1. Transformation and Immortalization
Chapter 18

2. 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

3. 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.

4. 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.

5. 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

6. 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

7. 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

8. 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

9. 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).

10. 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

11. 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

12. Immortalization with Viral genes
Mammalian cells transfected or retrovirally infected with immortalizing gene before they enter senescence
Extends proliferative life span for 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

13. 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

14. 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.

15. 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

16. 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

17. 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

18. 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

19. 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.

20. 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

21. 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?

22. 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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