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Alternative lengthening of telomeres, telomerase, and cancer
Cancer Letters 194 (2003) 155–162 www.elsevier.com/locate/canlet
Alternative lengthening of telomeres, telomerase, and cancer Roger R. Reddel* Children’s Medical Research Institute, 214 Hawkesbury Road, Westmead, Sydney, NSW 2145, Australia Received 6 September 2002; received in revised form 30 October 2002; accepted 30 October 2002
Abstract Telomere length may be maintained in cancer cells by telomerase or an alternative lengthening of telomeres (ALT) mechanism. Low levels of telomerase activity have been detected in some normal somatic cells and presumably some types of normal cells also have low levels of an ALT-like activity. It is hypothesized here that inherited abnormalities of these and other aspects of telomere maintenance may contribute to cancer and ageing. The telomere length maintenance mechanisms are similar in that activation of each is associated with immortalization. They may also confer other properties on cancer cells, however, and the nature of these additional properties may be different for telomerase and ALT. It is expected that these similarities and differences will have implications for prognosis and treatment. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Telomerase; hTERT; Cancer; Alternative lengthening of telomeres; Senescence; Immortalization; Cancer; Ageing; Down syndrome; Werner syndrome; Ataxia telangiectasia; Dyskeratosis congenita
1. Introduction Telomeres are tracts of repetitive DNA at each chromosome end. In normal human cells, the telomeres contain up to 15 kb of tandem repeats of the hexanucleotide, TTAGGG [1], and this amount decreases by an average of 50– 150 base pairs per cell cycle [2]. It is thought that this telomere shortening process ultimately limits the number of times a cell can divide [3], and acts as a powerful tumor suppressor mechanism. The great majority of cancers escape from the limitations on proliferation imposed by normal telomere shortening via activation of a telomere length maintenance mechanism (TMM) (reviewed in Ref. [4]). In most cancers, * Tel.: þ61-2-9687-2800; fax: þ 61-2-9687-2120. E-mail address: [email protected] (R.R. Reddel).
telomere length is maintained by an enzyme, telomerase, that synthesizes telomere repeat sequences to replace those that are lost during DNA replication [5]. Some cancers that are telomerase-negative maintain the length of their telomeres by one or more mechanisms referred to as alternative lengthening of telomeres (ALT) [6]. Similarly, cells that become immortalized in vitro escape from the normal limitations on proliferative capacity by activating telomerase [7] or ALT [8] (Fig. 1). An understanding of the roles of the TMMs in cancer depends on an understanding of their roles in normal telomere biology. It seems likely that there will be both similarities and differences between the roles of telomerase and ALT. This review contains a summary of some existing information about telomerase and ALT and speculation regarding the
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. doi: 1 0 . 1 0 1 6 / S 0 3 0 4 - 3 8 3 5 ( 0 2 ) 0 0 7 0 2 - 4
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Fig. 1. Telomere length maintenance and immortalization. When the telomere lengths of proliferating normal cells become marginal, the cells undergo senescence, which includes permanent withdrawal from the cell cycle. This barrier to proliferation may be breached temporarily by loss of function of key tumor suppressor genes, but the telomeres eventually become critically short and the cells enter crisis. Escape from these limitations on proliferation may be provided by activation of a TMM, telomerase or ALT (reviewed in Ref. [4]). Telomerase activation often results in stably short telomeres, or in telomere lengthening and stabilization in the normal length range. ALT results in a large increase in average telomere length, but there are very short and very long telomeres present within individual cells.
similarities and differences in their contributions to the cancer cell phenotype.
2. Telomere length maintenance mechanisms in cancer Telomerase [9] is a ribonucleoprotein complex that includes an RNA template molecule (usually referred to as TR or TER; encoded by the TERC gene) and a catalytic subunit (TERT) that has reverse transcriptase activity (reviewed in Ref. [10]). Other subunits include a protein, dyskerin, that binds to TER [11]. By reverse transcribing its RNA template moiety, telomerase synthesizes telomeric DNA to compensate for the loss of telomeric sequences during DNA replication [10]. A survey of published data on telomerase activity in a wide variety of human cancers concluded that approximately 85% of all cancers are telomerase-positive [5].
