John P. Murnane

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.

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.

Characterization of a human gene with sequence homology to Saccharomyces cerevisiae SIR2.

The proteins encoded by the SIR1, SIR2, SIR3 and SIR4 genes in yeast repress transcription at the mating type loci and telomeres. Among the SIR genes, SIR2 is the most evolutionarily conserved, and a number of genes with homology to SIR2 have been identified. In addition to transcriptional silencing, the product of SIR2 gene (Sir2p) has been shown to be involved in DNA repair and suppression of rDNA recombination. In the present study, the complete sequence of a human gene, SIR2L, with homology to the yeast SIR2 gene is presented. Comparison of the predicted sequence of the protein encoded by the SIR2L gene (SIR2Lp) with Sir2p or other proteins with homology to Sir2p reveals 20-33% overall identity and four highly conserved regions, the significance of which is unknown. SIR2L codes for a 2.1kb transcript which is expressed in various human tissues. The expression level of the transcript is found to be relatively high in the heart, brain and skeletal muscle tissues and low in lung and placenta. The intracellular location of SIR2Lp was visualized by fusion to the Green Fluorescent Protein or with a FLAG-tag. The results indicate that unlike Sir2p in yeast, SIR2Lp in human cells is found primarily in the cytoplasm. Using a mammalian inducible expression system, we also observed that unlike SIR2 in yeast, overexpression of SIR2L in human cancer cells has no effect on cell growth. Thus, although the human SIR2L gene appears to be related to the yeast SIR2 gene, it does not appear to have similar functions.

The Loss of a Single Telomere Can Result in Instability of Multiple Chromosomes in a Human Tumor Cell Line

Spontaneous telomere loss has been proposed as an important mechanism for initiating the chromosome instability commonly found in cancer cells. We have previously shown that spontaneous telomere loss in a human cancer cell line initiates breakage/fusion/bridge (B/F/B) cycles that continue for many cell generations, resulting in DNA amplification and translocations on the chromosome that lost its telomere. We have now extended these studies to determine the effect of the loss of a single telomere on the stability of other chromosomes. Our study showed that telomere acquisition during B/F/B cycles occurred mainly through translocations involving either the nonreciprocal transfer or duplication of the arms of other chromosomes. Telomere acquisition also occurred through small duplications involving the subtelomeric region of the other end of the same chromosome. Although all of these mechanisms stabilized the chromosome that lost its telomere, they differed in their consequences for the stability of the genome as a whole. Telomere acquisition involving nonreciprocal translocations resulted in the loss of a telomere on the donor chromosome, which consequently underwent additional translocations, isochromosome formation, or complete loss. In contrast, telomere acquisition involving duplications stabilized the genome, although the large duplications created substantial allelic imbalances. Thus, the loss of a single telomere can generate a variety of chromosome alterations commonly associated with human cancer, not only on a chromosome that loses its telomere but also on other chromosomes. Factors promoting telomere loss are therefore likely to have an important role in generating the karyotype evolution associated with human cancer.

Chromosome Instability as a Result of Double-Strand Breaks near Telomeres in Mouse Embryonic Stem Cells

ABSTRACT 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.

Cell cycle regulation in response to DNA damage in mammalian cells: A historical perspective

Cell cycle delay has long been known to occur in mammalian cells after exposure to DNA-damaging agents. It has been hypothesized that the function of this delay is to provide additional time for repair of DNA before the cell enters critical periods of the cell cycle, such as DNA synthesis in S phase or chromosome condensation in G2 phase. Recent evidence that p53 protein is involved in the delay in G1 in response to ionizing radiation has heightened interest in the importance of cell cycle delay, because mutations in p53 are commonly found in human cancer cells. Because mammalian cells defective in p53 protein show increased genomic instability, it is tempting to speculate that the instability is due to increased chromosome damage resulting from the lack of a G1 delay. Although this appears at first glance to be a highly plausible explanation, a review of the research performed on cell cycle regulation and DNA damage in mammalian cells provides little evidence to support this hypothesis. Studies involving cells treated with caffeine, cells from humans with the genetic disease ataxia telangiectasia, and cells that are deficient in p53 show no correlation between G1 delay and increased cell killing or chromosome damage in response to ionizing radiation. Instead, G1 delay appears to be only one aspect of a complex cellular response to DNA damage that also includes delays in S phase and G2 phase, apoptosis and chromosome repair. The exact mechanism of the genomic instability associated with p53, and its relationship to the failure to repair DNA before progression through the cell cycle, remains to be determined.

