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Telomere maintenance and cancer? look, no telomerase
Author: A. A. Neumann
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"� 2002 Nature Publishing Group PERSPECTIVES 17. Moyer, A. Psychosocial outcomes of breast-conserving surgery versus mastectomy: a meta-analytic review. Health Psychol. 16, 284?298 (1997). 18. Fallowfield, L. J. et al. Psychological outcomes of different treatment policies in women with early breast cancer outside a clinical trial. BMJ 301, 575?580 (1990). 19. Meyer, T. J. & Mark, M. M. Effects of psychosocial interventions with adult cancer patient: a meta analysis of randomised experiments. Health Psychol. 14, 101?108 (1995). 20. Sheard, T. & Maguire, P. The effect of psychological interventions on anxiety and depression in cancer patients: results of two meta-analyses. Br. J. Cancer 80, 1770?1780 (1999). 21. Fallowfield, L. et al. Positive epoetin alfa effect on quality of life in anemic cancer patients receiving chemotherapy: results from a randomized placebo?controlled trial. Eur. J. Cancer 35, 1461 (1999). 22. Coates, A. & Gebski, V. On the receiving end. VI. Which dimensions of quality-of-life scores carry prognostic information? Cancer Treat. Rev. 22 (Suppl. A), 63?67 (1996). 23. Earlam, S. et al. Relation between tumor size, quality of life, and survival in patients with colorectal liver metastases. J. Clin. Oncol. 14, 171?175 (1996). 24. Maisey, N. R. et al. Baseline quality of life predicts survival in patients with advanced colorectal cancer. Eur. J. Cancer 38, 1351?1357 (2002). 25. Fraser, S. C. A. et al. A daily diary for quality of life measurement in advanced breast cancer trials. Br. J. Cancer 67, 341?346 (1993). 26. Brady, M. J. et al. Reliability and validity of the Functional Assessment of Cancer Therapy-Breast quality-of-life instrument. J. Clin. Oncol. 15, 974?986 (1997). 27. Coster, S. & Fallowfield, L. The impact of endocrine therapy on patients with breast cancer: a review of the literature. Breast 11, 1?12 (2002). 28. Klastersky, J. & Paesmans, M. Response to chemotherapy, quality of life benefits and survival in advanced non-small cell lung cancer: review of literature results. Lung Cancer 34 (Suppl. 4), S95?S101 (2001). 29. Feeny, D. et al. Multi-attribute health-status classification systems: Health Utilities Index. Pharmacoeconomics 7, 490?502 (1995). 30. Rabin, R. & de Charro, F. EQ-5D: a measure of health status from the EuroQol Group. Ann. Med. 33, 337?343 (2001). 31. Carr-Hill, R. Current practice in obtaining the ?Q? in QALYs: a cautionary note. BMJ 303, 699?701 (1991). 32. Gelber, R. D., Goldhirsch, A. & Cole, B. F. Evaluation of effectiveness: Q-TWiST. The International Breast Cancer Study Group. Cancer Treat. Rev. 19 (Suppl. A), 73?84 (1993). 33. Griffin, A. M. et al. On the receiving end. V: Patient perceptions of the side effects of cancer chemotherapy in 1993. Ann. Oncol. 7, 189?195 (1996). 34. Lachaine, J. et al. Cost-effectiveness and quality of life evaluation of ondansetron and metoclopramide for moderately emetogenic chemotherapy regimens in breast cancer. Crit. Rev. Oncol. Hematol. 32, 105?112 (1999). 35. Velikova, G. et al. Automated collection of quality-of-life data: a comparison of paper and computer touch- screen questionnaires. J. Clin. Oncol. 17, 998?1007 (1999). 36. Fayers, P. M. & Machin, D. in Quality of Life: Assessment, Analysis and Interpretation 404 (John Wiley & Sons, Chichester, UK, 2000). Online links DATABASES Cancer.gov: http://www.cancer.gov/cancer_information/ breast cancer | colorectal cancer | lung cancer | melanoma LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ erythropoietin-? FURTHER INFORMATION QOLID.com: http://www.QOLID.com Access to this interactive links box is free online. NATURE REVIEWS | CANCER VOLUME 2 | NOVEMBER 2002 | 879 Telomere maintenance and cancer ? look, no telomerase Axel A. Neumann and Roger R. Reddel OPINION Activation of a telomere maintenance mechanism seems to be indispensable for the immortalization of human cells. Most cancers and cancer cell lines maintain their telomeres via telomerase. In some cancers, however, telomeres are maintained in the absence of telomerase activity by one or more mechanisms that are known as alternative lengthening of telomeres (ALT). Successful telomere-targeted anticancer therapy might therefore require a combination of telomerase and ALT inhibitors, emphasizing the importance of understanding the molecular details of telomere maintenance mechanisms in immortal cells and their repression in normal cells. Telomeres are the ends of linear chromo- somes, and in most eukaryotes they contain tandem arrays of a GT-rich nucleotide repeat sequence ? 