Download Telomere dynamics in cancer progression and prevention

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COMMENTARY
Cells from the telomerase knockout mouse immortalize with an approximately ten million-fold greater
frequency than human cells. In this commentary, Wright and Shay discuss the implications of this difference between
mice and men and its relationship to cancer.
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Telomere dynamics in cancer progression and prevention:
fundamental differences in human
and mouse telomere biology
other stress responses, including genotoxic
There is increasing evidence that telomerase
drugs (bleomycin or etoposide16), overexis an important component of human canWOODRING E. WRIGHT &
HAY
cer, whereas eliminating telomerase in
pression of oncogenes (H-ras V12; ref. 17),
JERRY W. S
mouse has no effects on tumor formation
or checkpoint factors (p16INK4a; ref. 18). If
for many generations. Is this an artifactual paradox or does it M1 represents a DNA damage response due to too-short telomhave important implications for human cancer? In this eres, then any stimulus producing DNA damage might mimic
Commentary, we will reflect on differences between human the senescent phenotype.
replicative aging and the cultured growth arrest of mouse cells,
Tissue culture represents a drastic departure from the in vivo
and how this affects the interpretation of data from the telom- environment and can lead to stress-response pathways that inerase-deficient mouse.
hibit cell division19. The replicative arrest in mouse cultures may
represent a response to the artificial ex vivo environment per se,
Replicative aging in human fibroblasts
from factors such as unrestrained mitogenic stimulation, DNA
Cellular senescence in human fibroblasts1 occurs in two phases, damage, oxidative damage or inadequate nutrient media.
mortality stages 1 and 2 (M1 and M2; ref. 2). M1, corresponding Mouse cells may be able to divide indefinitely in an appropriate
to normal replicative senescence, probably occurs when a few serum-free medium20. The behavior of MEFs deficient in certain
telomeres are sufficiently short that the ends are not fully DNA damage responses (lacking Ku80, the protein that is mumasked from being recognized as requiring double strand break tant in Ataxia Telangiectasis (ATM), or BrCA2, the protein that
repair3. Cell division is then blocked by factors associated with is mutant in many breast cancers) is consistent with this interboth acute and chronic DNA damage checkpoint activities, such pretation. These cells overexpress p53 and p21Cip1 and become
as p53 and p16INK4a/pRB (refs. 4, 5). If these checkpoints are growth-arrested after only 3–4 doublings21–24. Because these mice
blocked by mutations or expression of viral oncogenes, cells di- are viable, it is unlikely that their “premature senescence” reprevide until M2. M2 (crisis) likely represents the consequences of sents any limitation on division within the organism. One exterminally short telomeres, where end-to-end fusions and chro- planation is that the reduced ability to correct the DNA damage
mosome breakage-fusion cycles result in apoptosis. Evidence produced by the culture environment leads to a more rapid actisupporting a direct involvement of telomeres in M1 and M2 in- vation of p53-dependent cell cycle checkpoints. Consistent
cludes prevention of M1 if human Telomerase Reverse with this interpretation, MEFs from mice lacking both ATM and
Transcriptase (hTERT, the catalytic component of human telom- p53 (ref. 25) or deficient in ATM and ARF (ref. 14) continue to
erase) is introduced in fibroblasts before senescence6,7, and the proliferate when explanted into culture. The demonstration
delay of M2 if telomeres are experimentally elongated by non- that the Rb pathway is not involved in the mouse cultured
specific mechanisms8 or eliminated by the introduction of growth arrest13, that telomere shortening is not involved12 (see
telomerase before crisis9–11. Normal human fibroblasts essentially below), and that abrogation of ARF/p53 is sufficient for indefinever spontaneously immortalize in culture because three inde- nite proliferation may explain why mouse cells “immortalize”
pendent mechanisms (the p53-pathway, the p16INK4a/pRB-path- with such a high frequency compared to human cells. We beway and a telomere-shortening pathway) have to be altered to lieve that the term “cellular senescence” is ill-suited to describe
the growth arrest that occurs after 10–15 doublings of mouse fipermit indefinite growth.
broblasts in culture or that is induced by H-ras in both mouse
and human cells17.
