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Telomeres are the end of linear chromosomes, responsible for chromosome stability and cell viability. It is well known that radiations are able to induce chromosome instability but it has not yet been investigated whether telomere structure is affected by the radiation exposure and if radiations with different quality act in a different way on telomeres. The effect of radiations with different quality on telomere structure and chromosome instability was analysed in human primary fibroblasts exposed to X rays or low-energy protons (28.5 keV μm(-1)). Telomere length was evaluated at different harvesting times from 24 h up to 360 h (15 days), whereas chromosome instability was evaluated in terms of sister chromatid exchanges (SCEs) (48 h from irradiation) and chromosome painting (360 h from irradiation). Results indicated a delayed telomere lengthening 360 h after X-ray treatment, whereas protons were able to induce such a lengthening shortly from irradiation as well as at longer harvesting times. Data obtained from chromosome instability analysis indicated an increase of SCE frequency only after proton irradiation, but, on the contrary, at the longer harvesting time chromosome painting analysis displayed a higher frequency of aberrations after X-ray treatment, suggesting a role of selective process against highly damaged cells.

To achieve a more complete understanding of the molecular mechanisms underlying tumor radioresistance, we established a radioresistant cell line from the human larynx squamous cell carcinoma cell line Hep-2 after long-term radiation induction. The biological features of the resulting cell lines were characterized. cDNA microarray technology was used to measure the alterations of gene expression in the radioresistant cells. We found that certain genes associated with DNA repair, cell cycle, apoptosis, etc. were significantly changed. In particular, genes related to telomeres, such as POT1, were significantly altered. Radioresistant cells had higher telomerase activity and longer telomeres than their parental cells. Our research suggests that telomere function is a novel hallmark of cellular radiosensitivity, and the mechanistic link between telomere maintenance and radiosensitivity may involve the genes and pathways we implicated in this study.

It is well established that high-LET radiations efficiently induce chromosome aberrations. However, data on the effect of protons on telomere maintenance, as involved in genomic stability, are scarce and contradictory. Here we demonstrate that high-LET protons induce telomere lengthening in human primary fibroblasts and that this elongation does not involve the telomerase enzyme, supporting the hypothesis that high-LET radiations are able to activate a telomerase-independent mechanism. In tumor cells that lack telomerase, one or more non-telomerase mechanisms for telomere maintenance are present, which are termed alternative lengthening of telomeres (ALT). Since ALT cells are characterized by recombinational events at telomeres, known as telomeric-sister chromatid exchanges (T-SCE), and colocalization of telomeres and premyelocytic leukemia protein (PML), we analyzed both T-SCE and PML. Our results show that high-LET protons induce a 2.5-fold increase of T-SCE and a colocalization of PML protein and telomeric DNA. Furthermore, our data show that the ALT pathway can be activated in human primary cells after induction of severe DNA damage. Thus, since telomeres are known to be involved in chromosome maintenance, the present work may contribute in the elucidation of the mechanism by which ionizing radiation induces genomic instability.

To examine the correlation between spontaneous or radiation-induced apoptosis and telomere shortening, G(0) human peripheral blood lymphocytes were irradiated with X-rays and analyzed for viability, apoptosis, and telomere length. Part of the lymphocytes was kept under liquid-holding conditions for 48 h, and then loaded onto Ficoll-Paque medium to separate apoptotic (high-density) from normal (normal-density) cells. Then all samples were examined for the same three end-points. To determine whether expression of p53 influences the telomere shortening associated with a spontaneous or radiation-induced apoptotic process, the lymphocytes were also analyzed for expression of p53 at 0 and 48 h recovery times (non-irradiated and irradiated samples) and after 2 weeks in liquid-holding conditions (non-irradiated sample). After 48 h in liquid-holding, the p53-dependent apoptotic lymphocytes in the irradiated cultures presented shortened telomeres. After a 2-week recovery time, non-irradiated cells showed a p53-dependent spontaneous apoptosis, but no telomere shortening. These results demonstrate that radiation-induced apoptosis correlates with shortened telomeres in G(0) human lymphocytes. Spontaneous and radiation-induced apoptosis are dependent on expression of p53. In contrast, p53 may not play an effective role in telomere shortening, because spontaneous apoptosis did not correlate with telomere shortening. As most tumours are compromised with respect to p53 function, our findings on the role of p53 in telomere shortening may prove critical for applying therapeutic modalities in the clinic, and may facilitate the design of agents that selectively disrupt telomere integrity in tumour cells., (2010 Elsevier B.V. All rights reserved.)

Objective: To investigate the mechanism of telomere shortening through 8-methoxypsoralen (8-MOP) and subsequent ultraviolet A (UVA) irradiation-induced photoaging model in human dermal fibroblasts (HDFs)., Methods: Photoaging model was established by 8-MOP + UVA in skin HDFs. Flow cytometer, enzyme cytochemistry, immunofluorescence, Western blot and Real-time PCR were employed., Results: The percentage of G1 blockage of 8-MOP + UVA group were higher than that of control group at 24, 48, 72 h and 7 d (61.4% +/- 1.5% vs. 32.8% +/- 1.5%, 69.5% +/- 2.2% vs. 44.9% +/- 2.3%, 88.2% +/- 1.6% vs. 59.8% +/- 1.4%, 90.7% +/- 2.5% vs. 68.5% +/- 2.6%, all P < 0.01). The expression of SA-beta-Gal of 8-MOP + UVA group were higher than that of control group at 24, 48, 72 h and 7 d (34.87% +/- 0.59% vs. 7.11% +/- 0.78%, 59.38% +/- 0.46% vs. 10.57% +/- 0.47%, 72.46% +/- 0.98% vs. 11.67% +/- 0.87%, 94.33% +/- 0.13% vs. 12.04% +/- 0.12%, all P < 0.01). 8-MOP + UVA treatment could significantly aggravate the oxidative DNA damages, the percentage of 8-oxo-dG positive cell of 8-MOP + UVA group (95.78% +/- 0.14%) were significantly higher than that of control group (7.69% +/- 0.09%, P < 0.01), 8-MOP group (9.76% +/- 0.11%, P < 0.01) and UVA group (35.29% +/- 0.14%, P < 0.05). 8-MOP + UVA treatment could accelerate the telomere shortening ,the relative length of telomere of 8-MOP + UVA group were 2.57 +/- 0.05 lower than that of control group (6.63 +/- 0.12, P < 0.01). The levels of P53, P21(WAF-1) and P16(INK-4a) of 8-MOP + UVA group were higher than that of control group (3.00 +/- 0.88 vs. 0.54 +/- 0.10, 2.50 +/- 0.51 vs. 0.42 +/- 0.06, 2.21 +/- 0.34 vs. 0.38 +/- 0.05, all P < 0.01)., Conclusion: 8-MOP + UVA-induced photoaging of HDFs can be mediated though the regulation of telomere and subsequent P53-dependent signaling pathways.

