Processing by MRE11 is involved in the sensitivity of subtelomeric regions to DNA double-strand breaks.

The caps on the ends of chromosomes, called telomeres, keep the ends of chromosomes from appearing as DNA double-strand breaks (DSBs) and prevent chromosome fusion. However, subtelomeric regions are sensitive to DSBs, which in normal cells is responsible for ionizing radiation-induced cell senescence and protection against oncogene-induced replication stress, but promotes chromosome instability in cancer cells that lack cell cycle checkpoints. We have previously reported that I-SceI endonuclease-induced DSBs near telomeres in a human cancer cell line are much more likely to generate large deletions and gross chromosome rearrangements (GCRs) than interstitial DSBs, but found no difference in the frequency of I-SceI-induced small deletions at interstitial and subtelomeric DSBs. We now show that inhibition of MRE11 3'-5' exonuclease activity with Mirin reduces the frequency of large deletions and GCRs at both interstitial and subtelomeric DSBs, but has little effect on the frequency of small deletions. We conclude that large deletions and GCRs are due to excessive processing of DSBs, while most small deletions occur during classical nonhomologous end joining (C-NHEJ). The sensitivity of subtelomeric regions to DSBs is therefore because they are prone to undergo excessive processing, and not because of a deficiency in C-NHEJ in subtelomeric regions.

The role of ATM in the deficiency in nonhomologous end-joining near telomeres in a human cancer cell line.

Telomeres distinguish chromosome ends from double-strand breaks (DSBs) and prevent chromosome fusion. However, telomeres can also interfere with DNA repair, as shown by a deficiency in nonhomologous end joining (NHEJ) and an increase in large deletions at telomeric DSBs. The sensitivity of telomeric regions to DSBs is important in the cellular response to ionizing radiation and oncogene-induced replication stress, either by preventing cell division in normal cells, or by promoting chromosome instability in cancer cells. We have previously proposed that the telomeric protein TRF2 causes the sensitivity of telomeric regions to DSBs, either through its inhibition of ATM, or by promoting the processing of DSBs as though they are telomeres, which is independent of ATM. Our current study addresses the mechanism responsible for the deficiency in repair of DSBs near telomeres by combining assays for large deletions, NHEJ, small deletions, and gross chromosome rearrangements (GCRs) to compare the types of events resulting from DSBs at interstitial and telomeric DSBs. Our results confirm the sensitivity of telomeric regions to DSBs by demonstrating that the frequency of GCRs is greatly increased at DSBs near telomeres and that the role of ATM in DSB repair is very different at interstitial and telomeric DSBs. Unlike at interstitial DSBs, a deficiency in ATM decreases NHEJ and small deletions at telomeric DSBs, while it increases large deletions. These results strongly suggest that ATM is functional near telomeres and is involved in end protection at telomeric DSBs, but is not required for the extensive resection at telomeric DSBs. The results support our model in which the deficiency in DSB repair near telomeres is a result of ATM-independent processing of DSBs as though they are telomeres, leading to extensive resection, telomere loss, and GCRs involving alternative NHEJ.

Replication Stress and Telomere Dysfunction Are Present in Cultured Human Embryonic Stem Cells.

Replication stress causes DNA damage at fragile sites in the genome. DNA damage at telomeres can initiate breakage-fusion-bridge cycles and chromosome instability, which can result in replicative senescence or tumor formation. Little is known about the extent of replication stress or telomere dysfunction in human embryonic stem cells (hESCs). hESCs are grown in culture with the expectation of being used therapeutically in humans, making it important to minimize the levels of replication stress and telomere dysfunction. Here, the hESC line UCSF4 was cultured in a defined medium with growth factor Activin A, exogenous nucleosides, or DNA polymerase inhibitor aphidicolin. We used quantitative fluorescence in situ hybridization to analyze individual telomeres for dysfunction and observed that it can be increased by aphidicolin or Activin A. In contrast, adding exogenous nucleosides relieved dysfunction, suggesting that telomere dysfunction results from replication stress. Whether these findings can be applied to other hESC lines remains to be determined. However, because the loss of telomeres can lead to chromosome instability and cancer, we conclude that hESCs grown in culture for future therapeutic purposes should be routinely checked for replication stress and telomere dysfunction.

