For human cells to achieve immortalization they must bypass multiple proliferative checkpoints and acquire a telomere maintenance mechanism to counteract the natural telomere attrition that results from the end-replication problem. A number of human tumors and cells immortalized in culture maintain their telomeres by a telomerase independent mechanism termed Alternative Lengthening of Telomeres (ALT). The available data indicate that ALT involves homologous recombination-mediated DNA replication and requires the activity of the MRE11/RAD50/NBS1 recombination complex. Increased levels of various types of telomere recombination events in ALT cells suggest that the cellular mechanisms which normally regulate recombination at mammalian telomeres have been lost. We review here the current literature regarding ALT and telomere biology and discuss possible mechanisms that have evolved in mammalian cells (primarily human) to inhibit deregulated homologous recombination at the telomeres and thus prevent telomere elongation and cellular immortalization.

Introduction

The chromosome ends (telomeres) of mammalian cells contain tandemly arrayed hexanucleotide repeats with the sequence 5'-TTAGGG-3'.¹ This telomeric DNA is mostly double-stranded, but it terminates in a single-stranded 3' overhang.² In human somatic cells, each telomere is 4-12 kb long and the single-stranded overhang contains 100-200 nucleotides (Fig. 1A). Telomeres need to be distinguished from double strand breaks (DSBs), to avoid being fused to each other by normal DNA repair mechanisms. This is achieved in part by the proteins that bind to telomeric DNA, forming a “cap” structure (Fig. 1B)(ref. 3 for review) Additionally, mammalian telomeres form a higher order structure by sequestering the 3' overhang in cis within the duplex telomeric DNA, resulting in a telomere loop (t-loop) that likely contributes to the capping mechanism.⁴

Figure 1

Human telomere components and structure. A) Graphic representation of the telomeric DNA in human cells, which is normally composed of 4-12 kb of G-rich repeats (TTAGGG in red, AATCCC in blue), culminating in a 100-200 nt 3' overhang. Open and t-loop configurations (more...)

Due to the end-replication problem,^5,6 the ends of linear chromosomes shorten with each round of DNA replication.⁷ In human somatic cells, the progressive telomere shortening that occurs with continued proliferation eventually results in the triggering of a replicative checkpoint. Telomere shortening and the structural changes that it presumably causes, leads to a DNA-damage checkpoint response at the telomere and induction of a permanent p53- and Rb-dependent growth arrest (i.e., replicative senescence).^8-10 Because this limits the proliferative capacity of somatic cells, including those that have accumulated oncogenic mutations, telomere shortening and replicative senescence are a potent tumor suppressor mechanism.

If senescence pathways are absent, due for example to loss of p53 and Rb function, cells will continue to divide until the telomeres become almost completely eroded, leading to crisis, a period characterized by rampant chromosome end-to-end fusions and cell death.¹¹ Formation of tumors is, in most cases, dependent on the evolution of cells that escape from the barriers that senescence and crisis present to unlimited proliferation. Cells that achieve this are referred to as “immortalized” and in all cases this requires the activation of a mechanism for preventing telomere shortening. In most cases this is accomplished by upregulating the activity of telomerase,^12,13 a ribonucleoprotein enzyme that adds new telomeric repeats to chromosome termini. Telomerase has an important role in cells of the germ line and in normal somatic biology, especially in those tissue compartments that depend upon extensive cellular proliferation. Nevertheless, in normal somatic cells telomerase is not expressed at sufficient levels to prevent telomere shortening and telomere length maintenance in many cancers requires dysregulated levels of telomerase. A substantial minority of immortalized cell lines and tumors are telomerase-negative, however, and in these cells telomere length maintenance can be achieved instead by a telomerase-independent mechanism, termed Alternative Lengthening of Telomeres (ALT).^14,15

ALT may resemble (or represent) the earliest telomere maintenance mechanism (TMM), which preceded the evolution of telomerase-dependent maintenance of chromosomal termini. While the possibility cannot be excluded that a low level of ALT-like activity occurs at normal mammalian telomeres, the telomere phenotype seen in ALT-positive immortalized cells and tumors is not found in normal cells. The current data strongly support ALT being a homologous recombination (HR)-mediated DNA replication mechanism, which occurs in the context of telomere instability resulting from loss of several controls over telomere function. Here we discuss the literature regarding telomere biology and ALT, with particular attention to the possible mechanisms that have evolved in mammalian cells (especially human) to prevent aberrant telomere maintenance by HR.

