Lecture 18

Question 1

define terminology: transformation, transfection, transduction, immortalization

Answer 1

transformation (bacterial): uptake of new genetic material
transfection (mammalian): uptake of new genetic material
transduction: uptake of new genetic material using a virus as a vehicle
transformation (mammalian): spontaneous or induced permanent phenotypical change resulting from a heritable change in DNA and gene expression. can be from infection of transforming virus, gene mutations, ionizing radiation, chemical carcinogens
immortalization: infinite lifespan, no contact inhibition, anchorage independent

Question 2

explain the connection between genetic instability, growth control defects, and malignancy

Answer 2

genetic instability is associated with (meaning depends on AND causes) growth control defects and malignancy. genetic instability lends advantage to cells with growth defects because they grow way more (no contact inhibition, density limitation, anchorage dependence). malignancy is associated with invasive tumor growth in vivo.

Question 3

describe assays that can be used to determine a transformed phenotype in cells (slide 6-7)

Answer 3

growth phenotype assays: grow beyond 100 PD, clone in agar/suspension, microscopy, measure saturation density, low serum, growth curve

genetic phenotype assays: sister chromatid exchanges, chromosome content, southern blotting, microarray, FISH chromosome paints, immunostaining

structural phenotype assays: immunostaining to visualize proteins

neoplastic phenotype assays: xenograft on nude mice or SCID mice, VEGF production, protease activity assay, filter well invasion assay

Question 4

describe events that a cell line undergoes from primary to continuous

Answer 4

primary cells are finite, genetically stable lines. they undergo senescence, crisis, and cell death after enough PDs.
senescence: specific genes are expressed, >10 genes, dominant, stops cell cycle progression, negatively regulates telomerase
crisis: telomere length shortening, replicative senescence
cell death: how do overcome crisis? deletion/mutations of senescence genes, oncogene expression, telomerase

cells that survive the crisis are continuous cell lines that are unstable genetically. they have genetic heterogeneity and aneuploidy because of higher spontaneous mutation rate in vitro and no elimination of mutated cells.

Question 5

explain the 5’ end problem and the function of telomerase

Answer 5

5’ end isn’t replicated all the way to the end because of the lack of primer to build on lagging strand. telomeres (protective caps of repetitive DNA sequences) are in place to maintain chromosome integrity, but shorten with each division. Telomerase is a protein with RNA template that extends telomeres to prevent shortening. Telomerase is only present in germ cells and embryonic stem cells

Question 6

describe the relationship between telomere length, senescence, and crisis

Answer 6

telomeres shorten with each replication. when they are critically short this causes senescence and crisis to prevent genetic damage

Question 7

describe cell culture methods used to generate immortalized or transformed cells

Answer 7

1. use cancer cells from a biopsy
2. use embryonic stem cells, have telomerase to prevent senescence
3. use induced pluripotent stem cells made by yamanaka factors which turn on telomerase
4. transfect or infect with viral oncogenes (CLASSIC method). viruses produce proteins that overcome cell cycle arrest (hTRT)

Question 8

list and describe three assays to test for cell tumorigenicity

Answer 8

Determine factors like

1. malignancy/tumor transplantation: implant cells in vivo in immune suppressed host. transformed cells form tumors
2. invasiveness: histological evaluation of tumor. chick heart assay by the tumor spheroid invading the chick heart forming organoids. placing in filter well inserts and monitoring for proteolytic enzyme release (?)
3. angiogenesis: tests if tumor cells release factors that stimulate migration in endothelial cells (VEGF, FGF-2, angiogenin)

Question 9

characteristics of transformed cells

Answer 9

immortalization: all transformed cells are immortal, but not all immortal cells are transformed as they can still have contact inhibition and anchorage dependence
Genetic instability: growth control defect with loss of contact inhibition, density limitations, and anchorage dependence. malignancy associated with growth of invasive tumors in vivo

Question 10

two possible telomere lengthening mechanisms

Answer 10

1. telomerase activation: hTERT (protein) and hTR (RNA) activated and lengthen telomeres
2. alternative lengthening of telomeres (ALT): recombination mediated. allows cancers to grow even if telomerase is inhibited