Some telomerase-negative tumors maintain the length of their telomeres by ALT [6]. The available evidence regarding the mechanism of ALT is consistent with a model in which a DNA strand from one telomere of an ALT cell anneals with the complementary strand of another telomere, thereby priming synthesis of new telomeric DNA using the complementary strand as a copy template [12] (Fig. 2A). Prior to crisis, some of the chromosome ends shorten so much that all of the telomere repeats are lost together with some of the subtelomeric region. This region contains a mixture of variant repeat sequences and some TTAGGG sequences. When ALT is activated residual TTAGGG repeats in the subtelomeric region may prime the addition of new telomeric DNA, with the net result being that the most distal variant subtelomeric repeats are replaced by TTAGGG [13] (Fig. 2B). The types of tumors and tumor cell lines in which ALT has been observed include osteosarcoma, soft
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Fig. 2. Recombination-mediated lengthening of telomeres in ALT cells. Available data are consistent with a mechanism in which telomeres use other telomeres (or extrachromosomal telomeric DNA) as a copy template. (A) One strand of a shortened telomere may invade another telomere and anneal with the complementary strand. This acts as a primer for extension of the invading strand using the invaded telomere as a copy template for DNA synthesis, after which the other strand can be filled in. Evidence for this mechanism was provided by inserting a DNA tag into telomeres of ALT cells and showing that the tag was copied on to other telomeres [12]. (B) If telomere shortening extends into the subtelomeric region that contains variant repeat sequences, recombination-mediated lengthening would be predicted to result in replacement of distal variant repeats by TTAGGG repeats. This prediction was confirmed by sequencing subtelomeric regions of cells before and after activation of ALT and escape from crisis [13].
tissue sarcoma, glioblastoma multiforme, renal cell carcinoma, adrenocortical carcinoma, breast carcinoma, non-small cell carcinoma of the lung, and ovarian carcinoma [6,14,15]; J. Henson and R. Reddel, unpublished data). Based on limited information, it appears that ALT is more often present in cell lines and tumors of mesenchymal origin than in those of epithelial origin; possible reasons for this have been discussed recently [16]. More extensive surveys need to be done to identify the tumor types in which ALT is most common. The following evidence indicates that some tumors possess both ALT and telomerase activity [6]. ALT cells have a telomere length phenotype characterized by extreme heterogeneity (i.e. the telomere lengths
range from very short to extremely long). Some tumors have both telomerase activity and the characteristic telomere length pattern [6]. It is not yet known whether this is due to intratumoral heterogeneity with some areas of the tumor being telomerase-positive and other areas having ALT activity, or whether these TMMs coexist within the same tumor cells. In vitro experiments have shown that the latter is possible in principle [17 – 20]. It has been observed in one case that when a treated telomerase-positive brain tumor recurred, the regrowth was telomerase-negative and had telomere lengths indicative of ALT (J. Royds, personal communication); although there are several possible explanations, the simplest would be that the original
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Table 1 Telomere-related abnormalities that could potentially contribute to oncogenesis Known or putative telomere abnormality
Known or hypothetical mechanism(s)
Predicted outcomes
Ia. Increased length maintenance: prevention of shortening
Activation of telomerase and/or ALT
Immortalization, cancer
Ib. Increased length maintenance: decreased shortening
Overactivity of telomerase or putative ALT-like activity in normal tissues Decreased nuclease access and/or activity
Clonal expansion of cells that acquire an oncogenic mutation, cancer
II. Accelerated shorteninga
Excessive nuclease access and/or activity Excessive recombination-mediated loss Inadequate telomerase or ALT-like activity, e.g. during development
Eventual failure of proliferation, especially in highly proliferative tissues; features of premature ageing; overgrowth of clones with oncogenic mutations
III. Abnormal repair eventsa e.g. end-to-end fusions
Excessive shortening Abnormal access of DNA repair complexes Other failure of ‘cap’ function
Genomic instability
a Mutations that result in a combination of II and III would be expected to result in syndromes with premature ageing and increased cancer susceptibility.
tumor was heterogeneous with regard to TMM and that it was a portion of the tumor with ALT activity that survived treatment and regrew.
