Transformation and Immortalization

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Role in cell line characterization/How will you

characterize a cell line?


Transformation is an event or series of events – depends
on and promotes genetic instability
Alters cell line properties – growth rate, mode of
growth, specialized product formation, longevity and
tumorigenicity
Whether culturing cells from neoplastic cells and not
normal blood vessel cells or inflammatory cells.
What is Transformation?
Implies a spontaneous or induced permanent
phenotypic change resulting from a heritable change in
DNA and gene expression

Artificial introduction of DNA or DNA transfer into


mammalian cells – Transfection

Transformation – result of infection with virus or


transfection with mutant ras genes or spontaneous
exposure to ionizing radiation or chemical carcinogens
What are the three major classes of phenotypic
change?
Immortalization – the acquisition of an infinite life span

Aberrant growth control – the loss of contact inhibition


of cell motility, density, limitation of cell proliferation and
anchorage dependence

Malignancy – Growth of invasive tumors in vivo


Genetic Instability
Human finite cell lines are stable
Mouse cell lines are genetically unstable and transform
easily
Continuous cell lines (tumorigenic) of all species are
unstable
Two causes of genetic heterogeneity
- Spontaneous mutation is higher in vitro
- Mutant cells cannot be eliminated without impairment in
their growth capacity
Chromosomal Aberrations
Genetic rearrangement – seen in chromosome counts
and karyotype analysis

Also determined by sister chromatid exchange assay

Variations in ploidy, frequency of individual


chromosomal aberrations and variations in chromosome
number – tumorigenic cell lines
Immortalization
Rodent cells are karyotypically normal at isolation and
undergo changes after 12 th generation

If maintained at a low cell density and not allowed to


remain at confluence for any length of time, they
remain sensitive to contact inhibition and density
limitation of growth

If allowed to remain at confluence for extended periods


– reduced contact inhibition- cells pile up – overgrowth-
will be seen in subsequent subcultures - tumorigenic
Control of Senescence
Senescence genes in finite cells negatively control cell
cycle progression
Regulates expression of telomerase – synthesizes
telomeric DNA
Deletions and/or mutations within senescence genes or
overexpression or mutation of one or more oncogenes
allows reexpression of telomerase
Control of Senescence
Immortalization involves both inactivation of senescence
and cell cycle regulatory genes such as Rb and p53
Product of SV40 LT gene – T antigen binds to Rb and
p53
Allows extended proliferative life span and restricts
DNA surveillance activity of genes
- Increases genomic instability
- Increases chances of mutations
Control of Senescence
Immortalization – does not imply development of
aberrant growth control and malignancy

- Retains contact inhibition of cell motility, density


limitation of cell proliferation and anchorage
dependence and are non-tumorigenic

- Some aspects of growth control are abnormal and likely


increase of genomic instability
Immortalization with Viral genes
SV 40 virus – immortalize cells with their large T (LT)
gene

Adenovirus E 1a, human papilloma virus (HPV) E6 and


E7 and Epstein-Barr virus (EBV)

Inhibits activity of genes such as CIP-1/WAF-1/p21, Rb,


p53 and p16
Immortalization with Viral genes
Mammalian cells transfected or retrovirally infected with
immortalizing gene before they enter senescence

Extends proliferative life span for 20-30 population


doublings
- Cells cease proliferation and enter crisis

- Up to several months in crisis – subset of immortal cells


overgrows

- Fraction of immortal cells obtained


Telomerase-Induced
Immortalization
Telomerase or terminal transferase – composed of two
subunits – RNA component (hTR) and a protein
catalytic subunit (hTERT)
hTR is expressed in normal and malignant tissues
hTERT is expressed in tumors, germ cell lines and
activated lymphocytes
Transfecting cells with hTERT extends life span of cell
line
- Some cells become immortal but not malignantly
transformed
Aberrant growth control
Tumorigenic cell lines exhibit
- Lower serum or growth factor dependence

- Form clones with higher efficiency

- Acquire autonomous growth control

- Autocrine growth control – secrete mitogens or receptors


Aberrant growth control by anchorage
independence
Transformation due to cell surface modifications in cell
surface glycoproteins and adhesion molecules
Leads to loss of cell-cell recognition – to disorganized
growth pattern, loss of contact inhibition of cell motility
and density limitation of cell proliferation
In vitro exp. – stirred suspension culture or semisolid
media as agar or methocel
Aberrant growth control by serum dependence
Lower serum dependence due to secretion of growth
factors by tumor cells
Allows them to enter into mitotic phases
Cause nontransformed cells to adopt transformed
phenotype and grow in suspension
Produce interleukins 1,2 and 3 along with colony
stimulating factors (CSF)
Hormones released
Aberrant growth control by Oncogenes
Overexpression of oncogenes
Modified receptors erb-B2 oncogene product, modified
G protein and overexpression of genes regulating stages
in signal transduction (src kinase) or transcriptional
control (myc, fos and jun)
It is permanently active and cannot be easily regulated
Tumorigenicity by malignancy
Transformation culminates into neoplastic cells
Malignant tumors – increased growth rate, reduced
anchorage dependence, more pronounced aneuploidy and
immortalization
All cell lineages present within a tumor need not have
same transformed properties
Same properties cannot be expressed in every tumor
Tumorigenicity by malignancy
Cells have developed the capacity to generate invasive
tumors if implanted in vivo into an isologous host or if
transplanted as a xenograft into an immune-deprived
animal.
Tumorigenicity by angiogenesis
Tumor cells release factors – VEGF, EGF-2 and
angiogenin – capable of neovascularization

Tumor cells produce proteolytic enzymes – plasminogen


activator – helps the tumor cells in not attaching to any
surface – help them invading other cells

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