Phenotypic Identifiers of ALT Cells

ALT is defined as telomere length maintenance that is not dependent on telomerase activity. It is currently not clear whether there is more than one ALT mechanism in mammalian cells and there is no assay for ALT activity. The existence of ALT was deduced from observations of telomere length maintenance over many hundreds of population doublings (PDs) in the absence of detectable telomerase activity.^15,16 Fortunately, it is not necessary to perform this type of experiment to determine whether a cell line or tumor utilizes ALT, because ALT-positive human cells can now be recognized on the basis of a number of hallmarks. Analysis of telomeric DNA from an ALT cell line by pulsed field gel electrophoresis and Southern blotting indicates that within a population of cells the telomeric DNA ranges from < 2 to > 50 kb in length, with a mean size that is usually around 20 kb.¹⁵ Telomere length heterogeneity is also obvious at the single cell level when observing metaphases from ALT cells by fluorescent in-situ hybridization (FISH) with telomere specific probes.¹⁷ This confirms that some telomeres are very long and, notably, that within the great majority of individual ALT cells there is a subset of chromosome ends that lack any discernable telomere signal. Telomeres in ALT cells are also in a very dynamic state, exhibiting sudden lengthening and shortening events.¹⁸ A substantial portion of the telomeric repeats in ALT cells is extrachromosomal and may be linear^19,20 or circular^21,22 in form. The extrachromosomal telomeric repeat (ECTR) circles (t-circles) are also heterogeneous in size, ranging from < 1 to > 50 kb and equivalent structures have not been observed in high abundance in normal cells or in telomerase positive mammalian cell lines.^21,22

Another hallmark of ALT cell lines and tumors is the presence of specialized promyelocytic leukemia nuclear bodies (PNBs), termed ALT-associated PNBs (APBs).²³ In addition to the usual PNB components, including PML and Sp100, APBs are defined by the presence of telomeric DNA and telomere binding proteins and also contain an assortment of DNA replication, recombination and repair factors (Table 1). Large, easily recognizable APBs are present in only a minority of cycling ALT cells,²³ most likely due to their enrichment during the G2 phase of the cell cycle.^24,25

Table 1

Known protein constituents of ALT-associated promyelocytic leukemia nuclear bodies.

Occurrence of ALT

The typical ALT phenotype has only been found in abnormal situations, including immortalized human cell lines, human tumors and tumors or cell lines derived from telomerase null mice, suggesting that this an anomalous telomere phenotype.^17,26 Up to 10% of all human cancers and a greater proportion of cells immortalized in culture, utilize the ALT TMM.^17,27 Immortalization via activation of ALT appears to occur readily in cells of some Li-Fraumeni Syndrome individuals (p53 +/mut) and in fibroblasts immortalized using the SV40 Large T antigen.²⁸ ALT is not often detected in carcinomas (tumors of epithelial origin), but, for reasons that are currently unknown, ALT occurs commonly in sarcomas (tumors of mesenchymal origin) and there are some types of sarcomas where more than 50% of tumors are ALT-positive.²⁹

Abundant Telomere Recombination in ALT Cells

The rapid dynamics of telomere length polymorphisms in ALT cells suggested that the TMM involves HR.¹⁸ HR-dependent DNA replication of telomeres (Fig. 2B) within ALT cells was demonstrated by following a neomycin resistance marker inserted within the telomere repeats, or immediately proximal to the telomere (i.e., in a subtelomeric location), in the GM847 (ALT) and HT1080 (telomerase positive) cell lines.³⁰ After many PDs, the telomeric neomycin marker was copied to different telomeres within ALT cells, but no movement was observed for the sub-telomeric marker over the same period. No movement of the telomeric marker occurred in the telomerase positive HT1080 cell line, suggesting that substantial ongoing copying of telomeric DNA to other telomeres occurs exclusively in ALT cells.