3. Telomere maintenance in normal cells Although the telomeres of normal somatic cells undergo progressive shortening, their integrity is maintained in several ways. The telomeric DNA sequence is recognized by specific binding proteins, these proteins interact with others, and together they form a putative cap structure that protects the chromosome end from degradation or from attempted DNA repair. The telomeres of normal and telomerasepositive cells form a loop structure [21] in which the chromosome end is effectively hidden. It has been proposed that ‘uncapping’ results in the telomere being detected by the cell as a DNA break, and that the probability of uncapping increases as the telomere shortens [22]. The concentration and activity of telomere binding factors may also influence the probability that uncapping will occur [23]. The evidence for this concept is currently indirect as there are no molecular markers of an uncapped telomere. Some normal cells have detectable levels of
telomerase activity. It is possible that this partially compensates for normal proliferation-associated telomere attrition, thus increasing the proliferative capacity of specific cell populations in various tissues that require a high cell turnover. There may even be occasions in which telomere lengthening occurs in normal cells. For example, the telomere length of Blymphocytes appears to increase during their transit through the germinal center of the lymph node [24]. Nevertheless, the overall long-term trend in all tissues that have been studied is that telomere lengths decrease with increasing time. In dyskeratosis congenita, there is deficiency of telomerase activity in somatic tissues due to mutations in the TERC or dyskerin genes, and this results in early proliferative exhaustion, especially in the bone marrow (reviewed in Ref. [25]). It seems highly likely that the cancers and cell lines that utilize ALT are subverting a normal mechanism, the nature of which is currently unknown. There are no normal cells that are known to have the telomere length heterogeneity characteristic of ALT cell lines and tumors, so it cannot be assumed that a normal counterpart of ALT has exactly the same activity as ALT. For the time being, therefore, it seems reasonable to refer to this putative process as ‘normal ALT-like activity’.
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In telomerase-null mice, B cells appear to undergo lengthening in the germinal centers, although the amount of lengthening is less than in wild-type mice [26]. This suggests that an ALT-like activity is present in at least some normal mammalian cells. It is important to note that non-telomerase TMMs are fully compatible with the life of eukaryotic multicellular organisms. Mosquitoes (Anopheles gambiae ) utilize a recombination-based mechanism [27] and the fruitfly, Drosophila melanogaster, and related Dipteran species use a retrotransposition-mediated TMM [28] instead of telomerase. An interesting and unexplained observation regarding telomere length is that overexpression of the specific binding factor, TRF2, in telomerasenegative normal cells results in accelerated telomere shortening [29]. The possible interpretations fall into two categories: increased TRF2 may speed up the shortening process (e.g. by increasing the access and/ or activity of a putative exonuclease [30,31] that recesses the C-rich telomere strand) or it may inhibit a normal process that partly counteracts shortening. It is currently unknown whether such a process exists, but it could be an ALT-like activity, or some other DNA repair mechanism. Interestingly, TRF2 overexpression also results in an alteration in the telomere length set point at which senescence is triggered [29]. It is possible that senescence occurs when the cell detects that one or more chromosome ends have fewer than a set number of telomere binding proteins attached and this may be determined by the combined effects of telomere length and telomere binding protein concentration.
4. How might telomere-related factors contribute to oncogenesis? These observations and speculations about telomere function lead to the following predictions regarding the possible contributions of telomere malfunction to disease, including cancer (Table 1). The most obvious contribution is when telomere length is maintained or even increased by the aberrant activation of a TMM, allowing the nascent cancer cells to breach the senescence barrier. A more subtle contribution (for which there is as yet no evidence) might be excessive telomerase or ALT-like activity in
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otherwise normal tissue compartments at some stage of development resulting in increased reserves of telomere length that permit abnormal clonal expansion when oncogenic mutations happen to occur. Increased telomere shortening is observed in a number of human conditions, including Down syndrome, Werner syndrome, ataxia telangiectasia and dyskeratosis congenita. For most of these conditions, the relationship between the genetic abnormality and the telomere defect is unknown. In principle, this could be due to (a) an exonuclease that has increased activity and/or increased access to the telomere, (b) excessive telomere loss due to recombinational events analogous to those that appear to be responsible for rapid loss of telomere sequence in yeast [32], or (c) inadequate telomere length compensation mechanisms (telomerase or putative normal ALT-like activity) at some time during development or post-natal life. An example of the latter is deficient telomerase activity in dyskeratosis congenita [25], as described in Section 3. Accelerated telomere shortening may also result from environmental stressmediated DNA damage, or an increased susceptibility to such damage [33,34]. Whatever the cause, an increased rate of telomere shortening may contribute