Telomere Position Effect and Silencing of Transgenes near Telomeres in the Mouse

ABSTRACT 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.

Telomere Loss as a Mechanism for Chromosome Instability in Human Cancer

Cancer cells commonly have a high rate of telomere loss, even when expressing telomerase, contributing to chromosome instability and tumor cell progression. This review addresses the hypothesis that this high rate of telomere loss results from a combination of four factors. The first factor is an increase in the frequency of double-strand breaks (DSB) at fragile sites in cancer cells due to replication stress. The second factor is that telomeres are fragile sites. The third factor is that subtelomeric regions are highly sensitive to DSBs, so that DSBs near telomeres have an increased probability of resulting in chromosome instability. The fourth factor is that cancer cells may be deficient in chromosome healing, the de novo addition of telomeres to the sites of DSBs, a mechanism that prevents chromosome instability resulting from DSBs near telomeres. Understanding these factors and how they influence telomere loss will provide important insights into the mechanisms of chromosome instability and the development of novel approaches for anti-cancer therapy. Cancer Res; 70(11); 4255-9. (c)2010 AACR.

Establishment and characterization of a permanent pSV ori−-transformed ataxia-telangiectasia cell line

A permanent ataxia-telangiectasia (A-T) cell line has been established from the fibroblast strain AT2SF after transfection with the bacterial plasmid pSV ori-, which contains replication origin-defective SV40 sequences. The original transfection frequency, as measured by transformed foci, was markedly reduced in two A-T strains when compared with either normal or xeroderma pigmentosum fibroblasts. As with SV40 virion-transformed fibroblasts, pSV ori--transformed cells entered a crisis phase, from which about one-fourth of the original clones from A-T and normal fibroblasts recovered. Both the pSV ori--transformed TAT2SF cell line and an SV40 virion-transformed AT5BI (GM5489) cell line retained their characteristic sensitivity to the lethal effects of ionizing radiation, as well as their X ray-resistant DNA synthesis. Southern blot analysis of cellular SV40 sequences demonstrated a single major integration site of pSV ori- in the AT2SF cells. In contrast, AT5BI cells transformed with SV40 virions demonstrated a high degree of heterogeneity of integrated viral sequences. Neither the TAT2SF nor the GM5489 transformed cell line contains any detectable freely replicating SV40 viral sequences, which are seen in many other semipermissive SV40-transformed cells.

Telomere instability in a human tumor cell line expressing a dominant-negative WRN protein

Werner Syndrome (WS) is an autosomal recessive disease characterized by premature aging and chromosome instability. The protein involved in WS, WRN, is a RecQ-type helicase that also has exonuclease activity. WRN has been demonstrated to bind to a variety of other proteins, including RPA, DNA-PKcs, and TRF2, suggesting that WRN is involved in DNA replication, repair, recombination, and telomere maintenance. In culture, WS cells show premature senescence, which can be overcome by transfection with an expression vector containing the gene for the catalytic subunit of telomerase. However, telomerase expression does not eliminate chromosome instability in WS cells, which led to the proposal that telomere loss is not the cause of the high rate of chromosome rearrangements in WS cells. In the present study, we have investigated how a WRN protein containing a dominant-negative mutation (K577M-WRN) influences the stability of telomeres in a human tumor cell line expressing telomerase. The results demonstrate an increased rate of telomere loss and chromosome fusion in cells expressing K577M-WRN. Expression of K577M-WRN results in reduced levels of telomerase activity, however, the absence of detectable changes in average telomere length demonstrates that WRN-associated telomere loss results from stochastic events involving complete telomere loss or loss of telomere capping function. Thus, telomere loss can contribute to chromosome instability in cells deficient in WRN regardless of the expression of telomerase activity.