Figure 2

ALT associated telomere recombination. A) T-loop junction resolution. i) Resolution of the t-loop junction in a NBS1 and XRCC3 dependent manner at sites denoted by arrows results in ii) a free t-circle and a shortened telomere. Branch migration at the (more...)

ALT telomeres also undergo abundant postreplicative exchanges, compared to non-ALT cells, as assayed by telomere specific chromosome-orientation FISH (CO-FISH).^31,32 While these exchanges are commonly referred to as telomere-sister chromatid exchanges (T-SCEs; Fig. 2C), it is possible that they may also arise from recombination with nonsister telomeres or ECTR elements. It has been proposed that unequal exchanges between telomeres could lead to telomere length changes.³³

Abundant t-circles also suggest that there is an elevated rate of intra-telomeric recombination-related events in ALT cells. It seems that t-circles arise from t-loops: the size of the t-circles in the GM847 ALT cell line closely correlates with the size of the loop portion of t-loops, as measured by electron microscopy.²¹ T-loop formation and stability are thought to require TRF2 function.^4,34 Expression in mammalian cells of a truncated allele of TRF2 lacking the basic domain (TRF^Δ-B) results in telomere rapid deletions (TRD) and formation of t-circles, suggesting that the TRF2 basic domain protects against improper resolution of t-loop junctions (referred to here as t-loop junction resolution; t-loop JR)(Fig. 2A).²² Consistent with this interpretation, the TRF2 basic domain interacts in vitro with four-way DNA junctions, regardless of whether the sequence consists of telomeric repeats.³⁵ Induction of t-circles by TRF^Δ-B is dependent on NBS1 and XRCC3 and RNAi knockdown of NBS1 or XRCC3 in several ALT cell lines diminishes t-circle abundance.³⁷ Recent experiments show that deletion of POT1A in the mouse also results in NBS1-dependent formation of t-circles.³⁶ Therefore, the rapid telomere shortening events¹⁸ and abundant t-circles seen in ALT cells suggest that, in these cells, improper resolution of the HR-intermediate structures represented by t-loop junctions occurs at an increased rate. Furthermore, it is likely that these t-loop JR events are mediated by NBS1 and XRCC3 and are repressed in non-ALT cells by proteins such as TRF2 and POT1.

There is no increase in genomic HR in ALT cells as compared to non-ALT controls,^38,39 suggesting a telomere-specific dysfunction rather than a general increase in recombination in these cells. ALT telomeres thus appear to be susceptible to three distinct types of recombination events: 1) HR-dependent DNA-replication telomere copying, 2) postreplicative exchanges and 3) HR-mediated t-loop junction resolution.

Possible ALT Mechanisms

Although HR-mediated copying of one telomere by another is the simplest explanation for the spread of a DNA tag from one telomere to others,³⁰ other types of elongation events may also occur as observed in the telomerase null Type II survivors from the budding yeast species Saccharomyces cerevisiae and Kluyveromyces lactis.^40-42 It was suggested that in these species rolling circle replication on t-circles served as the initial lengthening event followed by HR spreading of the newly elongated telomere to other chromosome ends (“roll and spread” model). Evidence supporting this hypothesis was obtained by transfecting exogenous t-circles into telomerase null K. lactis cells.^43,44 The result was extension of the chromosomal telomeres with repetitive units of exogenous t-circle DNA and spreading of this sequence to other chromosome termini. Moreover, it was shown that a single elongated telomere is sufficient to drive extension of the other shortened telomeres in K. lactis cells.⁴⁵ Physical evidence of roll and spread was also documented in the mitochondria of Candida parapsilosis which contain a linear genome capped by telomeres maintained in a telomerase independent manner.⁴⁶ Because all ALT cell lines examined so far contain t-circles (refs. 21,22 and C. Fasching and R. Reddel, unpublished data), it seems feasible that roll and spread could also occur in mammalian ALT cells. Additional possibilities for telomere elongation mechanisms in ALT cells include DNA replication primed from the terminal hydroxyl group of the 3' overhang within the t-loop, or via HR with ECTR DNA molecules. Presumably, once the generation of long tracts of telomeric DNA by one or more of the above means results in a threshold quantity of telomere sequence being attained, the reservoir of telomeric DNA that this constitutes will permit ongoing telomere maintenance by HR.

