Transcript Biology of Cancer - Tunghai University
Chapter 10 Eternal Life: Cell Immortalization and Tumorigenesis
-10.1 ~ 10.9 May 1, 8 & 15, 2007
10.1 Normal cell populations register the number of cell generations separating them from their ancestors in the early embryo
- Normal cells have a limited proliferative potential.
- Cancer cells need to gain the ability to proliferate indefinitely – immortal.
- The immortality is a critical component of the neoplastic growth program.
Figure 10.2
The Biology of Cancer
(© Garland Science 2007) Escape from senescence and become immortal or even further transformed to malignant cells.
“Hayflick limit” of fibroblasts in the culture
Replicative senescence
in vitro
senescence associated acidic β-galactosidase
proliferating human senescent cells in culture fibroblasts
- “fried egg” morphology - remain metabolically active, but lost the ability to re-enter into the active cell cycle - the downstream signaling pathways seem to be inactivated Figure 10.3
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senescence associated β-galactosidase (lysosomal β-D galactosidase)
proliferating cells senescent cells Figure 10.3
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Young and old keratinocytes in the skin
Figure 10.4b
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(© Garland Science 2007) Keratinocyte stem cells in the skin lose proliferative capacity with increasing age.
Cancer cells and embryonic stem cells share some replicative properties
- Embryonic stem (ES) cells show unlimited replicative potential in culture and are thus immortal.
- The replicative behavior of cancer cells resembles that of ES cells.
- Many types of cancer cells seem able to proliferate forever when provided with proper
in vitro
culture conditions.
- HeLa cells ( He nrietta La cks, 1951): - the 1 st human cell line and 1 st established in culture human cancer cell line - derived from the tissue of cervical adenocarcinoma
10.2 & 3 Cells need to become immortal in order to form cancers
Two regulatory mechanisms to govern the replicative capacity of cells: 1.
senescence
- Cumulative physiologic stress over extended periods of time halts further proliferation. These cells enter into a state of
senescence
.
cumulation of oxidative damage contributes to senescence, e.g., r eactive o xygen s pecies (ROS), DNA damage 2.
crisis
- Cells have used up the allowed “quota” of replicative doublings. These cells enter into a state of
crisis
, which leads to apoptosis.
Cell populations in crisis show widespread apoptosis
Figure 10.10
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(© Garland Science 2007)
Cell senescence does occur
in vivo
senescence-associated β-galactosidase (SA-β-gal) Treatment of lung cancer with chemotherapeutic drugs appear to induce senescence in tumor cells Figure 10.9c
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10.4 The proliferation of cultured cells is limited by the telomeres of their chromosomes
Barbara McClintoch discovered (1941) specialized structures at the ends of chromosomes, the
telomeres
, that protected chromosomes from end-to-end fusions.
(She also demonstrated movable genetic elements in the corn genome, later called
transposons
.) - Nobel prize in Physiology & Medicine in 1983
Telomeres detected by f luorescence
i n s itu
h ybridization (FISH)
telomeric DNA
In an extreme form, all the chromosomes of the cell fused into one giant chromosome.
Figure 10.11b
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(© Garland Science 2007) The telomeres lose their protective function in cells that have been deprived of TRF2, a key protein in maintaining normal telomere structure.
Mechanisms of breakage-fusion-bridge cycles
2 sister chromatids during the G2 phase of the cell cycle Figure 10.14a
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Figure 10.14b, c
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(© Garland Science 2007) truncation translocation aneuploidy
Telomeric DNA shortens progressively as cells divide
Telomeres lose 50 to 100 bp of DNA during each cell generation.