Telomere dysfunction and chromosome instability.

The ends of chromosomes are composed of a short repeat sequence and associated proteins that together form a cap, called a telomere, that keeps the ends from appearing as double-strand breaks (DSBs) and prevents chromosome fusion. The loss of telomeric repeat sequences or deficiencies in telomeric proteins can result in chromosome fusion and lead to chromosome instability. The similarity between chromosome rearrangements resulting from telomere loss and those found in cancer cells implicates telomere loss as an important mechanism for the chromosome instability contributing to human cancer. Telomere loss in cancer cells can occur through gradual shortening due to insufficient telomerase, the protein that maintains telomeres. However, cancer cells often have a high rate of spontaneous telomere loss despite the expression of telomerase, which has been proposed to result from a combination of oncogene-mediated replication stress and a deficiency in DSB repair in telomeric regions. Chromosome fusion in mammalian cells primarily involves nonhomologous end joining (NHEJ), which is the major form of DSB repair. Chromosome fusion initiates chromosome instability involving breakage-fusion-bridge (B/F/B) cycles, in which dicentric chromosomes form bridges and break as the cell attempts to divide, repeating the process in subsequent cell cycles. Fusion between sister chromatids results in large inverted repeats on the end of the chromosome, which amplify further following additional B/F/B cycles. B/F/B cycles continue until the chromosome acquires a new telomere, most often by translocation of the end of another chromosome. The instability is not confined to a chromosome that loses its telomere, because the instability is transferred to the chromosome donating a translocation. Moreover, the amplified regions are unstable and form extrachromosomal DNA that can reintegrate at new locations. Knowledge concerning the factors promoting telomere loss and its consequences is therefore important for understanding chromosome instability in human cancer.

High-throughput screen to identify compounds that prevent or target telomere loss in human cancer cells.

Chromosome instability (CIN) is an early step in carcinogenesis that promotes tumor cell progression and resistance to therapy. Using plasmids integrated adjacent to telomeres, we have previously demonstrated that the sensitivity of subtelomeric regions to DNA double-strand breaks (DSBs) contributes to telomere loss and CIN in cancer. A high-throughput screen was created to identify compounds that affect telomere loss due to subtelomeric DSBs introduced by I-SceI endonuclease, as detected by cells expressing green fluorescent protein (GFP). A screen of a library of 1832 biologically-active compounds identified a variety of compounds that increase or decrease the number of GFP-positive cells following activation of I-SceI. A curated screen done in triplicate at various concentrations found that inhibition of classical nonhomologous end joining (C-NHEJ) increased DSB-induced telomere loss, demonstrating that C-NHEJ is functional in subtelomeric regions. Compounds that decreased DSB-induced telomere loss included inhibitors of mTOR, p38 and tankyrase, consistent with our earlier hypothesis that the sensitivity of subtelomeric regions to DSBs is a result of inappropriate resection during repair. Although this assay was also designed to identify compounds that selectively target cells experiencing telomere loss and/or chromosome instability, no compounds of this type were identified in the current screen.

Telomeres acquire distinct heterochromatin characteristics during siRNA-induced RNA interference in mouse cells.

Telomeres are protective structures present at the ends of linear chromosomes and consist of simple repeating-DNA sequences and specialized proteins [1, 2]. Integrity of the telomeres is important in maintaining genome stability[1-6]. RNA interference(RNAi) involves short double-stranded RNA (21-23 nucleotides long), termed short interference RNA(siRNA), resulting in the downregulation of genes with cognate sequences [7-9]. During transient siRNA-induced RNAi in mouse fibroblast cultures, we found significant reversible changes related to the telomeres. Telomeres acquired distinct heterochromatin features. There were increased bindings of Argonaute-1 (AGO1), telomeric repeat-binding factor 1(TERF1), and heterochromatin protein 1beta (HP1beta) on the telomeres. Histone H3 (lysine 9) was hypermethylated at the telomeres. The chromosome ends also were associated with an unidentified RNA. During RNAi, expression of a transgene inserted adjacent to the telomere was downregulated. In addition, the concentration of a group of heterogeneous high-molecular-weight RNA containing telomeric repeat sequences was increased, and this RNA formed a small number of transient, discrete nuclear foci. Our findings suggest that telomeres participate actively in the siRNA-induced RNAi process. These responses of telomeres to the RNAi process might partially account for the off-target effects of RNAi.