Figure 3

Factors that are known or thought to contribute to the ALT phenotype. Known associations with the ALT phenotype are signified by solid lines and more speculative associations are shown by dotted lines. APBs, t-circles and T-SCEs are known to associate (more...)

Although it is often assumed that APBs serve as sites of telomere extension in ALT cells, this remains to be fully validated. Evidence that is consistent with this notion includes the observation that BrdU incorporation within APBs is caffeine sensitive suggesting ATM- or ATR-dependent DNA synthesis, possibly in response to a DNA damage signal.⁴⁷ The periodic association and dissociation of chromosomal telomeres and APBs seen in live cell imaging experiments suggests APBs may colocalize with telomeres for extension.⁴⁸ Morever, when ALT is inhibited (see below) the percentage of APB positive cells decreases (ref. 49 and Z. Zhong and R. Reddel, unpublished data).

Genes Involved in ALT

It is becoming apparent that the relationship between DNA repair proteins and human telomeres is complex (ref. 50 for review). In this light and considering the phenotypic characteristics of ALT cells, almost any protein involved in telomere function, HR, DNA damage response and repair, DNA replication, or APBs could be involved in ALT. In Type II S. cerevisiae telomerase-null survivors, which have telomeres that phenotypically resemble those of ALT-positive human cells, the HR protein Rad52 and epistasis group members Rad50 and Rad59, as well as the RecQ Sgs1 helicase are required for telomere maintenance.^41,51-53 There are no data regarding human RAD52 involvement in ALT, although this seems likely given its function in HR. While the Sgs1 protein is the only RecQ helicase in S. cerevisiae, human cells contain several orthologs, with the Werner Syndrome helicase (WRN) drawing considerable attention in the telomere field. Mutation in the WRN helicase leads to a premature aging phenotype (ref. 54 for review) that is recapitulated in mouse models only in the context of telomere dysfunction in late generation telomerase null (TERC -/-) mice.^55,56 WRN telomere functions appear to include unwinding of G-quartets created in the lagging strand during DNA synthesis, that if left unresolved lead to sister chromatid loss and genomic instability due to chromosome fusions.^57,58 ALT tumors form in late generation (G5) TERC -/- WRN -/- mice following loss of p53 function.⁵⁹ Thus, WRN is not essential for ALT and this conclusion is supported by the observation that the W-v human Werner Syndrome cell line is a typical ALT line.⁶⁰ This does not exclude the possibility that other RecQ family members are required for ALT.

Recent experiments have, however, implicated the complex containing human MRE11, RAD50 and NBS1 (MRN) proteins as being required for ALT activity. Over-expression of the PNB component, Sp100, led to the sequestration of MRN in Sp100 microbodies via interaction with NBS1.⁴⁹ Long term expression of Sp100 resulted in ALT suppression for >80 PD in the IIICF/c ALT cell line as characterized by a steady decrease in telomere lengths consistent with natural telomere attrition and reduction of APBs. In a follow up study, individual components of the MRN complex were knocked down by long term expression of shRNAs in the IIICF/c ALT cell line (Z. Zhong and R. Reddel, unpublished data). Knockdown of NBS1 resulted in suppression of ALT, as characterized in the above study for >70 PD, although variable results were observed in individual clones. Similar ALT suppression was seen in clones following knockdown of RAD50 and to a lesser extent following knockdown of MRE11. However, knockdown of RAD50 or MRE11 also resulted in a reduction of NBS1, or NBS1 and RAD50 levels, respectively. Thus, it is difficult to draw conclusions about the contributions of the components of the MRN complex to the ALT mechanism. Interestingly, the MRN complex associates with normal telomeres, suggesting that there must be a mechanism to control its function and thus to prevent ALT-like activity.⁶¹

Another recent study elucidated a series of genes required for APB formation.⁶² Following the observation that methionine restriction enhances the abundance of APB positive cells, a screen for APB genes was carried out by transfecting siRNAs prior to methionine restriction. In this study, the telomere proteins TRF1, TRF2, RAP1 and TIN2, PML and all three components of the MRN complex were shown to be required for APB formation. Therefore, these proteins may be required for ALT, while the DNA response and repair protein 53BP1 was shown to be dispensable.

