transformation and immortalization

8/14/2019 Transformation and Immortalization

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Transformation and

ImmortalizationChapter 18


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Role in cell line characterization/How

will you characterize a cell line? Transformationis 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 cellsand

not normal blood vessel cells or inflammatorycells.


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What is Transformation? Implies a spontaneous or induced permanent

phenotypic changeresulting from a heritable change in

DNA and gene expression

Artificial introduction of DNA or DNA transfer into

mammalian cellsTransfection

Transformation

result of infection with virus or

transfection with mutant ras genes or spontaneous

exposure to ionizing radiation or chemical carcinogens


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Genetic Instability Human finite cell lines are stable

Mouse cell lines are genetically unstable andtransform easily

Continuous cell lines (tumorigenic) of all speciesare unstable

Two causes of genetic heterogeneity

- Spontaneous mutation is higher in vitro- Mutant cells cannot be eliminated without

impairment in their growth capacity


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Chromosomal Aberrations Genetic rearrangementseen in chromosome

counts and karyotype analysis

Also determined by sister chromatid exchangeassay

Variations in ploidy, frequency of individualchromosomal aberrationsand variations inchromosome numbertumorigenic cell lines


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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 toremain at confluence for any length of time, theyremain sensitive to contact inhibition and densitylimitation of growth

If allowed to remain at confluence for extended periodsreduced contact inhibition- cells pile upovergrowth-will be seen in subsequent subcultures - tumorigenic


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Control of Senescence Senescence genes in finite cellsnegatively

control cell cycle progression

Regulates expression of telomerase synthesizes

telomeric DNA

Deletions and/or mutationswithin senescence

genes or overexpression or mutationof one or

more oncogenes allows reexpression oftelomerase


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Control of Senescence Immortalization involves both inactivation of

senescenceand cell cycle regulatory genes suchas Rb and p53

Product of SV40 LT gene

T antigen binds to Rband p53

Allows extended proliferative life span andrestricts DNA surveillance activity of genes

- Increases genomic instability

- Increases chances of mutations


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Control of Senescence Immortalizationdoes not imply development of

aberrant growth control and malignancy

- Retains contact inhibition of cell motility, densitylimitation of cell proliferation and anchorage

dependence and are non-tumorigenic

- Some aspects of growth control are abnormaland likelyincrease of genomic instability


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Immortalization with Viral genes SV 40 virusimmortalize 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


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Immortalization with Viral genes Mammalian cells transfected or retrovirally infected with

immortalizing genebefore they enter senescence

Extends proliferative life span for 20-30 population

doublings- Cells cease proliferation and enter crisis

- Up to several monthsin crisis subset of immortal

cells overgrows

- Fractionof immortal cells obtained


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Telomerase-Induced Immortalization Telomerase or terminal transferasecomposed of two

subunitsRNA 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 malignantlytransformed


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Aberrant growth control Tumorigenic cell lines exhibit

- Lower serum or growth factor dependence

- Form cloneswith higher efficiency

- Acquire autonomous growthcontrol

- Autocrine growthcontrol secrete mitogens orreceptors


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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

todisorganized growth pattern, loss of contact

inhibition of cell motility and density limitation of

cell proliferation In vitro exp.stirred suspension culture or

semisolid mediaas agar or methocel


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Aberrant growth control by serum

dependence Lower serum dependence due to secretion of

growth factorsby 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 3along with

colony stimulating factors (CSF)

Hormonesreleased


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Aberrant growth control by Oncogenes Overexpression ofoncogenes

Modified receptors erb-B2 oncogeneproduct,

modified G proteinandoverexpression of genes

regulating stagesin signal transduction (src

kinase) or transcriptional control (myc, fosand

jun)

It is permanently active and cannot be easilyregulated


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Tumorigenicity by malignancy Transformation culminates into neoplastic cells

Malignant tumorsincreased 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 everytumor


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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 xenograftinto an immune-deprived animal.


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Tumorigenicity by angiogenesis Tumor cells release factors VEGF, EGF-2 and

angiogenincapable 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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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 inany transformed cell strain?


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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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