Telomeric Double Strand Breaks in G1 Human Cells Facilitate Formation of 5' C-Rich Overhangs and Recruitment of TERRA.

Telomeres, repetitive nucleoprotein complexes that protect chromosomal termini and prevent them from activating inappropriate DNA damage responses (DDRs), shorten with cell division and thus with aging. Here, we characterized the human cellular response to targeted telomeric double-strand breaks (DSBs) in telomerase-positive and telomerase-independent alternative lengthening of telomere (ALT) cells, specifically in G1 phase. Telomeric DSBs in human G1 cells elicited early signatures of a DDR; however, localization of 53BP1, an important regulator of resection at broken ends, was not observed at telomeric break sites. Consistent with this finding and previously reported repression of classical non-homologous end-joining (c-NHEJ) at telomeres, evidence for c-NHEJ was also lacking. Likewise, no evidence of homologous recombination (HR)-dependent repair of telomeric DSBs in G1 was observed. Rather, and supportive of rapid truncation events, telomeric DSBs in G1 human cells facilitated formation of extensive tracks of resected 5' C-rich telomeric single-stranded (ss)DNA, a previously proposed marker of the recombination-dependent ALT pathway. Indeed, induction of telomeric DSBs in human ALT cells resulted in significant increases in 5' C-rich (ss)telomeric DNA in G1, which rather than RPA, was bound by the complementary telomeric RNA, TERRA, presumably to protect these exposed ends so that they persist into S/G2 for telomerase-mediated or HR-dependent elongation, while also circumventing conventional repair pathways. Results demonstrate the remarkable adaptability of telomeres, and thus they have important implications for persistent telomeric DNA damage in normal human G1/G0 cells (e.g., lymphocytes), as well as for therapeutically relevant targets to improve treatment of ALT-positive tumors.

Loss of TGFß signaling increases alternative end-joining DNA repair that sensitizes to genotoxic therapies across cancer types.

Among the pleotropic roles of transforming growth factor-ß (TGFß) signaling in cancer, its impact on genomic stability is least understood. Inhibition of TGFß signaling increases use of alternative end joining (alt-EJ), an error-prone DNA repair process that typically functions as a "backup" pathway if double-strand break repair by homologous recombination or nonhomologous end joining is compromised. However, the consequences of this functional relationship on therapeutic vulnerability in human cancer remain unknown. Here, we show that TGFß broadly controls the DNA damage response and suppresses alt-EJ genes that are associated with genomic instability. Mechanistically based TGFß and alt-EJ gene expression signatures were anticorrelated in glioblastoma, squamous cell lung cancer, and serous ovarian cancer. Consistent with error-prone repair, more of the genome was altered in tumors classified as low TGFß and high alt-EJ, and the corresponding patients had better outcomes. Pan-cancer analysis of solid neoplasms revealed that alt-EJ genes were coordinately expressed and anticorrelated with TGFß competency in 16 of 17 cancer types tested. Moreover, regardless of cancer type, tumors classified as low TGFß and high alt-EJ were characterized by an insertion-deletion mutation signature containing short microhomologies and were more sensitive to genotoxic therapy. Collectively, experimental studies revealed that loss or inhibition of TGFß signaling compromises the DNA damage response, resulting in ineffective repair by alt-EJ. Translation of this mechanistic relationship into gene expression signatures identified a robust anticorrelation that predicts response to genotoxic therapies, thereby expanding the potential therapeutic scope of TGFß biology.

Isolation, nucleotide sequencing and genomic comparison of a Novel SXT/R391 ICE mobile genetic element isolated from a municipal wastewater environment.