Telomeric repeats preserve genome integrity by stabilizing chromosomes, a function that appears to be important for both cancer and aging. In view of this critical role in genomic integrity, the telomere's own integrity should be of paramount importance to the cell. Ultraviolet light (UV), the preeminent risk factor in skin cancer development, induces mainly cyclobutane pyrimidine dimers (CPD) which are both mutagenic and lethal. The human telomeric repeat unit (5'TTAGGG/CCCTAA3') is nearly optimal for acquiring UV-induced CPD, which form at dipyrimidine sites. We developed a ChIP-based technique, immunoprecipitation of DNA damage (IPoD), to simultaneously study DNA damage and repair in the telomere and in the coding regions of p53, 28S rDNA, and mitochondrial DNA. We find that human telomeres in vivo are 7-fold hypersensitive to UV-induced DNA damage. In double-stranded oligonucleotides, this hypersensitivity is a property of both telomeric and non-telomeric repeats; in a series of telomeric repeat oligonucleotides, a phase change conferring UV-sensitivity occurs above 4 repeats. Furthermore, CPD removal in the telomere is almost absent, matching the rate in mitochondria known to lack nucleotide excision repair. Cells containing persistent high levels of telomeric CPDs nevertheless proliferate, and chronic UV irradiation of cells does not accelerate telomere shortening. Telomeres are therefore unique in at least three respects: their biophysical UV sensitivity, their prevention of excision repair, and their tolerance of unrepaired lesions. Utilizing a lesion-tolerance strategy rather than repair would prevent double-strand breaks at closely-opposed excision repair sites on opposite strands of a damage-hypersensitive repeat., Competing Interests: The authors have declared that no competing interests exist.

Iodine-125 radioprobing is based on incorporation of radioiodine into a defined position in a nucleic acid molecule. Decay of (125)I results in the emission of multiple, low-energy Auger electrons that, along with positively charged residual daughter nuclide, produce DNA strand breaks. The probability of such strand breaks at a given nucleotide is in inverse proportion to the distance from the (125)I atom to the sugar of that nucleotide. Therefore, conclusions can be drawn about the conformation or folding of a DNA or RNA molecule based on the distribution of (125)I decay-induced strand breaks. Here we describe in detail the application (125)I radioprobing for studying the conformation of quadruplex structures, and discuss the advantages and limitations of the method.

Epidemiological studies have demonstrated age differences among human adults in susceptibility to radiation, with cancer cases attributable to radiation being more frequent for older individuals at time of exposure. In addition to the notion that susceptibility increases because of progressive decline in DNA monitoring and immunosurveillance, telomere function is now emerging as a new and important factor in modulating cellular and organism sensitivity to ionizing radiation. The link between telomeres and radiosensitivity is well-documented in humans, but the causal events remain elusive. In this paper, it is shown that irradiated human epithelial cells with short dysfunctional telomeres derived from normal mammary gland display elevated DNA damage. An approach identifying the specific chromosomes with critically shortened telomeres in each donor has allowed us to conclude that short dysfunctional telomeres in human epithelial cells join radiation-induced DNA broken ends, thus interfering with their efficient repair. These findings argue against telomeres participating as sensors or transducers of DNA damage, as previously suggested. Rather, our current findings give support to the idea that dysfunctional telomeres, by acting as an additional joining option, reduce the repair fidelity of DNA broken-ends induced by radiation throughout the genome. In the mammary gland, age-dependent telomere attrition due to epithelial turnover, together with the accretion of checkpoint deficiencies, might render the accumulation of short dysfunctional telomeres. This implies that the risks associated with mammography screening could be higher than previously assumed. Our results have the possibility of imprinting a temporal dimension onto radiation sensitivity, namely, that shortened telomeres in aged cells may more easily compromise normal tissue function in the elderly.

UV-induced melanogenesis (tanning) and "premature aging" or photoaging result in large part from DNA damage. This article reviews data tying both phenomena to telomere-based DNA damage signaling and develops a conceptual framework in which both responses may be understood as cancer-avoidance protective mechanisms.Journal of Investigative Dermatology Symposium Proceedings (2009) 14, 25-31; doi:10.1038/jidsymp.2009.9.

Poly(ADP-ribosyl)ation is a post-translational modification of proteins involved in the regulation of chromatin structure, DNA metabolism, cell division and cell death. Through the hydrolysis of poly(ADP-ribose) (PAR), Poly(ADP-ribose) glycohydrolase (PARG) has a crucial role in the control of life-and-death balance following DNA insult. Comprehension of PARG function has been hindered by the existence of many PARG isoforms encoded by a single gene and displaying various subcellular localizations. To gain insight into the function of PARG in response to irradiation, we constitutively and stably knocked down expression of PARG isoforms in HeLa cells. PARG depletion leading to PAR accumulation was not deleterious to undamaged cells and was in fact rather beneficial, because it protected cells from spontaneous single-strand breaks and telomeric abnormalities. By contrast, PARG-deficient cells showed increased radiosensitivity, caused by defects in the repair of single- and double-strand breaks and in mitotic spindle checkpoint, leading to alteration of progression of mitosis. Irradiated PARG-deficient cells displayed centrosome amplification leading to mitotic supernumerary spindle poles, and accumulated aberrant mitotic figures, which induced either polyploidy or cell death by mitotic catastrophe. Our results suggest that PARG could be a novel potential therapeutic target for radiotherapy.

Purpose: A repeated, non-coding, DNA sequence d(TTAGGG)(n) is present in the telomeric ends of all human chromosomes. These repeats can adopt multiple inter- and intra-molecular non-B-DNA conformations that may play an important role in biological processes. We applied (125)I -radioprobing to assess the conformation of the human telomeric DNA fragment in a complex with the quadruplex-specific drug - cationic porphyrin TMPyP4., Material and Methods: Synthetic DNA oligonucleotides containing the telomeric sequence were labeled with (125)I. The probability of DNA breaks caused by decay of (125)I is inversely related to the distance between the radionuclide and the sugar unit of the DNA backbone; hence, the conformation of the DNA backbone can be deduced from the distribution of breaks., Results: The obtained data indicate that the telomeric oligonucleotides predominantly fold into an intramolecular quadruplex conformation in the presence of TMPyP4. We propose a mixed-type (3 + 1) conformation of telomeric quadruplex in a complex with the cationic porphyrin TMPyP4 in solution. Binding of the porphyrin overrides the counterion effect on quadruplex conformation., Conclusions: We have demonstrated that (125)I radioprobing can be successfully applied not only to determine folding in G-quadruplexes, but also to reveal the mode of quadruplex interaction with small ligands.

