Changes in Telomere Length in Leukocytes and Leukemic Cells after Ultrashort Electron Beam Radiation.

Application of laser-generated electron beams in radiotherapy is a recent development. Accordingly, mechanisms of biological response to radiation damage need to be investigated. In this study, telomere length (TL) as endpoint of genetic damage was analyzed in human blood cells (leukocytes) and K562 leukemic cells irradiated with laser-generated ultrashort electron beam. Metaphases and interphases were analyzed in quantitative fluorescence in situ hybridization (Q-FISH) to assess TL. TLs were shortened compared to non-irradiated controls in both settings (metaphase and interphase) after irradiation with 0.5, 1.5, and 3.0 Gy in blood leukocytes. Radiation also caused a significant TL shortening detectable in the interphase of K562 cells. Overall, a negative correlation between TL and radiation doses was observed in normal and leukemic cells in a dose-dependent manner. K562 cells were more sensitive than normal blood cells to increasing doses of ultrashort electron beam radiation. As telomere shortening leads to genome instability and cell death, the results obtained confirm the suitability of this biomarker for assessing genotoxic effects of accelerated electrons for their further use in radiation therapy. Observed differences in TL shortening between normal and K562 cells provide an opportunity for further development of optimal radiation parameters to reduce side effects in normal cells during radiotherapy.

Shortened telomere length in peripheral blood leukocytes is associated with cumulative radioactive iodine doses in patients with differentiated thyroid carcinoma.

BACKGROUND: Telomere length is associated with cancer risk and cancer aggressiveness. Radioactive iodine (RAI) therapy for thyroid cancer has raised concerns for second primary malignancy (SPM) in patients with high cumulative doses. The association between RAI dose and peripheral blood leukocyte telomere length was examined. METHODS: A total of 425 patients were included who underwent total thyroidectomy and were followed up for at least 1 year with or without RAI treatment. The relative telomere length (RTL) of the patients was assessed via a quantitative polymerase chain reaction amplification method. RAI doses were divided into five groups on the basis of cumulative dose, and a comparison was made among these groups. RESULTS: The number of patients with RAI treatment was 287 (67.5%), and the cumulative RAI dose was 3.33 GBq (range, 1.11-131.35 GBq). The mean RTL was significantly shorter in the highest RAI group (>22.2 GBq) compared to both the no-RAI and lower dose groups. The association between RAI dose and RTL was positive in the lower RAI group (1.1-3.7 GBq) and negative in the highest RAI group in both univariate and multivariate analyses. We observed 59 (13.9%) SPMs and 20 (4.7%) mortalities, and RTL did not show a significant risk effect for all-cause, thyroid cancer-specific, or SPM-specific mortality. CONCLUSIONS: In patients with thyroid cancer who underwent total thyroidectomy, peripheral blood leukocyte telomere length exhibited a significant association with cumulative RAI dose higher than 22.2 GBq. These results suggest the possibility of telomere length shortening in patients who undergo high-dose RAI treatment.

RELATIVE TELOMERE LENGTH OF PERIPHERAL BLOOD LYMPHOCYTES AND STRUCTURAL AND FUNCTIONAL STATE OF THE LEFT VENTRICLE MYOCARDIUM IN CLEAN-UP WORKERS OF THE CHORNOBYL ACCIDENT WHO SUFFERED FROM STENOTIC CORONARY ATHEROSCLEROSIS. / VIDNOSNA DOVZhYNA TELOMER LIMFOTsYTIV PERYFERYChNOÏ KROVI TA STRUKTURNO-FUNKTsIONAL'NYI STAN MIOKARDA LIVOGO ShLUNOChKA SERTsIa V UChASNYKIV LIKVIDATsIÏ NASLIDKIV ChORNOBYL'S'KOÏ AVARIÏ ZI STENOZUIuChYM ATEROSKLEROZOM KORONARNYKh ARTERII.

