Mol Cell Biochem (2014) 396:129–135 DOI 10.1007/s11010-014-2149-5
Changes in telomere length distribution in low-dose X-ray-irradiated human umbilical vein endothelial cells Jing-Zhi Guan • Wei Ping Guan • Toyoki Maeda Naoki Makino
•
Received: 19 March 2014 / Accepted: 11 July 2014 / Published online: 25 July 2014 Ó Springer Science+Business Media New York 2014
Abstract Ionizing radiation (IR) is known to be a cause of telomere dysfunction in tumor cells; however, very few studies have investigated X-ray-related changes in telomere length and the telomerase activity in normal human cells, such as umbilical vein endothelial cells (HUVECs). The loss of a few hundred base pairs from a shortened telomere has been shown to be important with respect to cellular senescence, although it may not be detected according to traditional mean telomere length [assessed as the terminal restriction fragment (TRF)] analyses. In the present study, a continuous time window from irradiation was selected to examine changes in the telomere length, including the mean TRF length, percentage of the telomere length, telomerase activity, apoptotic rate, and survival rate in HUVECs from the first day to the fourth day after the administration of a 0.5-Gy dose of irradiation. The mean TRF length in the irradiated HUVECs showed shorter telomere length in first 3 days, but they were not statistically significant. On the other hand, according to the percentage analysis of the telomere length, a decreasing
Jing-Zhi Guan, Wei Ping Guan and Toyoki Maeda have authors equally contributed to this article. J.-Z. Guan The 309th Hospital of Chinese People’s Liberation Army, Beijing 100091, China W. P. Guan Nanlou Neurology Department, Chinese PLA general hospital, Beijing 100853, China T. Maeda (&) N. Makino The Division of Cardiovascular, Respiratory and Geriatric Medicine, Kyushu University Beppu Hospital, Beppu, Oita 874-0838, Japan e-mail: [email protected]
tendency was noted in the longer telomere lengths (9.4–4.4 kb), with a significant increase in the shortest telomeres (4.4–2.3 kb) among the irradiated cells versus the controls from the first day to the third after irradiation; no significant differences were noted on the fourth day. These results suggest that the shortest telomeres are sensitive to the late stage of radiation damage. The proliferation of irradiated cells was suppressed after IR in contrast to the non-irradiated cells. The apoptotic rate was elevated initially both in IR- and non-IR-cells, but that of IR-cells was maintained at an elevated level thereafter in contrast to that of non-IR-cells decreasing promptly. Therefore, a 0.5Gy dose of IR induces persistent apoptosis leading to an apparent growth arrest of the normal HUVECs. Keywords Telomere length distribution Telomerase Vascular endothelial cell X-ray Ionizing irradiation
Introduction Human cells undergo senescence growth arrest, when their telomeres reach a critical length [1]. Telomere instability causes chromosome fusion [2, 3], which may be one mechanism of radiation-induced genomic instability. Accelerated senescence is also associated with telomere shortening, telomere dysfunction, and/or alterations in the telomerase activity [4–8]. Endothelial cells play a key role in the inflammatory response. The modulation of apoptosis by ionizing radiation in endothelial cells is very important with respect to anticancer radiotherapeutic protocols. Previous studies have demonstrated that the replication of normal somatic cells is finite and that, after a critical number of cell divisions, the cells reach a state in which further division
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cannot occur, termed ‘‘replicative senescence,’’ as DNA replication during mitosis is incomplete, thus resulting in a loss of terminal 50–200 bp in telomeres per cell division [9]. Ionizing radiation causes DNA damage in the form of single- and double-strand breaks [10]. Reports of senescence arrest after irradiation are contradictory, with some authors suggesting induction of the telomerase activity [4– 6], others reporting a senescence expression without net telomere shortening [7], and still others demonstrating a reduction in the telomere activity [8]. Radiosensitive cells exhibit severely shortened telomeres [11, 12]. Additionally, IR is widely used in anticancer therapy. Therefore, the relationship between telomere stability and the telomerase activity in normal HUVECs is important. This background led us to investigate the response of IR-related arrest of senescence in the telomeres of normal cells and the side effects of IR anticancer therapy in order to determine, whether there is a relationship between telomere length and radiosensitivity in normal human endothelial cells. A previous study reported that HUVECs display high sensitivity to ionizing radiation and that a high number of genes with very important functions in endothelial cells demonstrate the highest level of upregulation at intermediate doses [13]. Therefore, in this study, we evaluated changes in the telomere length and telomerase activity in HUVECs following X-ray treatment at an intermediate dose of 0.5 Gy.
