm (i.e., the entire UVC region) is absorbed by the Earths
upper atmosphere, and does not reach the Earths surface. The UV light that
does reach the Earths surface comprises primarily (ca. 95%) UVA wavelengths, with the remainder (ca. 5%) comprising the shorter wavelength
(295320 nm) UVB radiation. This chapter will focus on the biological effects
of UVB and UVA radiation, as these are the most biologically relevant [1, 2].
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134
( a)
HN
O
NH2
NH
NH2
NH
N
N
T<>T
N
N
Deamination
O
HN
O
T<>C
(b)
C<>C
NH2
HN
NH2
C<>T
O
NH
N
T<>U
cis,syn
cis,anti
trans,syn
trans,anti
Scheme 1. Structures (a) of the cyclobutane pyrimidine dimers (P<>P) and the possible diastereoisomers (b) of T<>T.
135
mixture of diastereoisomers (Scheme 1b) is generated that differ in the orientation of the two pyrimidine rings relative to the cyclobutane ring, and on the
relative orientations of the C5C6 bonds in each pyrimidine base. In doublestranded DNA in its natural configuration (i.e. in the B form), where the dimer
involves two adjacent pyrimidine bases on the same strand, only the syn isomers can be generated, and the cis isomer is greatly preferred over the trans
isomer [12]. In single-stranded or denatured DNA, the trans,syn isomer
becomes more prevalent due to the increased flexibility of the DNA backbone.
Dimer formation (typically the cis,syn or trans,anti isomers) between the two
strands of double-stranded DNA can also be detected in trace amounts in aqueous solutions with UVC irradiation [16]. However, in situations where a different DNA conformation is adopted (e.g., in 80% ethanol or in the dry state)
the incidence of inter-strand dimers dramatically increases, as evidenced by
the increased proportion of anti isomers [16].
The P<>P dimers themselves are only mildly photoactive but under irradiation with UVC light the dimerization can be reversed by photo-induced splitting of the cyclobutane ring, to yield the original monomer bases [17, 18].
There are also a number of DNA repair processes invoked in vivo; in many
organisms these include photolyase enzymes that are activated upon exposure
to UV light (reviewed in [9, 13]).
For P<>P dimers that contain cytosine a further reaction can occur; due to
the saturation of the C5C6 bond in these products, they undergo deamination
via hydrolysis of the C4 amino group to yield a carbonyl function (Scheme 1)
[19, 20]. This results in the formation of uracil-containing products, for example, T<>C becomes a T<>U dimer. This has implications in their mutagenic
properties, as discussed in the section Mutagenicity of the DNA lesions [21].
136
TT o x e t a n e
O
HN
O
HN
O
N
NH
N
UV
NH
N
OH
HN
UVB
(6-4)TT
O
N
TT D e w a r i s o m e r
HN
O
OH
O
N
O
HN
(unstable intermediate)
O
NH
NH
N
NH2
HN
O
N
N
TC azeti d i ne
(unstable intermediate)
(6-4)TC
Scheme 2. Mechanism of formation of (6-4)TT and (6-4)TC dimers, and the isomerization of (6-4)TT
to its Dewar isomer.
As with the P<>P dimers, (6-4)PP adducts containing cytosine (and the corresponding Dewar isomers) can undergo deamination reactions to yield uracilcontaining adducts [24]. However, deamination can only occur when the cytosine residue is on the 5' side of the dimer, as the transfer of the amine group to
the 5' base during adduct formation when cytosine is in the 3' position prevents
deamination from occurring.
NH2
UV
N
N
137
Deamination
N
HO
Cy t o s i n e
Cytos ine
h y d r ate
NH
HO
Uracil
h y d r a te
Scheme 3. The formation of cytosine photohydrate and its deamination to uracil hydrate.
of this species has been proposed to involve the nucleophilic addition of H2O to
a low-lying vibrational level of the first excited singlet state [25]. This product
is, however, unstable [25] and escaped detection in UV-exposed DNA for many
years [26]. As with the dimeric compounds where the C5C6 bond of cytosine
is saturated, this material undergoes deamination to yield the uracil analogue
(Scheme 3) [25]. The increased stability of the latter product has allowed the
quantification of this material in isolated and cellular DNA [27]. These materials are, however, only minor products with their yields ca. 100 and 1000 times
lower, in isolated and cellular DNA, respectively, than the P<>P adducts.
Spore photoproduct
The spore photoproduct (Scheme 5) has been detected on exposure of bacterial spores to UV light [33, 34], but this material is only generated in high
138
(a)
N
N
N
UV
2 x Adenine
NH2
N
NH2
[2+2]
i n t e r m e d i ate
N
N
NH2
NH
OH
N
N
NH2
N
N
N
NH
HN
CN
N
H
Po r s c h k e
pho top rod uct
(b )
CH3
NH2
N
HN
O
N
N
TA p h o t o a d d u c t
(via [2+2] intermediate)
Scheme 4. Mechanism of formation of the photoinduced adenine dimers (a) and the structure of the
thymine adenine photoadduct (b).
HN
O
NH
N
yields in vitro by irradiating dry, isolated DNA with UVC light [35]. This
dimeric material arises from addition of the methyl group of one thymine
residue to the C5 position of a neighboring thymine. As this product requires
139
anhydrous conditions for its formation, it is of little relevance in most cellular environments.
H
N
O
HN
H2N
8 - o x o - Gu a
H
N
H
N
H2N
CHO
Fa pyG ua
O
HN
O
H2N
NH
Ox a z o l o n e
CH3
OH
H
OH
140
141
approach has been expanded to allow the yield of inter-strand dimers, spore
photoproducts, and pyrimidine photohydrates to be determined in isolated or
cellular DNA [16, 27, 35, 39, 40]. These studies consistently show that T<>T
is the most common dimeric product formed by UVB radiation, followed by
similar yields of T<>C and (6-4)TC (with ca. half the frequency of T<>T formation) [40]. Interestingly, the overall yield of TT and TC dimers are very similar, but the proportion of P<>P:(6-4)PP differs for the two classes of dimers
with ca. 10:1 for TT dimers, and 1:1 for TC dimers, respectively. The dimeric
adducts [P<>P or (6-4)PP] at CT and CC sites were detected in much lower
yields (from 5 to 10 times in cellular DNA) than the TT and TC lesions. In cellular studies, the total ratio of P<>P:(6-4)PP lesions was 3:1 [40], which is
similar to that detected by other methods [41]. In isolated DNA, Dewar
adducts were detected at low levels, but these were not detected in cellular
DNA [40]. Similarly, oxidized bases such as 8-oxo-Gua are relatively minor
products of UVB radiation, with yields that are two orders of magnitude lower
than the P<>P dimers [42, 43].
In contrast to UVB exposure, exposure of cellular DNA to UVA radiation
results in much lower levels of direct damage, with the observed lesions
appearing to be predominantly mediated via sensitized reactions (reviewed in
[12]). Thus, the major products generated by UVA would be expected to be
oxidized photoproducts of purine bases such as 8-oxo-Gua. These materials
are indeed present at higher levels in DNA exposed to UVA than UVB [43],
but recent studies have shown that P<>P dimers are the major products of
UVA damage, with these present at threefold greater levels than 8-oxo-Gua
[43]. Despite being the most prevalent lesion, the P<>P dimers are formed at
lower levels by UVA than UVB. Interestingly, the pattern of damage induced
by UVA is different to that given by UVB, with T<>T lesions predominating,
together with ca. 10% T<>C lesions [43, 44]. It has been shown that UVA
radiation and aromatic ketone sensitizers (e.g., benzophenone) give a similar
spectrum of damage in vitro [43], suggesting that unknown sensitizers within
cells are responsible for the formation of these lesions via sensitized reactions,
rather than direct UV absorption by DNA. The prevalence of T<>T lesions is
probably a consequence of the more facile triplet energy transfer from
endogenous sensitizers to T than C residues. In addition to these products,
UVA radiation can also induce strand breaks [43]. These observations are consistent with a significant role for 1O2-mediated reactions in UVA-induced
DNA damage.
Exposure of cells to simulated sunlight, gives a damage spectrum that is
similar, but not identical, to that observed with UVB [43]. Thus, the majority
of DNA damage by sunlight is probably induced by direct UVB absorption.
However, UVA-induced photosensitized reactions also play a role, with the
levels of T<>T dimers and oxidized purine bases present at higher levels than
with UVB alone. A further consequence of simulated sunlight is that the yield
of (6-4)PP Dewar isomer lesions are increased relative to UVB irradiation, as
UVA light readily promotes the isomerization reaction [22, 23, 43].
142
143
144
cystine. This results in reduction to give the disulfide radical anion (RSSR )
and the corresponding Trp and Tyr radical-cations (Trp+ and Tyr+ ; reviewed
in [2]). These radical-cations rapidly deprotonate to give the neutral indolyl
radical and phenoxyl radical, respectively (Scheme 7a and 7b) [53]. In contrast, the triplet of Phe undergoes direct photo-dissociation to yield a benzyl
radical (Scheme 7c) [54].
The indolyl and phenoxyl radicals from Trp and Tyr, respectively, undergo
further reactions [53, 55]. In the case of the indolyl radical, these include reaction with O2 to give a peroxyl radical at position C-3 on the indolyl ring, which
can undergo further hydrogen atom abstraction reactions (Scheme 7a) [56].
The phenoxyl radical of Tyr can undergo dimerization (via C-O and C-C linkages) to yield di-tyrosine products and hydrogen atom abstraction reactions
(see Scheme 7b; reviewed in [55]).
The disulfide radical anions (RSSR ) formed via reaction of 3Trp or 3Tyr
with cystine can readily dissociate, in a reversible reaction, to give the anion
(a)
3 Tr p
HN
+O2
HN
HN
Ind o l y l rad ic al
( b)
3 Ty r
NH
x2
HN
HO
HN
Di - t y r o s i n e
c ro s s lin k s
Ty ro sy l rad i c al
NH
OH
HN
HO
(c)
3P he
CH2
HN
Scheme 7. Reactions of the triplet species formed by UV radiation of (a) Trp, (b) Tyr, and (c) Phe.
145
(RS) and a radical (RS ), or react with O2 to give the superoxide radical (O2 )
and regenerate the parent disulfide (RSSR) [4, 55]. Both O2 and thiyl radicals
undergo further reactions. The former primarily undergoes disproportionation
to H2O2 and O2 (spontaneously or catalyzed by superoxide dismutase) or oneelectron reduction reactions of metal ions. The thiyl radicals typically react
with a thiyl anion to regenerate a disulfide radical anion, or with O2 to give a
thiyl peroxyl radical (RSOO) (reviewed in [55]). The resulting thiyl peroxyl
radicals revert to thiyl radicals, or can isomerize to a sulfonyl radical
[RS(=O)O ] and hence give rise to sulfonic (RSO3H) and sulfinic acids
(RSO2H) (reviewed in [57, 58]).
In addition to the reactions described above, other molecules can also
undergo rapid electron transfer reactions with the triplets of Tyr, Phe and Trp.
Thus, 3Tyr can be rapidly quenched by electron transfer with O2, His, and Cys
(reviewed in [1]). In each case 3Tyr is converted to the phenoxyl radical, probably via the radical-cation and subsequent rapid deprotonation. The partner is
converted to the radical anion, which undergoes further reactions (e.g., O2 )
[55]. 3Phe also reacts rapidly with O2 to yield O2 [4].
Formation of 3Trp, 3Tyr and 3Phe also commonly occurs via sensitization
mechanisms, where light absorption by cellular chromophores followed by
energy transfer gives the triplet species that behave as outlined above. Triplet
state chromophores can also induce direct electron/hydrogen atom transfer
reactions. One-electron oxidation occurs primarily at Trp and Tyr as these side
chains are the most readily oxidized, with Tyr the ultimate sink for oxidizing equivalents. One-electron reduction can occur at Cys, and also at carbonyl
and protonated amine sites, although there is evidence for the rapid transfer of
free electrons within protein structures with cystine groups being the ultimate sink for reducing equivalents. The transfer of oxidizing and reducing species within proteins and peptides has been the subject of considerable study,
and has been recently reviewed [55, 59]. Direct hydrogen atom abstraction
reactions mediated by high-energy triplet states of chromophores can occur
with most protein side chains. These reactions usually yield carbon-centered
radicals (or thiyl radicals from Cys) [55, 59].
146
HO
H2O
HN
HN
zyl radicals [53]. With Tyr, direct cleavage of the phenolic -O-H bond can
occur, yielding the phenoxyl radical, a proton and a hydrated electron [2].
The hydrated electron (eaq) produced by direct photo-ionization can add
rapidly to O2, to give O2 , which can, in turn, induce further protein damage.
eaq-mediated addition to free carboxyl groups (e.g., the C terminus, or
Asp/Glu side chains) and amine groups (e.g., the N terminus, or Lys side
chains) results in deamination and H elimination. Hydrated electrons also
react with cystine to give the disulfide radical anion (RSSR ), and with peptide backbone carbonyl groups yielding a radical anion that can subsequently
give rise to backbone cleavage [4, 55, 61, 62].
Protein photoproducts induced by 1O2 reactions
As the rate constants for reaction of 1O2 with protein side chains are higher
than those with most other cellular targets [7], and proteins are present in most
biological systems at particularly high concentrations, these are major targets
for 1O2 (reviewed in [3, 4, 6]). The majority of reactions of 1O2 with proteins
occur via reactions that result in chemical change, rather than quenching pathways that result in relaxation to ground state O2 without inducing protein damage. Of the common amino acids present in proteins, Trp, His, Tyr, Met and
Cys, react with 1O2 at significant rates at physiological pH values [37]. At
high pH, where Arg and Lys are in their neutral (unprotonated) forms, photooxidation also occurs at these residues [63]. Other amino acids can also be
consumed as a result of indirect photo-oxidation processes, due to further reactions of 1O2-induced intermediates at the above residues. These reactions have
been implicated in cross-linking/aggregation of proteins [5]. The mechanisms
and products that arise from 1O2 reaction with these reactive side chains are
reviewed below.
Reaction of 1O2 with tryptophan residues
Reaction of 1O2 with Trp gives both N-formylkynurenine (and hence kynurenine via hydrolysis) and 3-hydroxypyrroloindoles, via the initial formation of
147
OH
HN
or
N
H
O
O
OH
3-- hydr o x y py r r o l oi n d ol e
N
H
O
O
O
N
H
H
N
H
Dio x etane
O
N
H
N- f o r my l k y nu r eni ne
NH2
HN
k y n ur en i ne
148
O
1O
2
HO
HO
OH
O
O
O
O
OH
OOH
OOH
HO
N
HOHICA
Scheme 10. Products of 1O2-mediated oxidation of Tyr.
149
O
N
HN
N
H
1O
2
O
N
O
N
H
and / or
HN
NH2
NH2
HN
H OH
N
N
N
H
H
N
HN
OH
HN
O
O
O
NH2 HN
OH HN
+ other products
Scheme 11. Products of 1O2-mediated oxidation of His.
150
cule of the parent to give two moles of the sulfoxide (R2S=O) [84] (reviewed
in [5]). With some sensitizers, other intermediates including a stable nitrogensulfur cyclic intermediate have been reported (reviewed in [5]). Subsequent
hydrolysis of this species yields 1 mol of sulfoxide and 1 mol of H2O2 [84].
This type of reaction may only be of significance with free Met, due to the
involvement of the free amino group.
With free Met, the stoichiometry of molecular O2:Met consumption is pH
dependent (reviewed in [6]). The stoichiometry and intermediates involved in
the corresponding reactions on proteins remain to be fully elucidated, although
it is clear that methionine sulfoxide can be a major product.
Reaction of 1O2 with cysteine and cystine residues
Rapid, but non-quantitative generation of the disulfide (RSSR) occurs when
free Cys reacts with 1O2 [5, 85]. Other products are formed, probably including cysteic acid (RSO3H), but these have not been fully elucidated [5].
It has been suggested that reaction of 1O2 with free cystine occurs via a
zwitterion (RS+ OO ), similar to that with Met [86]. This species probably
reacts with a further molecule of cystine yielding two molecules of the monosulfoxide RSS(=O)R. The occurrence of these reactions in proteins, where the
3-D structure is likely to constrain the cystine molecules, remains to be determined.
151
152
sequently oxidize susceptible residues on other proteins (and thereby give rise
to enzyme inactivation [101, 102]), can deplete low-molecular-mass antioxidants [103], and can also give rise to the formation of oxidized DNA bases,
strand breaks and DNA-protein adducts [104106]. Decomposition of these
peroxides to radicals may also initiate lipid oxidation chain reactions and
thereby result in significant membrane damage.
153
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159
Abstract. Over the past 20 years there has been increasing evidence that cells and the progeny of cells
surviving a dose of ionizing radiation can exhibit a wide range of effects inconsistent with the level of
dose received. Recently, the cause of these delayed effects has been ascribed to so-called bystander
effects, occurring in cells not directly hit by an ionizing track, but which are influenced by signals
from irradiated cells. These effects are not necessarily deleterious, although most of the literature deals
with adverse delayed effects. What is important to consider is what, if anything, these effects mean for
what is still the central dogma of radiobiology and radiation protection, i.e., that DNA double-strand
breaks are the primary radiation-induced lesion that can be quantifiably related to received dose, and
which determine the probability that a cancer will result from a radiation exposure. In this chapter we
review the history of radiation biology which led to the DNA paradigm. We explore the issues and the
evidence which are now challenging the view that dose deposition in DNA is all important. We conclude that in the low-dose region, the primary determinant of radiation exposure outcome is the genetic and epigenetic background of the individual and not the dose. This effectively dissociates dose from
effect as a quantitative relationship, but it does not necessarily mean that the effect is unrelated to DNA
damage somewhere in the system.
Key words: Bystander effects, genomic instability, radiation, radiation carcinogenesis, radiobiology.
160
ed. Unconditional acceptance of paradigms can lead to an uncritical acceptance of dogma. Similarly, unless we regard the scientists responsible for developing the theories as charlatans, we must acknowledge they formed their conclusions based on solid, painstaking research. Therefore, we shall attempt to
review the field critically after giving a brief historical perspective.
Classical radiobiology really started in the 1940s with the almost simultaneous publication of two books on mechanisms of action of ionizing radiation
on living cells. Lea published Actions of radiations on living cells in 1946
[1] and Timofeeff-Ressovsky and Zimmer published Das Trefferprinzip in
der Biologie in 1947 [2]. Both authors noted that there had been much radiation research on the chemical effects of ionizing radiation on pure substances
in the gaseous, liquid and solid states, and on aqueous solutions. These experiments of necessity involved what would be considered now as extremely high
doses. In the absence of our modern sophisticated knowledge of molecular and
cellular biology, books on radiobiology wrote about the actions of radiation on
the chemical constituents of living systems. Lea decided that the actions of
radiation on dilute aqueous solutions could be described as indirect actions
since most of the molecules of solute that reacted had not been excited or ionized directly by the radiation, but their reaction followed excitation or ionization of the solvent molecules. Lea noted that, although the total energy dissipated by the radiation per gram of solution did not vary with the concentration,
the energy dissipated in the solute per gram of solution was proportional to the
concentration, and with dilute solutions was only a minute fraction of the total
energy dissipated per gram of solution. Lea concluded we see that the weight
of solute reacting is proportional not to the energy dissipated directly in the
solute alone, but to the energy dissipated in the solution altogether. However,
Lea also noted that, at sufficiently low concentrations, the ionic yield did not
remain constant but diminished with diminution of the dose.
At this time there was a hypothesis of activated water following irradiation.
This was an intermediary body of finite life, which caused reactions in many
solutes. With regard to the total reaction, Lea was able to state that one of the
characteristics of indirect action was that it was always proportional to dose.
Early radiobiologists took it for granted that the biological effects of ionizing radiation were due to the chemical changes induced by the radiation, but
noted there was a problem in explaining why marked biological changes were
produced by doses of radiation that produced only small degrees of chemical
change.
There were various hypotheses to explain this, including cell poisons (products of cellular decomposition) and activated water mechanisms (depending on
the sensitivity of enzymes in a cell being greater than enzymes in a concentrated solution). One hypothesis was that the direct action of radiation was
inversely proportional to the molecular weight, and that this could be related
to the localization of the radiation damage (e.g., in chromosomes).
This led into the concept of target theory, also initially known as
Treffertheorie and discussed in the book by Timofeeff-Ressovsky and
161
162
date target. Equally logical was the idea that reproductive failure had to mean
that the genome was the target. Thus, the nucleus was assumed to contain or
be the target and many experiments supported this view (reviewed in [4]).
However, as Tikvah Alper pointed out [5], it is more correct to consider the
entire replicative machinery in the cell as a target, in that an energy deposition
in any sensitive organelle essential for cell replication will cause reproductive
death. The focus on DNA and later on DNA double-strand breaks (DSBs) can
be traced to mathematical interpretation and formalism of target theory by
Kellerer and Rossi [6] and by Chadwick and Leenhouts [7] among others, and
also to classic experimental evidence correlating DSBs in a quantitative fashion with dose [8]. This correlation still forms the mechanistic justification for
the use of a linear, no-threshold model in radiation protection. Later investigations of repair of DSBs served to consolidate the evidence that these were the
critical lesion caused by radiation, which, if left unrepaired or if mis-repaired,
led to the observable and dose-dependent biological consequences [9]. The
central importance of DSBs dominated radiobiology during the 1970s and
1980s, although other targets were suggested such as membranes [10, 11].
These are reviewed in the next section. Critically, for the development of the
field in general, unrepaired DNA DSBs were considered lethal and cells containing them did not reproduce. Quantitative assays of cell survival deemed
that cells that reproduced five to six times were survivors, in that they did
not contain a lethal lesion [12]. How the conceptual leap from survivor to
undamaged, perfectly normal cell occurred is not clear, but the assumption
is made in Elkind and Whitmores book [12] that cells that have survived to
form a colony are normal, and will not show any effect of the progenitor irradiation. The evidence was there that this was not as simple as it seemed, but it
appears to have been largely disregarded [1318]. The only serious challenge
to the DNA DSB paradigm came from proponents of various types of repair
models or pool models [1921]. These suggested that the final expression of
the dose-response relationship was determined more by how the cells coped
with the damage than with the quantitative amount of energy deposition or
consequent DSBs. Cellular fitness was an issue and proponents of these
models placed great emphasis on the low-dose effects, which predominated in
the shoulder region of the semi-log plot of the traditional dose-response curve
[22]. These models still regarded the cell survival curve as an inactivation
curve, in that increasing radiation doses inactivated increasing numbers of
cells. The theory was that inactivation could be reduced by various intervention strategies.
163
a few hundred dealing with membrane or mitochondrial damage, and less than
20 dealing with effects involving other organelles such as lysosomes, Golgi
apparatus, ribosomes and endoplasmic reticulum. When reviewing radiation
effects in cellular organelles, it is important to stress that the cell is an integrated functional unit, operating in multicellular organisms, within a tissue
structure. Experiments looking at specific effects in specific organelles must
be analyzed in context. What happens in one situation cannot be taken as necessarily being indicative of a global mechanism. This is particularly true at low
doses of exposure, where hierarchical controls of survival and response at the
tissue level, dictate much of what happens at the level of the individual cell.
This is discussed later in the chapter.
Most work on cellular organelles other than the nucleus relates to radiationinduced membrane damage or compromised membrane function (reviewed in
[2225]). Often, however, membrane damage is seen as an indirect effect.
