HC7 - Immortalization Flashcards
serial passaging for
determining the maximum number of divisions in vitro
exceeding the number or allowed doublings leads to
cells ceasing to proliferate or enter apoptosis
senescence
inability to re-enter the cell-cycle
scenescent cells remain
metabolically active
appearence senescent cells
fried egg like > large cytoplasm
marker senescent cells
beta-galactosidase
number of replecative doublings depend on
- species from which the cells are
- tissue of origin
- age of the donor organism
embryonic stem cells with proper nutrients are
immortal
immortal
unlimited replicative potential
immortality is a feature of
the cell lineage, not the individual cell
cancer cells resemble
embryonic stem cells (ECS)
HeLa cells (1951)
immortal cancer cells from aggresive cervical adenocarcinoma
tumor growth is not
exponential > 2000 generations in colon carinomas instead of 40
limited division potential can
inhibit tumorigenesis
amount of divisions is limited by (2)
- accumulation of physiological stress
- recording number of cell divisions
in senescence the expression of the following CDK inhibitors is increased
P16^INK4A, p21^cip1, p53 > often mutated in cancers
reduction of culture atmosphere oxygen concentration leads to a
substantial increase in replicative lifespan in vitro
hyperoxia leads to
progressive breakdown of mitochondrial function and accumulation of ROS
physiological stress for cells in culture
culturing cells on plastic and unsuitable medium
senescence of human cells eliminated by
expression of SV40 large T antigen
SV40 T large antigen inactivates
pRb and p53 > control cell survival pathway
Ki67
proliferation marker
triggers for senescence (2)
- hyperoxia
- DNA damage
senescent cells release
pro-inflammatory cytokines > can stimulate tumorigenesis
proliferation of human cells is limited by
telomere length
chromosome
long DNA molecule
chromosomes are linear because of
telomeres
telomeres
protective shields for chromosomal ends
telomeres prevent
end-to-end fusion
hexanucleotide sequence
5’-TTAGGG-3’
size hexanucliotide sequence
5-10.000 bp > shortens each growth-and-division cycle
short telomeres cannot protect ends and leads to
crisis and apoptosis
no protection of ends chromosome can lead to
breakage-fusion-bridge cycle
G-rich strand telomere has a
3’ overhang of telomeric DNA
T-loop
invades double-stranded piece of telomere > 3 stranded DNA = big
D-loop
displacement loop > 3 stranded DNA
T-loops are bound by
shelterin complex
no ends sticking out
protection from degradation
shortening of telomeres > end replication problem because of (2)
- the need for a RNA primer on 3’ lagging strand
- under-replication of parental strand because primer cannot sit precisely at the end of template strand
exonucleases cause
telomere erosion
telomere erosion
50-100 bp lost / division
short telomeres enter
BFB cycle
protection from BFB cycle for cells is to enter crisis to
prevent transformation
cancer cells can escape crisis by
overexpressing telomerase
hTERT
catalytic subunit of telomerase in human cells
telomerase-independent alternative lengthening of telomeres (ALT)
interchromosomal copying mechanism
interchromosomal DNA transfer
uneven crossing over between 2 chromosomes