Methods Cell culture and X-irradiation For the experiment, triplicate independent samples were obtained. HUVECs were seeded at 3 9 105 cells/ml and precultured until approximately 80 % of confluence in a 5 % CO2 atmosphere at 37 °C in complete endothelial cell basal medium-2 (EGM-2) (containing growth factors and enriched with 2 % fetal calf serum) (Cambrex Bio Science Walkersville, Walkersville, MD, USA). At that point, the cells in the culture dishes were irradiated with X-ray at a dose of 0.5 Gy. The X-rays were delivered from an X-ray generator ((SOF-TEX M-150WE, Japan). The cells in the culture dishes were placed on an irradiation stage 30 cm from the radiation source, and the corresponding rate of administration was 2 Gy/min. The cells were then incubated for four days after which they were collected. The culture medium was changed every 24 h (2 ml/50 cm2) and then removed, and the cells were washed with Ca2?free phosphate-buffered saline (PBS-). Subsequently, the cells were immersed in 3 ml of 0.1 % trypsin for less than 2 min, after which 3 ml of medium was added to the dish,
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and the cells were collected via pipetting. The HUVECs were maintained under subconfluent conditions at all times. The number of viable cells determined based on the exclusion of trypan blue was calculated on days 0, 1, 2, 3, and 4 after IR exposure. All experiments were performed using cells of passage 4. Telomere detection Telomere detection was performed as previously described [14–16]. Briefly, Aliquots of DNA (1 lg) were digested at 37 °C with 3 U Msp I for 2 h. The digests (20 ll) were electrophoresed and transferred to a positively charged nylon membrane (Roche Diagnostics, Mannheim, Germany) by the conventional Southern blotting method. The blotted membranes were hybridized to a 500-bp long (TTAGGG)n (n = 80–90) digoxigenin (dig)-labeled telomere probe. The membrane was then incubated with antidigoxigenin-AP-specific antibody. The telomere probe was visualized by CSPD (provided with the kit). The membrane was then exposed to Fuji XR film with an intensifying screen. The smears of the autoradiogram were captured on an Image Master, and the telomere length was assessed quantitatively. Terminal restriction fragment (TRF) analysis The mean TRF was estimated using the formula R(ODibackground)/R(ODi-background/Li) [17], where ODi is the chemiluminescent signal and Li is the length of the TRF fragment at position i. In brief, the intensity (photo-stimulated luminescence: PSL) was quantified as follows: each telomeric sample was divided into grid squares as follows according to the molecular size ranges: 23.1 9.4, 9.4 6.6, 6.6 4.4, and 4.4 2.3 kb (Fig. 1a). The percent of PSL in each molecular weight range was measured (%PSL = intensity of a defined region-background 9 100/total lane intensity-background). Senescence-associated b-galactosidase (SA-b-Gal) expression The cells were washed in PBS, fixed for 10 min at room temperature in 2 % formaldehyde/0.2 % glutaraldehyde, rinsed in PBS, and then incubated at 37 °C (no CO2) with fresh SA-b-Gal staining solution. The staining solution consisted of 1 mg X-gal, per ml solution, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, 150 mM NaCl, and 40 mM citric acid and sodium phosphate at pH 6.0. This solution was left on the cells for 12 h to achieve the maximum staining. Hundred cells were scored from each well (plate) using a light microscope.
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horseradish peroxidase-conjugated antibodies (Chemicon) and the ECL detection system as previously described [19]. Statistical analysis Assays were repeated three times and analyzed statistically. Intergroup comparisons were performed using an independent samples t test and one-way ANOVA. Paired samples were compared using the paired t test. Significance was defined as p values of B0.05. Group data are expressed as mean ± standard deviation. Statistical analyses were performed using the SPSS 10.0 software package (SPSS, Chicago, IL).
Fig. 1 The change of the mean telomere lengths (kb) of HUVECs after 0.5 Gy X-irradiation. Pale and dark gray columns depict controls and irradiated subjects, respectively. Asterisk means p value \0.05 in statistical significance of the comparison between control and irradiated condition
Telomerase activity Telomerase activity was examined by means of a modified telomerase repeat amplification protocol (TRAP) method [18] with TeloChaser (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. Briefly, the substrate oligonucleotide is added to 0.5 mg protein extract. If telomerase is present and active, telomeric repeats (GGTTAG) are added to the 30 end of the oligonucleotide. After amplification, the PCR products were resolved on a 12 % polyacrylamide gel, stained with ethidium bromide, and detected using a FLA 5000 system (Fuji Film, Tokyo, Japan). The intensities of the bands were quantified with Image J (NIH). According to the manufacturer’s instructions, the telomerase activities were calculated and presented as Total Product Generated (TPG). Western blot and other analyses Cells from a dish were homogenized with 100 ll lysis buffer (100 mM Tris pH 6.8; 4 % SDS; 20 % glycerol containing the protease inhibitor M phenylmethanesulfonyl fluoride, 0.1 mM; leupeptin, 0.1 ll; and aprotinin, 0.1 ll). Gel electrophoresis was used to separate 10 lg protein on a 10 % SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose membranes (162–0112, Bio-Rad Labo-ratories, Hercules, California) blocked with 5 % dry milk or blocking solution for Western blot (Roche). Membranes were blocked and incubated with antibodies against telomerase reverse transcriptase (TERT) (Rockland), TRF1 (Imgenex), TRF2 (Cell Signaling), or b-actin (Santa Cruz Biotechnology). Detection was performed with secondary
