Terry Fox Laboratory, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada, V5Z 1L3; and Department of Medicine, University of British Columbia,Vancouver, British Columbia, Canada, V6T 2B5
THE observation that almost all malignant cancers
have telomerase activity has been explained by the
assumption that telomerase is crucial for the progression of malignancy. Surprisingly, cells from mice without a functional telomerase RNA gene and no telomerase activity are indistinguishable from normal cells in different transformation and immortalization assays. However, detailed analysis of chromosomes in telomerase null cells has
revealed multiple defects that point to the role of telomerase in normal biology and raise well-defined questions
for future research.
Telomere Biology
Telomeres are specialized structures at the end of chromosomes composed of DNA and proteins that are essential
for maintaining the stability of eukaryotic genomes (for
review see references 4, 34). The major functions of telomeres are (a) to protect chromosome ends from recombination, fusion, and degradation (cap function), (b) to position and move chromosomes during various stages of the
meiotic and mitotic cell cycle (anchor function), and (c) to
counter telomere erosion resulting from incomplete replication of chromosome ends (end-replication "buffer" function). The latter is essential because conventional DNA
polymerases are unidirectional and cannot copy all bases
at the 3 Murine Models
The putative tumor-suppressor function and cell senescence function of telomeres have long been challenged by
observations in the mouse (22; for review see reference
21). On average, telomeres are 5-10 times longer in murine cells than in human cells, and yet murine cells show
much more rapid senescence in culture. Nevertheless, like
in human cells, measurable telomerase activity is upregulated in mouse tumors (3, 6, 7, 9), suggesting a role for telomerase in murine tumor formation after all. These and
other conflicting data have added to a large degree of confusion regarding the role of telomeres and telomerase in cellular senescence and tumor formation in mouse and
man. In view of this situation, data from mice without telomerase were eagerly awaited by investigators both inside
and outside the telomere field. Such data have now been
obtained by Blasco et al. (5), who developed mice in which
the telomerase RNA template gene has been removed
from the germ line using standard gene knock-out (KO)1
techniques. The results obtained with (the cells from) the telomerase KO mice contain valuable lessons for anyone interested in telomere biology.
The Hidden Phenotype of Telomerase KO Mice
As expected, no telomerase could be detected in cells derived from homozygous KO animals, supporting the notion of a single telomerase RNA gene in the murine genome. The fact that the KO mice were born alive and
apparently normal was the first surprise, since it indicates
that telomerase is not essential for maintaining telomeres
in somatic (stem) cells of renewing tissues such as skin, gut,
and blood during development and normal steady-state tissue homeostasis. It has been suggested that cellular defects may be demonstrated upon challenges to such tissues or
with aging of the mice (24), but details of such defects are
currently not known. If the cells of self-renewing tissues
are affected, it will be important to distinguish between
the absence of telomerase and a decreased proliferative
potential (resulting from overall shorter telomeres; see below) as primary defects. The observation that the telomerase null mice were fertile (resulting in multiple subsequent generations of KO animals) was the second surprise, initially suggesting that telomerase was not necessary to
maintain telomeres in the germ line. Furthermore, cells from
telomerase-deficient mice were as efficiently transformed
into immortal and in vivo tumor-forming cells as cells from
telomerase-positive animals, demonstrating conclusively
that, in the mouse, telomerase is not an essential requirement for the establishment of cell lines, oncogenic transformation, or tumor formation. The surprises did not end
there. All of the above could still be explained by assuming that telomere shortening was perhaps occurring at a
very slow rate in cells known to have very long initial telomeres (22), perhaps together with alternative pathways for
telomere maintenance in immortal tumor cells (see below). The studies by Blasco et al. (5) have revealed that the
situation is more complex. Continued inbreeding has produced up to six generations of KO animals. Although conventional telomere length analysis by Southern essentially failed to detect telomere shortening, clear differences in telomere length between cells from subsequent generations of
KO animals were observed using quantitative fluorescence
in situ hybridization (Q-FISH) (25, 36). The Q-FISH observations on the ends of individual chromosomes in telomerase KO cells has provided some answers to several of
the long-standing questions about telomeres in the mouse
and provide a focus for future studies. Such observations are summarized below.