“Cultured growth arrest” in mouse fibroblasts
There is no evidence for a similar counting mechanism in the
growth arrest of cultured mouse cells. Like wild-type mouse Telomere hypothesis predicts tumors in mTR–/– mice
cells, telomerase negative mTR–/– mouse embryo fibroblasts Blasco et al. showed normal tumor formation in mTR–/– mouse
(MEFs) stop dividing after only 10–15 doublings, long before ap- cells lacking telomerase activity12. These authors discussed difpreciable telomere erosion occurs12. Nonetheless, there remain ferences between human and mouse cells and clearly stated
parallels to M1 in human cells. In particular, p53 (but not Rb) that their observations might not apply to human cancer.
function is required for the replicative arrest of MEFs13–15. However, in our view, it is important to also highlight how
Biochemical markers associated with human M1 and mouse these results are entirely consistent with the telomere hypothegrowth arrest reflect the accumulation of negative growth regu- sis. The postulated involvement of telomerase in cancer is to
lators (such as p16INK4a, p21Cip1) and factors (such as the senes- overcome the proliferative limits (the number of divisions, not
cence-associated β-galactosidase) induced during stress the rate of cell divisions) imposed by too-short telomeres, and
responses. These so-called senescence markers are very non-spe- thus to permit sufficient divisions to accumulate the (usually)
cific. Similar phenotypes can be mimicked by DNA damage and many mutations needed for malignancy. Telomerase would
NATURE MEDICINE • VOLUME 6 • NUMBER 8 • AUGUST 2000
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COMMENTARY
not be needed in cancers requiring only a few mutations, or in
organisms in which telomere shortening did not limit divisions. Blasco et al. showed that mTR–/– mouse cells “immortalized” (escaped from cultured growth arrest) with a frequency
equivalent to wild-type cells and divided at least 200–300
times12. This established that the growth inhibition after 10–15
doublings in cultured mouse cells is not due to short telomeres
and that mouse cells do not use a telomere-based mechanism
that considerably limits the number of divisions. If the function of telomerase in cancer is to overcome proliferative limits
and if telomere shortening is not used to count mouse cell divisions, then mouse cells lacking telomerase should have a perfectly normal ability to form tumors (see Figure). The results
demonstrate that telomere shortening is not the cause of what
has been termed mouse cellular senescence and that telomerase is not needed for unanticipated biological functions essential for embryonic development or viability. However,
rather than challenging the telomere hypothesis for the involvement of telomerase in human cancer, their observations
are entirely consistent with the model.
A difference between mouse and human crisis
In human fibroblasts, M2 (crisis) produces a barrier to further
proliferation, preventing premalignant cells with mutations
in both the p53 and pRB pathways that have bypassed M1 by
accumulating the additional mutations needed to become malignant. In mTR–/– mice, genomic instability caused by tooshort telomeres and p53 mutations contribute to cancer
formation26, which has been interpreted as an indication that
the same thing might be happening in human cancer.
However, analysis of sixth generation mTR–/– mouse fibroblasts
indicates that they overcome short telomeres with a frequency
so much higher than human cells as to make the comparison
questionable. Fifth generation mTR–/– Ink4a–/– mouse cells have
an appreciably reduced cloning efficiency in a Myc/RAS cotransfection assay and their subcloning efficiency is increased
after restoring telomerase activity by replacing mTR (ref. 27).
This shows that, in the presence of wild-type p53, fifth generation mTR–/– mice had sufficiently short telomeres to produce
a checkpoint growth arrest. However, sixth generation mTR–/–
p53–/–mice had a subcloning efficiency that approaches 100%
(59% compared to the 89% subcloning efficiency after providing mTR and restoring telomerase activity)26. This contrasts
with a frequency of approximately 10-7 of escape from crisis in
telomerase-silent human cells lacking p53 (and pRB) 28.
Human cells can also use non-telomerase based mechanisms
(alternative lengthening of telomeres (ALT), probably using
recombination) to maintain telomeres. The frequency of 10-7
represents the combined frequency of both telomerase-based
and alternative mechanisms for overcoming M2. Thus, there is
a magnitude difference of seven orders in the frequency with
which human and mouse fibroblasts lacking both telomerase
and p53 are able to overcome the consequences of terminally
short telomeres.
Human mammary epithelial cells (HMEC) immortalize with
a frequency that is higher than that for fibroblasts29.
LiFraumeni HMECs that have silenced the p16/pRB pathway30
and are p53+/– immortalize with a frequency of 5 x 10-6 (ref. 31),
whereas normal HMECs in which both pathways are inactivated by viral oncogenes escape crisis with a frequency of
about 10-5 (ref. 29). Thus, even in epithelial cells that have neither p16/pRb nor p53, the frequency of escape from crisis is
850
Telomere Interpretations in Mice and Men
Hypothesis 1
Telomere shortening limits the number of divisions in human cells, and
telomerase bypasses this limit by maintaining telomere length.