to premature failure of proliferative tissues and may, therefore, cause features of premature ageing, but this could also facilitate overgrowth of clones that have acquired an increased proliferative capacity due to oncogenic mutations. It also seems possible that telomeres may contribute to disease and especially to cancer, by involvement in abnormal repair events resulting, for example, in end-to-end fusions [35]. In some cases, the primary abnormality could increase telomere shortening leading to attempted repair. It is unclear whether there are naturally occurring mutations that result in end-to-end fusions in the absence of telomere shortening, although the possibility has been demonstrated in vitro by transduction of cells with dominant negative TRF2 constructs [23]. The expected result of end-to-end fusions would be chromosome breakage during subsequent cell divisions, and hence, genomic instability and cancer predisposition. Any genetic abnormalities that cause accelerated telomere shortening and permit abnormal DNA repair events at the telomere might be expected to result in premature ageing syndromes,
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that are accompanied by an increased cancer susceptibility. It will be of interest to determine whether Werner syndrome [36,37] is an example of this. Evidence is accumulating that telomerase may contribute to tumorigenesis by activities in addition to telomere maintenance (reviewed in Ref. [38]). In a mouse mammary cancer model, it was noted that telomerase was upregulated prior to significant telomere shortening [39], suggesting that some other function of telomerase was being exploited by the tumors. Transgenic mice with TERT overexpression in their keratinocytes were more susceptible than wild-type littermates to chemical carcinogen-induced skin carcinogenesis, even though the keratinocyte telomeres were of normal length, indicating that telomerase promotes proliferation in cells in which telomere length is not limiting [40]. It has recently been shown that, under conditions of nutrient and oxygen depletion, ALT cells (SV40-transformed human fibroblasts) transduced with an hTERT expression construct had a growth advantage over control ALT cells [20]. Although ALT-immortalized human fibroblasts are capable of full tumorigenic transformation upon overexpression of an activated ras oncogene [41,42], ras-induced immortalization of one such ALT cell line was facilitated by transduction with an hTERT expression construct, even when the encoded hTERT was modified in such a way that it had no effect on telomere length [20]. Overexpression of hTERT in a telomerase-positive erythroleukemia cell line resulted in resistance to apoptosis-induced by serum deprivation and double-stranded DNA break inducing agents, but not against DNA synthesis inhibitors; this effect, however, was not dissociated experimentally from an effect on telomere length [43]. Collectively, these data suggest the possibility that TERT may affect cellular functions other than telomere length, and that although telomerase and ALT appear to be equivalent in their ability to support immortalization, their contributions to tumor growth and survival in vivo may differ.
5. Telomere maintenance mechanisms and cancer treatment Transduction of telomerase-positive cell lines with
dominant negative TERT constructs causes decreased telomerase activity and may result in apoptosis or senescence, sometimes very rapidly [44 – 48]. Repression of telomere maintenance in ALT cell lines also results in cell death or senescence [49,50]. These data suggest that TMM inhibitors may be a useful form of cancer treatment. There is still a question as to how many population doublings it will take for such inhibitors to act, and whether it will usually be essential to combine them with treatments that produce a rapid decrease in tumor bulk. The low level of telomerase in most normal tissues suggests that telomerase inhibitors may be relatively non-toxic, although the consequences of deficient telomerase activity in individuals with dyskeratosis congenita suggest that it will not be possible to administer such inhibitors continuously for very extended periods. The likely side-effects of ALT inhibitors are unknown. A predicted consequence of the existence of ALT in a substantial minority of human tumors is that telomerase inhibitors will be ineffective in this tumor subset, and that it may be useful to develop inhibitors of ALT. In addition, although it has not yet been observed in cell culture experiments [44,45,51,52], it seems likely that the use of telomerase inhibitors for treatment of telomerase-positive tumors will provide a selective advantage to tumor cells which activate an ALT mechanism, and that ALT inhibitors may be useful for preventing their emergence. Similarly, it may be necessary to prevent the emergence of telomerase-positive cells in tumors treated initially with ALT inhibitors.
6. Conclusions The available evidence suggests that it is worth testing TMM inhibitors as cancer therapeutics. The observations indicating that telomerase and ALT may confer different properties on tumor cells under the adverse conditions that exist in vivo make it important to investigate the prognostic implications of these TMMs in tumors. In addition, if it is found that the type of TMM affects response to current cancer treatments, it may be necessary in future to stratify clinical trials of cancer treatments according to TMM.
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Acknowledgements Work in the author’s laboratory was supported by the Carcinogenesis Fellowship of The Cancer Council NSW, and project grants from the National Health and Medical Research Council of Australia and the Virtual Research Institute of Aging of Nippon Boehringer Ingelheim. The author thanks Elizabeth Collins and Axel Neumann for the figures.