Integrative Conjugative Elements (ICE's) of the SXT/R391 family have largely been detected in clinical or environmental isolates of Gammaproteobacteria, particularly Vibrio and Proteus species. As wastewater treatment plants accumulate a large and diverse number of such species, we examined raw water samples taken from a municipal wastewater treatment plant initially using SXT/R391 family integrase gene-specific PCR probes to detect the presence of such elements in a directed approach. A positive amplification occurred over a full year period and a subsequent Restriction Fragment Length Polymorphism (RFLP) analysis revealed a very limited diversity in the treatment plant examined. Samples demonstrating positive amplification were cultured using Vibrio and Proteus selective media and PCR amplification tracking was utilized to monitor SXT/R391-ICE family containing strains. This screening procedure resulted in the isolation and identification of a Proteus mirabilis strain harbouring an ICE. Whole-genome sequencing of this ICE containing strain using Illumina sequencing technology revealed a novel 81 kb element that contained 75 open reading frames on annotation but contained no antibiotic or metal resistance determinants. Comparative genomics revealed the element contained a conserved ICE core with one of the insertions containing a novel bacteriophage defence mechanism. This directed isolation suggests that ICE elements are present in the environment without apparent selective pressure but may contain adaptive functions allowing survival in particular environments such as municipal wastewater which are reservoirs for large bacterial phage populations.

Defining the mutation signatures of DNA polymerase θ in cancer genomes.

DNA polymerase theta (POLQ)-mediated end joining (TMEJ) is a distinct pathway for mediating DNA double-strand break (DSB) repair. TMEJ is required for the viability of BRCA-mutated cancer cells. It is crucial to identify tumors that rely on POLQ activity for DSB repair, because such tumors are defective in other DSB repair pathways and have predicted sensitivity to POLQ inhibition and to cancer therapies that produce DSBs. We define here the POLQ-associated mutation signatures in human cancers, characterized by short insertions and deletions in a specific range of microhomologies. By analyzing 82 COSMIC (Catalogue of Somatic Mutations in Cancer) signatures, we found that BRCA-mutated cancers with a higher level of POLQ expression have a greatly enhanced representation of the small insertion and deletion signature 6, as well as single base substitution signature 3. Using human cancer cells with disruptions of POLQ, we further show that TMEJ dominates end joining of two separated DSBs (distal EJ). Templated insertions with microhomology are enriched in POLQ-dependent distal EJ. The use of this signature analysis will aid in identifying tumors relying on POLQ activity.

Telomerase-dependent and -independent chromosome healing in mouse embryonic stem cells.

Telomeres play an important role in protecting the ends of chromosomes and preventing chromosome fusion. We have previously demonstrated that double-strand breaks near telomeres in mammalian cells result in either the addition of a new telomere at the site of the break, termed chromosome healing, or sister chromatid fusion that initiates chromosome instability. In the present study, we have investigated the role of telomerase in chromosome healing and the importance of chromosome healing in preventing chromosome instability. In embryonic stem cell lines that are wild type for the catalytic subunit of telomerase (TERT), chromosome healing at I-SceI-induced double-strand breaks near telomeres accounted for 22 of 35 rearrangements, with the new telomeres added directly at the site of the break in all but one instance. In contrast, in two TERT-knockout embryonic stem cell lines, chromosome healing accounted for only 1 of 62 rearrangements, with a 23 bp insertion at the site of the sole chromosome-healing event. However, in a third TERT-knockout embryonic stem cell line, 10PTKO-A, chromosome healing was a common event that accounted for 20 of 34 rearrangements. Although this chromosome healing also occurred at the I-SceI site, differences in the microhomology at the site of telomere addition demonstrated that the mechanism was distinct from that in wild-type embryonic stem cell lines. In addition, the newly added telomeres in 10PTKO-A shortened with time in culture, eventually resulting in either telomere elongation through a telomerase-independent mechanism or loss of the subtelomeric plasmid sequences entirely. The combined results demonstrate that chromosome healing can occur through both telomerase-dependent and -independent mechanisms, and that although both mechanisms can prevent degradation and sister chromatid fusion, neither mechanism is efficient enough to prevent sister chromatid fusion from occurring in many cells experiencing double-strand breaks near telomeres.