Treatment of malignancies with chemotherapeutic drugs and/or radiotherapy is designed to eliminate the disease by depriving the tumor cell of its reproductive potential. Frequently, the desired effect of cell killing is achieved through the promotion of apoptosis; however, accumulating evidence suggests that apoptosis may not be the exclusive or even primary mechanism whereby tumor cells lose their self-renewal capacity after radiation or drug treatment, particularly in the case of solid tumors. While failure to undergo apoptosis in response to chemotherapeutic drugs or radiation may represent a mechanism of drug and radiation resistance, particularly in the case of leukemias and lymphomas, it is gradually being recognized that in the case of solid tumors, loss of reproductive capacity can occur through alternative pathways including reproductive cell death or mitotic catastrophe, through autophagic cell death, and as described below, through a terminally arrested state similar to replicative senescence. Studies building upon the phenomenon of replicative senescence in normal cells approaching the limit of their reproductive potential have identified a comparable senescence-like arrest as a component of the tumor cell response to chemotherapeutic drugs and radiation. This response, which has been termed "premature senescence", "senescence-like growth arrest", "stress-induced premature senescence", and "accelerated senescence", can also result from supraphysiological mitogenic signaling, sub-optimal culture conditions, and ectopic expression of oncogenes. Here, we will use the term "accelerated senescence" in our consideration of the morphological, biochemical, and molecular aspects of treatment-induced senescence, its relationship to classical replicative senescence, its prevalence in clinical specimens and the implications of accelerated senescence for the outcome of cancer therapy.

Telomeres, the ends of eukaryotic chromosomes, have a variable length among individuals and cell types. While studies in telomerase-deficient mice and cells showed an inverse correlation between telomere length and radiosensitivity, it is less clear whether this remains true in telomerase-proficient cells. To gain insight into this topic, we studied radiosensitivity in three telomerase immortalized fibroblast clones derived from the same cell line and characterized by different telomere length. In two clones, cen3tel4 and cen3tel5, the mean terminal restriction fragment length was approximately 13 and 10 kb, respectively and in the third clone, cen3pci16, it was approximately 4 kb, which is lower than in senescent fibroblasts. To test radiosensitivity, we determined survival to gamma-rays and the induction of chromosomal aberrations after irradiation. Neither the LD50, the gamma-ray dose that reduces survival to 50%, nor the frequency of aberrations detected in the three cell lines showed an inverse correlation with telomere length. In particular, the cen3pci16 cells, which have very short telomeres, did not show a higher sensitivity to irradiation or a greater frequency of chromosomal abnormalities compared to the other two cell lines. Our results suggest that, in the presence of telomerase activity, short telomeres are stabilized and do not cause an increase in radiosensitivity.

A relationship between telomeres and radiosensitivity has been established by several studies based on non-mammalian model systems, mouse models, and few human genetic diseases. However, the relationship has not been proven in human carcinoma cells, which have more clinical significance than these other models. The present study aims to determine whether telomere length is related to radiosensitivity in human carcinoma cells, and to examine the influence of tissue or genetic background. Two HEp-2 larynx squamous carcinoma cell lines, eight hepatocellular carcinoma cell lines, and five breast cancer cell lines were used. Telomere length was determined by terminal restriction fragment (TRF) Southern blot analysis and cell survival was measured by a colony-forming assay. Our results indicated that there was a significant negative correlation of telomere length and radiosensitivity in the same tissue-derived cell lines, with or without the same genetic background. Thus, telomere length may be used as a promising tool to predict the radiosensitivity of human carcinomas.

Genomic stability is maintained by telomeres, the end terminal structures that protect chromosomes from fusion or degradation. Shortening or loss of telomeric repeats or altered telomere chromatin structure is correlated with telomere dysfunction such as chromosome end-to-end associations that could lead to genomic instability and gene amplification. The structure at the end of telomeres is such that its DNA differs from DNA double strand breaks (DSBs) to avoid nonhomologous end-joining (NHEJ), which is accomplished by forming a unique higher order nucleoprotein structure. Telomeres are attached to the nuclear matrix and have a unique chromatin structure. Whether this special structure is maintained by specific chromatin changes is yet to be thoroughly investigated. Chromatin modifications implicated in transcriptional regulation are thought to be the result of a code on the histone proteins (histone code). This code, involving phosphorylation, acetylation, methylation, ubiquitylation, and sumoylation of histones, is believed to regulate chromatin accessibility either by disrupting chromatin contacts or by recruiting non-histone proteins to chromatin. The histone code in which distinct histone tail-protein interactions promote engagement may be the deciding factor for choosing specific DSB repair pathways. Recent evidence suggests that such mechanisms are involved in DNA damage detection and repair. Altered telomere chromatin structure has been linked to defective DNA damage response (DDR), and eukaryotic cells have evolved DDR mechanisms utilizing proficient DNA repair and cell cycle checkpoints in order to maintain genomic stability. Recent studies suggest that chromatin modifying factors play a critical role in the maintenance of genomic stability. This review will summarize the role of DNA damage repair proteins specifically ataxia-telangiectasia mutated (ATM) and its effectors and the telomere complex in maintaining genome stability., (Copyright 2008 S. Karger AG, Basel.)

Telomeres are the very ends of the chromosomes. They can be seen as natural double-strand breaks (DSB), specialized structures which prevent DSB repair and activation of DNA damage checkpoints. In somatic cells, attrition of telomeres occurs after each cell division until replicative senescence. In the absence of telomerase, telomeres shorten due to incomplete replication of the lagging strand at the very end of chromosome termini. Moreover, oxidative stress and accumulating reactive oxygen species (ROS) lead to an increased telomere shortening due to a less efficient repair of SSB in telomeres. The specialized structures at telomeres include proteins involved in both telomere maintenance and DNA repair. However when a telomere is damaged and has to be repaired, those proteins might fail to perform an accurate repair of the damage. This is the starting point of this article in which we first summarize the well-established relationships between DNA repair processes and maintenance of functional telomeres. We then examine how damaged telomeres would be processed, and show that irradiation alters telomere maintenance leading to possibly dramatic consequences. Our point is to suggest that those consequences are not restricted to the short term effects such as increased radiation-induced cell death. On the contrary, we postulate that the major impact of the loss of telomere integrity might occur in the long term, during multistep carcinogenesis. Its major role would be to act as an amplificator event unmasking in one single step recessive radiation-induced mutations among thousands of genes and providing cellular proliferative advantage. Moreover, the chromosomal instability generated by damaged telomeres will favour each step of the transformation from normal to fully transformed cells.

The photophysical properties of stacked G-tetrads in diverse systems, including concentrated solutions of 5'-guanosine monophosphate (5'-GMP), polyguanylic acid (poly(G)) and the G-rich oligodeoxynucleotide sequence characteristic of human telomeric DNA, are probed by ps-TRIR and compared to those of the monomeric 5'-GMP.