Germ Cells Need Telomerase to Maintain
Telomere Length
The first lesson from Q-FISH is that telomerase is essential to maintain telomere length in the germ line. Without
telomerase, telomere repeats are lost at a variable rate of
2-7 kb per generation of mice. Assuming a loss of 75-150
or 100 bp/cell division (1, 16, 29), this translates into 20-70
cell divisions from germ cell to germ cell in subsequent
generations of animals. It has been estimated that sperm
cells undergo an average of 62 cell divisions from the zygote, whereas from zygote to oocyte takes on average only
25 divisions (14). The agreement between experimental data and theoretical predictions is striking and strongly
supports the original suggestion that telomere shortening
in somatic cells results from the absence of telomerase in
such cells (10).
Uncapped Chromosomes?
The next surprise is related to telomeres apparently lacking TTAGGG repeats altogether. Chromosomes without
detectable TTAGGG on at least one end were observed at
increased frequencies from generation 2 and higher (several per metaphase spread in later generations). That such
ends are unstable is demonstrated by the increasing frequency of aneuploid cells and end-to-end associations of
chromosomes with each subsequent generation of the KO
mice. This observation provides direct and formal proof
that chromosome ends without TTAGGG are unstable and
predisposed to chromosomal abnormalities, as was predicted (18; for review see reference 12). More surprising is
that such uncapped ends are so readily observed. Using
plasmids with TTAGGG inserts of variable size, the sensitivity of Q-FISH for the detection of TTAGGG repeats has been determined to be in the order of a few hundred
base pairs or less (36). If sensitivity is similar for chromosomes in metaphase spreads, ends without detectable
Q-FISH signals may contain less than a few hundred base
pairs of TTAGGG. What is the history and what are the
implications of such apparently uncapped chromosome ends? Did or do such ends signal a prolonged but transient
cell cycle arrest as in yeast (see below), or are they at least
temporarily ignored, perhaps similarly to uncapped ends
of Drosophila chromosomes (27)? If loss of TTAGGG by
itself is not sufficient to make chromosomes become fusogenic and/or recombinogenic, what other factors are involved in this transition?
Uncapped Chromosomes: Are Adaptive
Responses Involved?
It has been shown that a single break at the end of a yeast
chromosome will trigger a RAD9-mediated cell cycle arrest
(30). Interestingly, in that study many of the yeast cells recovered from the initial arrest without repairing the damaged chromosome and resumed cell divisions. Although
uncapped chromosomes in yeast were destined for eventual loss, they were apparently no longer recognized by the
RAD9 pathway, or this pathway was shut off. A similar adaptation of signal transduction systems may apply to cells
of telomerase KO mice. It is possible that cells without TTAGGG on one or more chromosomes, perhaps also after
a transient cell cycle arrest, adapted to signaling by uncapped
chromosomes, and this possibility warrants further investigation. In any event, it seems that murine cells with one or
more uncapped chromosomes can divide for many times
with or without an initial arrest. Indeed, normal development and even fertility does not seem to be immediately affected by the presence of such uncapped chromosomes.
Telomere Elongation by Recombination?
Conceivably, chromosomes without TTAGGG repeats could
also be "healed" by recombinational repair. Based on studies of yeast (for review see reference 35), many alternative
scenarios for recombinational telomere repair are possible. Uncapped chromosomes in yeast can acquire a new
telomere by homologous recombination, both reciprocal
and nonreciprocal, via RAD52-mediated gene conversion (32). Nonreciprocal recombination or gene conversion between telomeres on homologous or nonhomologous chromosomes (as depicted in Fig. 1) could occur in mammalian
cells as well. Indeed, the overall long telomeres in the
mouse could favor the use of such a nonreciprocal recombination: a critically short telomere in a murine cell could
perhaps easily recombine with remaining long telomeres
on other chromosomes. As the overall telomere length decreases in subsequent generations of the telomerase KO
animals, the efficiency of this pathways could decrease, possibly with the increased frequencies of chromosomal abnormalities seen at later generations as a result.