The objective was to analyze the relative telomere length (RTL) of peripheral blood lymphocytes depending onmyocardium structural and functional state in emergency workers (EW) of the Chornobyl accident who suffered fromcoronary arteries stenotic atherosclerosis. MATERIALS AND METHODS: There were examined 60 male EW who operated at the Chornobyl nuclear power plant at1986 and 25 male non-irradiated persons (control group - CG) with coronary heart disease (CHD). Everyone EW andCG patients were almost healthy before the accident. During the period 2016-2021, they underwent a comprehen-sive clinical and laboratory examination, echodopplercardiographic examination and determination of RTL by fluo-rescent hybridization in situ using laser flow cytometry. RESULTS: EW almost did not differ from CG according to its clinical characteristics, the presence of risk factors,indices of systolic and diastolic heart functions, as well as RTL. The analysis of variance showed that RTL was influ-enced by the fact of irradiation in combination with obesity (p = 0.020). At normal body weight, RTL average valuein CG was significantly higher than in EW (p = 0.023). According to the results of hierarchical cluster analysis of twovariables as RTL and end-diastolic volume normalized by body surface area (EDV/BSA), EW and CG patients togeth-er were divided into two subgroups. The first subgroup (1st cluster) differed from the second (2nd cluster) by signi-ficantly larger average values of left ventricle (LV) EDV and end-systolic volume (ESV) as well as EDV/BSA andESV/BSA, LV myocardial mass (MM) and MM/BSA, reduced ejection fraction (EF). In patients of the 1st cluster telom-eres were significantly shorter than in the 2nd one (10,3 ± 1.7 vs. 14.3 ± 2.0 at p = 0.000). The increase of myocar-dial mass and LV wall thickness caused the development of its hypertrophy. The number of people with hypertrophyLV was significantly higher among patients of the 1st cluster (91.6 vs. 67.2 %, p < 0.001) due to eccentric hypertro-phy LV. Accordingly, concentric hypertrophy LV was more common among patients in the 2nd cluster (24.6 vs. 4.2 %at p < 0.01). Patients of the 1st cluster was characterized by a more severe course of heart failure. CONCLUSIONS: In patients who suffered from CHD with stenotic atherosclerosis of the coronary arteries and wereexposed to radiation 30-35 years earlier, having normal body weight, there was a reduction in telomere. Hierarchicalcluster analysis proved to be a good tool that allows by the value of RTL and EDV/BSA to separate the group ofpatients with the most severe clinical course of CHD and LV systolic dysfunction among patients with the samepathology.

Twins, Telomeres, and Aging-in Space!

BACKGROUND: The landmark National Aeronautics and Space Administration Twins Study represented an integrated effort to launch human space life science research into the modern age of molecular- and "omics"-based studies. As part of the first One-Year Mission aboard the International Space Station, identical twin astronauts Scott and Mark Kelly were the subjects of this "out of this world" research opportunity. Telomeres, the natural ends of chromosomes that shorten with cell division and a host of lifestyle factors and stresses, are key molecular determinants of aging and aging trajectories. METHODS: We proposed that telomere length dynamics (changes over time) represent a particularly relevant and integrative biomarker for astronauts, as they reflect the combined experiences and environmental exposures encountered during spaceflight. Telomere length (quantitative polymerase chain reaction and telomere fluorescence in situ hybridization) and telomerase activity (quantitative polymerase chain reaction -telomere repeat amplification protocol) were longitudinally assessed in the space- and earth-bound twins. Chromosome aberrations (directional genomic hybridization), signatures of radiation exposure, were also evaluated. RESULTS: The twins had relatively similar telomere lengths before spaceflight, and the earth-bound twins' telomeres remained relatively stable over the course of the study. Surprisingly, the space twins' telomeres were longer during spaceflight, and upon return to Earth shortened rapidly, resulting in many more short telomeres after spaceflight than before. Chromosomal signatures of space radiation exposure were also elevated during spaceflight, and increased inversion frequencies persisted after spaceflight, suggestive of ongoing genome instability. CONCLUSION: Although the definitive mechanisms underlying such dramatic spaceflight-associated shifts in telomere length remain unclear, improved maintenance of telomere length has important implications for aging science and improving healthspan for those on Earth, as well.

Telomere shortening as a stress-related biomarker in children exposed to maternal chronic stress in utero measured 7 years after the Great East Japan Earthquake.

Seven years after the Great East Japan Earthquake, we investigated telomeres as a potential biomarker of maternal chronic stress in children according to the timing of exposure to the disaster. The subjects were children aged 5-9 years living in Rikuzentakata, Japan. Relative telomere length (rTL) was measured with PCR in saliva samples. The partial regression coefficient of the rTL was significantly shorter in the group of children conceived after the disaster than in the children who were in utero on the day of the disaster. Telomere length should be investigated as a biomarker for assessing disaster-related trauma in future studies.