Membranes are considered to be an important site of radical formation due to
the lipids, which enable peroxy-radical formation [11]. These radicals are
extremely toxic, causing DNA breakage. More recently, there has been consideration of the role of membrane channels and membrane-bound proteins in
the modulation of radiation damage [25]. Again however, DNA damage is
what is being modulated. Some possible indicators that membrane damage per
se is determining cellular outcome following exposure are the reports by
Gulbins and Kolesnick [26] and Lucero et al. [27] that ionizing radiation
among other stimuli can affect raft formation in cell membranes, leading to
transmembrane signaling. Benderitter et al. [28] show multiple changes in the
phospholipids content of cell membranes after irradiation and consider that it
must be considered as a critical target. Differences in radiation response
between cells that can communicate through gap junctions and those that cannot [29] also support a direct role for cell membrane damage in determining
response. Response here is tissue response, not individual cell response, thus
the argument is semantic since it pivots on how radiation damage is definedas
a targeted dose deposition or as a final cellular outcome.
The literature concerning mitochondria is interesting. Mitochondria are crucial for oxidative metabolism, and thus have roles in energy production for
repair, but are also the major site for the generation of oxidative stress. There
are thousands of mitochondria in cells, which are derived almost exclusively
from the maternal contribution to the zygote. Mitochondria contain their own
DNA and have their own genes controlling key cellular functions but do not
repair DNA damage very well [30, 31]. They have been well studied as candidates for expression of ionizing radiation damage and apoptosis. Most of the
studies are concerned with the oxidative stress aspects of mitochondrial radiation damage, for reviews see [3133] but some studies have actually looked at
mitochondrial DNA damage [34]. The problem is that with so many mitochondria in a cell, propagation of mutations in mitochondrial DNA is considered unlikely as a mechanism of fixation of DNA damage at the cellular level.
It is very likely that mitochondrial responses, including initiation of apoptosis
164
and generation of reactive oxygen species (ROS), are key determinants of radiation outcome, but whether mitochondria are targets in the classical sense,
in that direct energy deposition within their structure is required, is not clear.
Lysosomes are important because they release degradative enzymes following cellular injury. Only six published studies of lysosomal activity following
irradiation could be found and these dealt mainly with increased numbers of
lysosomes occurring after high-dose exposure [10, 3537]. Direct damage to
lysosomes has not been reported but the generation of lipid peroxy radicals or
inflammatory responses involving lysosomes has been seen [38, 39].
In the very few studies of radiation effects involving the Golgi apparatus,
the emphasis is on the response of Golgi-related enzymes to radiation [4045].
The Golgi apparatus is critical for intracellular trafficking of repair proteins
and the trans-Golgi-network (TGN) has important functions in controlling
repair, cell cycle progression, cytokinesis and genome stability, and it is likely
that studies of the role of the Golgi apparatus in these processes will increase.
The fundamental role of the TGN in the cytoskeleton, particularly the assembly and disassembly of the microtubules, means that compromised Golgi function will impact on outcome following radiation exposure [43]. This is likely
to be a secondary effect of oxidative stress, changed energy budgeting or
induction of apoptosis pathways, and not due to primary energy deposition in
Golgi structures [44].
Another cellular organelle that shows changed activity following irradiation
is the endoplasmic reticulum (ER) [4652]. This organelle is probably continuous with the nuclear membrane, and is a site of protein synthesis, assembly
and degradation [51]. The ER is the organelle in which newly synthesized
secretory and transmembrane proteins form their proper tertiary structure by
post-translational modification, folding, and oligomerization. However, many
of these proteins are unfolded or misfolded by extracellular or intracellular
stimuli. The accumulation of misfolded proteins constitutes a risk for living
cells. Eukaryotic cells possess at least three different mechanisms to adapt to
ER stress and thereby survive: (1) translational attenuation to limit further
accumulation of misfolded proteins; (2) transcriptional activation of genes
encoding ER-resident chaperones; and (3) the ER-associated degradation
(ERAD) pathway to restore the folding capacity. If the cells are exposed to
prolonged or strong ER stress, the cells are destroyed by apoptosis. Recent evidence indicates that ER stress signaling pathways are mediated in part by several protein kinases and play an important role in the pathogenesis of neurodegenerative disorders. There has been considerable interest recently in ER
stress, thought to arise from the physical overload of the ER with misfolded
proteins after exposure to cellular insults requiring repair or activation of
defense response pathways [52]. Papers considering ionizing radiation as the
cellular insult are few [4952], but clearly this site is important because mutations, particularly point mutations induced by ionizing radiation damage in
DNA, are likely to lead to protein misfolding and consequent ER stress and
apoptosisa type of cellular constipation. This again raises the issue of the
165
New evidence for radiation effects in the absence of DNA strand break
induction in the cell showing the effect
The fundamental paradigm shift from DNA-centered to response-driven radiobiology really started with the growing realization that all survivors are not
equally normal or healthy, and that progeny of irradiated survivors exhibit
many differences when compared to their unirradiated counterparts. The literature has references to this as early as 1964 [1318], but the major lines of evidence came from the late 1980s and early 1990s when demonstrations by
Seymour et al., Gorgogo and Little, Born and Trott, Mendonca et al., Streffer
et al., Kadhim et al. and Marder and Morgan [5359] using entirely different
systems and endpoints, all showed a high frequency of non-clonal effects in
the distant progeny of irradiated cells. These were variously called lethal mutations, genomic instability, delayed reproductive death or chromosomal instability, but basically all pointed to a persistence of expression of radiation damage in distant progeny of irradiated cells, deemed by conventional dogma of
the time to have survived the dose. A further challenge to the neat association
of DNA DSBs with outcome occurred in the 1990s with several papers showing that unirradiated cells that received signals from, or were in proximity to,
irradiated cells, but in which no energy deposition had occurred, could demonstrate effects similar to those seen in irradiated cells [6066]. The effects were
also transmissible to progeny [6769]. These changes in unirradiated cells
known (perhaps incorrectly) as bystander effects, make it difficult to talk of
targets for radiation interaction unless the response to the energy deposition
can be assessed at the level of the population of cells rather than the individual cell. This whole field has been extensively reviewed [7074], but it is
important to point out that, again, the historical evidence for what were then
known as abscopal and clastogenic effects was in the literature since 1954
[7589]. The current popularity of all these non-targeted or bystander
effects is attributable to the fact that they predominate in the low-dose region
of the survival curve [90], and thus may have major implications for the understanding of mechanisms of radiation action following low-dose exposures. It
is also because modern molecular biological techniques and sophisticated cell
culture methods permit the detection of effects at these low doses, which could
not be seen with less sensitive assays or the clonogenic cell lines available.
That being said, it is clear that in the pre genomic instability/bystander era
166
very good and careful research was done both using cell cultures and animal
models (reviewed in textbooks such as Lea, Timofeeff-Ressovsky and Zimmer,
Elkind and Whitmore, Bacq and Alexander, Alper, and Hall [1, 2, 5, 12, 91,
92]. Critical analysis of the data is required to try to integrate the old with
the new. In particular, primary sources of data need to be reexamined rather
that reliance on reviews, because often there is a tendency to go from the particular to the general if it fits current theories. A good example of this is the
dogma that there is an equal effect per fraction, i.e., that between doses there
is full recovery of cells so that they respond to the next dose as if never irradiated. Very rigorous work by Elkind and Sutton [12, 93] defined this split-dose
effect, which became known as Elkind Recovery. The theory fitted many of
the mathematical predictions and confirmed prevailing ideas that cells accumulated sub-lethal (single-strand breaks) DNA damage that, if time was
allowed, could be completely repaired, restoring cells to their pre-irradiation
state. Higher doses led to increased frequencies of lethal DSBs, and thus the
recovery effect was confined to the shoulder region of the curve and did not
alter the terminal slope of the curve [94]. Logically and experimentally, this
was a generalization containing many assumptions from particular data sets
generated from particular cell lines. The initial experiments were done with
high doses and with CHO cells, which have a high plating efficiency. Many
data did not fit, e.g., McNally et al. [95] showed that the iso-effect per fraction
broke down after more than five fractions, Bryant [96] showed decreased sensitivity after split-dose irradiation and Alper suggested that in vivo the survival
of clonogenic stem cells in tissues cannot be measured at all [5]. An alternative interpretation of the iso-effect per fraction data could be that a residual
damage effect is cancelled out by an adaptive response involving availability
of induced repair enzymes. This would obviously reach a limit, whereas the
residual damage would continue to accumulate, leading to apparent initial
adherence to an iso-effect law followed by increasing deviation as fraction
number increased. This example is presented merely to suggest that the data
are the data, it is the interpretation of the data that needs critical review.
167
mitochondria have DNA [99]. In the medium transfer experiments, broad field
irradiation of cells is used to produce the signal, so DNA damage cannot be
excluded as a cause of signal production here. Here clearly DNA damage is not
necessary to make cells respond to the signal by showing increased mutations
and induced genomic instability since signal recipients were not in the radiation field at all. The use of endonucleases produced contradictory data, Chang
and Little [100] concluded that endonucleases could result in delayed effects,
while Limoli et al. [101] cut DNA at several different sites and got no induction of chromosomal instability. They concluded that cutting DNA per se was
not sufficient to produce delayed effects in their system. Work with Auger
electrons [102] suggested that these strand-breaking electrons were operating
via indirect mechanisms. Radical scavengers that prevent radical damage to
DNA also reduce delayed effects [103, 104].
Some of the most convincing evidence that DNA damage is necessary, however remotely, comes from experiments by Nagazawa and Little and by
Mothersill et al. [105, 106] showing that bystander signals from DNA repairdeficient cells are more toxic, in the sense that they produces greater effects
than their wild-type line. Morgans group also have evidence that chromosomally unstable colonies derived from cells with rearrangements produce a
more toxic DIE factor than their wild-type relatives [107]. Other evidence
comes from the ability of DNA strand-breaking chemicals such as bleomycin,
but not DNA intercalating agents such as cis platinum, to produce delayed
effects [108]. Radical scavengers also reduce delayed effects [109111]. Work
reported very recently by Suzuki et al. [112] using the H2AX assay shows that
signals form irradiated cells can induce H2AX activity in unirradiated
bystander cells. Thus, it would appear that DNA damage is associated with the
production of delayed effects. The question is whether this damage is just a
manifestation of free radical damage [112116] or whether among the DNA
damage specific lesions such as DSBs or clustered damage is required [117,
118]. This question has not been satisfactorily resolved.
168
many areas of biology and involves, for example, cytokines, ceramides and
other molecules associated with intercellular communication in tissues. This is
outside the scope of the present review but is discussed extensively in [121]
and is considered in [122] in relation to radiation response. The idea has not
gained much favor in radiobiology, probably because of the association of dose
(energy deposition) with damage in a one-to-one relationship, but it makes
sense that at low doses where cellular energy supplies are adequate to cope
with the damage caused by energy deposition, there would be coordination
among the cells in a population or tissue to prevent energy wastage on nonimportant or unrepairable lesions, and a shuttling of cellular energy into the
most productive areas. Again this type of preferential distribution of available
resources is not novel and is well established in other areas of biology [123].
Obviously, once the damage burden exceeds a certain level in a tissue or cell
population, other mechanisms are needed to salvage tissue or population functionality. Figures 1 and 2 represent an attempt to conceptualize these ideas.
This analysis would predict late effects as a consequence of population tolerance of a certain amount of damage, which could be dealt with later at a time
when population reserves of energy and substrates needed for apoptosis or
repair were replenished. Thus, the time component of response becomes established in the context of energy supply and the kinetics of the coping mecha-
169
nisms. It is often forgotten that repair processes take time. Protein synthesis
is complex and substrate mobilization is not instantaneous [124]. Cells have a
limited capacity at the purely physical level of space in cellular organelles,
such as the ER as discussed previously and in reviews [125, 126]. At the population level, disposal of dead cells and general clean-up is an issue. Effects
in progeny or transgenerational effects would occur when cell division
occurred before repair was complete.
170
deleterious mutations. In any case the repair of these breaks places a burden on
cellular energy and protein production systems. The alteration in energy utilization patterns and the associated enhanced demand for protein synthesis carries with it further generation of ROS due to the higher metabolic rate.
Questions remain concerning the driver of the oxidative stress, how it persists
in distant progeny and most importantly, why it is not selected against in systems where enhanced cell death, deleterious mutations and reduced growth
occur. Clearly, the ROS driver must be continuously present and must be acting at the population level not at the cell level. A candidate driver is the
bystander signal pathway. Most of the evidence comes from radiobiology
because only in this system, can cells be uniquely targeted. The important
point is that bystander signals induce genomic instability-like effects, and
appear to do this by elevating ROS in the recipient cells [130, 131]. The recipient cells in turn produce bystander signals, which could both temporarily and
spatially distribute and maintain the instability phenotype in a population. The
key question of why this mechanism exists at all and why genomic instability
persists at roughly the same level in distant progeny is without an answer at
present. The phenomenon occurs at doses of radiation, which do not normally
kill cells. The bystander effect means that perfectly healthy, non-exposed cells
are showing effects which should only occur if they received a dose of radiation. This has prompted us to consider the possibility that the process is beneficial at some level of organization that supercedes the individual cell. Many
of the features of genomic instability suggest a permanently enhanced tolerance for mutations or plasticity in the progeny leading to enhanced frequencies
of chromosome damage or death in the population [132]. This hypothesis was
and still is very popular with Russian radiobiologists, and unfortunately the
discussion has not been very accessible to scientists in the West as much of it
was secret and, if published, was in Russian. Timofeeff-Ressovsky and colleagues [133] showed for the first time that the frequency of surviving cells
carrying chromosome abnormalities did not increase, while the mitotic index
did increase, as a result of exposing resting cells to low-dose irradiation, suggesting perhaps that an enhanced division rate was compensating for death (by
apoptosis?) of cells carrying mutations. They also defined cascade mutagenesis in yeast, which is very similar to what we call genomic instability and is
again evidence of plasticity of the genome occurring after exposure to low
doses [134139]. We suggest that genomic instability may represent part of
the mechanism by which adaptation to altered environmental conditions is
achieved at the population level. This natural selection is assumed to result
from selection among the biodiverse population for those which happen to be
best suited to the new challenge. We extend this hypothesis to suggest that the
change in environmental conditions actively liberates the exposed population
from the tight controls needed to maintain the stability of the DNA and consequent fitness of the population in its previous environment. We suggest a
two-stage mechanism; part 1 involves the release of cells from the tight control which previously terminated any showing abnormalities in the genome,
171
Is the central dogma challenged or is the system just more complex than
previously recognized?
To summarize, this paper reviews the historical data leading to the conclusion
that DNA damage, and, more particularly, DSBs are the critical site of energy
deposition that causes death, mutation and chromosome aberration. It then
considers whether is conclusion, upon which our radiation protection legislation is based, is challenged by the growing realization that at low doses much
of the damage ascribed to radiation does not require deposition of ionizing
radiation in the cell showing the effect. We conclude that at low doses, it is
likely, but by no means certain, that direct DNA damage is necessary somewhere in the system, to trigger the non-targeted effects measured in unexposed
cells, but the evidence that a DBS is required is weak. We stress the importance
of radiation response and of the emergent properties of tissues and cell populations in determining the final outcome. The consequences of this more complex situation are that the uncertainty at low doses is probably not resolvable,
and that, as shown in Figures 1 and 2, any outcome has a probability of occurring, which cannot be defined given our present understanding of these mechanisms.
Acknowledgements
We wish to acknowledge our funding agencies in Ireland (Science Foundation Ireland and Irish Cancer
Research), and in Canada, (the Canada Chair program and the National Science and Engineering
Research Council). We also wish to acknowledge the late Tikvah Alper, whose inspiration and willingness to look at and analyze data which did not fit laid the way for much of this field of research.
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The Channing Laboratory, Brigham and Womens Hospital, 181 Longwood Avenue, Boston, MA
02115, USA
Molecular Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA
Abstract. Oncogenes encoded by human tumor viruses play integral roles in the viral conquest of the
host cell by subverting crucial and relatively non-redundant regulatory circuits that regulate cellular
proliferation, differentiation, apoptosis and life span. Human tumor virus oncoproteins can also disrupt pathways that are necessary for the maintenance of the integrity of host cellular genome. Some
viral oncoproteins act as powerful mutator genes and their expression dramatically increases the incidence of host cell mutations with every round of cell division. Others subvert cellular safeguard mechanisms intended to eliminate cells that have acquired abnormalities that interfere with normal cell division. Viruses that encode such activities can contribute to initiation as well as progression of human
cancers.
Key words: Aneuploidy, centrosomes, cervical cancer, human papillomavirus, tumor suppressor,
viral oncogene.
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al mutagenesis, where integration of the provirus causes high-level dysregulated expression of a cellular proto-oncogene or disruption of a tumor suppressor. More frequently, however, retroviruses pick up cellular proto-oncogene sequences during their replication cycles. Since this process is generally
associated with concomitant deletion of viral coding sequences, many oncogenic retroviruses are intrinsically defective for completing the infectious life
cycle, and require normal helper viruses for replication. Replication of retroviral genomes involves the viral reverse transcriptase enzyme that lacks proofreading mechanisms, and thus is considerably more error-prone than host
chromosome replication. Moreover, the acquired host cell-derived sequences
do not contribute to the viral life cycle and are not subject to the same degree
of mutational restriction as the viral genome. Hence they will accumulate
mutations at a significantly higher rate than the remainder of the retroviral
genome. In rare cases, the resulting expression of specific mutated versions of
such retrovirally transduced cellular genes can endow the infected host cell
with a growth advantage relative to the surrounding uninfected cells, and a
tumor may form (reviewed in [4]).
Even though the concept of oncogene activation and transmission by retroviruses has not been clearly documented in human cancers, the recognition
that retrovirally transmitted oncogenes represent specifically altered versions
of cellular proto-oncogenes had a major impact on our understanding of carcinogenic mechanisms. In human cancers, proto-oncogenes are frequently
mutated and activated through cell intrinsic mechanisms, including point
mutations, gene amplification, gene fusion, or alterations that lead to increased
mRNA or protein stability. Moreover, activating mutations of oncogenes isolated from human cancers are often identical to those originally discovered
with retrovirally activated oncogenes [5].
Oncogenes of human tumor viruses are virally encoded genes that play
integral roles for the viral life cycle. To fulfill their roles in the viral life cycle,
human tumor virus oncogenes target critical cellular regulatory circuits,
including cellular proto-oncogenes and tumor suppressor pathways, and cause
their activation or inactivation, respectively.
Some viral oncogenes also subvert cellular processes that are necessary for
maintaining genomic integrity of the host cell. Hence, some human tumor
viruses also contribute to human carcinogenesis by creating a cellular milieu
that is conducive for the generation and accumulation of activating mutations
of cellular oncogenes and/or inactivating mutations of tumor suppressors in the
host genome. Such viruses contribute not only to initiation but also to progression of human cancer.
181
scribed, and the complementary strand does not contain any coding information. The papillomavirus genome can be divided into three parts. The early
coding region encodes approximately seven open reading frames (ORFs).
Individual early ORFs are denoted by the letter E and a number according
to their relative molecular size. The lower the number, the longer the corresponding ORF. The late coding region consists of two L ORFs, which
encode the viral capsid proteins. The non-coding region contains multiple cis
regulatory elements that modulate viral transcription and genome replication
(Fig. 1A) (reviewed in [6]). Approximately 200 different human papillo-
Figure 1. (A) Schematic depiction of the double-stranded circular genome of high-risk HPV-16. Only
one of the two DNA strands is actively transcribed and contains all the coding information. The different early (E) and late (L) open reading frames are encoded using each of the different possible
phases of translation as indicated by the concentric arcs. The long control region (LCR) does not contain extensive coding potential but contains various cis elements that are necessary for the regulation
of viral transcription and replication. The position of the major early promoter within the LCR is indicated by an arrow. See text for details. (B) Representation of integrated HPV LCR/E6/E7 sequences
in cervical cancer lines. The HPV genes are expressed from the viral promoter within the LCR (indicated by an arrow) expression is dysregulated due to transcriptional and non-transcriptional mechanisms. See text for details.
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maviruses (HPVs) have been identified, and additional types likely exist
(reviewed in [7]). HPVs display a pronounced tropism for squamous epithelial
cells, and approximately 30 HPVs specifically infect mucosal epithelia. The
mucosal associated HPVs are classified as high-risk and low-risk according to the propensity for malignant progression of the lesions that they cause.
Low-risk HPVs cause benign warts, which have an extremely low risk for
malignant progression. In contrast, infections with high-risk HPVs account for
more than 99% of all cervical carcinoma. Worldwide, in excess of 470,000 cervical cancer cases are newly diagnosed each year, and cervical cancer remains
a leading cause of cancer death in young women. Since no HPV-specific therapies exist, there are very limited regimens for treatment of late stage invasive
cervical cancer, and the death rate has remained unacceptably high at approximately 30% (reviewed in [8]). Cervical cancer incidence is much lower in
countries where there is broad access to preventive cytology-based screening
programs that allow for detection of potentially pre-cancerous high-risk HPVassociated squamous intraepithelial lesions (SILs). In the US cervical carcinoma accounts for approximately 6% of all cancer cases (13500 per year), and
remains frequent in medically underserved segments of the populations.
Approximately 20% of human oral cancers, particularly oropharyngeal carcinomas, are also high-risk HPV positive [9]. A fraction of other anogenital tract
malignancies such as penile cancer in males and vulvovaginal cancers in
females (reviewed in [10]), as well as anal carcinomas that frequently occur in
AIDS patients (reviewed in [11]), are also associated with high-risk HPV
infections. Even though preventive vaccination strategies using recombinant
empty capsid particles yielded promising results [12], it will be decades before
they might have a major impact on the incidence of HPV-associated disease
(reviewed in [13]).
Due to the small size of their genomes, HPVs do not encode key, rate-limiting replication enzymes, and thus these viruses have adopted a parasitic
replication strategy to exploit the cellular DNA replication machinery.
Establishing and maintaining a cellular environment conducive for viral
genome synthesis is paramount since the HPV life cycle is tightly linked to
the differentiation status of the infected keratinocyte. The squamous epithelium is a multilayered organ and only the basal layer contains undifferentiated,
actively dividing cells. These cells are the initial targets for infection, and
HPVs gain access to these cells either through an injury or at the squamocolumnar transformation zone where basal-like epithelial cells are more readily accessible. Expression of late genes and production of viral capsids, however, only occurs in differentiated epithelial cells. In a normal squamous
epithelium, cellular differentiation and proliferation are tightly coupled
processes, and cells terminally withdraw from the cell division cycle when
they undergo differentiation. To allow for viral genome replication in these
growth-arrested cells, HPVs encode regulatory proteins that can uncouple
these processes (reviewed in [6]). Consistent with this notion, high-risk HPV
E6 and E7 proteins functionally compromise the p53 and retinoblastoma
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Tetrasomy/multinucleation
Morphological examination of HPV-associated cervical lesions revealed the
presence of distinct nuclear abnormalities, including enlarged nuclei as well
as multinucleation (reviewed in [38]). Enlarged nuclei are a hallmark of
increased ploidy and numerical chromosomal abnormalities. Lesions caused
by high-risk HPV infections, but not those associated with low-risk HPV
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infections, showed an increased degree of tetrasomy [39, 40]. Ectopic highrisk HPV E6 or E7 expression can each independently induce tetraploidization both in actively dividing basal and intrinsically growth-arrested
suprabasal cells [41, 42]. The mechanism of tetrasomy induction by HPV
oncoproteins has not been delineated. Expression of high-risk HPV E6 may
cause increased ploidy and multinucleation through inactivation of the p53
tumor suppressor [43]. Interestingly, however, the ability of HPV E7 to
induce tetrasomy is unrelated to the capacity to inactivate the retinoblastoma
tumor suppressor [42].