Chromosomal Abnormalities in Telomerase KO Cells
The presence of the Robertsonian fusions observed in
later generations of the telomerase KO mice deserves special mention (Fig. 2). Given that telomeres on the short P
arm of acrocentric murine chromosomes are, in general, significantly shorter than q arm telomeres (36), a gradual loss
of telomere repeats would be expected to predispose to this
type of chromosomal abnormality. Are the Robertsonian
chromosomes in the telomerase KO mouse stable? If the
primary fusion event was indeed between two different uncapped chromosomes, the prediction would be that they
are not, as such chromosomes are expected to have two
functional centromeres (23). Further studies of these Robertsonian chromosomes should reveal whether they represent
unique cytogenetic abnormalities in telomerase KO mice.
Implications for Models of Tumor Growth and
Telomerase Inhibition Therapy
The observation that telomerase appears completely dispensable for tumorigenesis in the mouse should be discussed in relation to models of tumor cell proliferation and
the possible use of telomerase inhibitors in cancer therapy
(17). Approximately a quarter of immortalized human cell
lines lack detectable telomerase activity (for review see
reference 8), indicating that alternatives to telomerase for
the maintenance of telomeres also exist in human cells. Furthermore, evidence is accumulating that in different somatic cell types, including tumor cells, telomerase may be
best correlated with proliferation rate as telomeres continue to shorten in many telomerase-positive cells (reviewed in 2). Apparently, measurable enzyme activity is
frequently not associated with elongation or static maintenance of telomere length, perhaps because telomeres are
inaccessible to telomerase in most somatic and tumor cells. The observations with the telomerase KO mice clearly
show that telomere shortening, lack of telomerase, and
even uncapped chromosomes are not incompatible with
continued and extensive proliferation. However, caution
in the extrapolation of the murine data to the human situation is warranted, in view of the well-known observation that human cells are less efficiently immortalized than murine cells. Could less efficient adaptive responses and/or
recombination (as shown in Fig. 1) explain the differences
in immortalization rates between the species? The observation that cells from M. spretus (with telomeres of comparable length to human cells) will spontaneously, be it less
efficiently, immortalize in culture (29) indicates that overall telomere length cannot be a major factor predisposing
cells towards spontaneous immortalization. Differences in
the signaling and/or processing of critically short telomeres between murine and human cells could conceivably
make continued proliferation in human cells more dependent on telomerase. In general, many questions about the
role of telomerase and recombination pathways in the proliferation, senescence, and immortalization of normal and
malignant cells from different murine and human tissues
remain. For this reason caution in extrapolating findings with murine cells too directly to human cells remains warranted. However, with the current rate of progress, answers to many of these questions should be available in the
near future.
end of a linear duplex (33), resulting in the slow
loss of genetic material from the ends of chromosomes with
each replication round. In all vertebrates, including humans, the most terminal DNA consists of extended (up to
100 kb) arrays of TTAGGG repeats (26, 28), and the end-replication "buffer" function is accomplished in two fundamentally different ways. In cells of the germ line and
most immortal cell lines, repeats are added to the 3
end
by telomerase, a multimeric enzyme with reverse transcriptase activity and an RNA template encoding for terminal repeats (for review see reference 15). In most if not
all normal somatic cells, telomerase is either not expressed
or not capable of extending chromosome ends, and as a result, telomere repeats are lost with each replication round
(10, 13, 16, 19). A minimum telomere length appears to be
required to maintain the structural integrity of the chromosomes. Shortening beyond this point has been implicated in replicative senescence of cells (for review see reference 17), and (re)activation of functional telomerase has
been proposed as an important step in the development of
tumors (11, 20; for review see reference 31). In this scenario, the loss of telomere repeats with each replication
round represents a mechanism to suppress the uncontrolled proliferation of premalignant cells in long-lived species.
Fig. 1.
Pathways to maintain telomere length. Telomerase, a
reverse transcriptase, can extend the 3 end of telomeres using an
RNA component complimentary to the G-rich repeats of the 3
strand. Are recombination pathways important to maintain telomere length in telomerase KO mice? Shown is nonreciprocal recombination proposed for telomere elongation in yeast (32).
Continued shortening of telomeres and abundant chromosome
ends without detectable telomere repeats in cells from telomerase KO mice suggest that such recombination pathways are
either unable to prevent overall telomere loss or are not active at
all. Each box represents a repeat unit. Hatched boxes represent
newly synthesized repeats. The figure was adapted from reference 32.
[View Larger Version of this Image (19K GIF file)]
Fig. 2.