5?TTAGGG3? in vertebrates 1,2 . Telomeric DNA and telomere-specific bind- ing proteins (reviewed in REF. 3), together, have an essential role in stabilizing chromo- some ends by forming a cap structure that protects chromosome ends from degradation and terminal fusions. Human telomeres are 5?15 kb long and are predominantly double stranded; however, they end in a 30?200 nucleotide single-stranded GT-rich 3? over- hang 4?6 . This 3? overhang has been shown to form a lariat structure ? referred to as a telomere (T)-loop ? by invading the dou- ble-stranded region of the telomeric DNA 7 (see FIG. 1). It was recognized in the early 1970s that the known DNA polymerases would be unable to replicate the very end of linear DNA molecules 8,9 ? a phenomenon known as the ?end replication problem? 10 . RNA-primed DNA synthesis of the lagging strand results in a terminal gap after degradation of the most distal primer. In addition to this, the single- stranded GT-rich 3? overhang at each telom- ere involves the action of a putative 5??3? exonuclease that degrades the 5? end of the underhanging CA-rich strand 4,5 . Both of these processes lead to shortening of the telomeric DNA template that is available for semiconservative replication in the next round of DNA synthesis. In most normal somatic cells, therefore, telomeres shorten with every cell division 11 . Conversely, for rea- sons described below, prevention of telomere shortening is crucially important for the development of most human cancers. Cancers might use more than one mecha- nism to prevent telomere shortening. What are these mechanisms, and how does their elucidation impact on our understanding of cancer biology and our ability to treat cancer? Cellular immortalization Many cancers contain cells that have an apparently unlimited capacity to proliferate, and acquisition of this property is referred to as immortalization. This contrasts with the finite proliferative capacity of normal somatic cells 12 . The early evidence that immortaliza- tion has a key role in the cancer phenotype came from two main lines of investigation. The first was in vitro/in vivo studies with hamster cells that were treated with chemical carcinogens, in which it was found that immortality was necessary but not sufficient for malignant transformation 13 . The second was from studies of human cells that had been transduced with the simian virus 40 (SV40) early-region genes, which sometimes results in immortalization. It was found that these cells could undergo malignant transfor- mation by an activated RAS oncogene, but only if they were immortalized 14?16 (FIG. 2). At that time, little was known about the molecular events in immortalization. Shortly after the discovery that normal human cells become senescent after a limited number of cell divisions 12 , it was shown that cells infected with the SV40 virus could tem- porarily evade senescence, but that the cul- ture soon entered a state that is referred to as ?crisis? in which the population stopped expanding 17 . It was subsequently shown that this effect of the SV40 virus could be repro- duced by transfecting cells with a plasmid that contains the viral early region 18 and that encodes at least two oncoproteins, which include the SV40 large T antigen that binds to the protein products of the TP53 and RB genes (reviewed in REF. 19). Sometimes, a cell expressing the SV40 early-region genes can bypass or escape from crisis, but this occurs in only 1 in 10 7 cells 20,21 . Although this is a � 2002 Nature Publishing Group 880 | NOVEMBER 2002 | VOLUME 2 www.nature.com/reviews/cancer PERSPECTIVES ative cell lines was much greater than in their pre-crisis counterparts and was greater than in