Tetrasomy is often regarded as a prelude to aneusomy, since, as discussed
in more detail later, such cells are more prone to mitotic errors when they
undergo additional rounds of cell division (reviewed in [44]). Tetraploidy arises in cells that undergo DNA synthesis without completing nuclear and cellular division, most frequently as a consequence of cytokinesis problems. It is
important to point out that a tetraploid cell will have to be able to successfully complete a subsequent cell division to generate aneuploid progeny. Cells
that re-encounter cytokinesis problems, however, will become polyploid
and/or multinucleated (reviewed in [44]). The emergence of cells with severe
nuclear abnormalities may be of relevance diagnostically, but since such cells
were generated through persistent cytokinesis defects, and thus are incapable
of undergoing full cell division, they represent abortive structures [48, 49] that
do not contribute to malignant progression.
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Figure 2. Examples of mitotic abnormalities in HPV-16 oncogene-expressing cells. HPV-16 E7 oncogene expression causes centrosome duplication errors, which can give rise to tripolar metaphases (A),
which can undergo anaphase progression (B). Multiple centrosomes (indicated by arrowheads) in
HPV-16 E7-expressing cells can undergo coalescence and form abnormal bipolar metaphases (C) and
anaphases (D). Highly irregular multipolar metaphase in HPV-16 E7-expressing cells (E). HPV oncogene-expressing cells also contain centrosome-independent mitotic abnormalities including anaphase
bridges (F). These may be caused by dicentric chromosomes that might have formed as a consequence
of breakage fusion bridge cycles. See text for details.
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Figure 3. Centrosome abnormalities in human tumors can arise by different mechanisms. HPV-16 E7
oncoprotein expression in primary human cells induces centrosome duplication errors that can lead to
mitotic abnormalities, chromosome missegregation and aneuploidy. The retinoblastoma tumor suppressor restricts DNA replication in normal human cells by forming a transcriptional repressor complex with members of the E2F transcription factor family. HPV E7 associates with pRB and induces
its proteolytic degradation. E2F transcription factors now act as activators of gene expression. Cyclin
E is a transcriptional target of E2F and results in increased cdk2 activity. Initiation of DNA synthesis
also generates a licensing signal that renders each of the centrioles competent for duplication.
Normal centrosome duplication is coupled to S-phase progression, and each maternal centriole serves
as a template for a single daughter. In addition to inducing aberrant S-phase progression, HPV-16 E7
expression uncouples centrosome duplication from the cell division cycle, either by retaining the
licensed state of the maternal centriole (persistent licensing), which causes the formation of multiple daughters from a single maternal template, or the newly synthesized daughters in E7-expressing
cells may be immediately licensed for duplication, causing formation of granddaughters [102]. Cells
that acquired supernumerary centrosomes will either enter a multipolar metaphase or an abnormal
bipolar metaphase when individual centrosomes coalesce and form a single mitotic spindle pole body.
Abnormal metaphase to anaphase progression may be restricted through a checkpoint, as there is an
eight- to tenfold difference between abnormal metaphases and anaphases in HPV-16 E7-expressing
cells. If such abnormal cells can complete nuclear and cellular division, aneuploid progeny may be
formed. There may be an additional checkpoint that constrains cell division of abnormal mitotic cells,
as it has been shown that some tumors remain largely diploid despite the presence of excessive numerical centrosome abnormalities and related mitotic abnormalities [103]. Cells that encounter cytokinesis defects may decondense their chromosomes and reenter a tetraploid G1-like state. Alternatively
cells may complete nuclear, but not cellular, division and become binucleated. Tetraploid cells may
reenter the cell division cycle causing additional reduplication of centrosomes. Centrosome abnormalities in HPV-16 E6-expressing cells do not arise in diploid cells, but accumulate in parallel ...
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mentioned previously, cells with abnormal centrosome numbers are not necessarily destined to undergo multipolar cell division. Indeed, we observed an
approximately tenfold difference in the incidence of multipolar metaphases
versus multipolar anaphases, suggesting that there may in fact be control
mechanisms that thwart progression of multipolar mitotic processes in diploid
cells (Fig. 3) [56]. As mentioned previously, multiple centrosomes can form a
single mitotic spindle pole body through centrosome coalescence (reviewed in
[57]). Under such conditions, a diploid cell may undergo bipolar, albeit potentially asymmetric, cell division with abnormal chromosome segregation
(Fig. 2). In such a scenario, one of the resulting daughters may gain chromosomal material and become aneuploid (Fig. 3). Centrosome coalescence and
associated mitotic abnormalities have indeed been observed in HPV oncogeneexpressing cells [58].
Boveris prediction that centrosome duplication errors in normal cells may
contribute to carcinogenesis [53] could not yet be proven in this system; however, recent studies with transgenic mice that express HPV-16 E6 or E7 separately yielded results that are at the very least consistent with his hypothesis.
Mice engineered to express HPV-16 E7 in basal epithelial cells developed
high-grade cervical dysplasia that progressed to frank cervical carcinomas. In
contrast, HPV-16 E6-expressing mice only developed low-grade cervical dysplasia, which failed to undergo malignant progression [59]. Not surprisingly, a
similar fraction of cells exhibited centrosome abnormalities in lesions of HPV
E6- or E7-expressing animals [59]. Hence, detection of centrosome abnormalities in a tumor per se cannot be used as a generic predictor of carcinogenic
progression, but the finding that transgenic HPV-16 E7-expressing animals
develop tumors is consistent with Boveris model that aberrant centrosome
duplication may contribute to tumorigenesis. In contrast, centrosome abnormalities that occur in cells with nuclear abnormalities may represent abortive
events triggered by persistent cytokinesis defects.
Even though pRB inactivation can give rise to mitotic abnormalities and
cytokinesis problems due to mitotic checkpoint abnormalities [60], expression of HPV-16 E7 can induce centrosome duplication errors in cells that lack
pRB as well as the related pocket proteins p107 and p130 [61]. Hence, this
ability of HPV-16 E7 is at least in part independent of the ability to target pRb
and/or p107 and p130. Strikingly, however, inhibition of cdk2 activity in E7expressing cells abrogates the ability of E7 to induce aberrant centrosome
synthesis, whereas it does not similarly affect normal centrosome duplication.
Treatment of E7-expressing cells with the small molecule cdk2 inhibitor
indirubin-3'-monoxime dramatically decreased the steady level of centrosome
abnormalities in E7-expressing cells, and strikingly reduced the degree of
aneuploidy in such cells [62]. Hence, whereas cdk2 activity may not be strictly necessary for cell division and centrosome duplication [63, 64], aberrant
cdk2 activity may cause aberrant centriole synthesis and centrosome abnormalities [62].
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The large tumor antigen (TAg) of SV40 forms a complex with the Nijmegen
breakage syndrome protein (NBS1) that plays a key role in modulating double-strand DNA break repair. This leads to aberrant replication of cellular and
viral genomes, resulting in polyploidy and increased SV40 genome copy numbers in infected cells [92]. In addition, SV40 TAg associates with bub-1 and
bub-3 mitotic checkpoint proteins, thereby disturbing mitotic fidelity [93].
Ectopic expression of large tumor antigens encoded by some JC human polyomavirus strains were also reported to trigger numeric and structural chromosome aberrations [94].
Hepatitis B virus is a human member of the hepadnaviridae family that
causes hepatitis. Chronic hepatitis can progress to cirrhosis and ultimately to
hepatocellular carcinoma. Progression is a slow process that often takes several decades to occur [95]. BV-associated liver cancers are genomically unstable
and the HBV X protein (Hbx) can induce supernumerary centrosomes and
multipolar spindles that are associated with defective mitoses and abnormal
chromosome segregation as well as formation of multinucleated cells and
micronuclei [96, 97]. Treatment of Hbx-expressing cells with antagonists of
the Ran GTPase interacting nuclear export receptor Crm1 [96] or an inhibitor
of mitogen-activated protein/extracellular signal-regulated kinase (MEK) 1/2,
reduced the incidence of centrosome abnormalities [97]. Interestingly, the adenovirus E1A oncoprotein was also reported to induce centrosome abnormalities through a pathway that depends on the integrity of Ran-dependent nucleocytoplasmic transport [98].
Hence, similar to high-risk HPVs, a number of other human tumor viruses
may not only contribute to initiation of tumorigenesis by targeting cellular control mechanisms such as the retinoblastoma or p53 tumor suppressor pathways
that would normally restrict proliferation in infected host cells, but also contribute to carcinogenic progression through induction of genomic instability.
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Concluding remarks
Studies with DNA tumor virus oncogenes have been instrumental in the discovery of critical growth regulatory pathways that control proliferation, apoptosis and differentiation in normal human cells. Based in part on these discoveries it has been possible to define a minimal set of regulatory nodes that are
rendered dysfunctional in almost any human solid tumor [27]. Tumorigenic
human cell populations can be generated when these pathways are disrupted in
vitro (reviewed in [30]). Unlike naturally occurring human tumors such artificially generated human tumor-like cells do not exhibit marked genomic instability [31]. Thus, it appears that genome destabilization may be a necessary
step to set the stage for carcinogenic progression. It is an exciting possibility
that the study of viral oncoproteins will once again be instrumental in discovering cellular pathways that control the genomic integrity, and that these studies will have important ramifications for our understanding of tumorigenic
pathways. Cellular processes that control genomic instability may also be
attractive targets for development of novel anticancer therapies. Inhibition of
genomic instability in early pre-malignant lesions may restrain malignant progression, whereas therapeutic interventions that lead to increased genomic
destabilization in later stage tumors may create genomic chaos [101] that
could interfere with clonal expansion and the viability of the tumor [29].
Acknowledgements
The work on HPV-induced genomic destabilization in K.M.s laboratory is supported by grants
CA81135 and CA66980 from the National Institutes of Health, and a grant from CYTYC Corporation.
C.L.N. is supported by NIH training grant T32 CA09031. S.D.s and A.D.s laboratory is supported by
a Special Innovation Award from the PNC Foundation and a Pilot Project Developmental Grant from
the University of Pittsburgh Cancer Institute.
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Abstract. A conceptual shift has occurred in recent years from considering cancer as simply a disease
of deregulated cell proliferation to a view that incorporates the aberrant control of apoptosis into the
equation. Apoptosis is an organized, genetically programmed cell death process by which multicellular organisms specifically destroy, dismantle and dispose of cells. In cancer cells, this tightly controlled process is suppressed by genetic lesions, allowing cancer cells to survive beyond their normal
life span even in hostile environments that are prone to hypoxia and lack many trophic factor supports.
In the last two decades, cancer researchers have made great strides in our understanding of the underlying molecular mechanism of apoptosis in chemoresistance generation and tumorigenesis. This
tremendous increase in our knowledge of apoptosis in tumors has greatly impacted our perspective on
carcinogenesis. Key regulators of apoptosis such as members of the Inhibitors of Apoptosis family and
Bcl-2 family have been shown to play a pivotal role in allowing most cancer cells to escape apoptosis. The identification of specific targets involved in the suppression of apoptosis in cancer cells has
facilitated the design and development of therapeutic strategies based on rational molecular approaches that aim to modulate apoptotic pathways. Many promising apoptosis-dependent strategies have
been translated into clinical trials in the continued assessment of regimens that can effectively eradicate cancers.
Key words: Apoptosis, bcl-2, death receptors, IAP, mitochondria, p53.
Background
In the 1960s, Lockshin and Williams introduced the term programmed cell
death to refer to a gene-directed form of cell death [1]. The term apoptosis
was coined in 1972 by Kerr, Wyllie and Currie to describe a form of ischemiainduced hepatic cell death. The term comes from the Greek (apo + ptosis) for
falling off and depicts a distinct morphology of dying cells characterized by
cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation [2]. The realization that apoptosis is a genetically invoked form of
cell death has impacted our understanding of proliferative and degenerative diseases because of the implication that tissue homeostasis can be controlled by
factors that regulate cell survival and death, as well as those that affect proliferation and differentiation. The fact that apoptosis is controlled by genetic programs renders it susceptible to disruption by mutations, and the acquired ability of cancer cells to evade apoptosis is one of the hallmarks of cancer [3, 4]. In
this chapter, we first delineate the unique morphology of apoptotic cells.
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However, since the process of apoptosis is very much dictated by genetic programs, the main thrust of this chapter is on the discussion of apoptosis in terms
of the underlying molecular mechanisms. We also address the various
approaches currently in use and under consideration to reactivate apoptosis for
use in anticancer therapy. Our objective is that readers will gain a greater appreciation of the importance of apoptotic mechanisms underlying cancer pathogenesis, and thereby appreciate the subsequent impact this may have on newer
modes of the medical management of tumors.
Introduction: apoptosis
Apoptosis is a gene-directed mechanism in which unnecessary or dangerous
cells are triggered to undergo self-destruction without injuring neighboring
cells or eliciting any associated inflammatory response [5]. The core apoptotic pathway was first described through genetic analysis in the nematode
Caenorhabditis elegans and subsequently found in species as diverse as
Drosophila melanogaster and humans [6]. In these multicellular organisms,
the apoptotic process is crucial for normal development, differentiation, tissue
physiology and defense against pathogens. The dysregulation of apoptosis is
intricately involved in the etiology and pathogenesis of many diseases, including AIDS, autoimmune disorders, neurodegenerative diseases and cancer.
In general, apoptosis can be divided into the initiation phase, the effector
phase, and the degradation phase [7]. In the initiation phase, a stimulus, either
extrinsic or intrinsic to the cell, triggers the apoptosis process. This stimulus
may arise from a variety of sources and some general inducers include radiation, UV, growth factor withdrawal and cytotoxic agents such as chemotherapeutic drugs. The potency of each of these stimuli to induce apoptosis, however, is cell-type dependent. Despite the differences in the initiation of apoptosis, the effector phase in which the apoptotic machinery is activated shares
common biochemical features (see the section The apoptotic machinery).
Once cells have committed to apoptosis, the degradation phase begins and the
process becomes irreversible. At this late stage, double-stranded breakdown of
DNA into nucleosomal segments is manifested as DNA laddering in gel electrophoresis [5]. This DNA laddering is a defining feature of apoptotic cell
death that contributes to the unique morphology of apoptotic cells.
203
tation. As the nuclear outline convolutes, the cytoplasm also condenses and
blunt blebs or protrusions appear on the plasma membrane. While the cytoplasm continues to condense, the cell disintegrates into the characteristic membrane-bound apoptotic bodies enclosing fragments of the nucleus. The integrity of the membrane encasing the apoptotic fragments is retained during the
course of apoptosis until they are engulfed by phagocytes in a contained
manner without eliciting an inflammatory response that might be harmful to
the surrounding tissues [2, 13].
204
Figure 1. Apoptotic pathways. Key regulators in both the extrinsic and the intrinsic apoptotic signaling pathways are highlighted. See text for details.
of largely distinct molecular interactions and utilize different caspases, but are
also interconnected at numerous steps and ultimately converge at the level of
effector caspase activation [19]. However, for the sake of simplicity, we shall
initially treat the two as being mutually exclusive.
Intrinsic pathway
The intrinsic pathway is activated in response to intracellular stress, such as
DNA damage, hypoxia, growth factor deprivation and some chemotherapeutic
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Extrinsic pathway
The extrinsic pathway, also known as the death receptor-induced pathway, is
initiated by the ligation of death receptors belonging to the tumor necrosis factor receptor (TNF-R) superfamily, such as Fas/APO-1/CD95 and TNF-R1
found on a variety of cells [19]. Members of the TNF-R family are characterized by a cytoplasmic death domain (DD) involved in protein-protein interactions that is essential for delivering apoptotic signals [31, 32]. Binding of ligands promotes oligomerization of the death receptors, and their cytoplasmic
domains then recruit DD-containing adaptor proteins FADD and TRADD via
DD-DD interactions, leading to the formation of a death-inducing signaling
complex (DISC) [3335]. FADD then causes the sequestration of the proenzyme forms of caspase-8 and -10 through the homotypic interaction of DDs
known as death effector domains (DEDs) to DISC [36, 37]. The proximityinduced activation of multiple caspase-8 molecules by DISC [38] in turn activates effector pro-caspase-3 [39], at which point the intrinsic and the extrinsic
pathways converge [40].
Evidently, caspases occupy a central role in the regulation of apoptosis in
both the intrinsic and the extrinsic pathways. The apoptotic process is, thus,
also tightly controlled by regulators of caspases. An important family of
endogenous caspase inhibitors, termed the inhibitors of apoptosis (IAPs), was
identified as a central regulatory factor that blocks the execution of apoptosis.
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Inhibitors of apoptosis
Although other proteins have been identified that inhibit initiator caspases,
only the IAPs (see Fig. 2) have been demonstrated to be endogenous direct
repressors of the terminal caspase cascade [41, 42]. In humans, members of
this family of proteins include neuronal apoptosis inhibitory protein (NAIP),
X-linked inhibitor of apoptosis (XIAP/hILP), cellular IAP1 (c-IAP1/HIAP2),
cellular IAP2 (c-IAP2/HIAP1), Survivin, Livin, testis-specific IAP (Ts-IAP)
and Apollon/BRUCE. The anti-caspase activity of IAPs may be attributed to
their characteristic 7080-amino acid baculoviral IAP repeat (BIR) domains.
XIAP, arguably the most potent IAP identified, possesses three BIR domains,
of which BIR3 is an inhibitor to the initiator caspase-9 and BIR2 an inhibitor
to effector caspase-3 and -7 [43, 44]. Moreover, some IAPs also contain a
RING domain that functions as E3 ubiquitin ligase, capable of recruiting target proteins to a complex containing an E2 enzyme for ubiquitin conjugation
and proteasomal degradation [45]. In particular, c-IAP2 and XIAP can trigger
the ubiquitination of caspase-3 and -7 [46, 47], suggesting that targeting of
caspases to the proteasome may be another anti-apoptotic mechanisms of the
IAPs. During the course of apoptosis, the caspase-inhibitory function of IAPs
is negated by antagonists Smac/DIABLO and Omi/Htra2, which normally
reside in mitochondria but are proteolytically processed and released into cytoplasm once a cell receives an apoptotic stress [48]. In addition, XIAP-associ-
Figure 2. Domain structure of the IAP family. BIR, baculovial IAP repeat; CARD, caspase recruitment domain; RING, RING zinc-finger; NOD, nucleotide-biding oligomerization domain.
207
ated factor 1 (XAF1) has been identified as an antagonist of XIAP that promotes apoptosis by allowing unrestricted caspase activity [49].
Thus, the determination of whether a cell commits to the apoptotic process
is tightly regulated, and is essentially a function of the severity and not merely the specificity of the apoptotic stimulus. As we shall see, it is this function
that researchers are aiming to exploit in making cancer cells more susceptible
to current modes of therapy (see section Therapeutic opportunities).
Bcl-2 family
The Bcl-2 family proteins may regulate apoptosis by altering the integrity of
the mitochondria and by controlling calcium homeostasis [5052]. Members
of the Bcl-2 family can be divided into three classes: (1) anti-apoptotic (Bcl-2,
Bcl-XL, Bcl-w and Mcl-1); (2) pro-apoptotic Bax-like (Bax, Bak, Bok/Mtd
and Bcl-XS); and (3) pro-apoptotic BH3-only (Bad, Bid, Bik/Nbk, BimL/Bod,
Hrk/DP5, PUMA/Bbc3, BNIP3, Noxa and Bmf) [51] (see Fig. 3). Through
interactions between various pro- and anti-apoptotic Bcl-2 family members,
calcium and mitochondrial protein release, including that of cytochrome c, is
regulated.
The reader will note that we mentioned earlier that the two apoptotic pathways would be treated as mutually exclusive. However, at this point we must
digress from that statement to provide a clearer picture of the complexity of
cross-talk between the two pathways, and how certain members of the Bcl-2
family play a significant and vital role in bridging the two. For example, in
response to Fas signals, these two death pathways might cross-talk via the
function of cytosolic Bid. The full-length p22 Bid is inactive and is a substrate
of caspase-8. Cleavage of p22 Bid gives rise to truncated p7/p15 Bid, exposing a glycine that is N-myristoylated, which enables the targeting of a complex
of p7 and myristoylated p15 fragments of Bid to the mitochondria [53]. Upon
activation, Bid induces intramembranous oligomerization of mitochondrionresident Bak [54], as well as oligomerization and integration of cytosolic Bax
in the outer mitochondrial membrane [55]. Multimers of Bak and Bax form a
proposed pore on the mitochondria for cytochrome c efflux, thereby inducing
caspase activation through the formation of apoptosomes [54, 5658]. It is,
thus, possible for an apoptotic stimulus acting through the extrinsic pathway to
induce activation of the intrinsic pathway as well. By contrast, Bcl-2 inhibits
apoptosis by preserving mitochondrial membrane integrity. Bcl-2 inserted into
the outer mitochondrial membrane may, by a mechanism that has yet to be elucidated, prevent Bax/Bak oligomerization and subsequent release of apoptogenic molecules from the mitochondria [59].
In addition to controlling mitochondrial apoptotic process, Bcl-2 family
proteins also regulate apoptosis by affecting calcium homeostasis. The ER is a
major organelle involved in intracellular calcium homeostasis and calcium signaling [50]. Calcium released from the ER can induce a prolonged increase in
208
Figure 3. Classification of the Bcl-family. BH refers to Bcl-2 homology domain. The BH3 domain in
the pro-apoptotic members is a ligand for the hydrophobic groove formed by the BH1-BH3 domains
of the anti-apoptotic members. The hydrophobic C terminus consists of a 1723-amino acid -helix
that anchors the protein in intracellular membranes.
209
calcium [50]. However, mouse embryonic fibroblasts deficient of pro-apoptotic proteins Bax and Bak have a much reduced calcium concentration in the ER
and are resistant to a variety of apoptotic stimuli [25], suggesting that the Bcl-2
family proteins may play a role in regulating the ER-mitochondria amplification loop of apoptotic signals.
p53
p53 is a multi-faceted tumor suppressor gene that is capable of inducing temporary growth arrest and DNA repair, irreversible growth arrest, terminal differentiation, or apoptosis in response to potentially oncogenic cellular stress
such as DNA damage [66]. Therefore, it is imperative that functional p53 be
present in vivo for tumor growth suppression [67]. The function of the p53
gene is lost by mutation in over 50% of human cancer and a loss of heterozygosity often accompanies tumor progression [68, 69]. Unlike many other
tumor suppressor genes, more than 85% of p53 mutations result in single
amino acid substitutions rather than deletions or frame shifts [70]. Most of the
missense mutations occur in the DNA binding core domain (amino acids
210
102292) region of p53 that is evolutionarily conserved between p53 and its
homologues from Drosophila and C. elegans. In human tumors, amino acid
residues that are essential for contact with DNA target sequence (two repeats
of PuPuPuC(A/T)(A/T)GpyPyPy; in which Pu is a purine and Py is a pyrimidine) are frequently found to be mutated [69]. In addition, mutations of
residues that do not contact DNA directly but are required for structural maintenance also cause disruption of the p53-DNA interaction. Frequently, mutations in one allele are sufficient to interfere with p53-dependent apoptosis by
a dominant negative mechanism since in most cases mutant p53 negates wildtype p53 function through heteromerization.