Observations
The shortest mouse telomeres are longer than the longest human
telomeres. mTR–/– cells “immortalize” with identical frequencies to
normal mouse cells.
Conclusions
Telomere shortening is not limiting the growth of normal mouse cells
and telomerase should not be needed for mouse tumor formation.
Hypothesis 2
Genomic instability from short telomeres in p53-null cells causes a net
increase in tumor formation.
Observations
increased foci due
frequency of escape
contribution to
to genomic instability
from crisis
tumor formation
mouse
2–3 fold
×
100%
=
2–3 fold
human
2–3 fold
×
10-7
=
2–3 x 10-7
Conclusions
In p53-null human cells, the estimated contribution of genomic
instability to tumor formation is small compared to the proliferative
block due to crisis. Crisis increases tumor formation in mice but
reduces tumor formation in humans.
four to five orders of magnitude greater than in mouse cells
lacking only p53. The quantitative differences in the ability of
human versus mouse cells to escape the consequences of short
telomeres is simply too great to imply that the net effect of crisis is to contribute to, rather than to inhibit, cancer.
Breakage-fusion-bridge cycles or non-disjunction events induced by inadequate telomere maintenance likely contributes
to genomic instability and is important in some aspects of malignant progression even after the reactivation of telomerase.
However, we believe that the ability of human cells to activate
telomerase-independent mechanisms of telomere maintenance
(ALT) is so low that one cannot use the mouse data to infer that
inhibiting telomerase in p53-null human cells might increase
genomic instability sufficiently to easily generate cells that escape crisis using the ATLT pathway26, 32. Recent data showing
that human cancer cells in culture die after the inhibition of
telomerase support our conclusion33, 34.
Evolution of telomere-based counting mechanisms
The mTR–/–mouse results indicate that telomere shortening
does not produce a cultured growth arrest after 10–15 doublings in normal mouse cells and does not restrict the development of mouse tumors12. Because telomere shortening does
not seem to limit cell divisions in all vertebrates, we would like
to consider a possible scenario for the evolution of telomerebased counting mechanisms. Telomeres much longer than the
few hundred base pairs seen in model organisms such as yeast
may have evolved with the expansion of genome sizes for facilitating chromosome alignment during meiosis. The much
longer telomeres in ciliate micronuclei compared to macronuNATURE MEDICINE • VOLUME 6 • NUMBER 8 • AUGUST 2000
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COMMENTARY
clei is consistent with this interpretation. Thus, many species
may have long telomeres for reasons independent of any use
in counting cell divisions. The heterogeneity in telomere sizes
on individual chromosomes might reflect part of the sorting
mechanisms by which homologous pairs of chromosomes are
brought together. The ability to repress telomerase in quiescent cells35 is likely to be an ancient phenomenon used to prevent telomere elongation (to maintain telomere length at the
appropriate size) in post-mitotic or very slowly dividing cells.
In some long-lived vertebrates, the additional ability to repress
telomerase in dividing cells may have evolved as an anticancer
mechanism to limit the proliferative capacity of somatic cells.
Because a limited proliferative capacity also limits the divisions available for maintenance and repair, it might be disadvantageous for short-lived vertebrates to use telomere
shortening as a counting mechanism. Thus, short-lived organisms with anticancer mechanisms (such as accuracy of DNA
replication and repair) sufficient to prevent cancer during
their lifespans of a few months or few years might have a selective advantage if they did not utilize this additional level of
control. Although further evidence is needed, we are prepared
to entertain the possibility that replicative senescence does
not exist in the mouse.
Conclusion
The evidence supporting the relevance of replicative senescence of human cells and telomere biology to human cancer is
now quite strong. The evidence linking replicative senescence
to human aging is controversial and requires additional studies. We believe the evidence against linking the cultured
growth arrest in mouse cells that has been termed “cellular
senescence” to human replicative aging is so strong that data
derived from studies of mouse telomere biology need to be interpreted very carefully.
Acknowledgments
We thank K. Collins, C.J. Sherr and R.A. Weinberg for reviewing the
manuscript. J.W.S. is a senior scholar of the Ellison Medical Foundation.
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Department of Cell Biology
University of Texas Southwestern Medical Center
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Dallas, Texas 75390-9039
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