Chromosomal abnormalities in embryonic fibroblasts derived from the
sixth generation of telomerase
null mice. Results were obtained by Q-FISH using peptide nucleic acid (PNA) probes
as described (25, 36). Yellow
and orange represent telomere signals obtained with
Cy3-labeled (CCCTAA)3
PNA, and blue represents
DAPI-stained chromosomal
DNA. Pseudocolors were assigned using Adobe Photoshop software (San Jose, CA).
Asterisks indicate metacentric
Robertsonian fusion products between acrocentric chromosomes. Number signs indicate chromosome arms without
detectable TTAGGG repeats.
[View Larger Version of this Image (39K GIF file)]
Received for publication 22 August 1997 and in revised form 19 September 1997.
1. Abbreviations used in this paper: KO, knock-out; Q-FISH, quantitative fluorescence in situ hybridization.1. | Allsopp, R.C., H. Vaziri, C. Patterson, S. Goldstein, E.V. Younglai, A.B. Futcher, C.W. Greider, and C.B. Harley. 1992. Telomere length predicts replicative capacity of human fibroblasts. Proc. Natl. Acad. Sci. USA. 89: 10114-10118 [Abstract]. |
2. | Autexier, C., and C.W. Greider. 1996. Telomerase and cancer: revisiting the telomere hypothesis. Trends. Biochem. Sci. 21: 387-391 |
3. | Bednarek, A., I. Budunova, T. Slaga, and C.M. Aldez. 1995. Increased telomerase activity in mouse skin premalignant progression. Cancer Res. 55: 4566-4569 [Abstract]. |
4. | Blackburn, E.H.. 1994. Telomeres: no end in sight. Cell. 77: 621-623 |
5. | Blasco, M.A., H.-W. Lee, M.P. Hande, E. Samper, P.M. Lansdorp, R.A. DePinho, and C.W. Greider. 1997. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell. 91: 25-34 |
6. | Blasco, M.A., M. Rizen, C.W. Greider, and D. Hanahan. 1996. Differential regulation of telomerase activity and telomerase RNA during multi-stage tumorigenesis. Nat. Genet. 12: 200-204 |
7. | Broccoli, D., L.A. Godley, L.A. Donehower, H.E. Varmus, and T. de Lange. 1996. Telomerase activation in mouse mammary tumors: lack of detectable telomere shortening and evidence for regulation of telomerase RNA with cell proliferation. Mol. Cell Biol. 16: 3765-3772 [Abstract]. |
8. | Bryan, T.M., and R.R. Reddel. 1997. Telomere dynamics and telomerase activity in in vitro immortalised human cells. Eur. J. Cancer. 33: 767-773 |
9. | Chadeneau, C., P. Siegel, C.B. Harley, W.J. Muller, and S. Bacchetti. 1995. Telomerase activity in normal and malignant murine tissues. Oncogene. 11: 893-898 |
10. | Cooke, H.J., and B.A. Smith. 1986. Variability at the telomeres of the human X/Y pseudoautosomal region. Cold Spring Harbor Symp. Quant. Biol. 51: 213-219 |
11. | Counter, C.M., H.W. Hirte, S. Bacchetti, and C.B. Harley. 1994. Telomerase activity in human ovarian carcinoma. Proc. Natl. Acad. Sci. USA. 91: 2900-2904 [Abstract]. |
12. | de Lange, T. 1995. Telomere dynamics and genome instability in human cancer. In Telomeres. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 265-293. |
13. | de Lange, T., L. Shiue, R. Myers, D.R. Cox, S.L. Naylor, A.M. Killery, and H.E. Varmus. 1990. Structure and variability of human chromosome ends. Mol. Cell Biol. 10: 518-527 |
14. | Drost, J.B., and W.R. Lee. 1995. Biological basis of germline mutation: comparison of spontaneous germline mutation rates in Drosophila, mouse and human. Environ. Mol. Mutagen. 25: 48-64 |
15. | Greider, C.W.. 1996. Telomere length regulation. Annu. Rev. Biochem. 65: 337-365 |