Telomeres were first recognized as a bona fide constituent of the chromosome based on their inability to rejoin with broken chromosome ends produced by radiation. Today, we recognize two essential and interrelated properties of telomeres. They circumvent the so-called end-replication problem faced by genomes composed of linear chromosomes, which erode from their termini with each successive cell division. Equally vital is the end-capping function that telomeres provide, which is necessary to deter chromosome ends from illicit recombination. This latter property is critical in facilitating the distinction between the naturally occurring DNA double-strand breaks (DSBs) found at chromosome ends (i.e., telomeres) and DSBs produced by exogenous agents. Here we discuss, in a brief historical narrative, key discoveries that led investigators to appreciate the unique properties of telomeres in protecting chromosome ends, and the consequences of telomere dysfunction, particularly as related to recombination involving radiation-induced DSBs.

Purpose: To investigate the link between radiosensitivity and telomere length in murine lymphoid cell line stocks that have similar genetic backgrounds but different radiosensitivities., Materials and Methods: We used two stocks from both the parental L5,178Y-R cell line and the repair-deficient radiosensitive subline, L5,178Y-S, to assess telomere length. We used terminal restriction fragment analysis and flow-fluorescence in situ hybridization (FISH) telomere length assessment to determine telomere lengths in the related radiosensitive and non-radiosensitive cell lines. Each cell line was further tested for retention of its original radiation response phenotype using cell growth assays after treatment with ionizing radiation., Results: One stock of L5,178Y-R cells had long telomeres, whereas the other stock had short telomeres. Likewise, one stock of L5,178Y-S cells had long telomeres, whereas the other stock had short telomeres. Telomere lengths in these cell lines were relatively stable for over 80 divisions in culture. Each cell line was confirmed to have retained its original radiosensitivity phenotype., Conclusion: We conclude that radiosensitivity is independent of telomere length in these genetically similar cell lines.

Background: Previous studies have demonstrated that telomeres in somatic cells are not randomly distributed at the end of the chromosomes. We hypothesize that these chromosome arm specific differences in telomere length (the telomere length pattern) may be actively maintained. In this study we investigate the existence and maintenance of the telomere length pattern in stem cells. For this aim we studied telomere length in primary human mesenchymal stem cells (hMSC) and their telomerase-immortalised counterpart (hMSC-telo1) during extended proliferation as well as after irradiation. Telomere lengths were measured using Fluorescence In Situ Hybridization (Q-FISH)., Results: A telomere length pattern was found to exist in primary hMSC's as well as in hMSC-telo1. This pattern is similar to what was previously found in lymphocytes and fibroblasts. The cells were then exposed to a high dose of ionizing radiation. Irradiation caused profound changes in chromosome specific telomere lengths, effectively destroying the telomere length pattern. Following long term culturing after irradiation, a telomere length pattern was found to re-emerge. However, the new telomere length pattern did not resemble the telomere length pattern observed before irradiation., Conclusion: Our findings indicate that a telomere length pattern does exist in mesenchymal stem cells and that the pattern is not actively re-established after destruction by irradiation.

During the past several years increasing evidence indicating that the proliferation capacity of mammalian cells is highly radiosensitive, regardless of the species and the tissue of origin of the cells, has accumulated. It has also been shown that normal bone marrow cells of mice have a similar radiosensitivity to other mammalian cells so far tested. In this study, we investigated the genetic effects of ionizing radiation (2.5-15 Gy) on normal human mesenchymal stem cells and their telomerised counterpart hMSC-telo1. We evaluated overall genomic integrity, DNA damage/repair by applying a fluorescence-detected alkaline DNA unwinding assay together with Western blot analyses for phosphorylated H2AX and Q-FISH was applied for investigation of telomeric damage. Our results indicate that hMSC and TERT-immortalized hMSCs can cope with relatively high doses of gamma-rays and that overall DNA repair is similar in the two cell lines. The telomeres were extensively destroyed after irradiation in both cell types suggesting that telomere caps are especially sensitive to radiation. The TERT-immortalized hMSCs showed higher stability at telomeric regions than primary hMSCs indicating that cells with long telomeres and high telomerase activity have the advantage of re-establishing the telomeric caps.

Human individuals often exhibit important differences in their sensitivity to ionising radiation. Extensive literature links radiation sensitivity with impaired DNA repair which is due to a lack of correct functioning in many proteins involved in DNA-repair pathways and/or in DNA-damage checkpoint responses. Given that ionising radiation is an important and widespread diagnostic and therapeutic tool, it is important to investigate further those factors and mechanisms that underlie individual radiosensitivity. Recently, evidence is accumulating that telomere function may well be involved in cellular and organism responses to ionising radiation, broadening still further the currently complex and challenging scenario.

Purpose: To evaluate the types of induced chromosome aberrations after the exposure of peripheral blood to gamma-rays by the simultaneous detection of all centromeres and telomeres; and to analyse the suitability of different radiation fingerprints for the assessment of radiation quality in cases of recent exposures., Material and Methods: Peripheral blood samples were irradiated at 2, 4 and 6 Gy of gamma-rays. Cytogenetic analysis was carried out by fluorescence in situ hybridization (FISH) technique with pan-centromeric and peptide nucleic acid (PNA)-telomeric DNA probes. Cells were analysed using a Cytovision FISH workstation, chromosome aberrations and the length of the acentric fragments were recorded., Results: The total number of the incomplete chromosome elements was 276. The ratio between incomplete elements and multicentrics was 0.38. The number of acentrics was 1096, 71% were complete acentrics, 15% incomplete acentrics, and 14% interstitial fragments. The relative length of complete, incomplete and interstitial acentrics fragments were 2.70 +/- 0.04, 1.91 +/- 0.07, and 1.42 +/- 0.04 respectively. The mean value of the F-ratio was 11.5 higher than the one, 5.5, previously obtained for alpha-particles. For the G-ratio there was no difference between gamma-rays and alpha-particles, 2.8 and 2.8 respectively. The mean value of the H-ratio for gamma-rays, 0.25, was lower than for alpha-particles 0.40., Conclusion: The results support that the percentage of incomplete chromosome aberrations depends on radiation type; low-linear energy transfer (LET) radiation would produces less incomplete aberrations than high-LET radiation. The F- and H-ratios seem to be good indicators of radiation quality, although a real estimation of the H-ratio is only possible using pan-telomeric probes.

Purpose: Chronic viral infection and combinations of chemotherapeutic drugs have been reported to accelerate telomere erosion. Here, we asked if chemoradiotherapy, using the single agent cisplatin, would accelerate telomere loss in head and neck cancer patients, and whether loss was linked to smoking status, age, gender, or stage of disease at diagnosis., Experimental Design: Blood samples were collected from 20 patients with squamous cell cancer of the head and neck before, during, and after chemoradiotherapy. Following DNA isolation from peripheral blood mononuclear cells, telomere length was measured by terminal restriction fragment analysis., Results: Chemoradiotherapy increased the rate of telomere erosion>100-fold. Telomere length before treatment in chemoradiotherapy patients was similar to age-matched controls. Although smokers began with significantly shorter telomeres, smoking status did not affect chemoradiotherapy-induced attrition, nor did gender or stage of disease. We also make the novel observation that a significantly greater telomere loss occurred in response to treatment in older patients, with those younger than 55 years losing an average of 400 bp of telomeric DNA compared with the 880 bp lost by those over 55 years., Conclusions: The lack of telomere length difference before treatment suggests that shortened telomeres may not be a risk factor for development of head and neck cancer in the age range we examined. Chemoradiotherapy caused a severe telomere length reduction in all patients. The significant difference seen in the elderly (P=0.018) suggests that chemoradiotherapy may have more severe effects on the replicative capacity of blood cells in older patients.

