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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
Ebooks File WHO Classification of Female Genital Tumours WHO Classification of Tumours World Health Organization Classification of Tumours International Agency For Research On Cancer All Chapters