Abnormal nuclear morphology is independent of longevity in a zmpste24-deficient fish model of Hutchinson-Gilford progeria syndrome (HGPS).

Lamin is an intermediate protein underlying the nuclear envelope and it plays a key role in maintaining the integrity of the nucleus. A defect in the processing of its precursor by a metalloprotease, ZMPSTE24, results in the accumulation of farnesylated prelamin in the nucleus and causes various diseases, including Hutchinson-Gilford progeria syndrome (HGPS). However, the role of lamin processing is unclear in fish species. Here, we generated zmpste24-deficient medaka and evaluated their phenotype. Unlike humans and mice, homozygous mutants did not show growth defects or lifespan shortening, despite lamin precursor accumulation. Gonadosomatic indices, blood glucose levels, and regenerative capacity of fins were similar in 1-year-old mutants and their wild-type (WT) siblings. Histological examination showed that the muscles, subcutaneous fat tissues, and gonads were normal in the mutants at the age of 1 year. However, the mutants showed hypersensitivity to X-ray irradiation, although p53target genes, p21 and mdm2, were induced 6 h after irradiation. Immunostaining of primary cultured cells from caudal fins and visualization of nuclei using H2B-GFP fusion proteins revealed an abnormal nuclear shape in the mutants both in vitro and in vivo. The telomere lengths were significantly shorter in the mutants compared to WT. Taken together, these results suggest that zmpste24-deficient medaka phenocopied HGPS only partially and that abnormal nuclear morphology and lifespan shortening are two independent events in vertebrates.

Radiobiological Effectiveness of Ultrashort Laser-Driven Electron Bunches: Micronucleus Frequency, Telomere Shortening and Cell Viability.

Laser-driven electron accelerators are capable of producing high-energy electron bunches in shorter distances than conventional radiofrequency accelerators. To date, our knowledge of the radiobiological effects in cells exposed to electrons using a laser-plasma accelerator is still very limited. In this study, we compared the dose-response curves for micronucleus (MN) frequency and telomere length in peripheral blood lymphocytes exposed to laser-driven electron pulse and X-ray radiations. Additionally, we evaluated the effects on cell survival of in vitro tumor cells after exposure to laser-driven electron pulse compared to electron beams produced by a conventional radiofrequency accelerator used for intraoperative radiation therapy. Blood samples from two different donors were exposed to six radiation doses ranging from 0 to 2 Gy. Relative biological effectiveness (RBE) for micronucleus induction was calculated from the alpha coefficients for electrons compared to X rays (RBE = alpha laser/alpha X rays). Cell viability was monitored in the OVCAR-3 ovarian cancer cell line using trypan blue exclusion assay at day 3, 5 and 7 postirradiation (2, 4, 6, 8 and 10 Gy). The RBE values obtained by comparing the alpha values were 1.3 and 1.2 for the two donors. Mean telomere length was also found to be reduced in a significant dose-dependent manner after irradiation with both electrons and X rays in both donors studied. Our findings showed a radiobiological response as mirrored by the induction of micronuclei and shortening of telomere as well as by the reduction of cell survival in blood samples and cancer cells exposed in vitro to laser-generated electron bunches. Additional studies are needed to improve preclinical validation of the radiobiological characteristics and efficacy of laser-driven electron accelerators in the future.

Telomere-Mitochondrion Links Contribute to Induction of Senescence in MCF-7 Cells after Carbon-Ion Irradiation.

The effects of carbon-ion irradiation on cancer cell telomere function have not been comprehensively studied. In our previous report cancer cells with telomere dysfunction were more sensitive to carbon-ion irradiation, but the underlying mechanisms remained unclear. Here we found that telomerase activity was suppressed by carbon-ion irradiation via hTERT down-regulation. Inhibition of telomere activity by MST-312 further increased cancer cell radiosensitivity to carbon-ion radiation. hTERT suppression caused by either carbon-ion irradiation or MST-312 impaired mitochondrial function, as indicated by decreased membrane potential, mtDNA copy number, mitochondrial mass, total ATP levels and elevated reactive oxygen species (ROS). PGC-1α expression was repressed after carbion-ion irradiation, and hTERT inhibition by MST-312 could further exacerbate this effect. Lowering the mitochondrial ROS level by MitoTEMPO could partially counteract the induction of cellular senescence induced by carbon-ion radiation and MST-312 incubation. Taken together, the current data suggest that telomere-mitochondrion links play a role in the induction of senescence in MCF-7 cells after carbon-ion irradiation.