Under normal conditions, p53 has a short half-life and is maintained at very
low levels by Mdm2-mediated degradation [71]. However, in response to
stress by DNA damage, hypoxia, oxidative stress and oncogene activation, p53
is stabilized and activated by post-translational modification [69]. In tumor
cells, transcriptionally inactive mutant p53 is unable to induce the expression
of the Mdm2 protein which would normally provide a feedback mechanism
that downregulates p53 protein levels [72]. Moreover, some p53 mutants
exhibit lower affinity for association with Mdm2 [73]. Hence, mutant p53 proteins that are impervious to these negative regulations accumulate to high levels in cancer cells and negate the functions of the wild-type protein.
Pathways through which p53 induces apoptosis may involve both transcriptional transactivation and transrepression of multiple p53-target genes, as well
as transcription-independent mechanisms that engage the mitochondrial-apoptotic pathways [70]. In general, apoptotic target genes of p53 may be divided
into two major categories: (1) proteins acting at the level of receptor signaling
for apoptosis, and (2) proteins acting downstream by activating apoptotic
effector proteins [74]. The former includes the insulin-like growth factor-1binding protein 3 (IGF-BP3), which induces apoptosis by blocking the IGF-1
survival signal [75] and Fas/APO-1/CD95, which functions in the T cell killing
triggered by anticancer drugs [76]. Essential downstream p53-targeted apoptotic effector proteins are primarily associated with mitochondrial changes,
including caspase-9 and its cofactor Apaf-1 in myc oncogene-induced apoptosis [77], and Bax, necessary for p53-mediated cell death in brain tumors [78].
In addition to acting as a regulatory gene coordinating the expression of many
proteins involved in apoptosis, recent research also suggests that p53 is
involved in mediating apoptosis at the mitochondrial level by directly and
physically interacting with the Bcl-2 member Bak, resulting in the release of
cytochrome c from the mitochondria [79, 80].
211
extremely aggressive tumor progression and poor clinical prognosis [83]. Bax
and Bak were found to be sufficient but not necessary for drug-induced apoptosis [84]. By contrast, increased copy numbers of the anti-apoptotic Bcl-XL
occurs in breast carcinoma, glioblastomas, and Hodgkin lymphoma and other
specific tumor types [52]. Similarly, the anti-apoptotic Bcl-2 protein is frequently overexpressed in many tumors including acute lymphoblastic
leukemia (ALL), precursor B-lymphoblastic leukemia/lymphoma and diffuse
large B cell lymphoma [52]. Bcl-2 is an antagonist to Bax and Bak and inhibits
mitochondrial membrane disruption, a mechanism that likely accounts for
drug resistance in Bcl-2-overpressing lymphomas [85].
Death receptors
Fas/APO-1/CD95 and TRAIL-R1/R2 are sensors on the cell surface that, upon
binding to their respective ligands, initiate the extrinsic apoptotic pathway (see
above). Fas ligand and TRAIL are components of a tumor surveillance mechanism that partakes in the killing of cancer cells by cytotoxic lymphocytes [99,
100]. Tumorigenic disruptions, found in the intrinsic pathway, may also occur
in the extrinsic pathway, albeit far less frequently. Fas is observed to be mutated and downregulated in lymphoid and solid tumors [101], whereas TRAILR1/R2 is mutated in metastatic breast cancers [102]. Suppression of the death
212
receptor pathway could allow immune escape and provide a survival advantage to tumor cells. This loss of function is also associated with resistance to
drug-induced cell death.
213
NF-B
The nuclear factor of B (NF-B) family is composed of a number of heterodimeric transcription factors that regulate the expression of over 200 genes
that are involved in the control of immune, inflammatory and stress responses,
as well as growth and apoptosis [122, 123]. The activity of NF-B is deregulated in many cancers, notably in B cell lymphomas [124]. Although NF-B
transcriptionally activates both anti- and pro-apoptotic genes, on a balance,
NF-B activation favors the suppression of apoptosis [123]. Key anti-apoptotic genes activated by NF-B include the IAPs and the anti-apoptotic members
of the Bcl-2 families [124, 125]. Since the IAPs and the anti-apoptotic members of the Bcl-2 families are crucial inhibitors to both the extrinsic and intrinsic death pathways, as expected, active NF-B can inhibit these pathways and
induce drug resistance in cancer cells [124].
Therapeutic opportunities
Given that apoptosis suppression is fundamental to cancer cell survival, it is
not surprising that components of the apoptotic pathway have emerged as
important therapeutic targets. A variety of antisense oligonucleotides, traditional small molecules, biologically active peptides, peptidomimetics, monoclonal antibodies and gene therapy pay loads have been incorporated into
strategies that target apoptotic pathways in cancer cells [64, 126130].
Although factors such as unexpected toxicities, poor pharmacokinetics, stability and oral bioavailability may limit the use of these compounds in anticancer
treatment, these apoptosis-based antitumor agents might still serve as precursor molecules for the development of more effective therapies.
The importance of Bcl-2 in tumor cells resistant to most cytotoxic anticancer drugs has propelled this anti-apoptotic gene to the forefront as a candidate for antisense oligonucleotides (ASONs)-based therapies. ASONs are
short pieces of DNA that hybridize to a specific target mRNA, thereby blocking its translation to a functional protein. In vitro experiments and xenograft
214
Conclusions
Cancer is the consequence of parallel pathways that lead to both inappropriate
cell proliferation and aberrant control of apoptosis. The inherent suppression
of apoptosis in cancer cells has emerged to be a fundamental mechanism of
tumor formation, progression and resistance to therapy. Advances made in elu-
215
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223
Vascular Biology Program, Childrens Hospital Boston, Karp Research Building 12.214, 300
Longwood Avenue, Boston, MA 02115,USA
2
Department of Surgery, Harvard Medical School, Boston, MA 02115 USA
Abstract. Angiogenesis, the process of new capillary formation from a pre-existing vessel plays an
essential role in both embryonic and postnatal development, in the remodeling of various organ systems, and in several pathologies, particularly cancer. In the last 20 years of angiogenesis research, a
variety of angiogenic regulators, both positive and negative, have been identified. The discovery of
several anti-angiogenic factors has led to the development of novel cancer therapies based on targeting a tumors vascular supply. A number of these new therapies are currently being tested in clinical
trials in the U.S.A. and elsewhere. A major advance in the field of anti-angiogenic therapy occurred
recently when the FDA approved Avastin (bevacizumab), the first solely anti-angiogenesis therapy
approved for treatment of human cancer. While it has long been appreciated that tumor growth and
progression are dependent on angiogenesis, it is only recently that progress has been made in elucidating the molecular mechanisms that regulate the earliest stage in the angiogenic program, the angiogenic switch. This checkpoint is characterized by the transition of a dormant, avascular tumor into an
active, vascular one. Anti-angiogenic therapies to date have essentially been designed to suppress the
neovasculature in established tumors. However, identifying the mechanisms that cause a tumor to
acquire an angiogenic phenotype may lead to the discovery of new therapeutic modalities and complementary diagnostics that could be used to block the angiogenic switch, thereby preventing subsequent tumor progression. In this chapter on the role of angiogenesis in cancer, we (1) provide an
overview of the process of angiogenesis with special regard to the molecules and physiological conditions that regulate this process, (2) review recent studies describing the use of anti-angiogenic
approaches in the treatment of a variety of human cancers, and (3) discuss the recent literature focused
on the study of the molecules and molecular mechanisms that may be regulating the initiation of the
angiogenic phenotype in tumors, and the clinical impact that this knowledge may have in the future.
Key words: Angiogenesis, angiogenic switch, cancer, diagnostics, therapeutics, tumor, VEGF.
Tumor angiogenesis
Introduction: the angiogenic process
Angiogenesis, or neovascularization, occurs when new capillaries sprout from
an existing vessel and develop in a directed manner towards a chemoattractant.
It has been hypothesized that the process of angiogenesis is controlled by a
balance between positive and negative regulators, and that angiogenesis proceeds only when the activity of angiogenic stimulators outweighs that of
inhibitors of angiogenesis. Therefore, the first stage of the angiogenic program
is the expression or overexpression of angiogenic factors with or without the
224
downregulation of angiogenesis inhibitors, creating a pro-angiogenic environment. This acquisition of the angiogenic phenotype, sometimes referred to as
the angiogenic switch [1], is discussed in greater detail in the final section of
this chapter. In the case of tumor angiogenesis, angiogenic factors diffuse from
the tumor, through the extracellular matrix and stimulate the endothelial cells
lining a nearby parent vessel to first degrade the basement membrane surrounding the vessel, and then to proliferate and migrate towards the tumor.
Matrix-degrading enzymes are upregulated in the endothelial cells, allowing
for the degradation of both the vascular basement membrane as well as the
extracellular matrix between the endothelial cells and the tumor. Capillary
tube formation occurs as these proliferating and migrating endothelial cells
begin to organize and form a capillary sprout off of the parent vessel, complete
with a patent lumen. This capillary continues to develop, mature and elongate
until it eventually invades and vascularizes the tumor. For a detailed review on
these angiogenic processes, the reader is referred to Klagsbrun and Moses,
1999 [2] and Carmeliet, 2003 [3]. The molecules and mechanisms involved in
these steps are described in detail below.
Angiogenesis is required for many stages of embryonic development such
as the establishment of the circulatory system and organ formation.
Postnatally, however, angiogenesis is restricted to specific physiological situations including endochondral ossification in bone development, as well as certain stages of the female reproductive cycle including corpus luteum formation
and endometrial remodeling during the menstrual cycle and formation of the
placenta during pregnancy. Angiogenesis is also a key component of many different pathologies, including ischemic conditions such as cerebral ischemia
and myocardial infarction, atherosclerosis, wound healing, diabetic retinopathy, psoriasis, rheumatoid arthritis and cancer.
The field of angiogenesis research was founded some 35 years ago when Dr.
Judah Folkman first characterized the process of angiogenesis and determined
that tumor growth was dependent on the process of neovascularization [4]. He
was the first to postulate that, for a tumor to grow and progress, it was necessary that it recruited its own vascular network. Due to the distance limitations
on oxygen diffusion in tissue, the tumor needs to recruit its own vascular network to reach a size greater than a few millimeters in diameter [5]. Once vascularized, the tumor can undergo rapid growth and progression towards a
metastatic phenotype. Angiogenic tumor vessels supply not only oxygen and
nutrients to the tumor and remove metabolic wastes [4], but recent data suggests that they also function as a source of growth factors, cytokines and hormones that the tumor cells can utilize [6], proteolytic activities that can promote an invasive phenotype [7, 8], as well as circulating endothelial cells and
endothelial progenitor cells (EPCs) that can be incorporated into the tumor
neovasculature [9, 10]. In addition, the tumor vasculature provides a conduit
for metastatic cancer cell dissemination as these cells intravasate through these
angiogenic vessels, enter the circulation and subsequently extravasate from a
distant capillary to establish metastatic foci [1113].
225
226
Figure 1. Interactions between VEGF family members and VEGF receptors and downstream consequences. Preferential binding has been reported between the various members of the VEGF family
and the known VEGF receptors. Each specific ligand/receptor interaction mediates a specific downstream effect on endothelial cells.
227
Figure 2. Positive and negative regulation of VEGF expression and activity. Several positive and negative regulators of VEGF have been identified. Regulation can occur at the level of transcription (1),
mRNA stability/translation (2), ligand binding (3), or signaling pathways downstream of
ligand/receptor interactions (4).
cytokines have been reported to stimulate VEGF expression including, but not
limited to, transforming growth factor- (TGF-) [60], PDGF [61], EGF [62],
insulin-like growth factor-1 (IGF-I) [63], interleukin-1 (IL-1) [64], IL-6 [65],
tumor necrosis factor (TNF-) [66], and FGF4 [67]. Nitric oxide (NO) is also
a potent inducer of VEGF expression [68, 69] and could potentially form an
autocrine loop with VEGF since VEGF upregulates NO synthesis [70, 71].
Transcription of VEGF is regulated by a variety of different mechanisms
(Fig. 2). Most notably, hypoxia is a strong stimulator of VEGF expression in
vitro and in vivo [72, 73]. The hypoxia-induced upregulation of VEGF is pri-
228
229
ure of blood vessel development due to inhibition of endothelial cell differentiation in VEGFR-2-deficient mice [119]. Endothelial cell differentiation and
vascular channel development are normal in VEGFR-3/ mice, but these animals die in utero due to lack of lumen formation in, and proper maturation of,
vessels [120]. NRP-1-deficient mice and zebra fish also exhibit an embryonic
lethal phenotype due to cardiovascular defects [121, 122], as do NRP-1 transgenic mice [123].
VEGF receptor expression was originally thought to be endothelial cell
specific [124]; however, various cell types have been shown to express VEGF
receptors including smooth muscle cells [125, 126], hematopoietic cells [127,
128], EPCs [5456, 129], osteoblasts [130, 131], and islet cells in the pancreas [132].
Interestingly, several types of cancer cells also express one or several of the
VEGF receptors in vitro and in vivo [133]. Breast cancer cells express
VEGFR-1 [134], VEGFR-2 [134, 135] and NRP-1 [136]; colon cancer cells
express VEGFR-2 [135] and NRP-1 [137]; esophageal cancer cells express
VEGFR-1, VEGFR-2 [138] and NRP-1 [137]; gallbladder cancer cells
express NRP-1 [137]; gastric cancer cells express NRP-1 [137, 139]; pancreatic cancer cells express VEGFR-1, VEGFR-2 [140, 141] and NRP-1 [137,
142] and NRP-2 [142]; and prostate cancer cells express VEGFR-1 [143145]
and VEGFR-2 [143, 145] and NRP-1 [146, 147]. Even cells from oncological
blood disorders express VEGF receptors. Non-Hodgkins lymphoma cells
express VEGFR-2 [135] as do leukemia cells [148].
The observation that tumor cells express VEGF receptors suggests that an
autocrine loop of VEGF signaling could occur in these cells since many of the
cell types that express VEGF receptors also express VEGF [136, 141,
143145, 148, 149]. Thus, it is likely that anti-angiogenic therapies that target
VEGF and its receptors may function to target both the endothelium as well as
the tumor cells themselves.
Angiogenesis inhibitors
Angiogenesis inhibitors have been divided into two classes: direct and indirect inhibitors. Direct inhibitors, as their name suggests, function directly on
endothelial cells either by arresting the proliferation of these cells, or by
inducing apoptosis. Indirect angiogenesis inhibitors target the signaling
induced by angiogenic stimuli either by sequestering the angiogenic factors
secreted by tumor cells or by blocking the downstream signal transduction
pathways that are activated upon the binding of these factors to their cognate
receptors on endothelial cells. Several selected endogenous angiogenesis
inhibitors with which we are particularly familiar are discussed below. A
more-detailed discussion of selected indirect angiogenesis inhibitors that
specifically target VEGF is presented in the second section of this chapter.
230
Thrombospondin
The first endogenous angiogenesis inhibitor to be identified was thrombospondin-1 (TSP-1) [150152]. TSP-1 is a secreted, homotrimeric glycoprotein that is associated with either the cell surface or the extracellular matrix
[153]. High levels of TSP-1 can be detected in the embryonic and developing
heart, brain, lung, liver, kidney, skeletal muscle and bone [154159]. It is
abundantly expressed in cartilage and bone with much higher expression in
osteoblasts than in chondrocytes [158, 159], and is among a group of cartilagederived inhibitors of angiogenesis purified from articular cartilage [160].
TSP-1 is a potent inhibitor of angiogenesis in vitro as well as in vivo.
Proliferation, migration and adhesion of endothelial cells in vitro are all
blocked by intact TSP-1 or fragments of TSP-1 [161164], and either TSP-1
or its fragments are capable of inducing apoptosis in endothelial cells [165,
166]. These effects of TSP-1 on endothelial cells are reportedly mediated
through TSP-1 binding to either CD36 [167, 168] or the v3 and 31 integrins independently [169, 170] or complexed with integrin-associated protein
(IAP) [171, 172].
Further evidence for the role of TSP-1 in cancer comes from data that
demonstrate that transcription of TSP-1 is activated by tumor suppressor genes
such as p53 [173175] and PTEN [176]. Conversely, TSP-1 expression is
downregulated by several oncogenes including c-myc [177], v-src [178, 179],
c-jun [180] and ras [177, 181, 182], hypoxia [182], as well as by the Id1 transcription factor [183]. Verification of the role of Id1 in negatively regulating
TSP-1 expression comes from Id1 knockout mice [183]. TSP-1 expression is
significantly increased in these mice, and tumor growth is markedly decreased
due to potent inhibition of angiogenesis.
Immunohistochemical data from several tumor models demonstrates that
TSP-1 protein levels are markedly reduced or non-existent in tumor tissues,
while TSP-1 protein is abundant in surrounding, adjacent normal tissue [182,
184], suggesting that expression of TSP-1 in the tissues adjacent to the tumor
may form a type of anti-angiogenic barrier [184].
Data from in vivo tumor models provides further evidence that TSP-1
inhibits angiogenesis and subsequent tumor growth. Genetic studies in which
TSP-1-null mice were crossed with mice deficient in p53 demonstrated that
melanoma tumors in these double knockout mice exhibited twice the growth
rate of tumors grown in mice deficient in p53 alone [185]. Mammary-specific
overexpression in mice prone to mammary tumors resulted in significantly
inhibited or no tumor growth due to decreased vascularity [186]. Conversely,
mammary-specific knockout of the TSP-1 gene in these same mice led to
increased incidence of tumors, coupled with increased growth rates and hypervascularity of resultant tumors due to increased VEGF/VEGFR-2 interaction.
Interestingly, elevated levels of active matrix metalloproteinases (MMPs),
zinc-dependent proteases that are involved in endothelial cell migration during
angiogenesis and in tumor invasivity (see below) were also observed in these
231
animals, while overexpression of TSP-1 in mammary epithelial cells negatively regulated the activity of MMPs. Further studies with cells from these mice
demonstrated that the interaction between TSP-1 and the proform of MMP-9
prevents the proteolytic cleavage of MMP-9 required to activate the enzyme
from its latent state [186]. MMP-9 has been shown to release heparin-binding
growth factors, such as VEGF from the extracellular matrix [187].
Taken together, these data suggest that the anti-angiogenic properties of
TSP-1 include inhibition of endothelial cell migration, proliferation and adhesion, and prevention of MMP-9 activation to, in turn, prevent the release of
angiogenic factors stored in the extracellular matrix.
Troponin I
Our laboratory first described the anti-angiogenic properties of troponin I
(TnI), a protein first characterized as an inhibitor of actomyosin ATPase during contractility of cardiac and skeletal muscle [188190]. TnI was purified
to homogeneity from bovine scapular cartilage using an in vitro angiogenesis assay that measured inhibition of endothelial cell proliferation to screen
the purification process, and the identity of TnI was verified by microsequencing. Since this was the first demonstration that TnI could be anti-angiogenic, the human TnI gene was cloned and expressed. Human TnI was found
to inhibit both bFGF- and VEGF-stimulated proliferation of endothelial cells
in vitro. TnI is also a potent inhibitor of in vivo angiogenesis, as evidenced
by inhibition of embryonic angiogenesis in the chick chorioallantoic membrane assay and of bFGF-induced angiogenesis in the mouse corneal pocket
assay [189].
More recent studies have focused on elucidating the mechanisms by which
TnI inhibits angiogenesis. TnI has been shown to bind the bFGF receptor and,
thus, may inhibit bFGF-induced angiogenesis by competing with the angiogenic factor bFGF for its receptor expressed on endothelial cells [191]. A 30amino acid peptide of TnI (pTnI) was recently shown to inhibit endothelial cell
proliferation and tube formation in vitro [192]. This peptide also inhibited in
vitro VEGF expression by the pancreatic cancer cell line CAPAN-1, and treatment of mice injected with CAPAN-1 cells with pTnI significantly decreased
the number of liver metastases compared to control animals [192]. Based on
these reports, TnI is currently in clinical development for use as an inhibitor of
solid tumor growth and metastasis.
TIMP-2/Loop 6
The MMPs, a multigene family of metal-dependent endoproteinases, are
required for extracellular matrix remodeling in vivo, and are therefore critically important enzymes in the processes of tumor angiogenesis, progression
232
and metastasis [187, 193195]. Our laboratory was the first to demonstrate
that inhibition of angiogenesis in vivo could be accomplished by inhibiting
MMP activity with an endogenous tissue inhibitor of MMPs (TIMP) [196,
197]. This work was subsequently confirmed by a number of other groups,
and demonstrated the key role that MMP activity played in successful neovascularization.
The TIMPs are important, endogenous negative regulators of MMPs.
Currently, four TIMP family members have been identified: TIMP-1, TIMP-2,
TIMP-3 and TIMP-4. Each of the TIMPs is capable of inhibiting the activity
of MMPs, and thus could negatively regulate angiogenesis by preventing
MMP-mediated migration of endothelial cells and tumor cells [188]. However,
of the known TIMPs, TIMP-2 is unique in that it has been shown to inhibit
endothelial cell proliferation as well [198], in contrast to, for example,
TIMP-1, which has been shown to modestly stimulate the proliferation of
many types of cells including endothelial cells and tumor cells [199, 200].
Therefore, it is possible that TIMP-2 could inhibit angiogenesis by inhibiting
both the proliferation and migration of endothelial cells in addition to its
MMP-inhibitory activity.
Little is known regarding the mechanism by which TIMP-2 inhibits
endothelial cell proliferation. One possibility is that TIMP-2 inhibits the
expression of VEGF. Overexpression of TIMP-2 in breast carcinoma cells was
associated with downregulation of VEGF expression both in vitro and in vivo,
resulting in decreased angiogenesis and tumor growth [201].
The ability of TIMP-2 to inhibit endothelial cell proliferation may be a
receptor-mediated event and studies have recently focused on the identification
of endothelial cell surface receptors for TIMP-2. It has been demonstrated that
the anti-proliferative activity of TIMP-2 was dependent on the cellular expression of 1 integrins [202], and further data showed that the 31 integrin is a
functional TIMP-2 receptor on endothelial cells.
To characterize the MMP-inhibitory and anti-proliferative activities of
TIMP-2 vis--vis its anti-angiogenic activity, our laboratory performed a series
of structure-function studies of TIMP-2 to determine the regions of the molecule responsible for its anti-angiogenic activity [203]. We first confirmed previous reports that the MMP-inhibitory activity was housed in the N-terminal
domain (designated T2N) [204, 205]. Using in vitro endothelial cell proliferation assays, MMP radiometric enzyme assays, and the in vivo angiogenesis
assays, the CAM assay and the mouse corneal pocket assay, we found that the
anti-proliferative activity was housed in the C-terminal domain (designated
T2C), a domain that lacks MMP-inhibitory activity [203]. Further structurefunction mapping of the C-terminal domain determined that the anti-proliferative, anti-angiogenic region of the T2C domain resided in Loop 6 of the
TIMP-2 molecule. Loop 6 was a potent inhibitor of endothelial cell proliferation in vitro, and inhibited in vivo angiogenesis in the CAM and corneal pocket assays [203]. To summarize, TIMP-2 contains two anti-angiogenic activities
that are independent of one another: MMP-inhibitory activity in the T2N and
233
anti-proliferative activity in the T2C. Furthermore, Loop 6, which is responsible for the anti-proliferative activity of T2C, represents a novel, small molecular weight inhibitor of angiogenesis [203].
Angiostatin
The phenomenon of spontaneous growth of dormant metastases after surgical
resection of certain types of primary tumors [206] led to the hypothesis that
perhaps the primary tumor was producing a circulating inhibitor that suppressed the growth of these dormant metastases. Murine tumor models using
Lewis Lung carcinoma were developed that could mimic this phenomenon.
Research to identify these tumor-derived inhibitors using this tumor system led
to the discovery of the angiogenesis inhibitor angiostatin [207].