normal cells (FIG. 3), indicating that the telomeres had undergone extensive lengthen- ing 32 . The increased telomere length was maintained over hundreds of population doublings in the persistent absence of detectable telomerase activity 32 . After technical reasons for this apparent lack of telomerase activity were excluded, it was deduced that there must be at least one non-telomerase mechanism of telomere length maintenance 32 ; these mechanisms were dubbed alternative lengthening of telomeres (ALT) 33 . Definitive evidence that telomerase was not involved in ALT was pro- vided by a study showing that some ALT cells lack expression of a subunit of telomerase (the RNA template, TERC), which is essential for telomerase activity 34 . Mouse cells that are null for the telomerase RNA gene, Terc,are also able to undergo immortalization 35 . Although ALT was defined as any telomerase- independent mechanism of telomere length maintenance, so far there is no clear evidence for the existence of more than one ALT mech- anism in human cells. ALT and cancer The original evidence for ALT came from studies of cell lines that were immortalized in vitro by SV40 and other means. Its rele- vance to cancer was indicated by the obser- vation that ALT cell lines that have been transduced with an activated RAS oncogene are tumorigenic 36,37 . The presence of ALT in human cancer samples showed most clearly that it is not an artefact of cell culture 38 .Of 57 human tumours, four were telomerase negative and had a significantly increased mean telomere length; the telomere length was also extremely heterogeneous, which is characteristic of ALT cells. Similarly, 4 out of 56 tumour cell lines had ALT. So, in this lim- ited survey, 8 out of 113 (7%) tumours and tumour cell lines had ALT 38 , compared with 35% of cell lines that were immortalized in vitro 39 . The reason for this discrepancy might be that most of the in vitro immortal- ized cell lines that were examined were of fibroblast (that is, mesenchymal) origin, whereas most human cancers are carcino- mas (that is, of epithelial origin; many nor- mal epithelia have low levels of telomerase activity). In support of this explanation, preliminary data indicate that sarcomas (which are of mesenchymal origin) are more likely to use ALT than are carcino- mas 38 , perhaps because carcinomas are derived from cells that have fewer barriers to upregulation of telomerase activity. cells proliferate 11,24,25 . The second was the dis- covery of the telomerase enzyme, which adds telomeric DNA onto the chromosomes of protozoa 26 . The telomeres of the SV40-trans- formed cells at crisis were even shorter than at senescence, but their length stabilized post- crisis, after telomerase activity commenced 23 . It was subsequently shown that transducing SV40-transformed cells with the catalytic sub- unit of telomerase (TERT) ? which switches on telomerase activity ? allows them to bypass crisis completely 27,28 and to be trans- formed by an activated RAS oncogene 29 . These observations showed that expression of telom- erase is a key event in the immortalization of SV40-transformed cells. Alternative lengthening of telomeres However, some SV40-immortalized cells have been found to be telomerase negative 30?32 . The mean telomere length of telomerase-neg- rare event, it is much less rare than sponta- neous immortalization, which is estimated to occur in less than 1 in 10 12 human cells 22 . It therefore seems clear that expression of the SV40 genes results in increased probabil- ity of a change that is responsible for immortalization. Telomerase and immortalization The nature of the change that occurs at immortalization was elucidated in a study showing that SV40-transformed cells expressed the enzyme telomerase on emer- gence from crisis, but not beforehand 23 . This key observation resulted from the convergence of two lines of investigation. The first was the development of the telomere hypothesis of senescence ? namely, that senescence is trig- gered when telomeres become short, and the experimental verification of the prediction that telomeres shorten progressively as normal RAD50 5? 3? 3? 