Leukocyte telomere shortening in grown-up patients with congenital heart disease.

BACKGROUND/OBJECTIVES: Children with congenital heart disease are exposed by repeated imaging to ionizing radiation, which may have important implications for lifetime health risks. Leukocyte telomere length (LTL), a reliable biomarker of genomic instability, is associated with increased risk of cancer and cardiovascular disease. We investigated LTL in grown-up patients with CHD (GUCHs) and a positive history of medical radiation exposure as well as the influence of functional polymorphisms of genes involved in DNA repair. METHODS: A group of 50 GUCH patients (26 males; age 25.2 ± 9.0 years) and 50 healthy age/gender-matched subjects (20 males; 27.0 ± 3.1 years) were enrolled. In GUCH patients, the cumulative exposure was estimated as effective dose (ED) in milliSievert. LTL was measured by quantitative RT-PCR. X-ray repair cross complementing-1 (XRCC1) and X-ray repair cross complementing-3 (XRCC3) SNPs (XRCC1Arg399Gln, XRCC1Arg194Tr and XRCC3 Thr241Met) were evaluated. RESULTS: GUCHs showed significantly shorter LTL compared with controls (1.0 ± 0.3 vs 1.3 ± 0.4, p = 0.001). A significant inverse correlation between LTL and cumulative radiological ED was observed (r = -0.34, p = 0.03). Patients with Thr/Met XRCC3 or Met/Met XRCC3 genotypes were significantly associated with a significantly shorter LTL compared with wild-type genotype (p = 0.01 for Thr/Met and p = 0.008 for Met/Met). Carriers of XRCC1 194Trp and XRCC3 241Met alleles presented a significant interaction with cumulative radiation dose exposure for LTL (both p interaction = 0.02). CONCLUSIONS: GUCH patients have LTL shortening, suggesting evidence of early biological aging. Common SNPs in DNA repair genes modify the effects of medical exposure to radiation LTL-related degenerative diseases.

Telomere length in Chernobyl accident recovery workers in the late period after the disaster.

The outcome of the Chernobyl nuclear power plant (CNPP) accident was that a huge number of people were exposed to ionizing radiation. Previous studies of CNPP clean-up workers from Latvia revealed a high occurrence of age-associated degenerative diseases and cancer in young adults, as well as a high mortality as a result of cardiovascular disorders at age 45-54 years. DNA tandem repeats that cap chromosome ends, known as telomeres, are sensitive to oxidative damage and exposure to ionizing radiation. Telomeres are important in aging processes and carcinogenesis. The aim of this study was to investigate the long-term effect of protracted ionizing radiation exposure on telomere length in CNPP clean-up workers. Relative telomere length (RTL) was measured in peripheral blood leukocytes of 595 CNPP clean-up workers and 236 gender- and age-matched controls using real-time quantitative polymerase chain reaction (q-PCR). Close attention was paid to participation year and tasks performed during the worker's stay in Chernobyl, health status, and RTL differences between subgroups. Telomere shortening was not found in CNPP clean-up workers; on the contrary, their RTL was slightly greater than in controls (P = 0.001). Longer telomeres were found in people who worked during 1986, in those undertaking 'dirty' tasks (digging and deactivation), and in people with cancer. Shorter telomeres appeared frequently in those with cataract, osteoporosis, atherosclerosis, or coronary heart disease. We conclude that the longer telomeres revealed in people more heavily exposed to ionizing radiation probably indicate activation of telomerase as a chromosome healing mechanism following damage, and reflect defects in telomerase regulation that could potentiate carcinogenesis.

Topical application of preparations containing DNA repair enzymes prevents ultraviolet-induced telomere shortening and c-FOS proto-oncogene hyperexpression in human skin: an experimental pilot study.