Using in vitro angiogenesis assays to monitor the purification process,
angiostatin was purified from the urine and serum of Lewis lung carcinomabearing mice [207]. Sequencing of purified angiostatin, a 38-kDa protein,
revealed that it is an internal fragment of plasminogen encompassing the first
four of the five kringle domains of the molecule. Angiostatin, but not intact
plasminogen, inhibited endothelial cell proliferation in vitro in a cell-specific
manner, and the anti-angiogenic activity of angiostatin could be blocked
through immunodepletion with angiostatin-specific antibodies. Angiostatin
was also effective in inhibiting embryonic angiogenesis in the CAM assay.
Furthermore, angiostatin was shown to inhibit the growth of primary tumors
and metastases in a variety of in vivo human tumor models. In a separate
report, it was demonstrated that angiostatin restricts the growth of micrometastases by increasing the rate of apoptosis in these tumors [208].
Reports in the literature suggested that tumor cells do not secrete angiostatin. Rather, it is hypothesized that tumors secrete a number of enzymes that
generate angiostatin through proteolysis of plasminogen [209]. A number of
molecules that release angiostatin from its parent molecule have been identified. Several members of the MMP family are able to cleave plasminogen in
such a manner as to produce angiostatin: MMP-2 [209, 210];
MMP-3/stromelysin [211], MMP-7 [212], MMP-9 [212, 213], and MMP12/human macrophage metalloelastase [214, 215]. In addition, both tissuetype plasminogen activator (tPA) and urokinase plasminogen activator (uPA)
can generate angiostatin from plasminogen [216].
The full complement of anti-angiogenic mechanisms utilized by angiostatin
is still being elucidated. Significant effort has been focused on the identification of receptors on the surface of endothelial cells that could mediate angiostatins anti-angiogenic activities. To date, several molecules have been identified as putative endothelial cell surface receptors for angiostatin, including the
ATP synthase F1 complex [217, 218], the v3 integrin [219], angiomotin
[220], and annexin II [221]. Angiostatin is currently being tested in a number
of human clinical trials for its antitumor effects.
234
Endostatin
Like angiostatin, endostatin is an internal fragment of a larger, parent molecule. Endostatin, a 20-kDa C-terminal fragment of collagen XVIII, is a potent
inhibitor of angiogenesis, while its parent molecule does not possess antiangiogenic activities [222]. Unlike the simple cleavage of angiostatin from
plasminogen via one enzyme, the generation of endostatin from collagen
XVIII appears to be a two-step process. An as-of-yet unidentified MMP activity is responsible for cleavage of the non-collagenous-1 (NC1) domain from
the parent molecule, which is then cleaved by an elastase activity that processes the NC1 domain into functional endostatin [223]. Other enzymes can also
perform this second digestion producing endostatin from the NC1 domain,
such as cathepsins L, K and B, MMP-3, MMP-9, MMP-12, MMP-13 and
MMP-20, as well as MMP-2 and MMP-14, although to a lesser degree [224,
225].
Endostatin was originally purified from the conditioned medium of hemangioendothelioma (EOMA) cells, and was shown to specifically inhibit
endothelial cell proliferation [222] and migration [226], and to induce apoptosis in endothelial cells [227].
Systemic treatment with recombinant endostatin was shown to drastically
inhibit the growth of multiple tumor types through decreased angiogenesis
coupled with increased apoptosis of tumor cells [222, 228]. Most remarkably,
however, cyclic rounds of endostatin treatment not only inhibited tumor
growth during the treatment stages, but after several rounds of treatment, the
tumors entered remission [229]. This suggested that endostatin might be an
effective anti-angiogenic cancer therapy without the induction of drug resistance, which is common to many chemotherapeutic agents.
A considerable amount of data suggests that at least one of the major activities of endostatin appears to be its inhibition of endothelial cell migration
[230]. Many of the observed activities of endostatin are consistent with this
premise. Endostatin can block the activation of MMP-2, an MMP shown to be
important in endothelial cell motility, by binding to the catalytic domain of
MMP-2 [231, 232]. Recently, in vitro endothelial cell assays coupled with
transcriptional profiling experiments demonstrated that endostatin inhibits
migration of endothelial cells via suppression of c-myc and other genes associated with cell migration [226, 233].
Endostatin may also inhibit endothelial cell migration by negatively regulating VEGF expression and/or activity. Endostatin treatment in the in vitro
mouse aortic ring assay and in in vivo murine tumor models, resulted in significant downregulation of VEGF mRNA and protein [234]. A direct interaction between endostatin and VEGFR-2 has been reported [235], and this interaction blocks VEGF-stimulated chemotaxis [236]. These negative effects of
endostatin on VEGF and VEGF signaling may explain the ability of endostatin
to block the VEGF-stimulated mobilization of circulating endothelial cells
(CECs), which express VEGFR-2 [237].
235
236
237
Avastin/bevacizumab
In February 2004, the US FDA approved the first anti-angiogenic cancer therapy for the treatment of cancer, Avastin/bevacizumab, and in doing so validated the anti-angiogenic approach to treating cancer [264266]. Avastin is a
recombinant, humanized VEGF-specific monoclonal antibody designed by
Genentech, Inc (San Francisco, CA) that has a high affinity for all of the VEGF
isoforms [269]. It has been shown to inhibit several of the VEGF-mediated
effects on endothelial cells including proliferation, vascular permeability and
angiogenesis. Avastin was demonstrated to inhibit tumor growth in several in
vivo tumor models [268, 270, 271] and displayed synergism with certain
chemotherapeutic agents, suggesting that it had potential clinical applications
[271, 272].
In phase I clinical trials, Avastin demonstrated no dose-limiting toxicity at
the doses tested, and did not potentiate the toxicity of chemotherapy [273,
274]. Avastin was tested as a single-agent drug or in combination with
chemotherapy in a number of phase II clinical trials for metastatic colorectal,
renal, lung, and breast cancers [275278]. A low-dose regimen of Avastin
combined with chemotherapy resulted in a marked increase in response rate of
metastatic colorectal and breast cancers compared to chemotherapy alone or
chemotherapy coupled with a high dose of Avastin [275, 277], while a more
traditional dose response was observed in the metastatic lung and renal cancer
trials [276, 278]. These data supported the study of Avastin in phase III trials.
238
Drug
Mechanism
Phase I
A6
CEP-7055
CP-547,632
HuMV833
Marimastat/low MolWt
Heparins/captopril
NM-3
PD-547
Suramin
VEGF-Trap
Vitaxin
Multi-functional
VEGFR antagonist
VEGFR antagonist
VEGF antagonist
Multi-functional
VEGFR antagonist
VEGFR antagonist
Growth factor antagonist
VEGF antagonist
v3 antagonist
Phase II
2-Methoxyestradiol
AG-013736
Angiostatin
Angiozyme
EMD 121974
Endostatin
IM682
PTK787/ZK222584
Thrombospondin-1
TNP-470
ZD6474
Multi-functional
VEGFR antagonist
Multi-functional
Tx Repressor of VEGFR-1
v3/v5 antagonist
Multi-functional
Decreased VEGF
VEGFR antagonist
Multi-functional
Multi-functional
VEGFR antagonist
Phase III
CC-5013
Neovastat
SU11248
Multi-functional
Multi-functional
VEGFR antagonist
Approved
Avastin
Tarceva
Thalidomide
VEGF antagonist
HER1/EGFR antagonist
Multi-functional
Two phase III clinical trials with Avastin met with opposite results. In phase
III trials with Avastin coupled with chemotherapy for treatment of refractory
breast cancer, a doubling of the response rate was observed, but no significant
increase in time to disease progression or survival were observed [279]. It was
reported that the advanced stage of this cancer may have precluded significant
efficacy of Avastin, and currently other clinical trials with Avastin for less
advanced metastatic breast cancer are ongoing
However, significant clinical success was observed in a second phase III trial
that studied the effects of Avastin coupled with IFL chemotherapy (irinothecan,
5-fluorouracil and leucovorin) on previously untreated metastatic colorectal
cancer [264266]. Significant improvements in response rate (45% versus
35%), duration of response (10.6 months versus 6.2 months), and progressionfree and overall survival were observed with Avastin/IFL treatment (mean of
20.3 months) compared to IFL alone (mean of 15.6 months) with minor, but
treatable, adverse events reported. Although the optimal dose and treatment
239
VEGF-Trap
The success of Avastin/bevacizumab has provided evidence that inhibition of
VEGF could serve as an effective strategy to treat human cancer. Another
potentially successful anti-angiogenic strategy that is designed to target VEGF
is VEGF-Trap. VEGF-Trap is a fusion of the extracellular, ligand-binding
domains of VEGFR-1 and VEGFR-2 to the Fc portion of human IgG1 [280].
Like Avastin, this molecule sequesters VEGF from its cell surface receptors,
but has a much higher affinity for VEGF than Avastin [280, 281].
Preclinical data suggests that VEGF-Trap can inhibit angiogenesis and
tumor growth in several in vivo tumor systems. VEGF-Trap was first shown to
be successful in inhibiting the growth of murine melanoma, human rhabdomyosarcoma, and rat glioma [280]. Treatment of these various tumors with
VEGF-Trap resulted in significant reduction of these tumors that could be
attributed to dramatically decreased vascularity due to blockage of tumorinduced angiogenesis. In fact the most inhibited of these tumors were essentially avascular. This inhibition of tumor growth could be achieved at doses
that were tenfold less than that of DC101, a VEGFR-2 neutralizing antibody
that was also successful in treating these same tumors [280].
These original findings have been confirmed in a number of reports studying the effects of VEGF-Trap on other tumor systems. VEGF-Trap was effective in inhibiting neuroblastoma in a murine xenograft model with efficacy
greater than that observed upon treatment with a monoclonal VEGF antibody
[282]. In fact, VEGF-Trap treatment has also been shown to regress pre-existing tumor vasculature, resulting in complete regression of established primary
tumors and metastasis [283287].
The efficacy of VEGF-Trap is currently being tested in phase I clinical trials
involving advanced tumors and non-Hodgkins lymphoma [288]. Preliminary
240
results from this dose-escalation study indicate that like Avastin, VEGF-Trap
does not induce toxicity and the maximum tolerated dose has not yet been
reached. The clinical results from this study should be released later this year.
Anti-angiogenic/low-dose/metronomic chemotherapy
One of the most interesting applications of anti-angiogenic therapy has come
from the discovery that low-dose, or metronomic scheduling of conventional
chemotherapeutic agents has the potential to be anti-angiogenic. Cytotoxic
chemotherapy is designed to indiscriminately induce apoptosis of proliferating
cells, i.e., tumor cells, and is administered at the clinically determined maximum-tolerated dose (MTD). Treatment regimens generally include short
courses of treatment followed by breaks to allow recovery, usually in a periodic or cyclical fashion. The indiscriminate death of normal proliferating cells
throughout the body (hematopoietic cells, hair follicles, intestinal lining etc.)
results in many side effects, which necessitate breaks between treatments.
The main targets of conventional chemotherapy are the tumor cells themselves. However, since these agents target dividing cells, in theory they should
also possess anti-angiogenic activity by killing endothelial cells stimulated to
proliferate by angiogenic factors. Another important facet of endothelial cells
that makes them ideal targets for chemotherapeutics is that they are essentially genetically stable, and, therefore, would not acquire drug resistance commonly observed in many types of cancer due to the genetic instability of can-
241
cer cells [229]. Much work has been done recently to enhance the efficacy of
traditional chemotherapeutics by modifying the treatment regimens to attack
not only the tumor cells but also the endothelial cells of the tumors vasculature in other words, anti-angiogenic chemotherapy.
The seminal research that literally opened this new field of cancer therapy
was first reported by Browder et al. [293]. Rather than treat tumors explanted
into mice with the conventional MTD-determined regimen, tumor-bearing
mice were treated with an anti-angiogenic schedule of chemotherapy delivery:
more continuous, lower doses of the chemotherapeutic agent, cyclophosphamide. The anti-angiogenic schedule of cyclophosphamide resulted in the
stabilization of cyclophosphamide-resistant Lewis lung carcinomas and
EMT-6/CTX breast carcinomas. This suppression of tumor growth in drugresistant tumors was attributed to increased apoptotic rates in the endothelial
cells of the tumor vasculature coupled with subsequently increased apoptosis
of the tumor cells themselves. This anti-angiogenic schedule also eradicated
cyclophosphamide-sensitive Lewis Lung carcinomas and L1210 leukemias
without drug-resistance, which is not possible with the MTD-based schedule.
Finally, the anti-angiogenic schedule of cyclophosphamide coupled with the
angiogenesis inhibitor TNP-470 not only suppressed tumor growth, but also
led to complete regression of these tumors. The importance of this work is that
it demonstrated that by altering the treatment schedule, treatment of
cyclophosphamide-resistant tumors with cyclophosphamide could still be
effective.
The question arises: Since most chemotherapeutic agents are capable of
inducing apoptosis of proliferating endothelial cells [294], why is it that MTDbased chemotherapy is not effective in attacking the tumor vasculature, while
anti-angiogenic or metronomic [295] chemotherapy is? It now appears that
the break period between doses in MTD-based scheduling allows for the
recovery of the vasculature [293]. The metronomic schedule arose as a means
to prevent the repair of the tumor vasculature by administering lowered doses
of chemotherapy more frequently. The effectiveness of metronomic scheduling of chemotherapy either alone or coupled with other anti-angiogenic agents
has been confirmed by several other reports from preclinical and clinical studies [296309].
While the most effective anti-angiogenic chemotherapy regimens must still
be evaluated on a case by case basis, these reports give some hope as to the
effectiveness of this anti-angiogenic strategy for treating cancer. Currently several clinical trials are underway to measure the effectiveness of metronomic
chemotherapy against many types of tumors [310314]. Recently, preclinical
studies using the RIP1-Tag2 mouse model of pancreatic tumorigenesis demonstrated that a combinatorial strategy involving MTD scheduling of chemotherapeutics, followed by a metronomic chemotherapy schedule coupled with antiVEGF was most effective in treating intractable end-stage pancreatic tumors
[315]. These data suggest that similar combinatorial strategies coupling anti-
242
243
Figure 3. In vivo model of the angiogenic switch. A tumor model that reliably recapitulates the angiogenic switch has been developed by our laboratory in which avascular tumor nodules (A) and vascular tumor nodules (V) can be harvested simultaneously for biochemical and molecular analyses to
characterize the molecular determinants of the angiogenic switch. Scale bar = 1 mm.
244
245
Ras
Ras is perhaps one of the best characterized oncogenes. The Ras proteins,
H-Ras, N-Ras, and two forms of K-Ras (K-Ras4A and K-Ras 4B) are GTPbinding proteins whose activity is regulated in a positive manner by guanine
nucleotide exchange factors (GEFs) and negatively through hydrolysis of GTP
by GTPase-activating proteins (GAPs) [327, 328]. Ras is in an inactive state
when bound to GDP and becomes activated when GEFs, such as Ras-GAP
[329], dissociate GDP from Ras allowing GTP to bind. Ras returns to an inactive state when GTPases, for example p120/GAP, hydrolyze the bound GTP to
form GDP [328]. GTP-bound, activated Ras is farnesylated and then localizes
to the inner surface of the cell membrane [330, 331], where it can act as an
effector of a number of activated receptor tyrosine kinases including the VEGF
and EGF receptors (Fig. 2) [332335]. The downstream activity of Ras is mediated through a series of effectors including, but not limited to, Raf serine/threonine kinases, MAP kinases and phosphoinositide 3-kinases (PI3Ks) [327].
Expression of mutant forms of Ras is one of the most common genetic
changes detected in human cancers, present in approximately 30% of all human
cancers [83, 336]. These mutations interfere with the ability of GAPs to
hydrolyze GTP bound to Ras, resulting in a constitutively active protein.
Oncogenic Ras mutations are associated with virtually every aspect of malignant transformation including proliferation, transformation, invasion and
metastasis [327, 328, 337]. In addition to Ras conferring a survival advantage
directly upon the cancer cells themselves, Ras and mutated Ras proteins are
also involved in promoting angiogenesis via a number of different mechanisms.
A link between Ras signaling and subsequent upregulation of VEGF expression has been clearly established [78, 8385, 177, 181, 338342].
Overexpression of K-ras and H-ras oncogenes in normal epithelial cell lines or
expression of mutant Ras in immortalized epithelial cells results in significant
upregulation of VEGF [83, 181], and expression of mutant Ras results in significantly increased VEGF expression in fibroblasts [181, 338] and endothelial
cells [84]. Inhibition of the Ras pathway via transfection and expression of a
246
247
V-Src
Another oncogene that may exert its oncogenic effects through activation of
the angiogenic switch via upregulation of VEGF and downregulation of TSP-1
is V-Src. V-src, the first described oncogene, encodes a tyrosine kinase that is
a downstream effector of several RTKs [363]. Treatment of tumors with a Src
tyrosine kinase inhibitor (AZM475271; AstraZeneca) results in significant
reduction in tumor volume and number of metastases due to decreased
microvessel density [364], suggesting that Src may be playing a role in the regulation of angiogenesis. Numerous reports indicate that expression of oncogenic V-Src upregulates expression of VEGF [8688, 346, 365] and suggest
that oncogenic mutations of V-Src might contribute to initiating the angiogenic
phenotype.
Overexpression of V-Src results in the activation of the VEGF promoter in
promoter reporter assays, indicating that downstream effectors of V-Src upregulate VEGF expression through increased transcription of the VEGF gene
[365]. Two transcription factors that activate VEGF transcription are reported
to be downstream of V-Src.
Earlier in this section the role of HIF-1-mediated VEGF expression in the
angiogenic switch was discussed. Cells that overexpress oncogenic V-Src display increased expression of HIF-1 with subsequent upregulation of VEGF
[87]. Conversely, inhibition of Src activity through either expression of a dom-
248
inant negative form of Src or through treatment with Src kinase inhibitors,
results in significantly decreased HIF-1 synthesis and VEGF expression even
during hypoxic conditions [86, 88, 366]. Taken together these results suggest
that V-Src may aid in hypoxia-mediated upregulation of VEGF by increasing
expression of HIF-1. It is also possible that oncogenic V-Src, by upregulating expression of HIF-1, can stimulate hypoxia-independent activation of the
HIF1 transcription factor resulting in increased VEGF expression.
Another transcription factor downstream of V-Src is signal transducer and
activation of transcription 3 (STAT3). STAT3 is a potent activator of VEGF
transcription through direct interaction with a STAT3 binding site in the VEGF
promoter [367369]. Expression levels of STAT3 are elevated in cells transformed with V-Src [370] and blocking STAT3 activity through expression of a
dominant negative STAT3 mutant or by treatment with STAT3 antisense
oligonucleotides inhibits V-Src-mediated VEGF expression [368]. Oncogenic
mutation and activation of V-Src may trigger a signaling cascade leading to
increased VEGF expression by activation of the transcription factors HIF-1
and STAT3.
Src may also mediate downstream effects of VEGF signaling, suggesting a
potential autocrine loop involving Src and VEGF. Src binds directly to activated VEGFR-2 in VEGF-stimulated endothelial cells [371, 372] and this
interaction between Src and VEGFR-2 has been reported to mediate VEGFinduced vascular permeability [371374]. Increased permeability of the tumor
vasculature could enhance perfusion of the tumor as well as increase the possibility that metastatic tumor cells could enter the circulation.
In addition to upregulating VEGF expression, V-Src may also promote a
pro-angiogenic environment through downregulation of TSP-1 [345].
Overexpression of V-Src in NIH3T3 cells resulted in significantly decreased
expression of TSP-1 [178], while transformation of Rat1 fibroblasts with V-Src
resulted in a 10- to 50-fold reduction in the level of TSP-1 transcripts compared to control cells [179].
p53
The oncogenes Ras and V-Src promote the initiation of the angiogenic phenotype by simultaneously activating expression of angiogenesis stimulators
(VEGF, bFGF, MMPs), while downregulating inhibitors of angiogenesis
(TSP-1). Certain tumor suppressor genes play a converse role in the angiogenic switch: they shift the balance in favor of inhibition of angiogenesis by
activating expression of negative regulators of angiogenesis concomitantly
with downregulating angiogenic factors. Therefore, the angiogenic switch cannot occur unless the activities of these tumor suppressors are neutralized
through mutation or deletion of the genes themselves. While Ras is one of the
best-characterized oncogenes, the most famous tumor suppressor gene is perhaps p53.
249
The p53 tumor suppressor was first identified and characterized for its role
in DNA repair [375]. p53 induces cell-cycle arrest or apoptosis in cells whose
DNA has been mutated or damaged, to prevent the inheritance of potentially
deleterious mutations, in particular mutations that might lead to oncogenic
transformation [376, 377]. Given its role as a critical checkpoint during the cell
cycle, it is not surprising that mutations in or deletion of the p53 gene have
been identified in over 50% of all human cancers [378]. In addition to its ability to induce cell cycle arrest and apoptosis, p53 may also suppress tumorigenesis through inhibition of angiogenesis. Direct correlations can be made
between wild-type p53 status and expression of TSP-1 in many types of human
cancer, and mutations in p53 are associated with decreased levels of TSP-1
[379, 380]. Conversely, a direct correlation exists between mutant p53 and
VEGF expression [381, 382].
Numerous reports indicate that p53 is a potent stimulator of TSP-1 expression. Transfection of wild-type p53 into human fibroblasts from Li-Fraumeni
patients or into human glioblastoma cells, both of which lack a wild-type p53
allele, resulted in significant increase in TSP-1 synthesis due to increased
activity of the TSP-1 promoter [173175]. Loss of both alleles of p53 as
fibroblasts progress towards a tumorigenic phenotype results in a 20-fold
decrease in secreted TSP-1 [383].
Data suggest that p53 can activate transcription of TSP-1 as well as repress
transcriptional activation of VEGF. Introduction of wild-type p53 into cells
that express mutant p53 results in decreased VEGF promoter activity using
promoter reporter assays [93, 365]. Loss of wild-type p53 alleles, which resulted in a 20-fold decrease in TSP-1, concomitantly resulted in a 4-fold increase
in VEGF secretion [383]. A separate study indicated that mutant p53 induced
VEGF expression through a protein kinase C pathway [384].
Wild-type p53 may exert its repression of VEGF through modulation of the
transcription factors HIF-1, and Sp1, and Src. p53 binds to HIF-1 and
mediates ubiquitination and eventual degradation of the protein, and loss of
p53 results in elevated levels of HIF-1 protein and HIF-1-mediated VEGF
expression [89]. Wild-type p53 can also bind to Sp1, preventing binding of the
transcription factor to the VEGF promoter [91], and sequestration of Sp1 may
be responsible for the decrease in VEGF promoter activity reported in Zhang
et al. [93]. Src-mediated upregulation of VEGF expression can be blocked by
wild-type p53 [365] even under hypoxic conditions [91].
250
may, one day, develop diagnostics that could detect the onset of neovascularization in an otherwise undetectable tumor through analysis of the presence or
absence of biomarkers specific for the angiogenic switch.
Recent proteomic studies in our laboratory, aimed at identifying tumor biomarkers in the urine of cancer patients, demonstrate that the presence of urinary
MMPs serve as a sensitive and specific human cancer biomarker. Our laboratory was the first to demonstrate that detection of MMPs in the urine of patients
is predictive of disease status in a variety of human cancers, including those outside of the urinary tract [385]. We have also reported a direct correlation
between the levels of MMPs, for example a disintegrin and metalloproteinase12 (ADAM-12), and stages of disease [386], and also that the levels of MMPs
present in the urine can serve as a monitor of therapeutic efficacy [387]. Given
this direct correlation between stage of disease and urinary MMP levels, and
given that MMP activity is one of the earliest and most sustained activities operative in the angiogenic program, we are currently investigating the possibility
that urinary MMPs may be predictive of the angiogenic switch, allowing us to
detect cancer much earlier than is possible with current diagnostic technologies.