5? TRF2 TRF2 TRF1 TRF1 TRF1 TRF1 TRF1 TRF1 TRF1 TRF2 TRF2 TRF2 TRF2 TANK1 TANK2 RAP1 hnRNPs A2-B1 E KU KU D A1 TIN2 NBS1 MRE11 T-loop D-loop Figure 1 | Telomeric DNA and protein assembly. Schematic representation of putative telomere T-loop structure with telomere-specific binding proteins. The single-stranded DNA at the end of the telomere is able to invade and anneal with part of the duplex DNA (thereby forming a displacement (D)-loop) in the same telomere, with the overall result being a telomere (T)-loop 7 . Several proteins bind specifically to telomeric DNA, and these recruit other proteins to the chromosome end. Activated RAS oncogene Malignant transformation Activation of TMM Loss of tumour suppressors Senescence Crisis Unlimited proliferation Normal Cell Figure 2 | Senescence and immortalization. Normal somatic cells permanently exit from the cell cycle (that is, become senescent) after a limited number of cell divisions. Cells might escape temporarily from the senescence barrier if they lose the function of key tumour-suppressor genes, especially TP53 and/or RB, but most will eventually die, at which stage the cell population is described as being in ?crisis?. Cells might bypass crisis and become immortalized (capable of unlimited proliferation) if a telomere maintenance mechanism ? telomerase or ALT ? is activated. Immortalized cells, but not their mortal predecessors, might be susceptible to malignant transformation by an activated oncogene such as RAS. � 2002 Nature Publishing Group PERSPECTIVES telomerase null by targeting the telomerase RNA gene, it was found that telomeric short- ening occurred and the cells eventually died. Survivors were found to maintain their telomeres by a mechanism that was depen- dent on RAD52, which encodes a protein that is involved in recombination 54 . In the period before activation of a survivor path- way, proliferation was enhanced if the DNA mismatch-repair pathway was defective 55 . A genome-wide survey of genes with altered expression after deletion of telomerase activ- ity identified several genes that remain upregulated in survivors, including the puta- tive meiotic recombination/DNA-repair gene MSC1 (REF. 56). An extensive survey of the published litera- ture on telomerase activity in human cancer concluded that 85% of all cancers are telom- erase positive 40 . It cannot be assumed, how- ever, that the remaining 15% must use an ALT mechanism: as argued in more detail else- where 41 , some malignancies (solid tumours and leukaemias) might not need any telomere maintenance mechanism. It is possible that solid tumours and leukaemias that have a rela- tively small number of crucial genetic changes, and/or a low cell turnover, can become clini- cally significant without immortalization 41 .It is important to determine how many of the telomerase-negative tumours use ALT. To complicate the situation further, how- ever, it seems that some tumours use both telomere maintenance mechanisms 38 . It is not yet known whether ALT and telomerase are sometimes co-expressed spontaneously within individual tumour cells, although cell-culture experiments in which telomerase is artificially switched on in ALT cells have shown, in princi- ple, that this is possible 42?44 and that telomerase activity can sometimes repress or mask the ALT phenotype 45 . One ALT cell line was more likely to become tumorigenic if transduced with expression constructs for both activated RAS and TERT than if transduced with RAS alone 46 . However, it is not yet known whether ALT and telomerase are sometimes co- expressed spontaneously within tumour cells. Another possible explanation for the presence of both telomere maintenance mechanisms within a tumour is intratumoral heterogeneity. In SV40-transformed fibrob- lasts, the probability that telomerase or ALT will become activated to allow escape from crisis is quite similar (ALT is activated in at least one-third of SV40-immortalized fibrob- last lines) 32 . If this is the case for some types of tumour cell, then it would not be surprising if ALT and telomerase are sometimes turned on in separate areas of the same tumour. Implications for cancer treatment When telomerase activity was downregulated in telomerase-positive cancer cells by expres- sion of a