16. | Harley, C.B., A.B. Futcher, and C.W. Greider. 1990. Telomeres shorten during ageing of human fibroblasts. Nature (Lond.). 345: 458-460 |
17. | Harley, C.B., N.W. Kim, K.R. Prowse, S.L. Weinrich, K.S. Hirsch, M.D. West, S. Bacchetti, H.W. Hirte, C.M. Counter, C.W. Greider, et al . 1994. Telomerase, cell immortality, and cancer. Cold Spring Harbor Symp. Quant. Biol. 59: 307-315 |
18. | Hastie, N.D., and R.C. Allshire. 1989. Human telomeres: fusion and interstitial sites. Trends Genet. 5: 326-330 |
19. | Hastie, N.D., M. Dempster, M.G. Dunlop, A.M. Thompson, D.K. Green, and R.C. Allshire. 1990. Telomere reduction in human colorectal carcinoma and with ageing. Nature (Lond.). 346: 866-868 |
20. | Kim, N.W., M.A. Piatyszek, K.R. Prowse, C.B. Harley, M.D. West, P.L.C. Ho, G.M. Coviello, W.E. Wright, S.L. Weinrich, and J.W. Shay. 1994. Specific association of human telomerase activity with immortal cells and cancer. Science (Wash. DC). 266: 2011-2015 |
21. | Kipling, D.. 1997. Telomere structure and telomerase expression during mouse development and tumorigenesis. Eur. J. Cancer. 33: 792-800 |
22. | Kipling, D., and H.J. Cooke. 1990. Hypervariable ultra-long telomeres in mice. Nature (Lond.). 347: 347-402 . |
23. | Kipling, D., H.E. Wilson, A.R. Mitchell, B.A. Taylor, and H.J. Cooke. 1994. Mouse centromere mapping using oligonucleotide probes that detect variants of the minor satellite. Chromosoma (Berl.). 103: 46-55 |
24. | Kolberg, R.. 1997. No neat endings yet in the tale of telomerase KO mice. J. NIH Res. 9: 24-26 . |
25. |
Lansdorp, P.M.,
N.P. Verwoerd,
F.M. van de Rijke,
V. Dragowska,
M.-T. Little,
R.W. Dirks,
A.K. Raap, and
H.J. Tanke.
1996.
Heterogeneity in
telomere length of human chromosomes.
Hum. Mol. Genet.
5:
685-691
|
26. | Lejnine, S., V.L. Makarov, and J.P. Langmore. 1995. Conserved nucleoprotein structure at the ends of vertebrate and invertebrate chromosomes. Proc. Natl. Acad. Sci. USA. 92: 2393-2397 [Abstract]. |
27. | Mason, J.M., and H. Biessmann. 1995. The unusual telomeres of Drosophila. Trends Genet. 11: 58-62 |
28. | Moyzis, R.K., J.M. Buckingham, L.S. Cram, M. Dani, L.L. Deaven, M.D. Jones, J. Meyne, R.L. Ratliff, and J.-R. Wu. 1988. A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proc. Natl. Acad. Sci. USA. 85: 6622-6626 [Abstract]. |
29. | Prowse, K.R., and C.W. Greider. 1995. Developmental and tissue-specific regulation of mouse telomerase and telomere length. Proc. Natl. Acad. Sci. USA. 92: 4818-4822 [Abstract]. |
30. | Sandell, L.L., and V.A. Zakian. 1993. Loss of a yeast telomere: arrest, recovery and chromosome loss. Cell 75: 729-739 |
31. | Shay, J.W., and W.E. Wright. 1996. Telomerase activity in human cancer. Curr. Opin. Oncol. 8: 66-71 |
32. | Wang, S.-S., and V.A. Zakian. 1990. Telomere-telomere recombination provides an express pathway for telomere acquisition. Nature (Lond.). 345: 456-458 |
33. | Watson, J.D.. 1972. Origin of concatameric T4 DNA. Nature New Biol. 239: 197-201 |
34. | Zakian, V.A.. 1995. Telomeres: beginning to understand the end. Science (Wash. DC). 270: 1601-1607 [Abstract]. |
35. | Zakian, V.A.. 1996. Telomere functions: lessons from yeast. Trends Cell Biol. 6: 29-33 . |
36. |
Zijlmans, J.M.,
U.M. Martens,
S.S. Poon,
A.K. Raap,
H.J. Tanke,
R.K. Ward, and
P.M. Lansdorp.
1997.
Telomeres in the mouse have large inter-chromosomal variations in the number of T2AG3 repeats.
Proc. Natl.
Acad. Sci. USA.
94:
7423-7428
|