Summary
It is now widely appreciated that sustained angiogenesis is a hallmark of successful tumor growth and progression. As anti-angiogenic therapies continue
to be tested and approved for clinical use, it becomes possible to begin to think
of cancer as a chronic, rather than as a terminal, disease. Given that the defining characteristics of angiogenesis inhibitors include their generally low cytotoxicity, with little to no induction of drug resistance, it is clear that anti-angiogenic treatments represent ideal candidates for long-term therapeutics. Antiangiogenic cancer therapy, coupled with the development of sensitive and specific diagnostic and prognostic cancer tests that can reproducibly monitor disease status and therapeutic efficacy, provides a clearly novel paradigm for the
detection and treatment of human cancer. Moreover, to the extent that these
detection systems are of sufficiently high sensitivity, one can begin to imagine
the day, in the not too distant future, when we will detect and suppress tumor
growth and progression at a point much earlier than is currently possible.
Acknowledgements
The authors acknowledge the support of NIH grants 2PO1CA45548, CA83106, AT00650-01 and
1P50 DK065298. The authors also gratefully acknowledge the expert administrative assistance of
Carol Figueroa.
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Department of Pathology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo
113-8421, Japan
2
Toxicology Group, Safety Research Laboratories, Dainippon Pharmaceutical Co., Ltd., 33-94,
Enoki-cho, Suita, Osaka 564-0053, Japan
Summary. Cancer is a heritable disorder of somatic cells. Carcinogenesis at the cellular level is like an
opened Japanese fan, because initiated cells grow in several directions and tumors suggest the edge of
the fan by having many gene abnormalities. We discuss here the primal force and gene networks (federal headship) in renal carcinogenesis. The Eker (Tsc2 mutant) rat model of hereditary renal carcinoma (RC) is an example of a Mendelian dominantly inherited predisposition to a specific cancer in an
experimental animal. Recently, we discovered a new hereditary renal carcinoma in the rat in Japan,
and the rat was named the Nihon rat. We suggest that its predisposing (Bhd) gene is a novel renal
tumor suppressor gene. We present these unique models as part of the study of problems in carcinogenesis; e.g., multistep carcinogenesis, cancer prevention and the development of the therapeutic treatments that can be translated to human patients, as well as how environmental factors interact with cancer susceptibility gene(s).
Key words: Bhd gene (Nihon) mutant, Erc gene, hereditary cancer, Knudsons two-hit, Tsc 1 gene,
Tsc2 gene (Eker) mutant, Niban gene.
Introduction
Environment and heredity both operate in the origin of human cancer. These
environment and genetic determinants of cancer can be classified into four
groups designated oncodems by Knudson in 1985 [1]. Oncodem 1 is the
irreducible background level of cancer due to spontaneous mutagenesis.
Oncodem 2 is environmentally induced cancer, the causative agents among
which are chemical carcinogens, radiation, or viruses. Oncodem 3 is basically
environmentally induced cancer, but there are genetically determined differences among persons, e.g., in relation to tendencies for activation or inactivation of carcinogens. Most human cancers are believed to belong to oncodem 2
and/or 3 (about 80%), for which the probability of the occurrence of the carcinogenic steps is increased, although the number of steps is not decreased.
Oncodem 1 would contain the 20% that would remain if environmental
induced cancers (oncodem 2 and/or 3) were prevented. Oncodem 4 is all
hereditary cancer. Hereditary cancers have been important in the understanding of carcinogenesis, and pointed to the first identification of a tumor
270
O. Hino et al.
General history of the Eker rat model of inherited renal cell carcinoma
After 1971, Knudson, at the Fox Chase Cancer Center, Philadelphia, was looking for an animal model of two-hit carcinogenesis that might parallel hereditary retinoblastoma in man, and found it in the Eker rat. The naturally occurring hereditary cancer in the rat had been described by Reidar Eker in Oslo in
1954 [4]. In 1983, Reidar Eker kindly sent some Eker rats to Knudson in
Philadelphia. Progress toward genetic linkage analysis was slow because few
genetic markers were available in the rat. The author (O.H.) went to the
Knudsons laboratory on an American Cancer SocietyEleanor Roosevelt
International Fellowship (UICC) from 1989 to 1991 and began to isolate rat
genetic markers [5]. Unfortunately, these initial linkage studies were unproductive. When the author (O.H.) returned to the Cancer Institute in Tokyo,
Knudson sent him some Eker rats, and they have been bred on a normal
LongEvans strain background at the Animal Facility of the Cancer Institute
since 1991. The author (O.H.) has since continued investigating them in
Tokyo, independently of Knudsons group in Philadelphia. Thus, Eker rat
research has been continued by a third generation of investigators (Oslo
PhiladelphiaTokyo). Finally, we and Knudsons group independently identified a germline retrotransposon insertion in the rat homolog of the human
tuberous sclerosis (TSC2) gene, resulting in aberrant RNA expression from the
mutant allele, 40 years after the original discovery of the Eker rat [68]. To the
best of our knowledge, this was the first isolation of a Mendelian dominantly
predisposing cancer gene in a naturally occurring animal model. We then constructed transgenic Eker rats with a wild-type Tsc2 gene and ascertained that
germline suppression of the Eker phenotype is possible for both embryonic
lethality of the homozygote and tumor predisposition in the heterozygote, and
Gastric carcinoma
Melanoma
cylindroma
Parathyroid cancer
BHD
(FLCN)
BMPR1A
BRCA1
BRCA2
CDH1
CDKN2A
CHEK2
CYLD
EXT1
EXT2
FH
HRPT2
INI1
(SNF5)
5q22.2
APC
22q11.23
1q31.2
1q43
11p11.2
8q24.1
16q12.1
22q12.1
9p21.3
16q22.1
13q13.1
17q21.31
10q23.2
17p11.2
Chromosome
localization
Chromatin remodeling
unknown
Fumarate hydratase
Cell adhesion
Unknown
Function
Table 1. Tumor suppressor genes responsible for autosomal dominantly inherited tumor predisposing diseases
E: Rhabdoid tumor,
O: embryonic lethality
unknown
Unknown
Unknown
O: embryonic lethality
Unknown
O: Lymphoma
O: lymphoma, sarcoma
O: embryonic lethality
O: embryonic lethality
O: embryonic lethality
O: embryonic lethality
Schwannoma, meningioma
(Neurofibromatosis type II)
Retinoblastoma, osteosarcoma
MEN1
MLH1
MSH2
MSH6
(GTBP)
NF1
NF2
PRKAR1A
PTCH
PTEN
RB
19p13.3
LKB1
13q14.2
10q23.31
9q22.32
17q24.2
22q12.2
17q11.2
2p16
2p21
3p22.3
11q13.1
Chromosome
localization
Table 1. (Continued)
Protein/lipid phosphatase
cAMP-dependent protein
kinase regulatory subunit
Cytoskeletal regulation
Unknown
Protein kinase
Function
E: medulloblastoma,
O: embryonic lethality
O: lymphoma
O: lymphoma, sterility
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O. Hino et al.
1p36.13
Pheochromocytoma, paraganglioma
Pheochromocytoma, paraganglioma
Pheochromocytoma, paraganglioma
Medulloblastoma
Hepatoma
SDHB
SDHC
SDHD
SMAD4
SUFU
TCF1
TP53
TSC1
TSC2
VHL
WT1
11p13
3p25.3
16p13.3
9q34.13
17p13.1
12q24.31
10q24.32
18q21.1
11q23.1
1q23.3
Chromosome
localization
Table 1. (Continued)
Transcription factor
Rheb-GAP
Tsc2 binding
Transcription factor
Transcription factor
Function
O: embryonic lethality
O: embryonic lethality
E O: lymphoma, osteosarcoma,
hemangiosarcoma
Unknown
Unknown
Unknown
BLM
FANCA
FANCC
FANCD2
FANCE
FANCF
FANCG
FANCL
NBS1
POLH
11q23
Leukemia, lymphoma
(Ataxia telangiectasia)
ATM
6p21.1
8q21.3
2p16.1
9p13.3
11p14.3
6p21.31
3p25.3
9q22.32
16q24.3
15q26.1
Chromosome
localization
RAD51/MRE11 complex
DNA damage repair
E3 ubiquitin ligase
unknown
DNA helicase
Function
Table 2. DNA repair-related genes responsible for recessively inherited tumor predisposing diseases
Unknown
unknown
unknown
unknown
O: hematopoietic defect,
mitomycin C-hypersensitvity
O: lymphoma
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O. Hino et al.
8q24.3
Leukemia
(Schwachman-Diamond syndrome)
RECQL4
SBDS
WRN
XPA
XPB
XPC
XPD
XPE
(DDB2)
XPF
XPG
13q33.1
16p13.12
11p11.2
19q13.32
3p25.1
2q14.3
19p13.2
8p12
7q11
Chromosome
localization
Table 2. (Continued)
DNA endonuclease
DNA endonuclease
DNA helicase
DNA binding
DNA helicase
DNA helicase
RNA metabolism
DNA helicase
Function
O: growth defect
O: growth defect
O: embryonic lethality
unknown
O: no obvious phenotype
unknown
12q14.1
Melanoma
CDK4
FAS
KIT
MET
RET
10q11.21
7q31.2
4q12
10q23.31
Chromosome
localization
FAS
Function
Table 3. Oncogene responsible for autosomal dominantly inherited tumor predisposing disease
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O. Hino et al.
Figure 1. Multi-stage renal carcinogenesis in the Eker (Tsc2 gene mutant) rat [32]. (1) Macroscopic
renal tumor. (2) Small regions of phenotypically altered renal tubules, which develop via atypical
hyperplasia into (3) adenomas, and finally, into (4) fully developed carcinomas
of the wild-type allele even in the earliest pre-neoplastic lesions, e.g., phenotypically altered renal tubules [28], supporting the hypothesis that a second,
somatic mutation (second hit) might be a rate-limiting step for renal carcinogenesis in the Eker rat model of dominantly inherited cancer, as well as indicating a tumor-suppressor function for the Tsc2 gene. Thus, heterozygosity is
not by itself a sufficient condition for the development of cancer, but only one
hit is enough to produce phenotypically altered renal tubules in the Eker rat.
Such a lesion may initially be benign, but continued proliferation virtually
ensures that other critical, though not rate-limiting, events will occur. Although
the initial event that triggers Eker rat renal cancer is a somatic mutation of the
Tsc2 wild-type allele, other genetic or epigenetic modifications may also contribute to tumor progression in multistep renal carcinogenesis (Fig. 2).
281
Figure 2. Fan model of renal carcinogenesis [2932]. Carcinogenesis is like an opened Japanese fan.
The primal force is two hits on the predisposing Tsc2 gene. Initiated cells grow then in many directions and accumulate different clinical features at the periphery. The edge of the fan then corresponds
to the diverse clinical features of the tumor cells.
AP-1 genes
We identified the highly expressed genes in Eker RCs as: the C3 gene encoding the third component of complement, the annexin II gene encoding the calpactin 1 heavy-chain, the Erc (expressed in renal carcinoma) gene, and the
fra-1 gene encoding a transcriptional factor activator protein 1 (AP-1) [33]. We
found that other members of the AP-1 transcriptional factors were involved in
the renal carcinogenesis in the Eker rat model [34]. Interestingly, using
immunohistochemistry, AP-1 proteins were shown to be highly expressed even
in the earliest pre-neoplatic lesions (e.g., phenotypically altered tubules).
Moreover, we transfected antisense oligonucleotides targeting AP-1 genes into
RCCs and demonstrated that their growth was strongly inhibited [34]. These
data suggest that expression of AP-1 genes might play a crucial role in renal
carcinogenesis in the Eker rat model.
We also elucidated an underlying regulatory mechanism of Tsc2 gene
expression, occurring bidirectionally via two Ets-transcription factor binding
sites, and found that Ets family proteins were highly expressed in the RCs [35].
These observations might be helpful in the search for the downstream molecular targets and the gene network of the Tsc2 gene.
Erc gene/MPF/mesothelin
After we determined the complete primary structure of rat Erc cDNA, we
showed that the putative rat Erc product has 56.1% identity with human
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O. Hino et al.
Niban gene
Recently, we also isolated a novel gene, which was named the Niban (the
second in Japanese) [37]. The Niban gene consists of 14 exons and is located
on rat chromosome 13, mouse chromosome 1, and human chromosome 1. The
expression of Niban was upregulated even in early pre-neoplastic lesions (phenotypically altered renal tubules) that developed in the renal carcinogenesis
models [38]. Niban might be a candidate marker for early stage renal carcinogenesis, and its molecular analysis might provide new insights into multistep
carcinogenesis in the kidney.
283
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285
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O. Hino et al.
Figure 3. Macrophotograph of a renal tumor from a Nihon (BHD gene mutant) rat. (1) Macroscopic
renal tumor (74 weeks old age; male). (2) Clear cell type. (3) Acidophilic cell type & mixed cell type.
(4) Papillary type.
287
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O. Hino et al.
Nihon rat
Transmission
Mendelian dominant
Mendelian dominant
Predisposing gene
BHD
TSC2
3 weeks
4 weeks
8 weeks
6 months
8 weeks
16 weeks
20 weeks
12 months
clear/acidophilic
or basophilic
chromophobic
or basophilic
Lesions
PA tubules
Hyperplasia
Adenoma
RC
Histology
Eker rat
289
Conclusions
Knudsons visionary two-hit model to explain hereditary and sporadic forms of
retinoblastoma led to the development of a new paradigm, indicating the inheritance of a mutant allele of a critical gene as a predisposing factor for the development of a malignant cell [2]. Vogelstein and Kinzler reviewed the multistep
nature of cancer and three to six mutations appear to be required to complete
the process, driving a wave associated with increases in tumor size, disorganization and malignancy [60]. Fidler et al. [61] addressed the progression of
tumors from a less- to a more-malignant phenotype, which results in tumor heterogeneity and the selection of cells with a more malignant (metastatic) phenotype, which in turn is likely due to the instability of the tumor cell genome.
We have proposed that carcinogenesis is diagrammatic similar to an opened
Japanese fan; the initiated cells grow in several directions, with clinical tumors
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O. Hino et al.
suggesting the edge of the fan since they have many gene abnormalities, and
genetic instability might also play a role [2932] (Fig. 2). The germline mutation is like a primal force, i.e., the initial gene of the abnormal networks of
gene expressions that are involved in tumor formation (federal headship of carcinogenesis). Our opened fan model of carcinogenesis might be pertinent, like
the two-hit and multi-hit mutational models, because it incorporates the
phenomena of genetic instability and of factors which alter gene expression.
Acknowledgements
This work was supported in part by Grants-in Aid for Cancer Research from the Ministry of
Education, Culture, Sports, Science and Technology of Japan, and the Ministry of Health, Labor and
Welfare of Japan.
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Genetics 9: 7074
9 Kobayashi T, Mitani H, Takahashi R, Hirabayashi M, Ueda M, Tamura H, Hino O (1997)
Transgenic rescue from embryonic lethality and renal carcinogenesis in the Eker rat model by
introduction of a wild-type Tsc2 gene. Proc Natl Acad Sci USA 94: 39903993
10 Hino O, Klein-Szanto AJP, Freed JJ, Testa JR, Brown DQ, Vilensky M, Yeung RS, Tartof KD,
Knudson AG (1993) Spontaneous and radiation-induced renal tumors in the Eker rat model of
dominantly inherited cancer. Proc Natl Acad Sci USA 90: 327331
11 Everitt JJ, Goldsworthy TL, Wolf DS, Walker CL. (1992) Hereditary renal cell carcinoma in the
Eker rat: a rodent familial cancer syndrome. J Urol 148: 19321936
12 Hino O, Mitani H, Katsuyama H, Kubo Y. (1994) A novel cancer predisposition syndrome in the
Eker rat model. Cancer Lett 83: 117121
13 Kubo Y, Mitani H, Hino O (1994) Allelic loss at the predisposing gene locus in spontaneous and
chemically induced renal cell carcinomas in Eker rat. Cancer Res 54: 26332635
14 Kubo Y, Kikuchi Y, Mitani H, Kobayashi E, Kobayashi T, Hino O (1995) Allelic loss at the tuberous sclerosis (Tsc2) gene locus in spontaneous uterine leiomyosarcomas and pituitary adenomas
in the Eker rat model. Jpn J Cancer Res 86: 828832
15 Hino O, Kobayashi T, Mitani H, Kubo Y, Tsuchiya H, Kikuchi Y, Nishizawa M, Hirayama Y
(1995) The Eker rat, a model of dominantly inherited cancer syndrome. Transplant Proc 27:
15291531
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293
Chemical Structure & Dynamics, Pacific Northwest National Laboratory, MS K8-88, 3335 Q Ave,
Richland, WA 99352, USA
2
Radiation Oncology Research Laboratory, BRB 7-011, and Greenebaum Cancer Center, University
of Maryland, Baltimore, Baltimore, MD 21201-1551
Abstract. Ionizing radiation is perhaps the most extensively studied human carcinogen. There have
been a number of epidemiological studies on human populations exposed to radiation for medical or
occupational reasons, as a result of protracted environmental exposures due to radiation accidents, or
after atomic bombings. As a result of these studies exposure to ionizing radiation has been unambiguously linked to cancer causation. While cancer induction is the primary concern and the most
important somatic effect of exposure to ionizing radiation, potential health risks do not only involve
neoplastic diseases but also somatic mutations that might contribute to birth defects and ocular maladies, and heritable mutations that might impact on disease risks in future generations. Consequantly
it is important we understand the long-term health risks associated with exposure to ionizing radiation.
Key words: Genomic instability, ionizing radiation, non-targeted effects, radiation carcinogenesis.
Introduction
According to the American Cancer Society, the United States can expect
1368030 new cases of cancer in 2004 [1]. Among the many carcinogens
Americans are exposed to, ionizing radiation will contribute to this statistic.
Humans live in a radiation environment. Ionizing radiation is in the air we
breathe, the earth we live on, and the food we eat. Man-made radiation adds to
this naturally occurring radiation level, thereby increasing the chance for
human exposure. For many decades the scientific community, governmental
regulatory bodies, and concerned citizens have struggled to estimate health
risks associated with radiation exposures, particularly at low doses. While cancer induction is the primary concern and the most important somatic effect of
exposure to ionizing radiation, potential health risks do not involve neoplastic
diseases exclusively, but also include somatic mutations that might contribute
to birth defects and ocular maladies, and heritable mutations that might impact
on disease risks in future generations. Consequently, it is important we understand the effect of ionizing radiation on cellular structures and the subsequent
long-term health risks associated with exposure to ionizing radiation.
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M. Sowa et al.
Radiation carcinogenesis
Ionizing radiation is perhaps the most extensively studied human carcinogen.
There have been a number of epidemiological studies on human populations
exposed to radiation for medical or occupational reasons, as a result of protracted environmental exposures due to radiation accidents, or after atomic
bombings (reviewed in [2]). As a result of these studies, exposure to ionizing
radiation has been unambiguously linked to carcinogenesis. While many types
of human cancer have been convincingly linked to radiation, there are a few
notable exceptions including chronic lymphocytic leukemia, Hodgkins disease, cervical cancer, and prostate cancer.
Cancer incidence is modified by the dose rate, the total dose of radiation
delivered, and the quality of radiation, with high linear energy transfer radiation, e.g., radon particles being more biologically effective than low linear
energy transfer radiation, e.g., x- or -radiation. In general radiation carcinogenesis is a stochastic effect. That is, the probability of an effect increases as
the dose increases, with no dose threshold. However, the severity of the effect
is not dose related, such that a high dose of radiation does not induce a worse
cancer than a low dose of radiation. It should be noted that there are also deterministic effects associated with radiation exposure. While these effects are
comparatively rare relative to the well-documented stochastic effects, deterministic effects indicate a threshold of dose and the severity of the effect is
dose related. Radiation induced cataracts are an example of a deterministic
effect.
There are a number of biological modifiers of radiation-induced cancer risk.
These include age at time of exposure, sex, and the target organ. In addition,
the cancer risk can be modulated by potential genetic susceptibility factors
such as polymorphisms in genes involved in cellular responses to DNA damage (reviewed in [3]). So while it is clear that radiation exposure can lead to
cancer, what is not clear is how radiation causes cancer. Cancer appears to
arise from the accumulation of multiple genetic abnormalities including gene
mutations, deletions, rearrangements and/or alterations in gene expression, as
well as chromosomal rearrangements and changes in chromosome number.
Radiation-induced cancers have a long latency period between exposure and
the appearance of the malignancy. The shortest period is for leukemia, with a
peak 57 years, but solid tumors show a longer latency period, anything from
10 to 50 years and the excess risk appears to be a lifelong elevation of the natural age-specific cancer risk [4].
Unfortunately for those studying the mechanisms of radiation-induced carcinogenesis and attempting to understand radiation-induced cancer risk, there
is no unique signature to cancers associated with radiation exposure. Instead,
radiation-induced cancers are similar to those occurring spontaneously.
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Figure 1. Schematic representation of radiation-induced genomic instability. Ionizing radiation initiates the instability phenotype either directly by hitting the target cell or indirectly via the secretion of
soluble factors or cell-to-cell gap junction-mediated communication from an irradiated cell to a nonirradiated cell. Once initiated, instability can manifest in the progeny of that cell during clonal expansion and is measured by multiple endpoints [41]. Cell clones showing induced instability can also
exhibit persistently elevated levels of reactive oxygen species [35, 36], which in turn can stimulate
changes in gene expression, and/or protein/enzyme levels [50]. The combination of increased reactive
oxygen species and subsequent altered cellular homeostasis provide protracted stimuli perpetuating
instability over time. Some unstable clones also generate soluble cytotoxic factors, such that media
from unstable clones is lethal when transferred to non-irradiated cells [31]. This death-inducing
effect results in the induction of DNA double-strand cleavage rapidly after transfer to recipient cells,
leading to chromosome changes, micronuclei formation and ultimately cell death [39]. The majority
of exposed cells die by apoptosis [39], which might result in lytic products from these dead and dying
cells contributing to the death-inducing effect and perpetuating instability over time [24]. The end
result is a heterogeneous population of cells containing multiple genomically rearranged subpopulations resulting from clonal expansion of a radiation-initiated cell. The phenotypes of radiationinduced genomic instability are similar to those described for tumor cells.
damage response in undamaged cells, although there is considerable inter-individual variation in both production and response.
Wright and colleagues [42] recently proposed an interesting and plausible
mechanism for delayed effects of radiation in vivo. They observed that
macrophages exhibited the phenotype of activated phagocytes after whole
body irradiation of mice. The characteristics of these macrophages are consistent with features of inflammatory responses known to have the potential for
both non-targeted bystander type responses and persisting damage, as well as
for conferring a predisposition to malignancy. Consequently, radiationinduced instability in vivo might reflect inflammatory-type responses to radiation-induced stress and injury. The observations of persistent inflammatory
activity in some of the A-bomb survivors [43] lends credence to the hypothe-
299
Conclusions
That exposure to ionizing radiation can cause cancer is a given fact, but how it
does so is not known. In a variety of cells in vitro [18] and model systems in
vivo [18], radiation can induce destabilization of the genome such that surviving cells acquire many of those characteristics associated with tumor cells. As
such radiation-induced genomic instability has been proposed as a very early,
if not an initiating event in radiation carcinogenesis. However, despite the
attraction of such a concept, a definitive link between induced instability and
carcinogenesis has yet to be established.
