BLM and SLX4 play opposing roles in recombination-dependent replication at human telomeres.

Alternative lengthening of telomeres (ALT) is a telomere lengthening pathway that predominates in aggressive tumors of mesenchymal origin; however, the underlying mechanism of telomere synthesis is not fully understood. Here, we show that the BLM-TOP3A-RMI (BTR) dissolvase complex is required for ALT-mediated telomere synthesis. We propose that recombination intermediates formed during strand invasion are processed by the BTR complex, initiating rapid and extensive POLD3-dependent telomere synthesis followed by dissolution, with no overall exchange of telomeric DNA.

Extensive Proliferation of Human Cancer Cells with Ever-Shorter Telomeres.

Acquisition of replicative immortality is currently regarded as essential for malignant transformation. This is achieved by activating a telomere lengthening mechanism (TLM), either telomerase or alternative lengthening of telomeres, to counter normal telomere attrition. However, a substantial proportion of some cancer types, including glioblastomas, liposarcomas, retinoblastomas, and osteosarcomas, are reportedly TLM-negative.

Alternative lengthening of telomeres in normal mammalian somatic cells.

Some cancers use alternative lengthening of telomeres (ALT), a mechanism whereby new telomeric DNA is synthesized from a DNA template. To determine whether normal mammalian tissues have ALT activity, we generated a mouse strain containing a DNA tag in a single telomere. We found that the tagged telomere was copied by other telomeres in somatic tissues but not the germline.

Variant repeats are interspersed throughout the telomeres and recruit nuclear receptors in ALT cells.

Telomeres in cells that use the recombination-mediated alternative lengthening of telomeres (ALT) pathway elicit a DNA damage response that is partly independent of telomere length. We therefore investigated whether ALT telomeres contain structural abnormalities that contribute to ALT activity. Here we used next generation sequencing to analyze the DNA content of ALT telomeres.

Extreme telomere length dimorphism in the Tasmanian devil and related marsupials suggests parental control of telomere length.

Telomeres, specialised structures that protect chromosome ends, play a critical role in preserving chromosome integrity. Telomere dynamics in the Tasmanian devil (Sarcophilus harrisii) are of particular interest in light of the emergence of devil facial tumour disease (DFTD), a transmissible malignancy that causes rapid mortality and threatens the species with extinction. We used fluorescent in situ hybridisation to investigate telomere length in DFTD cells, in healthy Tasmanian devils and in four closely related marsupial species.

Five dysfunctional telomeres predict onset of senescence in human cells.

Replicative senescence is accompanied by a telomere-specific DNA damage response (DDR). We found that DDR+ telomeres occur spontaneously in early-passage normal human cells and increase in number with increasing cumulative cell divisions. DDR+ telomeres at replicative senescence retain TRF2 and RAP1 proteins, are not associated with end-to-end fusions and mostly result from strand-independent, postreplicative dysfunction.

Normal mammalian cells negatively regulate telomere length by telomere trimming.

In human cancer cells with telomeres that have been over-lengthened by exogenous telomerase activity, telomere shortening can occur by a process that generates circles of double-stranded telomeric DNA (t-circles). Here, we demonstrate that this telomeretrimming process occurs in cells of the male germline and in normal lymphocytes following mitogen-stimulated upregulation of telomerase activity. Mouse tissues also contain abundant t-circles, suggesting that telomere trimming also contributes to telomere length regulation in mice.

Spontaneous occurrence of telomeric DNA damage response in the absence of chromosome fusions.

Telomere dysfunction is typically studied under conditions in which a component of the six-subunit shelterin complex that protects chromosome ends is disrupted. The nature of spontaneous telomere dysfunction is less well understood. Here we report that immortalized human cell lines lacking wild-type p53 function spontaneously show many telomeres with a DNA damage response (DDR), commonly affecting only one sister chromatid and not associated with increased chromosome end-joining.

Control of telomere length by a trimming mechanism that involves generation of t-circles.

Telomere lengths are maintained in many cancer cells by the ribonucleoprotein enzyme telomerase but can be further elongated by increasing telomerase activity through the overexpression of telomerase components. We report here that increased telomerase activity results in increased telomere length that eventually reaches a plateau, accompanied by the generation of telomere length heterogeneity and the accumulation of extrachromosomal telomeric repeat DNA, principally in the form of telomeric circles (t-circles). Telomeric DNA was observed in promyelocytic leukemia bodies, but no intertelomeric copying or telomere exchange events were identified, and there was no increase in telomere dysfunction-induced foci.