The exposure to ultraviolet radiation (UVR) is one of the most important risk factors for skin aging and increases the risk of malignant transformation. Telomere shortening and an altered expression of the proto-oncogene c-FOS are among the key molecular mechanisms associated with photoaging and tumorigenesis. Photolyase from A. nidulans and endonuclease from M. luteus are xenogenic DNA repair enzymes which can reverse the molecular events associated with skin aging and carcinogenosis caused by UVR exposure. Therefore, the purpose of this study was to investigate whether the topical application of preparations containing DNA repair enzymes may prevent UVR-induced acute telomere shortening and FOS gene hyperexpression in human skin biopsies. Twelve volunteers (Fitzpatrick skin types I and II) were enrolled for this experimental study, and six circular areas (10 mm diameter) were marked out on the nonexposed lower back of each participant. One site was left untreated (site 1: negative control), whereas the remaining five sites (designated sites 2-6) were exposed to solar-simulated UVR at 3 times the MED on four consecutive days. Site 2 received UVR only (site 2: positive control), whereas the following products were applied to sites 3-6, respectively: vehicle (moisturizer base cream; applied both 30 minutes before and immediately after each irradiation; site 3); a traditional sunscreen (SS, SPF 50) 30 minutes before irradiation and a vehicle immediately after irradiation (site 4); a SS 30 minutes before irradiation and an endonuclease preparation immediately after irradiation (site 5); a SS plus photolyase 30 minutes before irradiation and an endonuclease preparation immediately after irradiation (site 6). Skin biopsies were taken 24 h after the last irradiation. The degree of telomere shortening and c-FOS gene expression were measured in all specimens. Strikingly, the combined use of a SS plus photolyase 30 minutes before irradiation and an endonuclease preparation immediately after irradiation completely abrogated telomere shortening and c-FOS gene hyperexpression induced by the experimental irradiations. We conclude that the topical application of preparations containing both photolyase from A. nidulans and endonuclease from M. luteus may be clinically useful to prevent skin aging and carcinogenesis by abrogating UVR-induced telomere shortening and c-FOS gene hyperexpression.

The role of telomere length modulation in delayed chromosome instability induced by ionizing radiation in human primary fibroblasts.

Telomere integrity is important for chromosome stability. The main objective of our study was to investigate the relationship between telomere length modulation and mitotic chromosome segregation induced by ionizing radiation in human primary fibroblasts. We used X-rays and low-energy protons because of their ability to induce different telomeric responses. Samples irradiated with 4 Gy were fixed at different times up to 6 days from exposure and telomere length, anaphase abnormalities, and chromosome aberrations were analyzed. We observed that X-rays induced telomere shortening in cells harvested at 96 hrs, whereas protons induced a significant increase in telomere length at short as well as at long harvesting times (24 and 96 hrs). Consistent with this, the analysis of anaphase bridges at 96 hrs showed a fourfold increase in X-ray- compared with proton-irradiated samples, suggesting a correlation between telomere length/dysfunction and chromosome missegregation. In line with these findings, the frequency of dicentrics and rings decreased with time for protons whereas it remained stable after X-rays irradiation. Telomeric FISH staining on anaphases revealed a higher percentage of bridges with telomere signals in X-ray-treated samples than that observed after proton irradiation, thus suggesting that the aberrations observed after X-ray irradiation originated from telomere attrition and consequent chromosome end-to-end fusion. This study shows that, beside an expected "early" chromosome instability induced shortly after irradiation, a delayed one occurs as a result of alterations in telomere metabolism and that this mechanism may play an important role in genomic stability.

Radiation-associated changes in the length of telomeres in peripheral leukocytes from inpatients with cancer.

PURPOSE: The telomere length of somatic cells shortens with age and with other endogenous and exogenous pathogenic factors. However, the effects of radiation therapy on telomere DNA of non-cancer tissue have not been thoroughly investigated. This study analyzed the telomere length of inpatients with cancer treated with radiation therapy to see whether the telomere lengths change in response to therapeutic radiation. MATERIALS AND METHODS: Twenty-five patients were enrolled in the study. The patients had lung cancer, prostate cancer, thyroid cancer, hepatoma, or rectal cancer. They received radiation therapy with a dose range of 15-74 Gy. The telomere lengths and telomere length distribution in peripheral leukocytes were analyzed by using a Southern blot-based method. RESULTS: The telomere length and the telomere length distribution of the peripheral leukocytes did not change after radiation therapy. However, there was a significant proportional decrease in the short telomere fraction (< 4.4 kb) per day and per Gy. CONCLUSIONS: This observation suggested that the telomere length distribution of peripheral leukocytes could be affected by radiation therapy, and that the effect of radiation tends to appear in cells with short telomeres. Radiation therapy-associated somatic telomere length change within a short range of time, about three months or shorter, can be detected by analyzing the mean telomere length and telomere length distribution.