Cellular senescence is a phenotype that is likely linked with aging. Recent concepts view different forms of senescence as permanently maintained DNA damage responses partially characterized by the presence of senescence-associated DNA damage foci at dysfunctional telomeres. Irradiation of primary human dermal fibroblasts with the photosensitizer 8-methoxypsoralen and ultraviolet A radiation (PUVA) induces senescence. In the present study, we demonstrate that senescence after PUVA depends on DNA interstrand cross-link (ICL) formation that activates ATR kinase. ATR is necessary for the manifestation and maintenance of the senescent phenotype, because depletion of ATR expression before PUVA prevents induction of senescence, and reduction of ATR expression in PUVA-senesced fibroblasts releases cells from growth arrest. We find an ATR-dependent phosphorylation of the histone H2AX (gamma-H2AX). After PUVA, ATR and gamma-H2AX colocalize in multiple nuclear foci. After several days, only few predominantly telomere-localized foci persist and telomeric DNA can be coimmunoprecipitated with ATR from PUVA-senesced fibroblasts. We thus identify ATR as a novel mediator of telomere-dependent senescence in response to ICL induced by photoactivated psoralens.

The protein products of several rad checkpoint genes of Schizosaccharomyces pombe (rad1+, rad3+, rad9+, rad17+, rad26+, and hus1+) play crucial roles in sensing changes in DNA structure, and several function in the maintenance of telomeres. When the mammalian homologue of S. pombe Rad9 was inactivated, increases in chromosome end-to-end associations and frequency of telomere loss were observed. This telomere instability correlated with enhanced S- and G2-phase-specific cell killing, delayed kinetics of gamma-H2AX focus appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase-specific DNA damage repair. Furthermore, mammalian Rad9 interacted with Rad51, and inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S and G2 phases of the cell cycle. Together, these findings provide evidence of roles for mammalian Rad9 in telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.

High-charge and energy (HZE) nuclei represent one of the main health risks for human space exploration, yet little is known about the mechanisms responsible for the high biological effectiveness of these particles. We have used in situ hybridization probes for cross-species multicolor banding (RxFISH) in combination with telomere detection to compare yields of different types of chromosomal aberrations in the progeny of human peripheral blood lymphocytes exposed to either high-energy iron ions or gamma rays. Terminal deletions showed the greatest relative variation, with many more of these types of aberrations induced after exposure to accelerated iron ions (energy 1 GeV/nucleon) compared with the same dose of gamma rays. We found that truncated chromosomes without telomeres could be transmitted for at least three cell cycles after exposure and represented about 10% of all aberrations observed in the progeny of cells exposed to iron ions. On the other hand, the fraction of cells carrying stable, transmissible chromosomal aberrations was similar in the progeny of cells exposed to the same dose of densely or sparsely ionizing radiation. The results demonstrate that unrejoined chromosome breaks are an important component of aberration spectra produced by the exposure to HZE nuclei. This finding may well be related to the ability of such energetic particles to produce untoward late effects in irradiated organisms.

Purpose: To assess the chromosomal breakpoint distribution in human peripheral blood lymphocytes (PBL) after exposure to a low dose of high linear energy transfer (LET) alpha-particles using the technique of multiplex fluorescence in situ hybridization (m-FISH)., Materials and Methods: Separated PBL were exposed in G0 to 0.5 Gy 238Pu alpha-particles, stimulated to divide and harvested approximately 48 - 50 hours after exposure. Metaphase cells were assayed by m-FISH and chromosome breaks identified. The observed distribution of breaks were then compared with expected distributions of breaks, calculated on the assumption that the distribution of breaks is random with regard to either chromosome volume or chromosome surface area., Results: More breaks than expected were observed on chromosomes 2 and 11, however no particular region of either chromosome was identified as significantly contributing to this over-representation. The identification of hot or cold chromosome regions (pter,p,cen,q,qter) varied depending on whether the data were compared according to chromosome volume or surface area., Conclusions: A deviation from randomness in chromosome breakpoint distribution was observed, and this was greatest when data were compared according to the relative surface area of each individual chromosome (or region). The identification of breaks by m-FISH (i.e., more efficient observation of interchanges than intrachanges) and importance of territorial boundaries on interchange formation are thought to contribute to these differences. The significance of the observed non-random distribution of breaks on chromosomes 2 and 11 in relation to chromatin organization is unclear.

Telomeres are specialised nucleoprotein complexes that serve as protective caps of linear eukaryotic chromosomes. The loss of the ends of the chromosomes due to these un-rejoined double strand breaks (DSBs) may not be lethal to the cell, but may instead result in the loss of functional telomeres, chromosome fusions and initiation of breakage/fusion/bridge cycle-induced chromosome instability. The telomeres also participate in the process of DNA repair, as evidenced by 'de novo' synthesis of telomere repeats at DSBs and by the capacity of telomeres to binding the essential components of the DNA repair machinery. Based on the observation that high-LET radiations efficiently induce chromosome aberrations, it was tested whether protons were able to affect telomere structure. Human primary fibroblasts (HFFF2) and mouse embryonic fibroblasts (MEFs) were irradiated with 4 Gy of 3 MeV protons at the radiobiology facility of the INFN-LNL. Experiments with X rays were also carried out. Cells were fixed after either 24 h or 15 d from treatment. A difference in average telomere length, measured by quantitative fluorescence in situ hybridisation (Q-FISH), between X rays and protons treatment was observed. X rays are able to modify telomere length in HFFF2 harvested at a later time. On the other hand, 3 MeV low-energy protons induced, both in HFFF2 and in MEFs, a significant increase in telomere length at short as well as at long harvesting time periods from treatment. These results seem to indicate that lesions characterised by different complexity, as those expected after low-energy protons and those induced by damage similar to that induced by sparsely ionising radiation, are able to modulate telomere elongation at different time periods.

Basic to virtually all relevant biological effects of ionizing radiation is the underlying damage produced in DNA and the subsequent cellular processing of such damage. The damage can be qualitatively different for different kinds of radiations, and the genetics of the biological systems exposed can greatly affect damage processing and ultimate outcome--the biological effect of concern. The accurate repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity and function. Incorrect repair of such lesions results in chromosomal rearrangements and mutations that can lead to cancer and heritable defects in the progeny of irradiated parents. We have focused on the consequent phenotypic effects of faulty repair by examining connections between cellular radiosensitivity phenotypes relevant for carcinogenesis after exposure to ionizing radiation, and deficiencies in various components of the non-homologous end-joining (NHEJ) system. Here we produced deficiencies of individual components of the DNA-dependent protein kinase (DNA-PK) holoenzyme (Ku86 and the catalytic subunit, DNA-PKcs), both singly and in combination, using RNA interference (RNAi) in human lymphoblastoid cell lines. Exposure of cells exhibiting reduced protein expression to either gamma rays or 1 GeV/nucleon iron particles demonstrated differential effects on telomere dysfunction and mutation frequency as well as differential effects between radiation qualities.