Acknowledgements
This work was supported by the Biological and Environmental Research Program (BER), U.S.
Department of Energy, Grant No. DE-AC06-76RLO (MSR) and DE-FG02-01ER63230 (WFM).
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303
Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, M5G
1X5 Canada
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto,
Ontario, M5G 1X5 Canada
3
Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto,
Toronto, ON, M5S 1A1, Canada
2
Abstract. Genetic, or genomic, instability refers to a series of observed spontaneous genetic changes
occurring at an accelerated rate in cell populations derived from the same ancestral precursor. This is
far from a new finding, but is one that has increasingly gained more attention in the last decade due to
its plausible role(s) in tumorigenesis. The majority of genetic alterations contributing to the malignant
transformation are seen in growth regulatory genes, and in genes involved in cell cycle progression
and arrest. Genomic instability may present itself through alterations in the length of short repeat
stretches of coding and non-coding DNA, resulting in microsatellite instability. Tumors with such profiles are referred to as exhibiting a mutator phenotype, which is largely a consequence of inactivating
mutations in DNA damage repair genes. Genomic instability may also, and most commonly, results
from gross chromosomal changes, such as translocations or amplifications, which lead to chromosomal instability. Telomere length and telomerase activity, important in maintaining chromosomal structure and in regulating a normal cells lifespan, have been shown to have a function in both suppressing and facilitating malignant transformation. In addition to such direct sequence and structural
changes, gene silencing through the hypermethylation of promoter regions, or increased gene expression through the hypomethylation of such regions, together, form an alternative, epigenetic mechanism leading to instability. Emerging evidence also suggests that dietary and environmental agents can
further modulate the contribution of genetic instability to tumorigenesis. Currently, there is still much
debate over the distinct classes of genomic instability and their specific roles in the initiation of tumor
formation, as well as in the progressive transition to a cancerous state. This review examines the various molecular mechanisms that result in this genomic instability and the potential contribution of the
latter to human carcinogenesis.
Keywords: Cancer, CIN, epigenetics, genomic instability, MIN, telomeres.
Introduction
The last two decades have seen tremendous gains in promoting the understanding of the process of tumorigenesis. The identification of specific cancer
susceptibility genes has provided evidence of an underlying genetic basis for
cancer. As such, the advent of tumorigenesis requires the accumulation of multiple mutations in a single cell, thus rendering it with a selective advantage in
the environment in which it is present. Clonal expansion of this more favorable
cell follows, and, over time and through accrual of additional advantageous
mutations, this cell proceeds to malignancy. Cancer results when the equilibrium between cell birth and cell death moves toward uncontrolled cell prolifer-
304
ation [1]. This shift is in part due to mutations in genes that regulate cell
growth, differentiation and death, such as oncogenes and tumor suppressor
genes. Mutations in genes that protect the integrity of the genome and mediate
DNA repair processes also contribute to carcinogenesis.
The theory of the evolution of cancer has been one of long-standing debate.
The predominant models of carcinogenesis include one of somatic evolution,
where a cell acquires multiple somatic genetic mutations, which eventually
lead to malignancy, or alternatively, a model based on the underlying instability in existence at the nucleotide or chromosomal level. The first argues that
the normal spontaneous rate of mutation and selection of advantageous clones
for expansion is sufficient to initiate the process of tumorigenesis [2, 3].
However, it fails to explain an important factor concerning cancer evolution,
which involves the observance of a large number of mutations in tumors,
despite a relatively low spontaneous mutation rate in normal cells [4]. The second proposed model of carcinogenesis argues for the role of an underlying
genomic instability in cancer, which sees changes in the DNA sequence or
chromosomal structure. This argument suggests that the mutator phenotype is
necessary for cancer progression, and that tumorigenesis is often initiated by
the inherent genomic instability within a cell, without which it is difficult to
explain the multiple mutations observed in cancers [4, 5]. This instability may
result from subtle mutations in DNA stability genes, leading to microsatellite
instability (MIN), also known as MSI, or, more commonly, from structural or
numerical changes in whole chromosomes, referred to as chromosomal instability (CIN). Another tumor instability category that confers neither the MIN
nor CIN phenotype has been recently proposed, and results from defects in the
base-excision repair (BER) pathway. A distinct mechanism contributing to the
mutator phenotype involves epigenetic mechanisms, which together with subsequent genetic hits, facilitate the progression towards malignancy [6].
Although the presence of either CIN or MIN has been well documented in the
majority of cancers, the speculation continues as to whether this instability is
necessary for the initiation of cancer or whether it is the consequence of a cancer phenotype.
In the sections that follow, we review the types of alterations that have been
observed in neoplastic cells, then examine the role of the two predominant
types of instability described to date and some of the proposed dominant
mechanisms leading to each of the instabilities, and their respective roles in
tumorigenesis. We conclude with a look at the role of dietary and environmental factors in carcinogenesis.
305
have been found to be associated with cancer, and interestingly, the mutation
class appears to dictate the cell type that will undergo malignant transformation. An example of this has been observed in thyroid carcinomas, whereby
two different classes of mutations, chromosomal translocation and nucleotide
substitution, in the RET kinase gene, result in different site-specific cancerspapillary thyroid and medullary thyroid carcinomas, respectively [7].
There are at least four main categories of genetic alterations that have been
observed in cancer cells and these are described below.
Chromosomal aneuploidy
Upon examining cancer cells under a microscope, early cytogeneticists
Hansemann and Boveri, made the salient observation that tumor cells often
contained more than the normal complement of 46 chromosomes [8, 9].
Aneuploidy is frequently observed in the vast majority of human cancers and
involves the gains and losses of whole chromosomes, as well as structural
aberrations, such as inversions, deletions and duplications. Most tumors have
actually been observed to lose more than half of their alleles. However in some
instances, duplication of the remaining chromosome is able to maintain the
chromosome number, albeit, retaining two copies of the same parental allele
[10]. This could be potentially problematic if the duplicated copy harbors a
detrimental mutation or is prone to genomic imprinting.
Chromosome translocations
The field of cytogenetics has enabled the visualization of two patterns of chromosomal translocations: simple translocations, which see discernable
rearrangements of chromosomes that result in specific cancers, and complex
306
translocations, which occur almost at random and are not common even
among tumors of the same histology. The latter is observed to occur in many
solid tumors and results in gains or losses of segments of chromosomes, while
the former reproducible form of translocations displays itself mainly in
leukemias and lymphomas [11].
Gene amplification
In some instances, gross chromosomal changes can lead to gene amplification.
This has been observed to occur in the late-stages of cancer progression and
usually manifests in oncogenes and genes involved in metabolism and inactivation of drugs, thus contributing to resistance [12]. Little is known about
exactly how these multiple copies of a genomic segment are amplified, but
they likely persist due to a defect in DNA damage signaling.
307
respectively, with the former complex also having the ability to assist in the
repair of insertion/deletions. Upon recognition and binding of one of these
complexes, there is recruitment of MLH1-complexes to the site, which recruit
additional proteins conducive to the repair [15].
Much of what is known about the development of cancer was discerned
from the hereditary forms of colon cancer, the most common syndrome of
which is hereditary non-polyposis colorectal cancer (HNPCC), or Lynch syndrome. Examination of colon tumors from HNPCC patients revealed a change
in length in microsatellite sequences, which are short (<150 nucleotides), 15
nucleotide repeated sequences, scattered throughout the genome. These microsatellite regions appeared to have undergone a change in length due to the
insertion or deletion of nucleotides. Further studies examining tumors from
HNPCC patients led to a possible mechanism by which such widespread MIN
evolved, implicating a defective DNA-MMR system. In 4570% of HNPCC
families, a germline defect in one of the key MMR genes, MLH1 or MSH2,
was identified [1618]. This deficiency in MMR in HNPCC patients occurs
through the inheritance of one mutated copy and subsequent loss or mutation
of the second normal copy. Every somatic cell in such an individual harbors a
mutation in one allele, therefore these individuals are more prone to developing tumors. Once the second allele is lost, the mutation rate of this cell increases by as much as 1000-fold compared with normal cells [19, 20]. This
increased mutation rate allows for mutations to begin to accumulate in other
important growth regulatory and cell cycle genes, particularly those containing
repeat sequences, which are more prone to nucleotide misincorporation errors
introduced by DNA replication polymerases. Examples of such genes include
TGFBR2, BAX, TCF4, AXIN2, PTEN [15, 21]. Hence, MMR deficiency
appears to exacerbate the number of mutations in a given cell, and therefore
plays an important role in tumorigenesis.
Base-excision repair
BER is responsible for excision repair of damaged DNA bases sustained from
endogenously formed products of normal cellular metabolic processes, such as
reactive oxygen species, methylation, deamination, and hydroxylation [22,
23]. Similar to the other repair pathways, BER is a multi-step process requiring the activities and interactions of several proteins. In BER, the damaged or
mispaired base is recognized and removed by DNA glycosylases, and, to date,
ten such proteins have been identified in humans [24]. Three of the common
glycosylases involved in the removal of 8-oxo-7,8-dihydro2'deoxyguanosine
(8-oxoG), or oxidized guanine, the most stable product of oxidative DNA
damage, are OGG1, MYH, and MTH1 [23]. When left unrepaired, 8-oxoG can
readily mispair with adenines leading to G:CT:A mutations [25], thus the
proper functioning of these enzymes and their partial redundancy indicate their
importance in protecting cells against the mutagenic effects of guanine oxida-
308
tion. Each of these enzymes has a different function: OGG1 is involved in the
removal of the oxidized guanine from its pairing to cytosine in the DNA helix,
MYH excises misincorporated adenines found opposite unrepaired 8-oxoG,
and MTH1 prevents oxidized nucleotide precursors from entering the
nucleotide pool prior to the incorporation into DNA. Unlike MMR and NER,
inherited BER deficiencies had not been documented in humans until recently
[26], and this was believed to be due to the redundancy function between the
different repair systems, as mouse knockout models with complete loss of various glycosylases presented with no apparent phenotype [27]. The discovery
that biallelic mutations in the BER DNA glycosylase MYH resulted in an
autosomal recessive form of adenomatous colorectal polyposis, a high-risk
phenotype that may lead to cancer, has dispelled such long-held beliefs [26].
MYH is capable of removing misincorporated adenines from A/G mismatches, and with less efficiency from A/C mismatches, in replicated DNA, and has
been shown to interact with the MSH2/MSH6 heterodimer, thereby indicating
a possible partnership between MMR and BER [28]. The discovery of MYH
involvement in a cancer phenotype occurred through examining the pattern of
somatic APC mutations within a family having three siblings affected with
colon cancer. Using DNA extracted from the tumors, the sequencing of APC
revealed numerous somatic mutations of which the majority (~83%) were
G:CT:A transversions, mutations which were confirmed to be quite uncommon when compared with the available data on somatic APC mutations [26,
29]. Further studies of MYH-deficient colorectal tumors indicated that the two
bases immediately following the somatically mutated guanine are almost
always two adenine residues. This specificity of mutations at GAA sites is
believed to reflect improper recognition and/or repair by the DNA glycosylases. Why defects in MYH seem to have a predilection for formation of
tumors in the colon and not other sites is not yet clear, but there is speculation
that the high incidence of GAA sites within APC, compared to other tumor
suppressor genes, and the high levels of oxidative damage in the colon, are
important contributing factors [23, 30]. Colorectal tumors arising due to MYH
mutations do not exhibit either CIN or MIN.
Nucleotide-excision repair
NER is responsible for the removal of various DNA lesions that arise from
exogenous agents, such as mutagens and carcinogens, as well as UV photoproducts, which include bulky DNA adducts [31]. There are at least three
hereditary syndromes that are associated with defects in NER: xeroderma pigmentosum (XP), Cockayne syndrome and trichothiodystrophy (TTD). Each is
characterized by neurological degeneration and sensitivity to sun exposure, the
latter being the additional event that facilitates progression to cancer. Only
patients with XP have been associated with the cancer phenotype but all have
a hypersensitivity to killing by UV and exhibit defective DNA repair. NER uti-
309
lizes over 30 proteins in humans and includes seven XP NER complementation groups (XP-A to XP-G), plus a variant form with normal excision repair.
Patients with XP have an approximate 1000-fold increased risk of developing
the skin cancers, basal cell carcinoma, squamous cell carcinoma and
melanoma [27].
Chromosomal instability
CIN is apparent in the majority of cancers. In some instances it precedes the
onset, and is in fact the underlying cause of certain cancers, such as in lymphomas and leukemias, but in other instances it appears that this instability is
a result of previous inherent mutations in genes involved in cell division and
differentiation and/or repair of chromosomal breaks occurring during the
process. Here, we examine some of these genes and the evidence that exists for
their involvement in this cancer phenotype.
CIN is more commonly observed in many human malignancies than MIN,
and involves the gains and losses of whole chromosomes. Loss of a maternal
or paternal allele, referred to as loss of heterozygosity (LOH), occurs quite frequently in tumors, and is often accompanied by a gain of the opposite allele
through its duplication. The types of gene alterations that may lead to this
observed phenotype have been extensively studied in yeast, and are involved
in processes such as chromosome condensation, sister-chromatid cohesion,
kinetochore structure and function, centrosome/microtubule formation and
dynamics, and cell cycle checkpoints, whereas in humans to date, only genes
involved in the latter process have been implicated in CIN [32, 33].
The mitotic spindle checkpoint, involved in the separation of chromatids
only when proper alignment along the mitotic spindle has occurred, appears to
play a role in the CIN observed in human cancers. Many studies have shown
that genes involved in this process sometimes carry sequence alterations or are
observed to have altered expression. Examples of such genes include, MAD2,
BUB1 and BUBR1, which appear to have decreased expression in breast and
colon cancers respectively [10, 34].
In addition to the mitotic spindle checkpoints role in CIN, another checkpoint, the DNA-damage checkpoint, plays a vital role in the observed chromosomal changes. This checkpoint is responsible for preventing cells containing DNA damage, sustained from various endogenous and exogenous sources,
from entering mitosis. The genes involved in DNA-damage checkpoint control
include ATM, ATR, BRCA1, BRCA2, and TP53, all of which are implicated in
carcinogenesis [34].
Aneuploidy, or a change in chromosome number, is often seen in tumors
with the CIN phenotype, and involves yet another component of cell division:
centrosomes. The mitotic spindles involved in chromosome segregation appear
to be multipolar in cancers and the centrosomes from which they are generated occur in greater than normal numbers. The basis for the latter remains a
310
311
DNA methylation
DNA methylation is the most commonly studied form of epigenetic inheritance, and was thought to have first evolved in bacteria as a defense against
foreign DNA. The equivalent methylation of cytosine residues in eukaryotes,
however, served a far greater role in the maintenance of normal cellular functions and processes, involving the regulation of gene expression and the silencing of repeat elements in the genome [43]. In a normal cell, the DNA methy-
312
313
314
Histone modification
Histones may undergo modifications such as acetylation, methylation, and
phosphorylation at their N-terminal tails, consequently affecting transcriptional regulation and chromatin stability [58]. Both aberrant methylation and
deacetylation, via histone deacetylases (HDACs), have been observed to play
a causal role in cancer development. Generally, acetylation of lysine residues
in the histones leads to transcriptional activation. Deregulated HDACs have
been observed to transcriptionally silence tumor suppressor genes in a variety
of cancers. Histone methylation, on the other hand, is able to serve both functions, and as this modification is believed to be irreversible, with no demethylase described to date, histone methylation appears to serve an important function in long-term regulation [50]. Thus, histone modification may affect the
regulation of expression of certain genes through chromatin remodeling. In
addition, histone deacetylation, histone methylation, and DNA methylation
may work together in accomplishing silenced gene expression [85, 86].
Genomic imprinting
Genomic imprinting and its contribution to cancer have recently attracted
greater attention. Genomic imprinting refers to the epigenetic modification of
maternal and paternal genomes during gametogenesis, resulting in the differential expression of the particular parental allele [6, 87]. In a variety of childhood and adult tumors, there is loss of imprinting (LOI), which leads to activation of growth-promoting imprinted genes, such as IGF2 [6, 88], as well as
silencing of potential tumor suppressor genes such as p57KIP2 and ARH1 [89,
90]. LOI has been observed in colon cancer cases displaying MIN, and has
also been found to occur even in the normal colonic mucosa of these patients,
315
316
Conclusion
The evolution of cancer is usually a long process, requiring multiple genetic
mutations sustained over many years. Genomic instability, whether at the
nucleotide or chromosomal level, is a common feature in the majority of
tumors, yet its precise role in cancer initiation and progression is not completely understood. As cells are constantly exposed to an ever-changing
microenvironment, their likelihood of coming into contact with potential
DNA-damaging agents increases. Therefore, it is possible that genetic instability, or increased mutation rate, allows genetic and epigenetic alterations to
accumulate during carcinogenesis to provide the tumor cells with a selective
advantage that allow them to persist in their environment. Although there has
been, and continues to be, much debate over whether an underlying genetic
instability is necessary for cancer initiation, it is certain that without it, tumorigenesis would be a much more prolonged process.
Acknowledgements
B.B. is the recipient of CIHR Grant No. MOP_64225 and crCIHRt -43821 from the Canadian
Institutes of Health Research. S.R. is the recipient of the Ontario Student Opportunity Trust Fund and
the Samuel Lunenfeld Research Institute Fellowship.
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321
Abstract. Intensive research efforts during the last several decades have increased our understanding
of carcinogenesis, and have identified a genetic basis for the multi-step process of cancer development. Tumors grow through a process of clonal expansion driven by mutation. Several forms of molecular alteration have been described in human cancers, and these can be generally classified as chromosomal abnormalities and nucleotide sequence abnormalities. Most cancer cells display a phenotype
characterized by genomic hypermutability, suggesting that genomic instability may precede the acquisition of transforming mutations in critical target genes. Reduced to its essence, cancer is a disease of
abnormal gene expression, and these genetic abnormalities contribute to cancer pathogenesis through
inactivation of negative mediators of cell proliferation (including tumor suppressor genes) and activation of positive mediators of cell proliferation (including proto-oncogenes). In several human tumor
systems, specific genetic alterations have been shown to correlate with well-defined histopathological
stages of tumor development and progression. Although the significance of mutations to the etiological mechanisms of tumor development has been debated, a causal role for such genetic lesions is now
commonly accepted for most human cancers. Thus, genetic lesions represent an integral part of the
processes of neoplastic transformation, tumorigenesis, and tumor progression, and as such represent
potentially valuable markers for cancer detection and staging.
Keywords: Chromosomal instability, genomic instability, microsatellite instability, tumor suppressor
gene, proto-oncogene.
322
mortality rates for different forms of human cancer predict that multiple mutations in specific target genes are required for the genesis and outgrowth of
most clinically diagnosable tumors [5]. In accordance with this prediction, it
has been suggested that tumors grow through a process of clonal expansion
driven by mutation [6], where the first mutation leads to limited expansion of
progeny of a single cell, and each subsequent mutation gives rise to a new
clonal outgrowth with greater proliferative potential. The idea that carcinogenesis is a multi-step process is supported by morphological observations of
the transitions between pre-malignant (benign) cell growth and malignant
tumors. In colorectal cancer (and some other tumor systems), the transition
from a benign lesion to a malignant neoplasm can be easily documented and
occurs in discernible stages, including benign adenoma, carcinoma in situ,
invasive carcinoma, and eventually local and distant metastasis [7]. Moreover,
specific genetic alterations have been shown to correlate with each of these
well-defined histopathological stages of tumor development and progression
[8]. However, it is important to recognize that it is the accumulation of multiple genetic alterations in affected cells, and not necessarily the order in which
these changes accumulate, that determines tumor formation and progression.
These observations suggest strongly that the molecular alterations observed in
human cancers represent integral (necessary) components of the process of
neoplastic transformation and tumor progression.
323
324
Chromosomal abnormalities
Chromosomal alterations in cancer include the gain or loss of one or more
chromosomes (aneuploidy), chromosomal rearrangements resulting from
DNA strand breakage (translocations, inversions, and other rearrangements),
and gain or loss of portions of chromosomes (amplification, large-scale deletion). The direct result of chromosomal translocation is the movement of some
segment of DNA from its natural location into a new location within the
genome, which can result in altered expression of the genes that are contained
within the translocated region. If the chromosomal breakpoints utilized in a
translocation are located within structural genes, then hybrid (chimeric) genes
can be generated. The major consequence of chromosomal deletion (involving
a whole chromosome or a large chromosomal region) is the loss of specific
genes that are localized to the deleted chromosomal segment, resulting in
changes in the copy number of the affected genes. Likewise, gain of chromosome number or amplification of chromosomal regions results in an increase
in the copy numbers of genes found in these chromosomal locations.
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326
this multi-step pathway [42]. Cells expressing the mutator phenotype accumulate mutations more rapidly than normal cells, and would therefore be more
likely to sustain mutations in critical genes required for enhanced growth and
tumorigenesis [43, 44].
327
328
absence of cell proliferation [43, 44], but would facilitate clonal expansion of
an altered clone in response to subsequent selection pressures [36]. Numerous
sporadic tumor types exemplify this form of instability, including sporadic colorectal tumors of the tumor suppressor pathway [65], or the microsatellite
mutator pathway [64, 66]. It can be envisioned that both chromosomal abnormalities and DNA sequence abnormalities could result from the expression of
either of these forms of genomic instability during neoplastic transformation.
329
instability and may loose a significant number (2550%) of alleles during neoplastic transformation and tumorigenesis [71, 73, 82, 83]. These large-scale
genomic changes may be due to some form of progressive chromosomal instability [84, 85]. Supporting this suggestion, gains and losses of multiple chromosomes occur in aneuploid colorectal cancer cell lines 10- to 100-fold more
frequently than in diploid cancer cell lines of the same histological subtype
[53, 86]. In other studies, the rate of LOH at marker loci proximal to a selectable gene (APRT) was increased 10-fold in colorectal cancer cell lines that
exhibit proficiency of mismatch repair (MMR) compared with cell lines that
lack MMR [87, 88]. In addition, numerous studies combine to show that aneuploid cancers exhibit highly variable karyotypes [67, 89], suggesting that new
chromosomal variations are produced in a progressive manner during tumor
outgrowth and evolution.
The absence of chromosomal instability in diploid cancers and/or cancers
that exhibit nucleotide sequence alterations, argues against a nonspecific
mechanism for chromosomal instability related to abnormal properties of neoplastic cells [2]. Further, the high rates of numerical chromosomal alterations
in aneuploid cells do not simply reflect the ability of these cells to survive
changes in chromosome number [53]. Tetraploid cells resulting from the
fusion of diploid cancer cells retain a stable tetraploid chromosome number
[53], suggesting that the presence of a nondiploid chromosome number does
not precipitate progressive chromosomal instability. Rather, the evidence supports the existence of a specific form of genetic instability in cancer cells that
results from dysfunction of normal chromosomal homeostasis producing
numerical chromosomal abnormalities. Several possibilities have been investigated, including the involvement of (i) mutant p53 protein, (ii) abnormal centrosomes, (iii) abnormal mitotic spindle checkpoint function, or (iv) abnormal
DNA-damage checkpoint function [2, 85, 90].
330
[101]. These observations suggest that loss of normal p53 function may contribute significantly to chromosomal instability in certain forms of cancer, but
does not represent the primary cause of this form of genomic instability.
331
cells, and that inactivation or dysregulation of one or more of them can lead to
abnormal centrosome number/function.