Telomere Capping and ALT Inhibition

A series of recent reports suggest functional telomeres are recognized by the DNA damage machinery during G2 and the action of HR is necessary to cap the chromosome ends before entering mitosis.^63,64 Furthermore, the RAD51D recombination protein interacts with telomeric DNA and its deletion leads to a telomere uncapping phenotype.⁶⁵ The MRN recombination complex, which is essential for ALT,⁴⁹ also functions at normal human telomeres.⁶¹ Therefore, normal cells need to achieve a fine balance where the beneficial capping-associated HR at the telomere is permitted while the HR-mediated telomere lengthening associated with ALT is inhibited.

Telomeres in budding yeast appear to become much more recombinogenic in the absence of proper capping. In Type II S. cerevisiae survivors, recombinational telomere elongation occurs predominantly on the shortest telomeres⁴¹ and telomeres in K. lactis only become recombinogenic after extreme shortening following telomerase inhibition.⁶⁶ An interpretation of these data is that as telomeres become very short, they bind far fewer telomere associated proteins, become uncapped and lose their ability to repress telomere HR. Consistent with this, inhibition of the function of the capping proteins Cdc13 in S. cerevisiae and Stn1 in K. lactis, or the telomere protein Rif2 in S. cerevisiae, leads to induction of Type II-like telomere recombination.^41,67,68 Uncapping of telomeres in K. lactis due to mutations in the telomeric DNA that diminish binding of the telomere protein Rap1 also result in increased telomere recombination, t-loop JR and t-circle generation. (refs. 42,69,70 and A. Cesare, M. McEachern and J. Griffith, unpublished).

Telomere capping in mammalian cells is a major function of TRF2. Following TRF2 disruption, mammalian cells exhibit a telomeric DNA checkpoint response⁷¹ coinciding with p53/Rb-dependent senescence⁹ or ATM/p53-dependent apoptosis.⁸ Decreased TRF2 function in the absence of p53 results in escape from cellular arrest and extensive chromosome fusions in a non-homologous end joining (NHEJ) dependent manner.^72,73 T-loop formation and stability are associated with TRF2 function and sequestration of the 3' overhang with a t-loop is proposed to hide the chromosome end from NHEJ.^4,34 POT1 in humans and POT1A and B in mice, also appear to have a role in telomere capping, although decreased levels of any of these proteins do not result in the same drastic uncapping phenotypes as inhibition of TRF2.^36,74-76

It seems likely that proper telomere capping in mammalian cells plays a role in suppressing the telomere length maintenance associated with ALT. As described above, improper resolution of t-loop structure in mammals is prevented by the basic domain of TRF2,²² and in mice by POT1A.³⁶ TRF2 also functions with the NHEJ protein Ku70 to inhibit T-SCE at mouse telomeres.⁷⁷ When chromosome fusions are prevented in mouse embryo fibroblasts (MEFs) by deletion of DNA ligase 4, deletion of both TRF2 and Ku70, but not either of these genes alone, causes a remarkable increase in the abundance of T-SCEs, similar to what is observed in ALT cells.⁷⁷ The increase in T-SCEs is prevented by TRF^Δ-B expression indicating that repression of t-loop JR and T-SCE are separate functions. POT1A and POT1B deletion in mice also results in an increase in T-SCE, although to much more modest levels than seen in ALT cells.^36,76 Thus, proteins associated with telomere capping inhibit two of the three types of telomere recombination events associated with ALT cells. It has not yet been determined if normal capping also inhibits lengthening of ALT telomeres by HR-dependent DNA replication.