dominant-negative TERT mutant, the cells became apoptotic 47,48 . This showed, in principle, that telomerase inhibitors could be very useful for treating cancer. Telomerase inhibitors will not be useful, however, for the minority of tumours that use ALT. In addi- tion, in telomerase-positive tumours it would be predicted that effective telomerase inhibitors will exert a very strong selection pressure for the emergence of resistant cells that use the ALT mechanism. Activation of ALT was not observed in cell-culture experi- ments in which telomerase-positive cell lines were treated with small-molecule inhibitors of telomerase or dominant-negative TERT mutants 47?50 , indicating that it is not a high frequency event. This might be a problem, however, in clinically significant tumours con- taining as many as 10 12 cells. It might, there- fore, be necessary to develop ALT inhibitors. For tumours that use both telomere mainte- nance mechanisms, treatment might need to be initiated with a combination of telomerase and ALT inhibitors. Both telomerase and ALT must access the telomere, but how this might be achieved is, at present, unknown. It would however, be surprising, if the sets of proteins that are involved do not at least partly overlap, so it might be possible to identify molecular targets for simultaneous inhibition of both telomere maintenance mechanisms. It is expected that telomerase inhibitors will be developed that have far fewer side effects than many of the cancer chemothera- peutic agents that are available at present. The inherited syndrome dyskeratosis congenita (DKC) is caused by a mutation in one of the components of telomerase, so individuals with DKC are deficient for telomerase activity (reviewed in REF. 51). The features of DKC include abnormalities of the skin and nails, and eventual failure of proliferation in the bone marrow, which indicates that telomerase is required for normal proliferative capacity in these somatic tissues. Despite this telomerase deficiency, onset of pancytopaenia in these individuals does not occur until a median age of 10 years 52 , which indicates that it might be relatively safe to administer telomerase inhibitors continuously for several years. The potential toxicity of ALT inhibitors cannot be predicted without knowing the role of ALT in normal cells. It is important to develop an assay that can determine whether an ALT-like mechanism might be active during meiosis, or might modulate telomere length in somatic cells in one or more tissue compart- ments. Interestingly, there is preliminary evi- dence indicating that an ALT-like activity might be responsible for lengthening telomeres of lymphocytes in telomerase-null mice 53 . Although this is an entirely speculative idea at present, inherited defects in the putative nor- mal counterpart of ALT might also contribute to features of premature ageing, which is anal- ogous to telomerase deficiency in DKC. Lessons from yeast A rational approach to inhibiting ALT requires an understanding of the mechanism. As is often the case in contemporary life sci- ences research, studies of yeast genetics have pointed the way. When budding yeast ? Saccharomyces cerevisiae ? was made NATURE REVIEWS | CANCER VOLUME 2 | NOVEMBER 2002 | 881 ALT Telomerase Population doublings 70 72 75 76 77 78 80 106 113 123 142 48 10 8 a b kb Figure 3 | Telomere-length phenotype of ALT cells. a | Telomere-specific fluorescence in situ hybridization (FISH) on metaphase chromosomes of ALT and telomerase-positive cells, illustrating the highly heterogeneous telomere lengths within individual ALT cells (yellow, telomere-specific probe; blue, DAPI-stained metaphase chromosomes). b | Terminal restriction fragment (TRF) length analysis of an ALT cell line before crisis (at population doubling (PD) 76) and after crisis (at PD 77), showing the temporal correlation between immortalization and occurrence of the ALT characteristic telomere-length pattern. Reproduced with permission from REF. 37 � American Association of Cancer Research. � 2002 Nature Publishing Group 882 | NOVEMBER 2002 | VOLUME 2 www.nature.com/reviews/cancer PERSPECTIVES a polymerase-chain-reaction-based method was used to sequence the subtelomeric