By imposing a limit on the proliferative lifespan of most somatic cells, telomere erosion represents an innate mechanism for tumor suppression and may contribute to age-related disease. A detailed understanding of the pathways that link shortened telomeres to replicative senescence has been severely hindered by the inability of current methods to analyze telomere dynamics in detail. Here we describe single telomere length analysis (STELA), a PCR-based approach that accurately measures the full spectrum of telomere lengths from individual chromosomes. STELA analysis of human XpYp telomeres in fibroblasts identifies several features of telomere biology. We observe bimodal distributions of telomeres in normal fibroblasts; these distributions result from inter-allelic differences of up to 6.5 kb, indicating that unexpectedly large-scale differences in zygotic telomere length are maintained throughout development. Most telomeres shorten in a gradual fashion consistent with simple losses through end replication, and the rates of erosion are independent of allele size. Superimposed on this are occasional, more substantial changes in length, which may be the consequence of additional mutational mechanisms. Notably, some alleles show almost complete loss of TTAGGG repeats at senescence.

The loss of telomere function can result in telomeric fusion events that lead to the types of genomic rearrangements, such as nonreciprocal translocations, that typify early-stage carcinogenesis. By using single-molecule approaches to characterize fusion events, we provide a functional definition of fusogenic telomeres in human cells. We show that approximately half of the fusion events contained no canonical telomere repeats at the fusion point; of those that did, the longest was 12.8 repeats. Furthermore, in addition to end-replication losses, human telomeres are subjected to large-scale deletion events that occur in the presence or absence of telomerase. Here we show that these telomeres are fusogenic, and thus despite the majority of telomeres being maintained at a stable length in normal human cells, a subset of stochastically shortened telomeres can potentially cause chromosomal instability. Telomere fusion was accompanied by the deletion of one or both telomeres extending several kilobases into the telomere-adjacent DNA, and microhomology was observed at the fusion points. This contrasted with telomere fusion that was observed following the experimental disruption of TRF2. The distinct error-prone mutational profile of fusion between critically shortened telomeres in human cells was reminiscent of Ku-independent microhomology-mediated end-joining.[Keywords: Telomere; telomerase; genomic instability; mutation; DNA repair; neoplasia] Supplemental material is available at http://www.genesdev.org.

We performed single-molecule telomere length and telomere fusion analysis in patients at different stages of chronic lymphocytic leukemia (CLL). Our work identified the shortest telomeres ever recorded in primary human tissue, reinforcing the concept that there is significant cell division in CLL. Furthermore, we provide direct evidence that critical telomere shortening, dysfunction, and fusion contribute to disease progression. The frequency of short telomeres and fusion events increased with advanced disease, but importantly these were also found in a subset of early-stage patient samples, indicating that these events can precede disease progression. Sequence analysis of fusion events isolated from persons with the shortest telomeres revealed limited numbers of repeats at the breakpoint, subtelomeric deletion, and microhomology. Array-comparative genome hybridization analysis of persons displaying evidence of telomere dysfunction revealed large-scale genomic rearrangements that were concentrated in the telomeric regions; this was not observed in samples with longer telomeres. The telomere dynamics observed in CLL B cells were indistinguishable from that observed in cells undergoing crisis in culture after abrogation of the p53 pathway. Taken together, our data support the concept that telomere erosion and subsequent telomere fusion are critical in the progression of CLL and that this paradigm may extend to other malignancies. (Blood. 2010;116(11): 1899-1907) IntroductionNonreciprocal translocations (NRTs) are considered to be key mutational events that can drive many types of malignancy. 1 The underlying mechanisms that result in these types of events can include, among others, deficiencies in double-strand break repair, 2 mitotic checkpoints, 3,4 and telomere dysfunction. 5 Telomeres play a key role in upholding genomic integrity; in the context of DNA damage checkpoint defects, cells in culture undergo crisis and have extensive telomere erosion, chromosomal fusion, and genomic rearrangements. 6,7 NRTs, as well as localized gene amplification, 8 can arise as a consequence of cycles of anaphase-bridging, breakage, and fusion initiated by the formation of dicentric chromosomes after telomere fusion. 9 This paradigm is exemplified in vivo by telomerase knockout mice, where short telomeres appear to drive the formation of tumors containing NRTs. 5 However, evidence for this phenomenon in humans is circumstantial. Numerous malignancies, including breast, prostate, colorectal, and chronic lymphocytic leukemia (CLL), [10][11][12][13][14][15] have been documented to exhibit shorter telomeres compared with normal tissues. These data are consistent with the expected levels of cell division during the progression to malignancy but do not indicate that telomeres become short enough to lose their end-capping function. Telomere fusion, as well as other chromosomal defects, can lead to the formation of anaphase bridges; in situ data show an increase in anaphase bridges, often interpreted as a surrogate marker for telomere f...

Telomere fusion is an important mutational event that has the potential to lead to large-scale genomic rearrangements of the types frequently observed in cancer. We have developed single-molecule approaches to detect, isolate and characterize the DNA sequence of telomere fusion events in human cells. Using these assays, we have detected complex fusion events that include fusion with interstitial loci adjacent to fragile sites, intra-molecular rearrangements, and fusion events involving the telomeres of both arms of the same chromosome consistent with ring chromosome formation. All fusion events were characterized by the deletion of at least one of the telomeres extending into the sub-telomeric DNA up to 5.6 kb; close to the limit of our assays. The deletion profile indicates that deletion may extend further into the chromosome. Short patches of DNA sequence homology with a G:C bias were observed at the fusion point in 60% of events. The distinct profile that accompanies telomere fusion may be a characteristic of the end-joining processes involved in the fusion event.