Human skin fibroblast telomeres are shortened after ultraviolet irradiation.

OBJECTIVE: Telomere length was used as a biomarker of cell senescence to explore the role of telomere shortening in photoageing induced by ultraviolet A (UVA) light. METHODS: Real-time polymerase chain reaction was used to determine telomere length in cultured human fibroblasts of different generations and after exposure to UVA at doses up to 10 000 mJ/cm(2). Twoway analysis of variance was used to determine whether passaging or UVA was the main factor contributing to telomere shortening. RESULTS: In nonirradiated cells, telomere length was inversely related to cell generation number. In fibroblasts exposed to UVA at a dose of 1000 or 10 000 mJ/cm(2), telomere length was significantly shorter than that of nonirradiated controls and was negatively related to UVA dose. CONCLUSIONS: Telomere length and subsequent cell viability may be affected by UVA irradiation. DNA damage caused by UVA irradiation may initiate the photoageing process and telomeres may be a useful new target for attempts to prevent photoageing.

Telomere loss, not average telomere length, confers radiosensitivity to TK6-irradiated cells.

Many and varied are the proposed mechanisms that lead to resistance to ionizing radiation treatment. Among them, an inverse relationship between telomere length and radioresistance has been recently advanced. Investigating such a relationship in TK6 lymphoblasts, we found that clones originating from cells survived to 4Gy of X-rays showed a significantly higher telomere length when compared with clones grown from untreated cells. The lengthening observed was not attributable to a radiation-induced increase in telomerase activity, as demonstrated by TRAP assay performed in the dose range of 1-10Gy. Given the evidence that TK6 whole population was characterized by heterogeneity in cellular mean telomere length and telomere loss, we tested the hypothesis that a process of selection may favour cells with longer telomeres (more radioresistant cells) following exposure to irradiation. In order to do this 15 independent TK6 clones were selected and characterized for telomere length and loss on the basis of q-FISH and flow-FISH analysis. Among the screened clones four characterized by long telomeres and four characterized by short telomeres were tested for their radiosensitivity by means of clonogenic assay. The results obtained showed that, in our experimental conditions (cellular model, radiation doses) no significant correlation was observed between radiosensitivity and mean telomere lengths, whereas a positive correlation was observed with respect to telomere loss. Overall, these results indicate that telomere loss and not mean telomere length plays a critical role in the phenomenon of radiosensitivity/radioresistance.

Telomere shortening and ionizing radiation: a possible role in vascular dysfunction?

PURPOSE: In recent years, growing epidemiological evidence has linked ionizing radiation exposure to cardiovascular atherosclerotic disease. However, there are still major gaps in the knowledge of the molecular mechanisms of radiation-induced vascular disease, especially for low-dose levels. Telomeres, repetitive DNA sequences of (TTAGGG)(n) located at the ends of eukaryotic chromosomes, play a role in regulating vascular aging, and shorter leukocyte telomere length has been demonstrated to predict cardiovascular disease and mortality. There is also evidence supporting the crucial role of telomeres in the formation of chromosome and chromatid aberrations induced by ionizing radiation. CONCLUSIONS: The purpose of the present paper is to review the recent advances in the biological mechanisms determining telomere length erosion after ionizing radiation exposure as well as to examine the hypothesis that telomere shortening may be the crucial mediator leading to detrimental vascular effects after ionizing radiation exposure.

Genistein induces growth arrest and suppresses telomerase activity in brain tumor cells.