The natural ends of linear chromosomes, the telomeres, recruit specific proteins in the formation of protective caps that preserve the integrity of the genome. Unprotected chromosomes induce DNA damage checkpoint cascades and ultimately lead to senescence both in mouse and man in a p53 dependent manner and initial telomere length setting therefore determines the proliferative capacity of each cell. Yet, only little information is available on telomere biology during embryonic development. We have previously shown that the p53 gene plays a crucial role in the development of malformations (exencephaly, gastroschisis, polydactyly, cleft palate and dwarfism) in control and irradiated mouse embryos. Here, we investigated telomere biology and the outcome of radiation exposure in wild type (p53+/+) and p53-mutant (p53+/-- and--/--) C57BL mouse foetuses irradiated at three different developmental stages. We show that telomeres are significantly shorter in malformed foetuses as compared to normal counterparts. In addition, our results indicate that the observed telomere attrition is primarily associated with p53-deficiency but is also modulated by irradiation, more specifically during the gastrulation and organogenesis stages. In conclusion, we formulate a hypothesis in which telomere shortening is linked to the absence of p53 in mouse foetuses and that when, in the presence of shorter telomeres, these foetuses are irradiated, the chance for the occurrence of developmental defects increases substantially.

Hypomorphic mutations which lead to decreased function of the NBS1 gene are responsible for Nijmegen breakage syndrome, a rare autosomal recessive hereditary disorder that imparts an increased predisposition to development of malignancy. The NBS1 protein is a component of the MRE11/RAD50/NBS1 complex that plays a critical role in cellular responses to DNA damage and the maintenance of chromosomal integrity. Using small interfering RNA transfection, we have knocked down NBS1 protein levels and analyzed relevant phenotypes in two closely related human lymphoblastoid cell lines with different p53 status, namely wild-type TK6 and mutated WTK1. Both TK6 and WTK1 cells showed an increased level of ionizing radiation-induced mutation at the TK and HPRT loci, impaired phosphorylation of H2AX (gamma-H2AX), and impaired activation of the cell cycle checkpoint regulating kinase, Chk2. In TK6 cells, ionizing radiation-induced accumulation of p53/p21 and apoptosis were reduced. There was a differential response to ionizing radiation-induced cell killing between TK6 and WTK1 cells after NBS1 knockdown; TK6 cells were more resistant to killing, whereas WTK1 cells were more sensitive. NBS1 deficiency also resulted in a significant increase in telomere association that was independent of radiation exposure and p53 status. Our results provide the first experimental evidence that NBS1 deficiency in human cells leads to hypermutability and telomere associations, phenotypes that may contribute to the cancer predisposition seen among patients with this disease.

Background & Objective: Many studies showed that telomere length and telomerase activity closely correlate with proliferation and malignant degree of tumor cells, and both of them might be involved in the repair of radiation-induced DNA damage. So it was speculated that telomere length and telomerase activity maybe correlate to radiosensitivity of carcinoma cells. This study was designed to investigate the correlations of telomere length and telomerase activity to radiosensitivity of human laryngeal squamous carcinoma cell line Hep-2., Methods: Hep-2 cells were irradiated with 0, 2, 4, 8, or 12 Gy of gamma-ray for 3 times. Survival cells were subcultured for 20 generations. Radiosensitivity index, survival fraction at 2 Gy (SF(2)), was measured by clone formation assay. Telomere length (mean length of telomere restriction fragments, TRF) was examined by Southern blotû telomerase activity (TA) was detected by polymerase chain reaction-based telomeric repeat amplification protocol (PCR-TRAP) coupled with ELISA., Results: After different doses of irradiation, SF(2) of Hep-2 cells was 0.47-0.64; TRF was 3.76-9.43 kb; TA was 1.761-2.606. Each parameter had significant differences among the survival progenies (P<0.05). SF(2) was positively correlated with TRF (r=0.921, P<0.01), and negatively correlated with TA (r=-0.929, P<0.01); TRF was negatively correlated with TA (r=-0.944, P<0.01)., Conclusion: Radiosensitivity of Hep-2 cells negatively correlates with telomere length, and positively correlates with telomerase activity, which suggest that both telomere length and telomerase activity may be used to predict cellular radiosensitivity.

The combination of the transient absorption measurement and DNA damage quantification by HPLC clearly demonstrated the preferential excitation of guanine and its decomposition in quadruplex DNA by UVB-irradiation.

We examined the delayed induction of telomere instability in hTERT-immortalized normal human fibroblast (BJ1-hTERT) cells exposed to X-rays. BJ1-hTERT cells were irradiated with 2 Gy of X-rays, and chromosome aberrations were analyzed 24 hours after irradiation and in the surviving cells 14 days after X-ray exposure. We found that the X-ray-surviving cells showed an increased frequency of chromatid gaps and breaks and chromosome fragments compared to the control cells. Furthermore, centromere- and telomere-FISH revealed that the frequency of telomere loss and duplication significantly increased in surviving cells compared to the control level. Because no induction of telomere abnormality was observed in cells 24 hours after irradiation, X-irradiation might not affect telomeres directly, but it specifically induces delayed telomere instability in normal human fibroblast cells.

The gene responsible for ataxia telangiectasia (AT) encodes ATM protein, which plays a major role in the network of a signal transduction initiated by double strand DNA breaks. To determine how radiation-induced genomic instability is modulated by the dysfunction of ATM protein, we examined radiation-induced delayed chromosomal instability in individual cell lines established from wild-type Atm(+/+), heterozygote Atm(+/-), and knock-out Atm(-/-) mouse embryos. The results indicate that Atm(-/-) mouse cells are highly susceptible to the delayed induction of telomeric instability and end-to-end chromosome fusions by radiation in addition to the elevated spontaneous telomeric instability detected by telomere fluorescence in situ hybridization (FISH). The telomeric instability was characterized by abnormal telomere FISH signals, including loss of the signals and the extra-chromosomal signals that were associated and/or not associated with chromosome ends, suggesting that Atm deficiency makes telomeres vulnerable to breakage. Thus, the present study shows that Atm protein plays an essential role in maintaining telomere integrity and prevents chromosomes from end-to-end fusions, indicating that telomeres are a target for the induction of genomic instability by radiation.