Figure 10.13b
The Biology of Cancer
(© Garland Science 2007)
telomere shortening chromosomes fuse apoptotic death
The length of telomeric DNA in cells (Southern blotting analysis)
Figure 10.13a
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10.5 Telomeres are complex molecular structures that are not easily replicated
Telomeric DNA: 5’-TTAGGG-3’ hexanucleotide sequence, tandemly repeated thousands of times Figure 10.16
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Telomeric DNAs
_____________________________________________________ Telomeric repeat Organism sequence _____________________________________________________ Yeasts
Saccharomyces cerevisiae
G 1-3 T
Schizosaccharomyces pombe
G 2-5 TTAC Protozoans
Tetrahymena
GGGGTT
Dictyostelium
G 1-8 A Plant
Arabidopsis
GGGTTTA Mammal Human GGGTTA ______________________________________________
Structure of the T-loop
Figure 10.17
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(© Garland Science 2007)
Multiple telomere-specific proteins bound to telomeric DNA
Figure 10.19
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Pot1 binds the single- and double-stranded telomeric DNA in order to stabilize its structure s.s. telomeric DNA
Figure 10.18
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Primers and the initiation of DNA synthesis
Primers and the initiation of DNA synthesis
this sequence is not replicated
10.6 Incipient cancer cells can escape crisis by expressing telomerase
Telomerase activity (elongate telomeric DNA) is clearly detectable in 85 to 90% of human tumor cell samples, while being present at very low levels in most types of normal human cells.
telomerase holoenzyme: 1. hTERT 2. hTR catalytic subunit RNA subunit ( At least 8 other subunits may exist in the holoenzyme but have not been characterized.)
h
uman
t
elomerase associated
R
NA (template for hTERT)
h
uman
t
e
lomerase
r
everse
t
ranscriptase Figure 10.23a
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(© Garland Science 2007)
The sequence of the catalytic subunit of telomerase is homologous to various reverse transcriptases ciliates: yeasts:
Figure 10.22a
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The TRAP assay (
t
elomeric
r
epeat
a
mplification
p
rotocol) a synthetic primer + dNTP (in cell lysates) Figure 10.21a
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The products of the TRAP reaction are analyzed by gel electrophoresis
heat treatment of cell lysates (inactivating telomerase activity)
Activation of telomerase activity following escape from crisis
Figure 10.24
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Sidebar 10.5 Oncoproteins and tumor suppressor proteins play critical roles in governing
hTERT
expression
- The mechanisms that lead to the de-repression of
hTERT
transcription during tumor progression in humans are complex and still quite obscure.
-
Multiple transcription factors appear to collaborate to activate the
hTERT
promoter.
- For example, the Myc protein (Section 8.9) and Menin (the product of the
MEN1
tumor suppressor gene), deregulate the cell clock.
Prevention of crisis by expression of telomerase
Figure 10.25
The Biology of Cancer
(© Garland Science 2007) HEK:
h
uman
e
mbryonic
k
idney cells
Sidebar 10.6 The role of telomeres in replicative senescence
- In cultured human fibroblasts, senescence can be postponed by expressing hTERT prior to the expected time for entering replicative senescence. - However, senescence is also observed in cells that still possess quite long telomeres.
Why?
Possible explanations: - When cells encounter cell-physiologic stress or the stress of tissue culture, telomeric DNA loses many of the single-stranded overhangs at the ends. The resulting degraded telomeric ends may release a DNA damage signal, thereby provoking a p53-mediated halt in cell proliferation that is manifested as the senescent growth state
Replicative senescence and the actions of telomerase
This is a still-speculative mechanistic model of how and why telomerase expression can prevent human cells from entering into replicative senescence.
Figure 10.26
The Biology of Cancer
(© Garland Science 2007)
10.7 Telomerase plays a key role in the proli feration of human cancer cells
- Expression of antisense RNA in the telomerase (+) HeLa cells causes them stop growing 23 to 26 days.
- Expression of the dn (
d
ominant
n
egative) hTERT subunit in telomerase (+) human tumor cell lines also causes them to lose all detectable telomerase activity and, with some delay, to enter crisis.
Suppression of telomerase results in the loss of the neoplastic growth in 4 different human cancer cell lines
(length of telomeric DNA at the onset of the experiment) Figure 10.27
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Telomerase activity and the prognosis of pediatric tumors
Figure 4.11
Figure 10.28
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10.8 Some immortalized cells can maintain telomeres without telomerase
- 85 to 90% of human tumors have been found to be telomerase-positive.
- The remaining 10 to 15% lack detectable telomerase activity, yet these cells need to maintain their telomeres above some minimum length in order to proliferate indefinitely. - These cells obtain the ability to maintain their telomeric DNA using a mechanism that does not depend on the actions of telomerase.