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Alternative lengthening of telomeres, telomerase, and cancer Roger R. Reddel* Children’s Medical Research Institute, 214 Hawkesbury Road, Westmead, Sydney, NSW 2145, Australia Received 6 September 2002; received in revised form 30 October 2002; accepted 30 October 2002
Abstract Telomere length may be maintained in cancer cells by telomerase or an alternative lengthening of telomeres (ALT) mechanism. Low levels of telomerase activity have been detected in some normal somatic cells and presumably some types of normal cells also have low levels of an ALT-like activity. It is hypothesized here that inherited abnormalities of these and other aspects of telomere maintenance may contribute to cancer and ageing. The telomere length maintenance mechanisms are similar in that activation of each is associated with immortalization. They may also confer other properties on cancer cells, however, and the nature of these additional properties may be different for telomerase and ALT. It is expected that these similarities and differences will have implications for prognosis and treatment. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Telomerase; hTERT; Cancer; Alternative lengthening of telomeres; Senescence; Immortalization; Cancer; Ageing; Down syndrome; Werner syndrome; Ataxia telangiectasia; Dyskeratosis congenita
1. Introduction Telomeres are tracts of repetitive DNA at each chromosome end. In normal human cells, the telomeres contain up to 15 kb of tandem repeats of the hexanucleotide, TTAGGG [1], and this amount decreases by an average of 50– 150 base pairs per cell cycle [2]. It is thought that this telomere shortening process ultimately limits the number of times a cell can divide [3], and acts as a powerful tumor suppressor mechanism. The great majority of cancers escape from the limitations on proliferation imposed by normal telomere shortening via activation of a telomere length maintenance mechanism (TMM) (reviewed in Ref. [4]). In most cancers, * Tel.: þ61-2-9687-2800; fax: þ 61-2-9687-2120. E-mail address: [email protected] (R.R. Reddel).
telomere length is maintained by an enzyme, telomerase, that synthesizes telomere repeat sequences to replace those that are lost during DNA replication [5]. Some cancers that are telomerase-negative maintain the length of their telomeres by one or more mechanisms referred to as alternative lengthening of telomeres (ALT) [6]. Similarly, cells that become immortalized in vitro escape from the normal limitations on proliferative capacity by activating telomerase [7] or ALT [8] (Fig. 1). An understanding of the roles of the TMMs in cancer depends on an understanding of their roles in normal telomere biology. It seems likely that there will be both similarities and differences between the roles of telomerase and ALT. This review contains a summary of some existing information about telomerase and ALT and speculation regarding the
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Fig. 1. Telomere length maintenance and immortalization. When the telomere lengths of proliferating normal cells become marginal, the cells undergo senescence, which includes permanent withdrawal from the cell cycle. This barrier to proliferation may be breached temporarily by loss of function of key tumor suppressor genes, but the telomeres eventually become critically short and the cells enter crisis. Escape from these limitations on proliferation may be provided by activation of a TMM, telomerase or ALT (reviewed in Ref. [4]). Telomerase activation often results in stably short telomeres, or in telomere lengthening and stabilization in the normal length range. ALT results in a large increase in average telomere length, but there are very short and very long telomeres present within individual cells.
similarities and differences in their contributions to the cancer cell phenotype.
2. Telomere length maintenance mechanisms in cancer Telomerase [9] is a ribonucleoprotein complex that includes an RNA template molecule (usually referred to as TR or TER; encoded by the TERC gene) and a catalytic subunit (TERT) that has reverse transcriptase activity (reviewed in Ref. [10]). Other subunits include a protein, dyskerin, that binds to TER [11]. By reverse transcribing its RNA template moiety, telomerase synthesizes telomeric DNA to compensate for the loss of telomeric sequences during DNA replication [10]. A survey of published data on telomerase activity in a wide variety of human cancers concluded that approximately 85% of all cancers are telomerase-positive [5].