332
Gene amplification
The amplification of specific chromosomal segments or genes have been documented in some cancers and in many cancer cell lines [48, 125], some of
which involve cellular proto-oncogenes, resulting in abnormal expression levels of the proto-oncogene products [126]. In general, gene amplification
occurs late in tumorigenesis associated with tumor progression and is the recognized mechanism through which many tumors acquire resistance to
chemotherapeutic agents. Thus, gene amplifications can profoundly affect
tumor behavior, and can have prognostic significance for some cancers, but
333
334
and proposed for others [131, 132]. The role of chromosomal translocation in
cancer pathogenesis is suggested to involve proto-oncogene activation by
repositioning of the gene adjacent to a heterologous genetic control element.
Evidence for this type of proto-oncogene activation includes studies of chromosome translocations in Burkitts lymphoma [133]. In this cancer, the c-myc
proto-oncogene is translocated from chromosome 8 to chromosome 14, proximal to the immunoglobulin enhancer sequences, resulting in abnormal constitutive expression of c-myc [58].
335
individual microsatellite loci [140]. In addition, two distinct patterns of microsatellite alteration have been described in human cancers that display MSI, and
specific microsatellite markers tend to be altered in a characteristic pattern
[141, 142]. The pattern of alteration observed at a specific microsatellite locus
may reflect the nature of the genomic instability displayed by a tumor. Several
factors influence the probability of mutation at a specific microsatellite locus:
(i) the type of repeated sequence (mononucleotide, dinucleotide, etc.), (ii) the
length of the microsatellite sequence (number of repeated units), (iii) the location of the microsatellite sequence within the genome, and (iv) the underlying
molecular lesion. Thus, no single type of microsatellite will be diagnostic for
MSI in all tumors. This is supported by the observation that numerous polyA
repeats are altered in various human cancers [143, 144], but not all neoplasms
that exhibit MSI demonstrate alterations in polyA sequences, and may only
show alterations in higher order repeats [145]. A direct relationship has been
observed between the length of polyA tracts and their mutation frequency
among genetically unstable tumors [146], consistent with the suggestion that
the probability of sustaining a mutation in an individual microsatellite
sequence is proportional to the length of its sequence [147]. Extensive comparison of the mutation of dinucleotide versus higher order repeat units (trinucleotide or tetranucleotide) in human tumors suggests that larger alleles are
more susceptible to mutation in genetically unstable tumors [148]. Studies
with cancer cell lines that harbor MMR gene mutations demonstrate instability of specific classes of microsatellites. Cells possessing a defect in hPMS2
exhibit instability of trinucleotide repeats [149], while cells deficient for
hMSH3 or hMSH6 demonstrate an inability to correct mismatches in dinucleotide (or higher order) repeats [150]. Furthermore, cells lacking hMSH
demonstrate minimal levels of dinucleotide instability, while cell lines lacking
hMSH2 or hMLH1 demonstrate profound dinucleotide instability [151]. In
addition, specific MMR gene mutations can affect the extent of hypermutability at microsatellite sequences [140]. The microsatellite mutation rate in cells
lacking hMLH1 and hMSH3 is tenfold greater than that of cells lacking hPMS2
and hMSH6 [140]. These observations suggest that individual MMR complexes exhibit specificity for certain types of mismatches, and that the MSI displayed by cancer cells may be directly related to the number [152] and nature
[140] of MMR gene mutations.
336
337
338
have utilized single gene transfer to correct MMR deficiency. Cancer cells that
harbor an hPMS2 mutation and display MMR deficiency [149] show increased
microsatellite stability and reduced mutation rate at the HPRT locus, and cell
extracts can perform strand-specific MMR following transfection with a wildtype hPMS2 gene [179]. Likewise, transfection of tumor cells with hMSH6
resulted in restoration of MMR, increased stability of the BAT26 polyA tract,
and reduction in the mutation rate at the HPRT locus [180].
339
340
Conclusions
A large amount of evidence has now accumulated suggesting a genetic basis
for the development of neoplastic disease in humans. However, the genetic
damage documented in human cancers includes both large-scale alterations
(chromosomal aberrations and ploidy changes) and DNA sequence alterations
(single nucleotide changes or alterations in short segments of DNA). In addition, the patterns of genetic damage within a single tumor can vary from a few
molecular alterations at specific loci to genome-wide mutations involving a
large number of loci. Several distinct forms of genomic instability may provide
the molecular basis for neoplastic transformation in humans. Cells undergoing
neoplastic transformation may accumulate genetic damage related to progressive genomic instability, or due to episodic genomic instability. Transforming
mutations could arise through either of these mechanisms, involving chromosomal alterations or sequence alterations (point mutations and/or MSI).
Although the significance of mutations to the etiological mechanisms of tumor
development has been debated, a causal role for genetic lesions in the genesis
of cancer is commonly accepted. Thus, genetic lesions represent an integral
part of the processes of neoplastic transformation, tumorigenesis, and tumor
progression, and as such represent potentially valuable markers for cancer
detection, diagnosis, staging, and prediction of clinical outcome [3, 4].
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Abstract. Epigenetic mechanisms are involved in critical nuclear processes such as transcriptional control, genome stability, replication and repair. Recent evidence suggests that changes in the epigenetic
repertoire can drive tumorigenesis. This review examines the latest experimental evidence that questions the mechanisms underlying the consequence of epigenetic changes in gene regulation and cancer development.
Key words: Cancer, chromatin, DNA methyltransferase, methylation, transcriptional silencing, tumor
suppressor gene.
Introduction
There are many ways in which genes are regulated, and the field of epigenetics has seen a recent surge of interest in the study of modifications of the
genome and histone tails to explain transcriptional competence. The term epigenetics refers to heritable changes in gene expression that are not the result of
changes in the DNA code. DNA methylation is the best studied of these mechanisms with CpG methylation recognized as a major component of gene
silencing in cancer [1]. Microinjection experiments using methylated gene
constructs indicate that transcriptional repression occurs once chromatin is
assembled [2]. Nuclease resistance in mammalian nuclei is due to CpG methylation, and this correlated with transcriptional repression mediated by methylCpG binding (MBD) proteins [3, 4]. It is not coincidental then that MeCP2, a
global transcriptional repressor, silences gene activity and binds to chromatin
in a methylation-dependent manner [5]. Before focusing on the impact of
DNA methylation in tumorigenesis, the relevance of epigenetic mechanisms
and transcriptional control is discussed.
352
A. El-Osta
353
354
A. El-Osta
Proposed function
Refs
HDAC1
[54]
HDAC2
[55]
DMAP1
[55]
pRB
[56]
MBD3
[57]
PCNA
[5860]
DNMT1
RUNX1/MTG8
[61]
p53
Transcriptional silencing
[62]
RGS6
[63]
SuV39H1
[64]
p33ING1
[65]
[28, 29]
Condensin
[66]
hSNF2H
Epigenetic regulation
[67]
Transcriptional silencing
[68, 69]
DNMT3a/DNMT3b
RP58
DNMT3L
HDAC1
reveal that the DNMT3L protein can mediate transcriptional repression by its
biochemical interaction with histone deacetylase. These observations suggest
the methylation machinery are connected with chromatin remodeling; however, the biggest challenge in the area is to determine the mechanisms by
which the determinants are localized and segregated on target genes. In the
next section, I discuss possible mechanisms that could explain aberrant DNA
methylation patterns in cancer.
355
ic instability in cancer [31, 32]. Almost two decades ago, studies demonstrated that reductions in genomic methylation are associated with cancer progression [33, 34]. One of the best-studied models of cancer development is tumor
suppressor gene silencing and has been studied in different contexts and diseases. For example, the retinoblastoma tumor suppressor gene is silenced by
CpG methylation [35]. Alternatively, demethylating agents such as azacytidine
have been used to induce promoter sequence hypomethylation and derepress
gene silencing [36]. Clearly, experimental evidence suggests that hypomethylation and hypermethylation events can be associated with tumor development.
However, hypermethylation of tumor suppressor genes and transcriptional
repression do not explain how determinants could be mistargeted in cancer
when hypomethylation is believed to be the primary cause of tumorigenesis. In
this section I consider recent advances to our knowledge of methylation-mediated mechanisms in cancer and examine both hyper- and hypo- methylation
events in cancer.
356
A. El-Osta
Figure 1. Model of methylation-mediated transcriptional regulation. Hypermethylation of the promoter sequence is dominant in silencing gene transcription. Methylated CpG sequences become
recruitment sites for methyl-CpG-specific proteins and are associated with HDAC and Sin3 co-repressors. Demethylation by 5adC reduces the silencing potential mediated by methylation and the robust
release of the co-repressor complex. Hyperacetylation of histone tails can be induced using HDAC
inhibitors such as TSA, thereby decondensing chromatin and allowing assembly of activator complexes that drive gene expression.
Enzyme
activity
Reduction in
methylation content
Gene expression
DNMT1/
DNMT3b/
DNMT1/3b/
96%
87%
99.9%
20%
3%
95%
None
None
Expressed
357
are central to carcinogenesis. Taken together, these results suggest a leukemiapromoting protein is directly associated with carcinogenesis by inducing gene
hypermethylation and the recruitment of DNMTs. These observations clearly
identify that DNA hypermethylation is associated with silencing of tumor susceptibility genes in several forms of cancer. However, direct proof that CpG
hypermethylation and transcriptional silencing are the primary mechanisms of
cellular transformation is currently lacking.
358
A. El-Osta
Conclusion
It is clear that the study of epigenetics continues to attract widespread interest,
both within basic and medical research. The future holds great promise and,
given these recent research findings, may lead to the development of new therapeutic tools based on the pharmaceutical reversal of the methylation signal
and/or regulation of the machinery responsible for methylation.
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363
Index
2-AAF 67, 70, 76, 81
Abbe 6
4-ABP 83
abscopal effect 165
achromatic lense 2
acute myeloid leukemia (AML) 29
acute promyelocytic leukemia 33,
34
adaptive response 169
5adC 355, 357
AFB1-induced DNA damage 82
aflatoxin 66
aflatoxin B1 (AFB1) 79
Ah gene 74
Akt 211
albinism 15
Alexander 166
alkylating agent 12
Alper 166
Alu 355
amine/amide 65, 68
2-amino-1-methyl-6-phenylimidazo
[4,5-b]pyridine (PhIP) 315
aminoazo dye 67, 68
anacardic acid 32
anaphase bridge 191
anaplasia 8, 10, 313
aneuploidy 51, 185, 192, 305, 309,
324, 328
angiogenesis 14, 57, 58
angiogenic process 223
angiogenic switch 242, 244
angiomotin 233
angiomyolipoma 277
angiostatin 233, 236
aniline 66
aniline cancer 67
aniline dye 12
annexin II 233
annexin II 281
anti-angiogenic drugs 238
anti-angiogenic therapy 240
antisense oligonucleotide (ASON)
213
AP-1 115, 117
AP-1 gene 281
APC 340
apochromatic lense 6
apolipoprotein E4 (ApoE4) 316
apoptosis 14, 114, 201, 333
apoptosome 205
apoptotic body 203
apoptotic resistance 76
apurinic/apyrimidinic (AP) site 111
aquaglyceroporin 101
ARH1 315
ARNT 73, 85
aromatic amines/amides 76
aromatic compound 65
aromatic hydrocabon 67
arsenate 100
arsenic 12, 97, 99, 111, 112, 115
arsenic excretion 100
arsenic pathology 101
arsenic-contaminated water source
100
arsenite 100, 106
arsenobetaine 101
arsenosugar 101
arylhydrocarbon receptor (AHR)
72, 73, 75, 78
arylnitrenium ion 70
As(III) 116
As(V) 116
ATM 309, 331, 332
ATM 55
364
Index
bleomycin 167
BLM 340
bone cyst 15
Boveri, Theodor 6, 9, 51, 188, 305,
322
Boyland, Eric 74
BRAF gene 36
Bragg-Peak ionization deposition
159
BRCA1 tumor suppressor 81
BRCA1 309, 330, 332
BRCA2 309, 330, 332
breast cancer 1 (AIB1) 31
Browder 241
Brown, Robert 2
BUB1 309
BUBR1 309
Burch 289
bystander 296
bystander effect 165, 170
bystander signal pathway 170
bystander signal-associated
consequence 167
cadherin 99, 113
cadmium 77, 97, 99, 102, 108, 111,
112, 116
cadmium-nickel 102
Caenorhabditis elegans 202
calcium homeostasis 207
cancer biomarker 250
cascade mutagenesis 170
caspase 203
caspase inhibitor 205
Castlemans disease 192
catenin 113
cathepsin 234
CBP 31
CDH1 313
cdk2 190
Cdk2 37
cell cycle checkpoint 184, 191
cell poison 160
cellular oncogene 16
centriole 185
Index
365
co-carcinogens 98
Cockayne syndrome 308
collagen XVIII 234
Comings 17
comparative genomic hybridization
(CGH) 52
condensin 354
connexin 113
Cohnheim 5
copper 97
Co-REST 33
CpG dinucleotide repeat 312
CpG hypermethylation 358
CpG methylation 351, 352
CpG site 77
Cr (III) 103, 107
Cr (IV) 112
Cr (V) 107
Cr (VI) 103, 107
Cr (III)-DNA adduct 103
(CREB)-binding protein (CBP) 28
crebbp gene 31
critical target 161
critical mutation 323
cross-link 151
Currie 201
cyclin D2 312
cyclin E 40
cyclin K 192
cyclobutane pyrimidine dimer (CPD)
110, 134
cyclophosphamide 241
CYP family 70
CYP 76
CYP1A1 73, 82, 83, 84, 86
CYP1A2 84
CYP1B1 73, 84
CYP2A6 84
CYP2E1 84
cysteine 150
cystine 150
cytochrome c 205, 208
cytochrome P450 68
cytochrome P450-dependent
monooxygenase (CYP) 68
366
cytokine 168
cytokinesis 185
cytoplasmic death domain (DD) 205
cytosine photohydrate 137
D5S107 337
DAB 67, 68
damage-specific glycosylase 111
DC101 239
ddm1 (decrease in DNA
methylation) gene 358
De Vries 14, 322
deacetylase 31
death receptor 211
decoy adduct 83
dedifferentiation 8, 10
demethylation of CpG 352
DES 77
Dewar isomer 140
Dewar valence isomers 135
2,6-diamino-4-hydroxy-5formamidoguanine (FapyGua)
139
1,2:5,6-dibenzanthracene 67
dicentric chromosome 311
diethylnitrosamine 279
differentiation 13, 201
differentiation status 182
4-dimethylaminoazobenzene (DAB,
butter yellow) 67, 68
dimethylarsine 106
dioxin TCDD 72
DMA 101
DMAP1 354
DMBA 78
DNA adduct 68, 87
DNA damage arrest 80
DNA damage checkpoint 332
DNA glycosylase 112, 307, 308
DNA ligase 109, 111
DNA methyltransferase 118
DNA methylation 40, 77, 116, 311,
330, 351
DNA methyltransferase family
(DNMT) 353
Index
Index
367
368
Index
Index
369
linker histone 25
Little 11
liver tumor 336
Livin 206
localized hypermethylation 118
Lockhart-Mummery 16
Lockshin 201
Loeb, L.A. 19
Loeb, Leo 11
Loop 6 233
loss of heterozygosity (LOH) 53,
278, 309
Lsh/ mouse 358
lymph 2
lymphoid specific helicase (Lsh)
358
lysosome 163, 164
mad1 192
MAD2 309
malignant progression 183
mammalian target of rapamycin
(mTOR) 283
MAP kinase 245
MAP kinase phosphatase-1 35
MAPK pathway 76
maspin 312
matrix metalloproteinase (MMP)
230, 231, 234236, 243
maturation arrest 58
MBD protein 352
MBD protein family 352355
MeCP1 352
MeCP2 352
megakaryocyte potentiating factor
(MPF)/mesothelin 282
mEH 84
MEK/ERK pathway 36
membrane channel 163
membrane-bound protein 163
mercapturic acid 70
mercury 97
metal carcinogenesis 98
metal complexes with amino acids,
proteins and DNA 113
370
metal-dependent matrix
endoproteinase 231
metalloid arsenic 77
metallothionein 34, 116, 117
metaphase 59
metaplasia 4
metastasis 57
methionine 149
methyl metabolites of arsenic 106
methyl methane-sulfonate (MMS)induced damage 111
methylation 98, 99
methylation-dependent chromatin
condensation 118
methyl-CpG binding (MBD) protein
351
3-methylcholanthrene 78
methylenetetrahydrofolate
reducatase (MTHFR) 316
methyltransferase SUVAR39H1 39
metronomic chemotherapy 240, 241
metylation-specific repressor
MeCP2 352
Mi-2 353
micronuclei 188, 296
micronucleus formation 192
microsatellite instability (MSI) 306,
334
microsatellite locus 335
microsatellite mutation rate 337
microtubule 185
Miller, James A. 68
mismatch repair (MMR) 109, 306
mismatch repair gene 337
missense mutation 324
mitochondrial DNA 163
mitochondrial outer membrane
permeabilization (MOMP) 205
mitogen-activated
protein/extracellular signalregulated kinase (MEK) 193
mitosis 6, 185
mitotic instability 18
mitotic spindle 185
MLH1 313
Index
Index
371
NuRD 33
O6-methylguanine-DNA
methyltransferase (MGMT) 112,
313
OGG1 307
olefine 65
oncodem 269
oncogene 17, 180
oral cancer 182
organoarsenic species 100
O-sulfonation 70
ovarian cancer 336
8-oxo-7,8-dihydroguanine (8-oxoGua) 139142
8-oxo-Ade 140
8-oxo-dG 106, 108
8-oxoG 307
8-oxoguanine DNA glycosylase 112
oxazolone 140
oxidized DNA photoproduct 139
oxoflatin 34
p107 190
P<>P dimer 135
P<>P 140
p16 40, 118
p130 190
p16/CDKN2A 313
p16/INK4b 313
p16INK4A 355
p21WAF1 34
p300 28, 30
P33ING1 354
p38 MAPK 35
p53 34, 55, 116, 182, 183, 188, 193,
209, 210, 228, 230, 248, 249, 313,
327, 329, 331333, 354
p57KIP2 315
paclitaxel 213
Paget 58
PAH 7476, 78, 79, 86
PAH-DNA adduct 78, 86
papillomavirus 180
paraffin 12, 66
372
parasitic theory 5
particulate Cr(IV) 104
particulate nickel 104
PCAF 32
PCNA 354
PDGF 227, 240
penile cancer 182
PER 71, 73
peroxynitrite 107
phakomatosis 277
phase-II enzyme 70
phenoxyl radical 144
phenyl butyrate 34
Philadelphia (Ph) chromosome 52
phorbol esters 35
phosphoinositide 3-kinase (PI3K)
245
phospholipase A2 74
photo-oxidation of DNA and
proteins 151
photo-oxidation of proteins 150,
151
photoproducts of purine bases 137
PI3K 211
PINX1 55
pipe smokers cancer 12
plasminogen 233
ploidy 184
plutionium 295
PML-RAR 356
PML-RAR translocation 34
polychlorinated phenol 72
polycomb response element 40
polycyclic aromatic hydrocarbon
(PAH) 75
polymerase chain reaction (PCR) 18
population genetics 18
Porshke photoproduct 137
Pot1 55
Pott, Percivall 66
PP1 37
practicable threshold 87
pRB 190, 354
programmed cell death 201
progression 99
Index
Index
373
Sin3 33
single-hit theory 14
singlet oxygen 132
slippage mechanism 336
Slye, Maud 11
SMYD3 40
SNF2 subfamily 358
snuff 66
snuff cancer 12
sodium butyrate 34
somatic mutation 14
Sp1 33, 246
Sp1 352
spectral karyotyping (SKY) 52
spindle checkpoint 309, 331
spontaneous mutation rate 325
spore photoproduct 137
squamocolumnar transformation
zone 182
Src 248
stealth property 80
stem cell 58
stereochemistry 78
STK15 330
stochastic effect 294
Stoler 18
strand slippage 336
SU5416 240
SULT 70
SULT1A1 84
SULT1A2 84
SULT2A1 84
sunscreen 133
superoxide dismutase 105, 116
survivin 38, 206
Sutton 166
SuV39H1 354
SUV39H1 40
SWI2/SNF2 family 353
synergistic effect 83
TANK1/2 55
tar 12
target theory 161
tax protein 192
374
TCDD 74
TCF-4 307, 340
telomerase 50, 5355, 111, 183,
310, 311
telomere 4951, 53
telomere dysfunction 310
telomeric protein 50
terminal differentiation 167
tetraploidy 185
tetrasomy 185
TGFBR2 307
TGFRII 339
therapeutic target 32
Thiersch 5
thioketene 71
thiyl radical 145
thrombospondin 230
thyroid hormone receptor (SMRT)
33
TIF2 29
time-integrated DNA adduct level
(TIDAL) 78
Timofeeff-Ressovsky 160, 166, 170
TIMP-2 242
TIMP3 355
TIN2 55
tissue inhibitor of MMP (TIMP)
232
TP53 77, 81, 86, 309
TP53 mutational hotspot 82
TPA 76
TRADD 205
TRAIL 213
TRAIL-R1/R2 211
transcriptional repressor domain
(TRD) 352
transcriptional silencing 358
transcription-coupled repair (TCR)
110
transdifferentiation 14
transferase 70
transforming growth factor- (TGF) 227
transgenerational instability 169
translesional synthesis 80
Index
transversion 142
TRF1 55
TRF2 55
trichostatin A (TSA) 352
trichothiodystrophy (TTD) 308
triplet state 143
trophic sentinel response 193, 194
troponin I (TnI) 231
3
Trp 145
tryptophan (Trp) 143
TSA 34, 355, 357
Tsc2 278, 281
Tsc2 gene 277
TSP-1 246, 247, 249
tuberous sclerosis (TSC2) gene 270
tubulin 18
-tubulin 34
tumor necrosis factor (TNF-)
227
tumor necrosis factor receptor (TNFR) 205
tumor suppressor 180, 311
tumor suppressor gene silencing
355
tumour heterogeneity 18
tumour progression 9, 18
two-hit model 270
3
Tyr 145
tyrosine (Tyr) 147
Tyzzer 14
ubiquitin 41, 206
ulcerative colitis 339
ultraviolet light 12
unifying hypothesis 327
uracil hydrate 137
UV irradiation 35, 131133
vascular endothelial growth factor
(VEGF) 34, 225, 234, 235, 237,
242244, 246, 247
VEGF gene 226
VEGF receptor 229
VEGFR tyrosine kinase inhibitor
240
Index
VEGF-Trap 239
v-Fes 36
VHL 313
vinblastine 18
viral oncogene (e.g. AgTsv40) 54
Virchow 3, 4, 68, 13, 14
virus 161
v-Mos 36
Vogelstein 17, 289
von Baer 4
von Hansemann 69, 18, 51, 305
von Hippel-Lindau (VHL) disease
gene 279
von Hippel-Lindau gene product
228
v-Raf 36
V-Src 36, 228, 230, 247
VX-680 39
Waldeyer 5
Weismann 7
Whitman 9, 14
Whitmore 166
WI-38 SV40 41
Williams 201
Willis 16
Wilms tumor 59, 278, 313
wood dust 66
wound healing 58
WRN/BLM 55
Wyllie 201
xenobiotic metabolizing enzyme
(XME) 69
xeroderma pigmentosum (XP) 10,
18, 308
xeroderma pigmentosum group A
(XPA) 110
XIAP 214
XME 79, 82
XPD 85
X-ray 12
XRCC1/3 85
Yamagiwa, Katsusaburo 67
375
Yamaguchi 12
Y-family polymerases 80
YY1 33
Zimmer 160, 166
zinc 110
zymogen 203
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