Telomere position effect and silencing of transgenes near telomeres in the mouse.

Reversible transcriptional silencing of genes located near telomeres, termed the telomere position effect (TPE), is well characterized in Saccharomyces cerevisiae. TPE has also been observed in human tumor cell lines, but its function remains unknown. To investigate TPE in normal mammalian cells, we developed clones of mouse embryonic stem (ES) cells that contain single-copy marker genes integrated adjacent to different telomeres. Analysis of these telomeric transgenes demonstrated that they were expressed at very low levels compared to the same transgenes integrated at interstitial sites. Similar to the situation in yeast, but in contrast to studies with human tumor cell lines, TPE in mouse ES cells was not reversed with trichostatin A. Prolonged culturing without selection resulted in extensive DNA methylation and complete silencing of telomeric transgenes, which could be reversed by treatment with 5-azacytidine. Thus, complete silencing of the telomeric transgenes appears to involve a two-step process in which the initial repression is reinforced by DNA methylation. Extensive methylation of the telomeric transgenes was also observed in various tissues and embryonic fibroblasts isolated from transgenic mice. In contrast, telomeric transgenes were not silenced in ES cell lines isolated from 3-day-old preimplantation embryos, consistent with the hypothesis that TPE plays a role in the development of the embryo.

Misrepair in Context: TGFß Regulation of DNA Repair.

Repair of DNA damage protects genomic integrity, which is key to tissue functional integrity. In cancer, the type and fidelity of DNA damage response is the fundamental basis for clinical response to cytotoxic therapy. Here we consider the contribution of transforming growth factor-beta (TGFß), a ubiquitous, pleotropic cytokine that is abundant in the tumor microenvironment, to therapeutic response. The action of TGFß is best illustrated in head and neck squamous cell carcinoma (HNSCC). Survival of HNSCC patients with human papilloma virus (HPV) positive cancer is more than double compared to those with HPV-negative HNSCC. Notably, HPV infection profoundly impairs TGFß signaling. HPV blockade of TGFß signaling, or pharmaceutical TGFß inhibition that phenocopies HPV infection, shifts cancer cells from error-free homologous-recombination DNA double-strand-break (DSB) repair to error-prone alternative end-joining (altEJ). Cells using altEJ are more sensitive to standard of care radiotherapy and cisplatin, and are sensitized to PARP inhibitors. Hence, HPV-positive HNSCC is an experiment of nature that provides a strong rationale for the use of TGFß inhibitors for optimal therapeutic combinations that improve patient outcome.

Telomeric transgenes are silenced in adult mouse tissues and embryo fibroblasts but are expressed in embryonic stem cells.

In addition to their role in protecting the ends of chromosomes, telomeres also influence the expression of adjacent genes, a process called telomere-position effect. We previously reported that the neo and HSV-tk transgenes located adjacent to telomeres in mouse embryonic stem cells are initially expressed at low levels and then become gradually silenced upon passage in culture through a process involving DNA methylation. We also reported extensive DNA methylation in these telomeric transgenes in three different tissues isolated from mice generated from one of these embryonic stem cell clones. In the present study, we demonstrate that embryo fibroblasts isolated from two different mouse strains show extensive DNA methylation and silencing of the telomeric transgenes. Consistent with this observation, we also demonstrate little or no detectable expression of the HSV-tk telomeric transgene in somatic tissues using whole body imaging. In contrast, both telomeric transgenes are expressed at low levels and have little DNA methylation in embryonic stem cell lines isolated from these same mouse strains. Our results demonstrate that telomere-position effect in mammalian cells can be observed either as a low level of expression in embryonic stem cells in the preimplantation embryo or as complete silencing and DNA methylation in differentiated cells and somatic tissues. This pattern of expression of the telomeric transgenes demonstrates that subtelomeric regions, like much of the genome, are epigenetically reprogrammed in the preimplantation embryo, a process that has been proposed to be important in early embryonic development. Disclosure of potential conflicts of interest is found at the end of this article.

Telomeres, chromosome instability and cancer.