- In the yeast
Saccharomyces cervisiae
, following inactivation of genes encoding subunits of the telomerase holoenzyme, the vast majority of the cells enter a state of crisis and die.
- Rare variants emerged from these populations of dying cells that used the
a
lternative
l
engthening of
t
elomerase ( ALT ) mechanism to construct and maintain their telomeres.
- This ALT mechanism is also used by the minority of human tumor cells that lack significant telomerase activity, e.g., 50% osteosarcomas and soft-tissue sarcomas and many glioblastomas.
The ALT (
a
lternative
l
engthening of
t
elomerase mechanism (or copy-choice mechanism) )
Figure 10.30
The Biology of Cancer
(© Garland Science 2007)
Exchange of sequence information between the telomeres of different chromosomes
neomycin resistant gene was introduced into the midst of the telomeric DNA Figure 10.29
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10.9 Telomeres play different roles in the cells of laboratory mice and in human cells
- Rodent cells, especially those of the laboratory mouse strains, express significant levels of telomerase throughout life .
- The double-stranded region of mouse telomeric DNA is as much as 30 to 40 kb long (~ 5 times longer than corresponding human telomeric DNA).
- Therefore, laboratory mice do not rely on telomere length to limit the replicative capacity of their normal cell lineages and that telomere erosion cannot serve as a mechanism for constraining tumor development in these rodents.
Sidebar 10.8 Long telomeres (in mice) do not suffice for tumor formation
- Transgenic mice expressing mTERT (
m
ouse homolog of
te
lomerase tumorigenesis even though the mouse cells in which this enzyme acts already possess very long (>30 kb) telomeres.
r
everse
t
ranscriptase) contributes to - Thus, the mTERT enzyme aids tumorigenesis through mechanisms other than simple telomere extension.
- Mouse cells can be immortalized relatively easily following extended propagation in culture. - Human cells require, instead, the introduction of both the SV40
large T
oncogene (to avoid senescence) and the
hTERT
gene (to avoid crisis).
SV40 and T antigens
- If the SV40 large T oncoprotein is expressed in human fibroblasts, these cells will continue to replicate another 10 to 20 cell generations and then enter crisis.
- On rare occasion, a small propotion of cells (1 out of 10 6 immortalized.
cells) will proceed to proliferate and continue to do indefinitely → becoming
SV40: the 40 th
s
imian
v
irus in a series of isolates papovavirus: papilloma, polyoma & vacuolating agent Table 3.1
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SV40 virus
formation of large cytoplasmic vacuoles prior to the death of the cell and the release of progeny virus particles - vacuolating agent Figure 3.10
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(© Garland Science 2007) SV40 launches a lytic cycle in monkey kidney cells (permissive host)
3.6 DNA Tumor virus genomes persist in virus transformed cells by becoming part of host cell DNA
double-stranded, circular DNA of SV40 (5 kb) Cell integration of SV40 genome into host cell DNA transformation and the by SV40 depend on the continued presence of T (tumor-associated) antigens .
Figure 3.11 & 18
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(© Garland Science 2007) - large T , middle T & small T Ags
SV40 large T antigen can circumvent senescence
HEK:
h
uman
e
mbryonic
k
idney cells Figure 10.8
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(© Garland Science 2007)
HEK 293
- HEK 293 cells were generated by transformation of normal
h
uman
e
mbryonic
k
idney (HEK) cells with adenovirus 5 DNA in the late 1977.
- HEK 293T cell line contains, in addition, the SV40 large T antigen, that allows for episomal replication of transfected plasmids containing the SV40 origin of replication. This allows for amplification of transfected plasmids and expression of the desired gene products.
mTR -/-
mice exhibit no telomerase activity in any of their cells. They are indistinguishable phenotypically from wild-type mice for at least 3 generations.
showing premature aging : - wasting away of muscle tissues & a hunched back The progeny in the 6th generation show a diminished capacity to heal wounds, to respond properly to mitogenic signals, and suffer from substantially reduced fertility and tissue atrophy (loss of cells).
Figure 10.31
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