Transformation and Immortalization Chapter 18

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. Mode of growth – attached or in suspension. The cell line properties should be considered by validating a cell line to know whether it is from a neoplastic component of tumor rather than normal cells. Since all these features are seen in normal cell lines and neoplastic cell lines too therefore one needs to have more criterion to confirm neoplastic cell lines. Aneuploidy, heteroploidy and tumorigenicity are regarded as positive indicators of malignant transformation

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 In microbiology, the term of transformation was used for the first time. It implies a change in phenotype that is dependent on uptake of new genetic material. Transformation can be because of intro. of a plasmid or result of infection too.

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 Transformation is associated with genetic instability and three major classes of phenotypic changes - One or all of these can be expressed in one cell strain. Immortalization can be achieved without grossly aberrant growth control and malignancy which are usually linked with tumorigenic cell line.

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 Cell lines are prone to genetic instability. Instability allows necessary mutations to become continuous and allows deletion or alteration in DNA surveillance genes such as p53 to happen

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 Many cells die but a few survive with an enhanced growth rate and give rise to continuous cell line

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 Telomeric DNA becomes shorter during a finite life span until chromosomal DNA can no longer replicate. It is expressed in germ cell, has moderate activity in stem cells and is absent in somatic cells

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 Increases mutations favorable to immortalization (upregulation of telomerase or downregulation of one of the telomerase inhibitors).

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 Human fibroblasts can also be transformed by using SV40

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 Telomeres play an essential role in chromosome stability and determining cellular life span. The cause of senescence is telomeric shortening, followed by telomeric fusion, formation of dicentric chromosomes and subsequent apoptosis

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 Autonomous growth control by overexpression of oncogenes or by deletions of suppressor genes.

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 Loss may contribute to a decrease in cell-cell and cell-substrate adhesion and to decreased requirement for attachment and spreading for cells to proliferate. Cancer cells have the ability to break loose, travel to other parts of the body and create new which tumors make them lethal, this is called metastasis – in vivo move to other parts of body as they have changes in fibronectin, CAMs, integrins etc

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 Growth factors are known as autocrine growth factors

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

Open book final exam questions for 20 points What is transformation? What is Immortalization? What are the two ways in which any organism can be transformed? What are the three major classes of phenotypic changes that can associate transformation? Is it mandatory for all three phenotypes to be seen in any transformed cell strain?

Open book final exam questions for 20 points What are the different changes that you will see in a tumorigenic vs non-tumorigenic cell strain? What role does senescence genes play in making a cell strain immortal? Do tumorous cells become anchorage independent? If yes, then what changes lead to its free form? How do the newly formed tumorous cells survive?

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