Genistein, a soy isoflavone, has been reported to exhibit multiple effects, such as inducing cell cycle arrest, triggering apoptosis, inhibiting the activation of NF(K) B and inactivating several signaling cascades in human cancer cells. In vivo studies demonstrating antiangiogenesis and antimetastatic effects of genistein have also been reported. Here, we demonstrate that genistein inhibits the growth of glioblastoma multiforme and medulloblastoma cells with different TP53 mutations and radio-responses by arresting the cells at G2/M phase of the cell cycle. The cell cycle arrest was found to be independent of DNA damage and such an arrest was sustainable for at least 10 days even after drug removal. Annexin V staining revealed absence of apoptotic or necrotic cell populations after genistein treatment. This supports the observation that genistein induces insignificant DNA damage and indicates that the cell cycle arrest triggered does not lead to cell death. Gene and protein expression studies reveal similar changes in the same pathways following treatment in the cell types tested. Genistein was also able to inhibit telomerase activity resulting in telomere shortening. Thus, we demonstrate, for the first time, that genistein induces growth arrest in association with telomerase inhibition in brain tumor cells via the suppression of TR- and TERT mRNA. By elucidating the mechanisms of anticancer effects after genistein treatment in brain tumor cells, there will be a premise for the incorporation of genistein dietary sources to complement radiotherapy in brain tumor patients.

Telomere dysfunction induced by chemotherapeutic agents and radiation in normal human cells.

The number of long-term survivors of patients with various malignancies (>5 years) is increasing mainly owing to advances in cancer therapeutics, but long-term side effects of the cancer treatment in this population have emerged as an important health and socio-economical issue. Telomeres and telomerase are known to be essential for regulation of cellular life-span and maintenance of genomic stability, and earlier studies have demonstrated that cancer patients who receive chemotherapy have shorter telomeres in their blood cells, indicating accelerated telomere erosion and a potential contribution of telomere loss to late side-effects. Little is currently known about the effect of chemotherapeutic agents and radiation on telomere dynamics including potential effects on telomere length, structure, function, telomerase activity, and telomere shelterin proteins in normal human cells. In the present study, we had addressed this issue experimentally. The treatment of normal human T lymphocytes and fibroblasts with chemotherapeutic agents doxorubicin (DOX) or etoposide (VP16) led to significant shortening of telomeres, down-regulation of telomerase activity, and diminished expression of telomerase reverse transcriptase (hTERT) and the telomere binding proteins TPP1 and POT1. More importantly, telomere dysfunction was observed in cells treated with DOX or VP16. Furthermore, all the above alterations were similarly found in the cells receiving γ-irradiation. Taken together, both chemotherapy and radiotherapy significantly impair telomere maintenance and function in normal human cells. Conceivably telomere dysfunction causes shortened life-span and genomic instability of normal human cells, and thereby contributes to tissue/organ damage and secondary malignancies in long-term survivors of cancer.

Telomere shortening alters the kinetics of the DNA damage response after ionizing radiation in human cells.

Studies of telomerase-deficient mice and human cell lines have showed that telomere shortening enhances sensitivity to ionizing radiation (IR). The molecular basis for this observation remains unclear. To better understand the connection between telomere shortening and radiation sensitivity, we evaluated components of the DNA damage response pathway in normal human fibroblasts with short and long telomeres. Late-passage cells with short telomeres showed enhanced sensitivity to IR compared with early-passage cells with longer telomeres. Compared with early-passage cells, late-passage cells had a higher baseline level of phosphorylated H2AX protein (γH2AX) before IR but diminished peak levels of H2AX phosphorylation after treatment with IR. Both the appearance and disappearance of γH2AX foci were delayed in late-passage cells, indicative of delayed DNA repair. In contrast to the situation with H2AX, ATM and p53 phosphorylation kinetics were similar in early- and late-passage cells, but phosphorylation of the chromatin-bound ATM targets SMC1 and NBS1 was delayed in late-passage cells. Because impaired phosphorylation associated with short telomeres was restricted to chromatin-bound ATM targets, chromatin structure was assessed. DNA from cells with short telomeres was more resistant to digestion with micrococcal nuclease, indicative of compacted chromatin. Moreover, cells with short telomeres showed histone acetylation and methylation profiles consistent with heterochromatin. Together our data suggest a model in which short telomeres induce chromatin structure changes that limit access of activated ATM to its downstream targets on the chromatin, thereby providing a potential explanation for the increased radiation sensitivity seen with telomere shortening.