Some telomerase-negative tumors maintain the length of their telomeres by ALT [6]. The available evidence regarding the mechanism of ALT is consistent with a model in which a DNA strand from one telomere of an ALT cell anneals with the complementary strand of another telomere, thereby priming synthesis of new telomeric DNA using the complementary strand as a copy template [12] (Fig. 2A). Prior to crisis, some of the chromosome ends shorten so much that all of the telomere repeats are lost together with some of the subtelomeric region. This region contains a mixture of variant repeat sequences and some TTAGGG sequences. When ALT is activated residual TTAGGG repeats in the subtelomeric region may prime the addition of new telomeric DNA, with the net result being that the most distal variant subtelomeric repeats are replaced by TTAGGG [13] (Fig. 2B). The types of tumors and tumor cell lines in which ALT has been observed include osteosarcoma, soft
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Fig. 2. Recombination-mediated lengthening of telomeres in ALT cells. Available data are consistent with a mechanism in which telomeres use other telomeres (or extrachromosomal telomeric DNA) as a copy template. (A) One strand of a shortened telomere may invade another telomere and anneal with the complementary strand. This acts as a primer for extension of the invading strand using the invaded telomere as a copy template for DNA synthesis, after which the other strand can be filled in. Evidence for this mechanism was provided by inserting a DNA tag into telomeres of ALT cells and showing that the tag was copied on to other telomeres [12]. (B) If telomere shortening extends into the subtelomeric region that contains variant repeat sequences, recombination-mediated lengthening would be predicted to result in replacement of distal variant repeats by TTAGGG repeats. This prediction was confirmed by sequencing subtelomeric regions of cells before and after activation of ALT and escape from crisis [13].
tissue sarcoma, glioblastoma multiforme, renal cell carcinoma, adrenocortical carcinoma, breast carcinoma, non-small cell carcinoma of the lung, and ovarian carcinoma [6,14,15]; J. Henson and R. Reddel, unpublished data). Based on limited information, it appears that ALT is more often present in cell lines and tumors of mesenchymal origin than in those of epithelial origin; possible reasons for this have been discussed recently [16]. More extensive surveys need to be done to identify the tumor types in which ALT is most common. The following evidence indicates that some tumors possess both ALT and telomerase activity [6]. ALT cells have a telomere length phenotype characterized by extreme heterogeneity (i.e. the telomere lengths
range from very short to extremely long). Some tumors have both telomerase activity and the characteristic telomere length pattern [6]. It is not yet known whether this is due to intratumoral heterogeneity with some areas of the tumor being telomerase-positive and other areas having ALT activity, or whether these TMMs coexist within the same tumor cells. In vitro experiments have shown that the latter is possible in principle [17 – 20]. It has been observed in one case that when a treated telomerase-positive brain tumor recurred, the regrowth was telomerase-negative and had telomere lengths indicative of ALT (J. Royds, personal communication); although there are several possible explanations, the simplest would be that the original
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Table 1 Telomere-related abnormalities that could potentially contribute to oncogenesis Known or putative telomere abnormality
Known or hypothetical mechanism(s)
Predicted outcomes
Ia. Increased length maintenance: prevention of shortening
Activation of telomerase and/or ALT
Immortalization, cancer
Ib. Increased length maintenance: decreased shortening
Overactivity of telomerase or putative ALT-like activity in normal tissues Decreased nuclease access and/or activity
Clonal expansion of cells that acquire an oncogenic mutation, cancer
II. Accelerated shorteninga
Excessive nuclease access and/or activity Excessive recombination-mediated loss Inadequate telomerase or ALT-like activity, e.g. during development
Eventual failure of proliferation, especially in highly proliferative tissues; features of premature ageing; overgrowth of clones with oncogenic mutations
III. Abnormal repair eventsa e.g. end-to-end fusions
Excessive shortening Abnormal access of DNA repair complexes Other failure of ‘cap’ function
Genomic instability
a Mutations that result in a combination of II and III would be expected to result in syndromes with premature ageing and increased cancer susceptibility.
tumor was heterogeneous with regard to TMM and that it was a portion of the tumor with ALT activity that survived treatment and regrew.