region of specific chromosome ends before and after immortalization of ALT cells 69 . Immediately proximal to the telomere, there is a region of the chromosome that contains a few TTAGGG repeats that are admixed with vari- ant repeats such as TGAGGG, TTGGGG and TCAGGG. According to the model, if severe shortening of the chromosome end occurs before crisis, then all of the telomere and part of the subtelomeric region might be lost; after ALT is activated, annealing of a residual sub- telomeric TTAGGG repeat with the comple- mentary sequence of another telomere that still retains some telomeric sequence will allow the synthesis of new telomeric sequence. The net result would be replace- ment of variant repeats with (TTAGGG) n sequence; this predicted outcome was observed 69 . synthesized sequence can then be converted to double-stranded DNA, resulting in a net increase of telomeric DNA. This is obviously very different from reciprocal recombina- tion, which results in exchange between chromosomes, but no net increase in total genetic material. Further indirect evidence that the ALT mechanism might involve recombination came from the finding that some PML bodies in the nuclei of ALT cells are unusual 37,66 .PML bodies are found in the nuclei of most normal cells, and are domains that contain accumula- tions of proteins that are involved in a wide variety of functions (reviewed in REF. 67). In every ALT cell line that has been examined so far, a proportion of the cells have PML bodies that contain telomeric DNA (at least some of which is extrachromosomal), telomeric bind- ing proteins, and proteins that are involved in DNA replication and recombination (FIG. 6). PML bodies with these contents have not been found in telomerase-positive or mortal cells, so they are referred to as ALT-associated PML bodies (APBs) 37 . Although the function of APBs is unknown at present, the presence of recombination proteins within them ? including RAD52, RAD51 and RAD50 ? confirmed the importance of testing the recombinational model of ALT. To test the proposed model, a DNA tag was inserted into telomeres of ALT and telomerase-positive cells 68 . According to the ALT model, when a tagged telomere is used by another telomere as a copy template and when the point of strand invasion is proximal (centromeric) to the tag site, the tag will be copied to the other telomere. In clonal popu- lations of ALT, but not telomerase-positive, cells it was found that the number of tagged telomeres increased during cellular prolifera- tion 68 . This proposed mechanism was also supported by the findings of a study in which Further analyses identified two classes of telomerase-null survivors 57 . Type I survivors undergo amplification of a subtelomeric sequence named Y?, and type II survivors have telomeres with lengths that are very het- erogeneous, but are increased overall (FIG. 4). Telomeres of the type II cells therefore resem- ble those of human ALT cells. At present, it is unknown whether any human cancers use an ALT mechanism that is analogous to that of type I yeast survivors. However, the chromo- some ends of telomerase-null mouse embry- onic stem cells that underwent spontaneous immortalization in culture contained an amplified non-telomeric sequence 58 . The type I and type II mechanisms are both dependent on RAD52, but other gene requirements vary. Type I requires RAD51, RAD54 and RAD57, whereas type II requires RAD50, RAD59 and SGS1 (REFS 59?64).The protein products of all of these RAD genes are involved in recombination. Sgs1 is a helicase of the RecQ class and has five human homo- logues, three of which ? WRN, BLM and RECQL4 ? are known to be mutated in familial syndromes with features of prema- ture ageing and increased cancer incidence (Werner, Bloom, and Rothmund?Thomson syndromes, respectively). Recombination Adding to the genetic evidence implicating recombination in the survival of telomerase- null yeast cells, Murnane and colleagues found striking increases and decreases in telomere length in a telomerase-negative human cell line that they attributed to telomeric recombination events 30 .A recom- bination-based model for ALT was proposed 65 in which one DNA strand of a