Telomeres are composed of repetitive G-rich sequence and an abundance of associated proteins that together form a dynamic cap that protects chromosome ends and allows them to be distinguished from deleterious DSBs. Telomere-associated proteins also function to regulate telomerase, the ribonucleoprtotein responsible for addition of the species-specific terminal repeat sequence. Loss of telomere function is an important mechanism for the chromosome instability commonly found in cancer. Dysfunctional telomeres can result either from alterations in the telomere-associated proteins required for end-capping function, or from alterations that promote the gradual or sudden loss of sufficient repeat sequence necessary to maintain proper telomere structure. Regardless of the mechanism, loss of telomere function can result in sister chromatid fusion and prolonged breakage/fusion/bridge (B/F/B) cycles, leading to extensive DNA amplification and large terminal deletions. B/F/B cycles terminate primarily when the unstable chromosome acquires a new telomere, most often by translocation of the ends of other chromosomes, thereby providing a mechanism for transfer of instability from one chromosome to another. Thus, the loss of a single telomere can result in on-going instability, affect multiple chromosomes, and generate many of the types of rearrangements commonly associated with human cancer.

The DNA damage response at dysfunctional telomeres, and at interstitial and subtelomeric DNA double-strand breaks.

In mammals, DNA double-strand breaks (DSBs) are primarily repaired by classical non-homologous end joining (C-NHEJ), although homologous recombination repair and alternative NHEJ (A-NHEJ), which involve DSB processing, can also occur. These pathways are tightly regulated to maintain chromosome integrity. The ends of chromosomes, called telomeres, contain telomeric DNA that forms a cap structure in cooperation with telomeric proteins to prevent the activation of the DNA damage response and chromosome fusion at chromosome termini. Telomeres and subtelomeric regions are poor substrates for DNA replication; therefore, regions near telomeres are prone to replication fork stalling and chromosome breakage. Moreover, DSBs near telomeres are poorly repaired. As a result, when DSBs occur near telomeres in normal cells, the cells stop proliferating, while in cancer cells, subtelomeric DSBs induce rearrangements due to the absence of cell cycle checkpoints. The sensitivity of subtelomeric regions to DSBs is due to the improper regulation of processing, because although C-NHEJ is functional at subtelomeric DSBs, excessive processing results in an increased frequency of large deletions and chromosome rearrangements involving A-NHEJ.

Minimal climate change impacts on natural organic matter forecasted for a potable water supply in Ireland.

Natural organic matter poses an increasing challenge to water managers because of its potential adverse impacts on water treatment and distribution, and subsequently human health. Projections were made of impacts of climate change on dissolved organic carbon (DOC) in the primarily agricultural Boyne catchment which is used as a potable water supply in Ireland. The results indicated that excluding a potential rise in extreme precipitation, future projected loads are not dissimilar to those observed under current conditions. This is because projected increases in DOC concentrations are offset by corresponding decreases in precipitation and hence river flow. However, the results presented assume no changes in land use and highlight the predicted increase in DOC loads from abstracted waters at water treatment plants.

Subjugation of TGFß Signaling by Human Papilloma Virus in Head and Neck Squamous Cell Carcinoma Shifts DNA Repair from Homologous Recombination to Alternative End Joining.

PURPOSE: Following cytotoxic therapy, 70% of patients with human papillomavirus (HPV)-positive oropharyngeal head and neck squamous cell carcinoma (HNSCC) are alive at 5 years compared with 30% of those with similar HPV-negative cancer. Loss of TGFß signaling is a poorly studied consequence of HPV that could contribute to patient outcome by compromising DNA repair. EXPERIMENTAL DESIGN: Human HNSCC cell lines (n = 9), patient-derived xenografts (n = 9), tissue microarray (n = 194), TCGA expression data (n = 279), and primary tumor specimens (n = 10) were used to define the relationship between TGFß competency, response to DNA damage, and type of DNA repair. RESULTS: Analysis of HNSCC specimens in situ and in vitro showed that HPV associated with loss of TGFß signaling that increased response to radiation or cisplatin. TGFß suppressed miR-182, which inhibited both BRCA1, necessary for homologous recombination repair (HRR), and FOXO3, required for ATM kinase activity. TGFß signaling blockade by either HPV or inhibitors released miR182 control, compromised HRR and increased response to PARP inhibition. Antagonizing miR-182 rescued the HRR deficit in HPV-positive cells. Loss of TGFß signaling unexpectedly increased repair by error prone, alternative end-joining (alt-EJ). CONCLUSIONS: HPV-positive HNSCC cells are unresponsive to TGFß. Abrogated TGFß signaling compromises repair by HRR and increases reliance on alt-EJ, which provides a mechanistic basis for sensitivity to PARP inhibitors. The effect of HPV in HNSCC provides critical validation of TGFß's role in DNA repair proficiency and further raises the translational potential of TGFß inhibitors in cancer therapy.