To determine the effects of a defect in NHEJ on the induction of genomic instability by radiation, we investigated X-ray-induced delayed chromosomal aberrations such as dicentrics and fragments in scid mouse cells. We found that radiosensitive scid mouse cells are more susceptible than wild-type mouse cells to the induction of delayed chromosomal aberrations when the cells are exposed to an equivalent survival dose of X-rays. Telomere FISH analysis revealed that radiation enhances the induction of telomeric fusions where telomeric sequences remain at the fused position (tel+ end-fusions), suggesting that radiation induces telomere dysfunction. Moreover, formation of the tel+ end-fusions was found to be enhanced in scid mouse cells, suggesting that DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a role in telomeric stabilization. Thus, the present study suggests that a cause of genomic instability is telomere dysfunction induced by radiation and that a defect in DNA-PKcs enhances the telomeric destabilization.

Several recent studies point to the possibility that telomere maintenance may constitute a potential genetic marker of radiosensitivity. For example, the human diseases ataxia telangiectasia and Nijmegen breakage syndrome, which are characterized by clinical radiosensitivity, show alterations in telomere maintenance. In addition, Fanconi's anemia patients, who are characterized by mild cellular radiosensitivity and in some cases marked clinical radiosensitivity, have altered telomere maintenance. Similarly, a correlation between telomere maintenance and cellular radiosensitivity was reported in a group of breast cancer patients. Another study demonstrated that radiosensitivity may be more pronounced in human fibroblasts with short telomeres than in their counterparts with long telomeres. Several mouse models including mice deficient in Ku, DNA-PKcs (Prkdc), Parp and Atm, all of which are radiosensitive in vivo, show clear telomere alterations. The link between telomere maintenance and radiosensitivity is also apparent in mice genetically engineered to have dysfunctional telomeres. Finally, studies using non-mammalian model systems such as C. elegans and yeast point to the link between radiosensitivity and telomere maintenance. These results warrant further investigation to identify the extent to which these two phenotypes, namely radiosensitivity and telomere maintenance, are linked.

Interstitial telomeric sites (ITSs) in chromosomes from DNA repair-proficient mammalian cells are sensitive to both spontaneous and radiation-induced chromosome breakage. Exact mechanisms of this chromosome breakage sensitivity are not known. To investigate factors that predispose ITSs to chromosome breakage we used murine scid cells. These cells lack functional DNA-PKcs, an enzyme involved in the repair of DNA double-strand breaks. Interestingly, our results revealed lack of both spontaneous and radiation-induced chromosome breakage at ITSs found in scid chromosomes. Therefore, it is possible that increased sensitivity of ITSs to chromosome breakage is associated with the functional DNA double-strand break repair machinery. To investigate if this is the case we used scid cells in which DNA-PKcs deficiency was corrected. Our results revealed complete disappearance of ITSs in scid cells with functional DNA-PKcs, presumably through chromosome breakage at ITSs, but their unchanged frequency in positive and negative control cells. Therefore, our results indicate that the functional DNA double-strand break machinery is required for elevated sensitivity of ITSs to chromosome breakage. Interestingly, we observed significant differences in mitotic chromosome condensation between scid cells and their counterparts with restored DNA-PKcs activity suggesting that lack of functional DNA-PKcs may cause a defect in chromatin organization. Increased condensation of mitotic chromosomes in the scid background was also confirmed in vivo. Therefore, our results indicate a previously unanticipated role of DNA-PKcs in chromatin organisation, which could contribute to the lack of ITS sensitivity to chromosome breakage in murine scid cells., (Copyright 2003 S. Karger AG, Basel)

Sister chromatid exchanges (SCEs) are symmetrical exchanges between newly replicated chromatids and their sisters. While homologous recombination may be one of the principal mechanisms responsible for SCEs, the full details of their molecular basis and biological significance remain to be elucidated. Following exposure to ultraviolet light B (UVB), mitomycin C (MMC) and cisplatin, we analyzed the location of SCEs on metaphase chromosomes in Chinese hamster CHO cells. The frequency of SCEs increased over the spontaneous level in proportion to the agent's dose. UVB-induced SCEs occurred frequently in telomere regions, as cisplatin-induced SCEs did, differing from MMC-induced ones. The remarkable difference of intrachromosomal distribution among the three mutagens may be attributed to the specificity of induced DNA lesions and structures of different chromosome regions. Telomeric DNA at the end of chromosomes is composed of multiple copies of a repeated motif, 5'-TTAGGG-3' in mammalian cells. Telomeric repeats may be potential targets for UVB and cisplatin, which mainly form pyrimidine dimers and intrastrand d(GpG) cross-links, respectively, resulting in SCE formation. UVB irradiation shortened telomeres and augmented the telomerase activity. The possible implications of the frequent occurrence of SCEs in telomere regions are discussed in connection with the maintenance of telomere integrity., (Copyright 2003 S. Karger AG, Basel)

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination and repair. In Saccharomyces cerevisiae, several mutants in the RFA1 gene encoding the large subunit of RPA have been isolated and one of the mutants with a missense allele, rfa1-D228Y, shows a synergistic reduction in telomere length when combined with a yku70 mutation. So far, only one mutant allele of the rad11(+) gene encoding the large subunit of RPA has been reported in Schizosaccharomyces pombe. To study the role of S.pombe RPA in DNA repair and possibly in telomere maintenance, we constructed a rad11-D223Y mutant, which corresponds to the S.cerevisiae rfa1-D228Y mutant. rad11-D223Y cells were methylmethane sulfonate, hydroxyurea, UV and gamma-ray sensitive, suggesting that rad11-D223Y cells have a defect in DNA repair activity. Unlike the S.cerevisiae rfa1-D228Y mutation, the rad11-D223Y mutation itself caused telomere shortening. Moreover, Rad11-Myc bound to telomere in a ChIP assay. These results strongly suggest that RPA is directly involved in telomere maintenance.

Normal somatic cells have a finite number of divisions, a limited capacity to proliferate. Human telomeres contain TTAGGG repeats which are considered a molecular clock marker. The gradual and progressive telomere shortening at each replicative cycle is associated, through the activation of pRB and p53 pathways and genomic instability, to the replicative senescence, a non-dividing state and widespread cell death. There is no information available about telomere status in individuals who live long and have been exposed to ionizing radiations (IR). To determine the telomere stability, we examined telomeres at metaphase, G2-type chromosome aberrations after IR treatment and karyotypic analysis of 15 individuals. Three individuals were above the age of 80 years and 1 among the 3 was estimated to have received more than 10 Gy of occupational exposure about 30 years back. The other 12 were cancer patients that had received more than 50 Gy of gamma-radiation for therapeutic purposes. No telomere instability or defective G2 chromosome repair was found in 3 individuals above the age of 80 years. Whereas, 3 out of 7 prostate and 1 out of 5 breast cancer patients showed higher G2-type chromosome damage as well as a high frequency of telomeric association (also known as chromosome end associations) along with frequent loss of telomeres. Present studies demonstrate that telomere stability along with normal G2 chromosome repair correlates with the longevity of human beings, whereas defective G2 chromosome repair and telomere instability correlate with the radiotherapy related late toxicity.