short telomere anneals to the complemen- tary strand of another telomere and acts as a primer for DNA synthesis (FIG. 5a). The newly Telomere repeat shortening Type I survivors Type II survivors Subtelomeric Y? repeat element Telomeric G-rich repeat ( ) n Figure 4 | Telomerase-null Saccharomyces cerevisiae type I and type II survivors. Schematic representation of the two telomere phenotypes in telomerase-deficient yeast (S. cerevisiae) survivors. Shortening of telomeric G-rich repeats results in cell death. Rare survivors either show amplification and dispersal of the subtelomeric Y? repeat elements (blue rectangles; type I survivors) or show elongation of the terminal G-rich sequence (red triangles; type II survivors) as a result of recombination. Adapted from REF. 83. a Inter-telomeric b Intra-telomeric d Extrachromosomal (linear) c Extrachromosomal (circle) Figure 5 | Recombination-mediated telomere lengthening in ALT cells. It is proposed that a terminal telomeric DNA strand within an ALT cell can invade other telomeric DNA and anneal to the complementary strand. Synthesis of new telomeric DNA sequence can be primed on the template to which the invading strand is annealed, and this can be converted to double-stranded DNA (not shown). Potential templates include another telomere (a), the proximal (that is the centromeric) region of the same telomere via T-looping (b), and extrachromosomal telomeric DNA that is either circular (c) or linear (d). � 2002 Nature Publishing Group PERSPECTIVES Telomerase and ALT: interchangeable? There is a sense in which the telomere maintenance mechanisms are equivalent: immortalization can be associated with the activation of either telomerase or ALT, and fully malignant tumours can use either mechanism. It seems inadvisable, however, to assume that they are completely equivalent, especially if telomerase does have other func- tions in the cancer cell. Also, telomeres that are maintained by telomerase or ALT might not be functionally equivalent. Despite their long mean telomere length, most ALT cells contain some very short telomeres; these might be more prone to end-to-end fusion events than telomerase-maintained telom- eres and so might be a source of genetic instability 82 . One of the questions that needs to be addressed is whether tumours of the same stage and grade that use either ALT or telomerase have the same prognosis. In addi- tion, the response of such tumours to exist- ing therapies needs to be examined: are there existing cancer chemotherapeutic agents to which tumours using one or other telomere maintenance mechanism are particularly sensitive or resistant? If yes, there might be some tumour types for which it will be nec- essary to stratify clinical trials according to telomere maintenance mechanism. It will also be important to determine whether an individual patient?s tumour has telomerase and/or ALT activity ? or neither ? when telomere maintenance inhibitors become available for cancer therapy. Axel A. Neumann and Roger R. Reddel are at the Cancer Research Unit, Children?s Medical Research Institute, 214 Hawkesbury Road, Westmead, Sydney, New South Wales 2145, Australia. Correspondence to R.R.R. e-mail: rreddel@cmri.usyd.edu.au doi: 10.1038/nrc929 1. Moyzis, R. K. et al. A highly conserved repetitive DNA sequence, (TTAGGG) n , present at the telomeres of human chromosomes. Proc. Natl Acad. Sci. USA 85, 6622?6626 (1988). 2. Blackburn, E. H. Structure and function of telomeres. Nature 350, 569?573 (1991). 3. de Lange, T. Protection of mammalian telomeres. Oncogene 21, 532?540 (2002). 4. Wellinger, R. J., Ethier, K., Labrecque, P. & Zakian, V. A. Evidence for a new step in telomere maintenance. Cell 85, 423?433 (1996). 5. Makarov, V. L., Hirose, Y. & Langmore, J. P. Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening. Cell 88, 657?666 (1997). 6. Wright, W. E., Tesmer, V. M., Huffman, K. E., Levene, S. D. & Shay, J. W. Normal human chromosomes have long G- rich telomeric overhangs at one end. Genes Dev. 11, 2801?2809 (1997). 7. Griffith, J. D. et al. Mammalian telomeres end in a large duplex loop. Cell 97, 503?514 (1999). 8. Olovnikov, A. M. [Principle of marginotomy in template synthesis of polynucleotides]. Doklady Akademii. Nauk. SSR 201, 1496?1499 (1971). 