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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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This project is funded by a grant awarded under the Presidents Community Based Job Training Grant as
implemented by the U.S. Department of Labors Employment and Training Administration (CB-15-162-06-60).
NCC is an equal opportunity employer and does not discriminate on the following basis:
against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability,
political affiliation or belief; and against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998
(WIA), on the basis of the beneficiarys citizenship/status as a lawfully admitted immigrant authorized to work in
the United States, or his or her participation in any WIA Title I-financially assisted program or activity.
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