Telomeres and chromosome instability.

Genomic instability has been proposed to play an important role in cancer by accelerating the accumulation of genetic changes responsible for cancer cell evolution. One mechanism for chromosome instability is through the loss of telomeres, which are DNA-protein complexes that protect the ends of chromosomes and prevent chromosome fusion. Telomere loss can occur as a result of exogenous DNA damage, or spontaneously in cancer cells that commonly have a high rate of telomere loss. Mouse embryonic stem cells and human tumor cell lines that contain a selectable marker gene located immediately adjacent to a telomere have been used to investigate the consequences of telomere loss. In both cell types, telomere loss is followed by either the addition of a new telomere on to the end of the broken chromosome, or sister chromatid fusion and prolonged breakage/fusion/bridge (B/F/B) cycles that result in DNA amplification and large terminal deletions. The regions amplified by B/F/B cycles can then be transferred to other chromosomes, either through the formation of double-minute chromosomes that reintegrate at other sites, or through end-to-end fusions between chromosomes. B/F/B cycles eventually end when a chromosome acquires a new telomere by one of several mechanisms, the most common of which is translocation, which can involve either nonreciprocal transfer or duplication of all or part of an arm of another chromosome. Telomere acquisition involving nonreciprocal translocations results in the loss of a telomere on the donor chromosome, which subsequently becomes unstable. In contrast, translocations involving duplications do not destabilize the donor chromosome, although they result in allelic imbalances. Thus, the loss of a single telomere can generate a wide variety of chromosome alterations commonly associated with human cancer, not only on the chromosome that originally lost its telomere, but other chromosomes as well. Factors promoting spontaneous telomere loss and the resulting B/F/B cycles are therefore likely to be important in generating the karyotypic changes associated with human cancer.

Chromosome instability as a result of double-strand breaks near telomeres in mouse embryonic stem cells.

Telomeres are essential for protecting the ends of chromosomes and preventing chromosome fusion. Telomere loss has been proposed to play an important role in the chromosomal rearrangements associated with tumorigenesis. To determine the relationship between telomere loss and chromosome instability in mammalian cells, we investigated the events resulting from the introduction of a double-strand break near a telomere with I-SceI endonuclease in mouse embryonic stem cells. The inactivation of a selectable marker gene adjacent to a telomere as a result of the I-SceI-induced double-strand break involved either the addition of a telomere at the site of the break or the formation of inverted repeats and large tandem duplications on the end of the chromosome. Nucleotide sequence analysis demonstrated large deletions and little or no complementarity at the recombination sites involved in the formation of the inverted repeats. The formation of inverted repeats was followed by a period of chromosome instability, characterized by amplification of the subtelomeric region, translocation of chromosomal fragments onto the end of the chromosome, and the formation of dicentric chromosomes. Despite this heterogeneity, the rearranged chromosomes eventually acquired telomeres and were stable in most of the cells in the population at the time of analysis. Our observations are consistent with a model in which broken chromosomes that do not regain a telomere undergo sister chromatid fusion involving nonhomologous end joining. Sister chromatid fusion is followed by chromosome instability resulting from breakage-fusion-bridge cycles involving the sister chromatids and rearrangements with other chromosomes. This process results in highly rearranged chromosomes that eventually become stable through the addition of a telomere onto the broken end. We have observed similar events after spontaneous telomere loss in a human tumor cell line, suggesting that chromosome instability resulting from telomere loss plays a role in chromosomal rearrangements associated with tumor cell progression.