9. Watson, J. D. Origin of concatemeric T7 DNA. Nature New Biol. 239, 197?201 (1972). 10. Levy, M. Z., Allsopp, R. C., Futcher, A. B., Greider, C. W. & Harley, C. B. Telomere end-replication problem and cell aging. J. Mol. Biol. 225, 951?960 (1992). Although these results strongly support a recombinational mechanism for ALT, they do not exclude the possibility that the telomeres might sometimes use copy tem- plates other than another telomere. It is not known whether telomeres in ALT cells form the T-loops that are observed in telom- erase-positive cells (FIG. 1), but if they do it seems possible that a telomere could use itself as a copy template (FIG. 5b). They could also use extrachromosomal telomeric DNA ? circular or linear ? as a copy template (FIG. 5c,d). A rolling circle can template an essentially unlimited amount of lengthen- ing, and there is evidence that telomerase- negative yeast can use such a mechanism of telomere elongation 70 . Human ALT cells are known to contain extrachromosomal telomeric DNA 37,71,72 , as are telomerase- negative insect cells 73 . Repression of ALT in normal cells ALT-mediated lengthening of telomeres depends on the availability of copy tem- plates (TTAGGG repeats, which are present in every telomere) and proteins that are involved in recombination and DNA syn- thesis. All of these are present in normal cells, so an obvious question is why the telomeres of normal somatic cells undergo shortening. Presumably, normal cells have a mechanism for repressing telomere length maintenance by ALT. The existence of such a mechanism was shown by fusing normal cells with ALT cells and showing that the ALT mechanism was repressed in the hybrids 74 . Some telomerase-positive cells also contain repressors of ALT 44,74,75 .An understanding of how this repression is achieved might also identify useful molecu- lar targets for cancer treatment. Why is telomerase needed at all? The recombinational and DNA synthesis machinery is much more ancient than telomerase. Telomerase is unique to eukary- otes, but conserved recombination proteins are found in bacteria and more primitive organisms. For example, human RAD51 has structural and functional homology to the bacterial RecA protein 76 . Another obvious question that therefore arises is why telom- erase is needed at all. There are a few eukaryotes that do not have telomerase, and maintain their telomeres, instead, by recom- bination (such as mosquitoes and midges 77,78 ) or retrotransposition (such as Drosophila and related Dipterans 79,80 ), but the overwhelming majority of eukaryotes do have telomerase. Maybe telomerase is a more readily controlled mechanism for telomere maintenance than ALT. A recent Opinion article 81 reviewed accumulating data from Terc-null mice and suggested that telomerase might have functions in addition to telomere maintenance that could provide a selective advantage to organisms (and cancers) that express this enzyme. NATURE REVIEWS | CANCER VOLUME 2 | NOVEMBER 2002 | 883 ALT cells TRF2 PML DAPI Merge Telomerase-positive cells TRF2 PML DAPI Merge Figure 6 | ALT-associated PML bodies. ALT cell lines and tumours contain PML bodies (multiprotein complexes that are found in the nuclei of most normal cells) that have some unusual contents: telomeric DNA and specific telomere-binding proteins, including TRF2. PML protein is found in all PML bodies. ALT- associated PML bodies (APBs; yellow merge) are visualized here by co-localization of PML (red immunostaining) and TRF2 (green). APBs are seen in ALT cells, but not in normal or telomerase-positive cells, and are therefore a useful marker for ALT. With appropriate validation, it is possible that this technique can be used to detect ALT in tumour specimens that have been stored as paraffin blocks, which is how most tumour samples are archived by pathologists. � 2002 Nature Publishing Group 884 | NOVEMBER 2002 | VOLUME 2 www.nature.com/reviews/cancer PERSPECTIVES 65. Reddel, R. R., Bryan, T. M. & Murnane, J. P. Immortalized cells with no detectable telomerase activity. A review. Biochemistry 62, 1254?1262 (1997). 66. 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