Telomere elongation involves intra-molecular DNA replication in cells utilizing alternative lengthening of telomeres.

Alternative lengthening of telomeres (ALT) is a telomere length maintenance mechanism based on recombination, where telomeres use other telomeric DNA as a template for DNA synthesis. About 10% of all human tumors depend on ALT for their continued growth, and understanding its molecular details is critically important for the development of cancer treatments that target this mechanism. We have previously shown that telomeres of ALT-positive human cells can become lengthened via inter-telomeric copying, i.

Amplification of telomerase reverse transcriptase gene in human mammary epithelial cells with limiting telomerase RNA expression levels.

Activation of telomerase is a crucial step during cellular immortalization, and in some tumors this results from amplification of the human telomerase reverse transcriptase (hTERT) gene. Immortalization of normal human cells has been achieved by transduction with hTERT cDNA under the control of a strong heterologous enhancer/promoter, but this is sometimes an inefficient process, with periods of poor growth or even crisis occurring before immortalization. Here, we showed that normal human mammary epithelial cells expressing exogenous hTERT amplified the transgene extensively and expressed high levels of hTERT mRNA and protein.

Disruption of telomere maintenance by depletion of the MRE11/RAD50/NBS1 complex in cells that use alternative lengthening of telomeres.

Immortalized human cells are able to maintain their telomeres by telomerase or by a recombination-mediated DNA replication mechanism known as alternative lengthening of telomeres (ALT). We showed previously that overexpression of Sp100 protein can suppress ALT and that this was associated with sequestration of the MRE11/RAD50/NBS1 (MRN) recombination protein complex by Sp100. In the present study, we determined whether MRN proteins are required for ALT activity.

Suppression of alternative lengthening of telomeres by Sp100-mediated sequestration of the MRE11/RAD50/NBS1 complex.

Approximately 10% of cancers overall use alternative lengthening of telomeres (ALT) instead of telomerase to prevent telomere shortening, and ALT is especially common in astrocytomas and various types of sarcomas. The hallmarks of ALT in telomerase-negative cancer cells include a unique pattern of telomere length heterogeneity, rapid changes in individual telomere lengths, and the presence of ALT-associated promyelocytic leukemia bodies (APBs) containing telomeric DNA and proteins involved in telomere binding, DNA replication, and recombination. The ALT mechanism appears to involve recombination-mediated DNA replication, but the molecular details are largely unknown.

Alterations in the p16(INK4a) and p53 tumor suppressor genes of hTERT-immortalized human fibroblasts.

Exogenous expression of the catalytic subunit of telomerase, hTERT, in a normal human foreskin fibroblast cell strain resulted in telomerase activity and an extended proliferative lifespan prior to a period of crisis. Three immortalized cell lines with stably maintained telomere lengths were established from cells that escaped crisis. Each of these cultures underwent a significant downregulation of p16(INK4a) expression due to gene deletion events.

Alternative lengthening of telomeres in mammalian cells.

Some immortalized mammalian cell lines and tumors maintain or increase the overall length of their telomeres in the absence of telomerase activity by one or more mechanisms referred to as alternative lengthening of telomeres (ALT). Characteristics of human ALT cells include great heterogeneity of telomere size (ranging from undetectable to abnormally long) within individual cells, and ALT-associated PML bodies (APBs) that contain extrachromosomal telomeric DNA, telomere-specific binding proteins, and proteins involved in DNA recombination and replication. Activation of ALT during immortalization involves recessive mutations in genes that are as yet unidentified.

Comparison of human mammary epithelial cells immortalized by simian virus 40 T-Antigen or by the telomerase catalytic subunit.

We directly compared two methods of immortalizing human mammary epithelial cells (HMECs). Cells were transfected with an expression plasmid either for hTERT, the catalytic subunit of telomerase, or for the simian virus 40 (SV40) early region genes. Under standard culture conditions, HMECs were not immortalized by hTERT unless they had spontaneously ceased expression of the p16(INK4a) tumor suppressor gene.