Telomeres cap chromosome ends, avoiding end-to-end fusions and subsequent chromosome instability. Telomeric functions and DNA repair pathways are closely related. Telomere dysfunction has been shown to result in hypersensitivity to ionizing radiation. In this study, we have used the telomerase knockout model to investigate how telomere shortening influences the correct repair of broken chromosomes. We show that the correct repair of double-strand breaks is impaired in telomerase knockout mice. The chromosomes with shortened telomeres fuse to radiation-induced breaks, interfering with the correct rejoining of the broken ends. This type of fusion is responsible for the increased chromosome instability observed in this mouse model, after exposure to ionizing radiation. Our finding may be important for understanding the increased radiation sensitivity associated with age in humans, as well as for comprehending the interindividual differences to the cytotoxic effects of radiation therapy in cancer patients.

Purpose: To compare telomere length in mouse strains with constitutional chromosomal aberrations generated either by exposure of parents to ionizing radiation, a chemical mutagen or arising spontaneously with that of the karyotypically normal mouse from the same genetic background., Materials and Methods: Telomere length was assessed in five independently derived strains of mouse with constitutional chromosomal aberrations and in the karyotypically normal control mouse using quantitative fluorescence in situ hybridization (Q-FISH). Bone marrow cells obtained directly from the animals were used for the analysis., Results: Chromosomal aberrations, one in each mouse strain, included three reciprocal translocations induced by ionizing radiation, one insertion induced by a chemical mutagen and one spontaneous Robertsonian translocation. There was no cytogenetically detectable loss of material in any of the strains and most mice were phenotypically normal. Telomeres were significantly shorter in all mouse strains with constitutional chromosomal aberrations in comparison with those originating from the karyatypically normal mouse from the same genetic background. Telomeres were significantly shorter at p-arms than at q-arms in all animals. The telomere length in individual chromosomes was variable and there was no single chromosome with consistently short telomeres in all animals., Conclusions: The presence of stable chromosomal aberrations, such as translocations or insertions, in the mouse genome may generate telomere shortening. This might have implications for understanding biological consequences or radiation-induced stable chromosomal aberrations.

To test the involvement of the telomeres in the senescent phenotype, we used telomerase-immortalized human foreskin fibroblasts (hTERT-BJ1). We exposed hTERT-BJ1 and parental BJ cells to either UVB or H(2)O(2) subcytotoxic stress(es). Both cell lines developed biomarkers of replicative senescence: loss of replicative potential, increase in senescence-associated beta-galactosidase activity, typical senescence-like morphology, overexpression of p21(WAF-1) and p16(INK-4a), and decreased level of the hyperphosphorylated form of pRb. Telomere shortening was slightly higher under stress for both BJ and hTERT-BJ1 but still much lower than that reported for other cell lines. We conclude that pathways alternative to telomere shortening must cause the appearance of the senescence phenotype.

We describe the design, synthesis and biophysical characterization of a novel DNA construct in which a folded quadruplex structure is joined to a standard double helix. Circular dichroism, gel electrophoresis, three-dimensional UV melting and differential scanning calorimetry were all used to characterize the structure. Rigorous molecular dynamics simulations were used to build a plausible atomic-level structural model of the DNA construct. This novel DNA construct provides a model for the duplex-quadruplex junction region at the end of chromosomal DNA and offers a system for the study of structure-selective ligand binding.

Human blood leukocytes were exposed to X rays to analyze the initial level of DNA breakage induced within different satellite DNA sequence areas and telomeres, using the DNA breakage detection-FISH procedure. The satellite DNA families analyzed comprised alphoid sequences, satellite 1, and 5-bp classical satellite DNA sequences from chromosome 1 (D1Z1 locus), from chromosome 9 (D9Z3 locus), and from the Y chromosome (DYZ1 locus). Since the control hybridization signal was quite different in each of the DNA targets, the relative increase in whole fluorescence intensity with respect to unirradiated controls was the parameter used for comparison. Irradiation of nucleoids obtained after protein removal demonstrated that the alkaline unwinding solution generates around half the amount of signal when breaks are present in the 5-bp classical DNA satellites as when the same numbers of breaks are present the genome overall, whereas the signal is slightly stronger when the breaks are within the alphoids or satellite 1 sequences. After correction for differences in sensitivity to the alkaline unwinding-renaturation, DNA housed in chromatin corresponding to 5-bp classical satellites proved to be more sensitive to breakage than the overall genome, whereas DNA in the chromatin corresponding to alphoids or satellite 1 showed a sensitivity similar to that of the whole genome. The minimum detectable dose was 0.1 Gy for the whole genome, 0.2 Gy for alphoids and satellite 1, and 0.4 Gy for the 5-bp classical satellites. Telomeric DNA sequences appeared to be maximally labeled in unirradiated cells. Thus telomeric ends behave like DNA breaks, constituting a source of background in alkaline unwinding assays.

Purpose: A telomere consists of the short tandem DNA repeats of (T2AG3)n localized to the distal ends of chromosomes. Telomere repeat binding factor 2 (TRF2) has been implicated in the protection of chromosome ends. Recently, it has been reported that the loss of TRF2 induces apoptosis by various stimuli or genetic technique, however, the effects of radiation are not known. Therefore, this study investigated the interaction between TRF2 and radiation., Materials and Methods: Western blot analysis, immunohistochemistry, and a DNA fragmentation assay for the detection of apoptosis were performed. The interaction between elastase and TRF2 was also investigated in vitro., Results: Western blot analyses and immunohistochemistry showed that gamma-rays induce the temporary accumulation and subsequent loss of TRF2 protein in the nuclei of irradiated HL60 cells. Following DNA fragmentation, the loss of TRF2 could be detected. TRF2 was broken down by elastase, which was translocated into the nucleus before the loss of TRF2., Conclusions: The results of the study showed that irradiation first induces activation of TRF2, consequently protecting the end of the chromosome. Subsequently, translocation of elastase into the nucleus results in the breakdown of TRF2 after DNA fragmentation has occurred.

Telomeres are tandem repeats of DNA associated with specific proteins. These structures cap eukaryotic chromosomes and maintain the integrity of the chromosome ends. In the germline, telomeres are maintained by the enzyme telomerase, but in normal somatic cells the enzyme's activity is low or undetectable. Human tumours, including squamous-cell carcinoma of the head and neck (SCCHN), need telomerase to maintain telomere function; inhibition of the enzyme can lead to apoptosis. Furthermore, because most tumour cells have very short telomeres, they are more likely to succumb to telomerase inhibition than normal cells. Telomerase is therefore a potential selective anticancer target. The telomere is also involved in the repair of DNA double strand breaks, and telomere dysfunction provokes radiosensitivity. In this review we consider whether manipulation of telomere function may selectively sensitise SCCHN to radiotherapy and discuss the possible pitfalls. We also assess how some conventional treatments may affect the subsequent use of telomerase inhibitors.