The connection between telomeric uncapping and abnormal HR events suggests that ALT cells might have capping dysfunction. An important component of mammalian telomere capping is believed to be the formation of t-loops that sequester the 3' overhang from the NHEJ machinery in the G1 phase of the cell cycle, when this form of DSB repair is most active. T-loops are formed in the GM847 ALT cell line²¹ and rampant chromosome fusions are not observed in ALT cells consistent with proper capping in G1. During S phase, it has been proposed that DNA replication opens up the t-loops, resulting in chromosome ends being recognized by the DNA damage response machinery in G2.⁶³ HR proteins are then proposed to function in capping, possibly by re-establishing the t-loop.⁶⁴ T-loop JR and T-SCE are both post-replicative events,^22,78 so if they are due to an ALT-associated capping dysfunction, it is likely that this occurs in the G2 phase of the cell cycle, when HR is most active. The observation that APBs are associated with G2 is consistent with ALT activity occurring during this period.²⁴

ALT activity results from the loss of an inhibitory function that is present in normal somatic cells and also in telomerase-positive cells. This was demonstrated by fusing GM847 ALT cells with HFF5 normal fibroblasts, or with HT1080 or T24 telomerase-positive cells and observing suppression of ALT activity.⁷⁹ This raises the possibility that both the putative ALT capping dysfunction and the derepression of ALT result simply from the loss of a specific cap component. All of the known telomere associated proteins are present in ALT cells, however and mechanisms of DNA metabolism show no obvious defects (although this has not been analyzed in detail). Long term TRF2 overexpression in the SUSM-1 ALT cell line did inhibit some phenotypic features of ALT cells in one study, supporting the concept that ALT cells have a telomere capping dysfunction, although a prolonged and complete ALT inhibition was not observed (L. Colgin and R. Reddel, unpublished data). Rather than loss of a single telomere cap component, it seems more likely that higher order control over the intricate functions of DNA repair and telomere capping are altered in ALT cells.

The persistence of very short telomeres in ALT cells may also contribute to aberrant telomere recombination in these cells, but does not appear to be essential. The short telomeres in ALT cells may be more prone to initiating recombination, especially HR-dependent DNA replication, as seen in budding yeast.⁶⁶ Consistent with this, the shortest telomeres are preferentially, but not exclusively, elongated in ALT cells.¹⁸ Expression of exogenous telomerase elongates the shortest telomeres in ALT cells, but does not usually repress the phenotypic characteristics of ALT,^80-82 including t-circles²¹ and post replicative exchanges,³² suggesting that ALT activity is not dependent on the presence of very short telomeres.

Telomere Structural Dysfunction Response

At uncapped telomeres in mammalian cells, the DNA damage response proteins γ-H2AX, 53BP1, Rad17-ser645 and ATM-ser1981 accumulate in telomere dysfunction induced foci (TIFs), suggesting the chromosome end is now recognized as a DSB.^10,71,83 This may result in p53- and Rb-dependent senescence and occurs in response to inhibition of TRF2 function,71 t-loop JR induction by TRF2-B,²² or in aging cells with shortened telomeres.¹⁰ Persistent t-loop JR and the short telomeres in ALT cells would thus be expected to induce a similar checkpoint response. Although TIFs are observed in ALT cells, no arrest occurs.(refs. 47,83 and A. Cesare and R. Reddel unpublished). This is possibly due to a lack of signal transduction as the majority of ALT cell lines and tumors are p53-deficient. Of the human ALT cell lines that have been examined to date, only one (U-2 OS) is known to retain functional wild-type p53 and a recent study of glioblastoma multiforme showed that, of 18 tumors using the ALT pathway, 14 (78%) were p53-deficient, while 26 of the 33 telomerase positive tumors (79%) had wild-type p53.⁸⁴ In mouse cells, p53 loss has been shown previously to be permissive for telomere dysfunction,⁸⁵ and telomere dysfunction induced by WRN deletion leads to ALT tumors following loss of p53 function.⁵⁹