The accumulation of genetic abnormalities in a developing tumor is driven, at least in part, by the need to overcome inherent restraints on the replicative life span of human cells, two of which-senescence (M1) and crisis (M2)-have been well characterized. Here we describe additional barriers to clonal expansion (M int ) intermediate between M1 and M2, revealed by abrogation of tumor-suppressor gene (TSG) pathways by individual human papillomavirus type 16 (HPV16) proteins. In human fibroblasts, abrogation of p53 function by HPVE6 allowed escape from M1, followed up to 20 population doublings (PD) later by a second viable proliferation arrest state, M int E6, closely resembling M1. This occurred despite abrogation of p21 WAF1 induction but was associated with and potentially mediated by a further ϳ3-fold increase in p16 INK4a expression compared to its level at M1. Expression of HPVE7, which targets pRb (and p21 WAF1 ), also permitted clonal expansion, but this was limited predominantly by increasing cell death, resulting in a M int E7 phenotype similar to M2 but occurring after fewer PD. This was associated with, and at least partly due to, an increase in nuclear p53 content and activity, not seen in younger cells expressing E7. In a different cell type, thyroid epithelium, E7 also allowed clonal expansion terminating in a similar state to M int E7 in fibroblasts. In contrast, however, there was no evidence for a p53-regulated pathway; E6 was without effect, and the increases in p21 WAF1 expression at M1 and M int E7 were p53 independent. These data provide a model for clonal evolution by successive TSG inactivation and suggest that cell type diversity in life span regulation may determine the pattern of gene mutation in the corresponding tumors.Human tumors develop by a process of clonal evolution mediated by the acquisition of successive molecular abnormalities and driven, at least in part, by the need to overcome the inherent controls which limit the proliferative life span of normal human cells (51).Two of these proliferative life span barriers (PLBs)-senescence and crisis-have been well characterized, particularly with respect to human fibroblast models. These cells normally undergo around 40 to 70 population doublings (PD) (depending on age of donor) after which, even in ideal culture conditions, they enter a stable proliferative arrest in which they remain viable for many months (27). Escape from this state of replicative senescence, or mortality stage 1 (M1) (48), can be conferred by expression of a variety of DNA tumor virus genes, including simian virus 40 (SV40) T and human papillomavirus type 16 (HPV16) E6 plus E7, which target a common set of cell cycle regulatory tumor suppressor gene products, notably p53 and pRb (14, 41). The resulting clones are capable of at least an additional 30 PD, after which further expansion is limited by a second PLB termed crisis (or M2), which is due not so much to decreasing proliferation as to increasing cell death. Escape from this state is associated with stabiliz...

Single telomere length analysis (STELA) of the XpYp telomere has revealed extensive allelic variation and ultra-short telomeres in senescent cells. Superimposed on end-replication losses are additional mutational events that result in large-scale changes in telomere length. In order to establish if the dynamics of the XpYp telomere are typical of human telomeres, here we describe an analysis using STELA of the telomeres of 2p, 11q, 12q, 17p and XpYp. The dynamics of telomere loss (erosion rates and stochastic length changes) was conserved among 2p, 11q, 12q and XpYp within the same cell strains and was dependent on the replicative kinetics of the cells in culture. However, of the telomeres analysed, the telomere of 17p was more stable with a striking paucity of large-scale length changes, and exhibited the shortest recorded allelic distribution (300 bp) in senescent cells and displayed a general, but not absolute, trend towards being the shortest telomere. Ectopic over-expression of hTERT homogenized both allelic and chromosome-specific telomeric distributions. However, telomerase-expressing cancer cells displayed both allelic variation and chromosome-specific telomere length, with 17p displaying the shortest allelic telomere length. Although other telomeres in the genome may share the properties of 17p, these data suggest that physiological levels of telomerase allow differential telomere length regulation and indicate the presence of cis-acting factors that govern both telomeric stability and chromosome-specific telomere length in the presence of telomerase.

Telomeres play a key role in upholding the integrity of the genome, and telomerase expression in spermatogonial stem cells is responsible for the maintenance of telomere length in the human male germline. We have previously described extensive allelic variation in somatic cell telomere length that is set in the zygote, the ultimate source of which may be the germline. This implies that despite telomerase activity, substantial telomere length variation can be generated and tolerated in the germline; in order to investigate this further, we have examined the nature of telomere length variation in the human male germline. Here, we describe an analysis of both genome-wide telomere length and single molecule analysis of specific chromosome ends in human sperm. We observed individual specific differences in genome-wide telomere length. This variation may result from genetic differences within the components that determine the telomere length setting of each individual. Superimposed on the genome wide telomere length setting was a stochastic component of variation that generates germ-cells containing severely truncated telomeres. If not re-lengthened during early embryogenesis, such telomeres may limit the replicative capacity of cells derived from the zygote and have the potential to create fusagenic chromosomes, unbalanced translocations and terminal micro-deletions. These data may have implications for the genetic determination of ageing, genetic disease and fertility.

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