3. Telomere maintenance in normal cells Although the telomeres of normal somatic cells undergo progressive shortening, their integrity is maintained in several ways. The telomeric DNA sequence is recognized by specific binding proteins, these proteins interact with others, and together they form a putative cap structure that protects the chromosome end from degradation or from attempted DNA repair. The telomeres of normal and telomerasepositive cells form a loop structure [21] in which the chromosome end is effectively hidden. It has been proposed that ‘uncapping’ results in the telomere being detected by the cell as a DNA break, and that the probability of uncapping increases as the telomere shortens [22]. The concentration and activity of telomere binding factors may also influence the probability that uncapping will occur [23]. The evidence for this concept is currently indirect as there are no molecular markers of an uncapped telomere. Some normal cells have detectable levels of
telomerase activity. It is possible that this partially compensates for normal proliferation-associated telomere attrition, thus increasing the proliferative capacity of specific cell populations in various tissues that require a high cell turnover. There may even be occasions in which telomere lengthening occurs in normal cells. For example, the telomere length of Blymphocytes appears to increase during their transit through the germinal center of the lymph node [24]. Nevertheless, the overall long-term trend in all tissues that have been studied is that telomere lengths decrease with increasing time. In dyskeratosis congenita, there is deficiency of telomerase activity in somatic tissues due to mutations in the TERC or dyskerin genes, and this results in early proliferative exhaustion, especially in the bone marrow (reviewed in Ref. [25]). It seems highly likely that the cancers and cell lines that utilize ALT are subverting a normal mechanism, the nature of which is currently unknown. There are no normal cells that are known to have the telomere length heterogeneity characteristic of ALT cell lines and tumors, so it cannot be assumed that a normal counterpart of ALT has exactly the same activity as ALT. For the time being, therefore, it seems reasonable to refer to this putative process as ‘normal ALT-like activity’.
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In telomerase-null mice, B cells appear to undergo lengthening in the germinal centers, although the amount of lengthening is less than in wild-type mice [26]. This suggests that an ALT-like activity is present in at least some normal mammalian cells. It is important to note that non-telomerase TMMs are fully compatible with the life of eukaryotic multicellular organisms. Mosquitoes (Anopheles gambiae ) utilize a recombination-based mechanism [27] and the fruitfly, Drosophila melanogaster, and related Dipteran species use a retrotransposition-mediated TMM [28] instead of telomerase. An interesting and unexplained observation regarding telomere length is that overexpression of the specific binding factor, TRF2, in telomerasenegative normal cells results in accelerated telomere shortening [29]. The possible interpretations fall into two categories: increased TRF2 may speed up the shortening process (e.g. by increasing the access and/ or activity of a putative exonuclease [30,31] that recesses the C-rich telomere strand) or it may inhibit a normal process that partly counteracts shortening. It is currently unknown whether such a process exists, but it could be an ALT-like activity, or some other DNA repair mechanism. Interestingly, TRF2 overexpression also results in an alteration in the telomere length set point at which senescence is triggered [29]. It is possible that senescence occurs when the cell detects that one or more chromosome ends have fewer than a set number of telomere binding proteins attached and this may be determined by the combined effects of telomere length and telomere binding protein concentration.
4. How might telomere-related factors contribute to oncogenesis? These observations and speculations about telomere function lead to the following predictions regarding the possible contributions of telomere malfunction to disease, including cancer (Table 1). The most obvious contribution is when telomere length is maintained or even increased by the aberrant activation of a TMM, allowing the nascent cancer cells to breach the senescence barrier. A more subtle contribution (for which there is as yet no evidence) might be excessive telomerase or ALT-like activity in
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otherwise normal tissue compartments at some stage of development resulting in increased reserves of telomere length that permit abnormal clonal expansion when oncogenic mutations happen to occur. Increased telomere shortening is observed in a number of human conditions, including Down syndrome, Werner syndrome, ataxia telangiectasia and dyskeratosis congenita. For most of these conditions, the relationship between the genetic abnormality and the telomere defect is unknown. In principle, this could be due to (a) an exonuclease that has increased activity and/or increased access to the telomere, (b) excessive telomere loss due to recombinational events analogous to those that appear to be responsible for rapid loss of telomere sequence in yeast [32], or (c) inadequate telomere length compensation mechanisms (telomerase or putative normal ALT-like activity) at some time during development or post-natal life. An example of the latter is deficient telomerase activity in dyskeratosis congenita [25], as described in Section 3. Accelerated telomere shortening may also result from environmental stressmediated DNA damage, or an increased susceptibility to such damage [33,34]. Whatever the cause, an increased rate of telomere shortening may contribute to premature failure of