Telomeric Epigenetic Modification

Mammalian telomeres are assembled into constitutive heterochromatin as defined by specific modifications to the basic tails of the histones H3 and H4.^86,87 In MEFs with a double knock out (RB and RBL1), or triple knock out (RB, RBL1 and RBL2) of the Rb family proteins, the telomeres display increasing length and heterogeneity with progressive passages, with a concomitant decrease in the constitutive heterochromatin marker histone H4 tri-methyl lysine 20.^86,88 In a similar experiment, passage of embryonic stem (ES) cells or MEFs deleted for the Suv39h1 and Suv39h2 histone methyltransferases also resulted in increased telomere length and heterogeneity, coinciding with a significant decrease in the heterochromatin markers di- and tri-methylated histone H3 lysine 9 and their interacting partners, Cbx3 and Cbx5, at MEF or ES cell telomeres.⁸⁷ Similar telomeric DNA phenotypes were observed following deletion of DNA methyltransferases, DNMT1 or DMNT3a and DMNT3b, which resulted in decreased sub-telomeric DNA methylation (TTAGGG telomeres lack the CpG methylation site) in ES cells.⁸⁹ Additionally, mutation of the DMNT family members was reported to increase post-replicative telomeric exchanges and APBs, suggestive of ALT.⁸⁹ Finally, the epigenetic changes associated with euchromatin were observed with telomere shortening in MEFs from late generation TERC -/- mice and these changes were accompanied by reported increases in T-SCEs and in the proportion of APB positive cells.^89,90

These data suggest telomere epigenetic modifications may also regulate telomeric HR and thus inhibit ALT. In the above studies investigating the knockout of epigenetic regulatory genes, the cells used retained telomerase activity, therefore the observed telomere length increase could conceivably be due to increased access of telomerase to chromosome ends. Furthermore, no evidence was obtained for recombination-mediated telomere extension in these cells. Nevertheless, it is an attractive concept that changes in chromatin state may occur at the telomere and that a euchromatic state may result in the telomere being more open to HR. Since many ALT cell lines have functional deficiencies in Rb family proteins (e.g., due to expression of SV40 large T antigen which binds to each of these proteins), they may also have similar epigenetic alterations at the telomeres. Observation of ALT t-loops by electron microscopy indicated that the loop portion of the t-loop were similar in size to loops seen in telomerase positive or mortal cells, suggesting that a significant portion of the telomere may remain outside the loop in ALT cells.²¹ Therefore exclusion of much of the telomere from the loop, together with a more open state, may leave this portion of the telomere more prone to recombination or invasion by an adjacent telomere 3' overhang, leading to a HR-mediated DNA replication event.

What is ALT and Why Does It Exist?

It has been postulated previously that a telomerase-independent TMM preceded telomerase in the course of eukaryotic evolution,⁹¹ though it remains to be determined if such a mechanism functions at telomeres in normal mammalian cells. It is an intriguing possibility that human cells, especially those that are not normally rapidly dividing, may use such a mechanism to repair an accidentally truncated telomere without having to express telomerase and thereby risk immortalization. However, if one or more such mechanisms exist, they would need to be tightly controlled to avoid the deleterious consequences of telomere recombination and the possibility of cellular immortalization. In this view, the ALT phenotype as observed in immortalized cell lines and cancers results from loss of the normal mechanisms for controlling this telomere repair mechanism and consequent telomere dysfunction. Presumably, loss of the normal control mechanisms is selected for in the context of tumor suppressor pathway deficiencies that allow continued propagation of cells with shortened telomeres that are then under selection pressure to acquire a TMM. In some cases, the TMM is provided by dysregulated telomerase activity and in others by dysregulated HR-mediated telomere lengthening, i.e., ALT activity. Repression of ALT activity in normal mammalian cells appears to be a function that is conserved in eukaryotes, suggesting that telomeres have evolved intricate systems to utilize HR for beneficial purposes (e.g., capping) while inhibiting HR-mediated events that have deleterious consequences. These processes of HR-related mechanisms at the telomere and their systems of control represent the outcome of a long period of development, which has occurred in the context of the co-development of telomere binding proteins and control systems for telomerase, from the earliest eukaryotes with linear chromosomes and repetitive telomere sequences through to mammalian cells.

Acknowledgements

Members of the CMRI Cancer Research Unit, Clare Fasching, Axel Neumann, Ze-Huai Zhong and Lorel Colgin are thanked for critical review of the manuscript. A.J.C. is supported by a Sir Keith Murdoch Fellowship from the American Australian Association and the USA National Science Foundation International Research Fellowship Program. Research in the authors' laboratory is supported by a Program Grant from the Cancer Council New South Wales and project grants from the National Health and Medical Research Council Australia.

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