proliferative tissues and may, therefore, cause features of premature ageing, but this could also facilitate overgrowth of clones that have acquired an increased proliferative capacity due to oncogenic mutations. It also seems possible that telomeres may contribute to disease and especially to cancer, by involvement in abnormal repair events resulting, for example, in end-to-end fusions [35]. In some cases, the primary abnormality could increase telomere shortening leading to attempted repair. It is unclear whether there are naturally occurring mutations that result in end-to-end fusions in the absence of telomere shortening, although the possibility has been demonstrated in vitro by transduction of cells with dominant negative TRF2 constructs [23]. The expected result of end-to-end fusions would be chromosome breakage during subsequent cell divisions, and hence, genomic instability and cancer predisposition. Any genetic abnormalities that cause accelerated telomere shortening and permit abnormal DNA repair events at the telomere might be expected to result in premature ageing syndromes,
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that are accompanied by an increased cancer susceptibility. It will be of interest to determine whether Werner syndrome [36,37] is an example of this. Evidence is accumulating that telomerase may contribute to tumorigenesis by activities in addition to telomere maintenance (reviewed in Ref. [38]). In a mouse mammary cancer model, it was noted that telomerase was upregulated prior to significant telomere shortening [39], suggesting that some other function of telomerase was being exploited by the tumors. Transgenic mice with TERT overexpression in their keratinocytes were more susceptible than wild-type littermates to chemical carcinogen-induced skin carcinogenesis, even though the keratinocyte telomeres were of normal length, indicating that telomerase promotes proliferation in cells in which telomere length is not limiting [40]. It has recently been shown that, under conditions of nutrient and oxygen depletion, ALT cells (SV40-transformed human fibroblasts) transduced with an hTERT expression construct had a growth advantage over control ALT cells [20]. Although ALT-immortalized human fibroblasts are capable of full tumorigenic transformation upon overexpression of an activated ras oncogene [41,42], ras-induced immortalization of one such ALT cell line was facilitated by transduction with an hTERT expression construct, even when the encoded hTERT was modified in such a way that it had no effect on telomere length [20]. Overexpression of hTERT in a telomerase-positive erythroleukemia cell line resulted in resistance to apoptosis-induced by serum deprivation and double-stranded DNA break inducing agents, but not against DNA synthesis inhibitors; this effect, however, was not dissociated experimentally from an effect on telomere length [43]. Collectively, these data suggest the possibility that TERT may affect cellular functions other than telomere length, and that although telomerase and ALT appear to be equivalent in their ability to support immortalization, their contributions to tumor growth and survival in vivo may differ.
5. Telomere maintenance mechanisms and cancer treatment Transduction of telomerase-positive cell lines with
dominant negative TERT constructs causes decreased telomerase activity and may result in apoptosis or senescence, sometimes very rapidly [44 – 48]. Repression of telomere maintenance in ALT cell lines also results in cell death or senescence [49,50]. These data suggest that TMM inhibitors may be a useful form of cancer treatment. There is still a question as to how many population doublings it will take for such inhibitors to act, and whether it will usually be essential to combine them with treatments that produce a rapid decrease in tumor bulk. The low level of telomerase in most normal tissues suggests that telomerase inhibitors may be relatively non-toxic, although the consequences of deficient telomerase activity in individuals with dyskeratosis congenita suggest that it will not be possible to administer such inhibitors continuously for very extended periods. The likely side-effects of ALT inhibitors are unknown. A predicted consequence of the existence of ALT in a substantial minority of human tumors is that telomerase inhibitors will be ineffective in this tumor subset, and that it may be useful to develop inhibitors of ALT. In addition, although it has not yet been observed in cell culture experiments [44,45,51,52], it seems likely that the use of telomerase inhibitors for treatment of telomerase-positive tumors will provide a selective advantage to tumor cells which activate an ALT mechanism, and that ALT inhibitors may be useful for preventing their emergence. Similarly, it may be necessary to prevent the emergence of telomerase-positive cells in tumors treated initially with ALT inhibitors.
6. Conclusions The available evidence suggests that it is worth testing TMM inhibitors as cancer therapeutics. The observations indicating that telomerase and ALT may confer different properties on tumor cells under the adverse conditions that exist in vivo make it important to investigate the prognostic implications of these TMMs in tumors. In addition, if it is found that the type of TMM affects response to current cancer treatments, it may be necessary in future to stratify clinical trials of cancer treatments according to TMM.
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Acknowledgements Work in the author’s laboratory was supported by the Carcinogenesis Fellowship of The Cancer Council NSW, and project grants from the National Health and Medical Research Council of Australia and the Virtual Research Institute of Aging of Nippon Boehringer Ingelheim. The author thanks Elizabeth Collins and Axel Neumann for the figures.
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