Results Effects of IR on the average telomere length in the HUVECs The telomere length, as assessed according to the TRF length, was measured in the HUVECs (Fig. 1). There was a tendency toward shortening of the TRF length in the IRcells compared to that observed in the control cells (Control vs. IR, 6.8 ± 0.2 vs. 6.1 ± 2.1, 7.2 ± 0.7 vs. 6.3 ± 0.8, and 7.1 ± 1.0 vs. 6.5 ± 1.4 kb) on the first through the third day after IR, followed by the fourth day when the tendency seemed to disappear (6.9 ± 0.6 vs. 6.9 ± 0.4 kb), although none of their difference was statistically significant (Fig. 1). Effects of IR on the percentage of telomere length in the HUVECs The result of the percentage analysis of the telomere length is shown in Fig. 2. No significant change of telomere length distribution was observed during 4 days in control cell. There was a slight decreasing tendency in the longest telomere length (23.1–9.4 kb) of IR-cells in the first 3 days. In the middle ranges (9.4–6.6 and 6.6–4.4 kb), the peak was observed on the third day, and the lowest percentage was observed on the fourth day. On the other hand, the shortest range (4.4–2.3 kb) increased on 1st and 2nd day, followed by the lowest on the third day, and its percentage re-increased on the fourth day (Fig. 2). Relationship between the survival rate and duration after IR The number of cells in the mass cultures was followed over 4 days. The non-irradiated cells grew exponentially, whereas the irradiated cells showed only minimal cell
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Fig. 2 The change of the telomere length distributions of HUVECs after 0.5 Gy X-irradiation. Pale and dark gray columns depict controls and irradiated subjects, respectively. Asterisk means p value \0.05 in statistical significance of the comparison between control
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and irradiated condition. The telomere length ranges are as follows: a 23.1–9.4 kb, b 9.4–6.6 kb, c 6.6–4.4 kb, and d 4.4–2.3 kb. Asterisk means a significant difference between IR-cells and control (p \ 0.05)
The apoptotic rate after IR The percentage of apoptotic cells was detected by SA-b– Gal staining (Fig. 4). The apoptotic rate (%) were as follows; Non-IR versus IR, 0.0 ± 0.0 versus 0.0 ± 0.0 on day 0, 2.4 ± 0.1 versus 2.2 ± 0.5 on day 1, 1.0 ± 0.6 versus 2.4 ± 0.1 on day 2.5 (p \ 0.05), and 0.0 ± 0.0 versus 2.5 ± 0.7 on day 4 (p \ 0.02); Both IR-cells and non-IRcells showed a similar increase of apoptotic rate (*2.5 %) on day 1. Thereafter, they showed different ways. IR-cells maintained the elevated level of apoptotic rate in contrast to non-IR-cells, in which it decreased to 0 % on day 4. Fig. 3 The cell numbers of HUVECs after 0.5 Gy X-irradiation. Viable (trypan blue-non- stained) cell numbers per dish are plotted
proliferation over this period (Fig. 3). These results indicate that a 0.5-Gy dose of IR induced growth arrest in the normal HUVECs, similar to the findings of previous studies [20].
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Effects of IR on the telomerase activity in the HUVECs We examined the effects of IR on the telomerase activity in the HUVECs. The cells were cultured for 4 days after IR, and the pellets were analyzed every day after IR using a TRAP assay. There were no significant differences between the groups (Fig. 5).
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Expression of telomere-associated proteins The protein expression levels of TERT, TRF1, and TRF2 were analyzed (Fig. 6). They showed no significant change after irradiation from day 0 to day 4.
Discussion Ionizing radiation does not induce significant overall telomere shortening in senescence cells, although it is possible that a single shortened telomere may be responsible for the senescence response [16, 21, 22]. However, it should be noted that the TRF (telomere length) assay evaluates only the average telomere length. Therefore, in the present study, we analyzed the effects of 0.5 Gy of X-rays on changes in the
Fig. 4 The ratio of senescence-associated b-galactosidase (SA-bGal) staining of HUVECs cultured after X-irradiation. The graph shows the percentages of IR-cells and non-IR-cells that were stained
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telomere length, including the mean TRF length, percentage of the telomere length, telomerase activity, the expression of telomere-associated proteins (TRF1 and TRF2), apoptotic rate, and survival rate in HUVECs. We found that the mean TRF length in the irradiated HUVECs exhibited no significant change, but a relatively decreasing tendency in the first 3 days. The changes of TL distribution after IR in this study suggested that cells containing the longest range of telomere were relatively conserved. It was also suggested that middlesized telomeres were shortened and entered the shortest range, and cells with the shortest telomeres increased for 2 days but some of them entered senescence stage and died on the third day, followed by the fourth day when the shortest range increased by shortening of middle-sized telomeres from the 3rd to 4th day, from the observation that middlesized range increased the best on the third day and sharply decreased on the fourth day, and the shortest range decreased on the third day and restored on the fourth day. Cell growth was suppressed from the third day after IR. In this period, the apoptotic rate of IR-cell population was maintained at a constantly elevated rate in contrast to non-IR-cell population. These observations characterized three size ranges of telomere. Cells containing the longest telomeres were relatively resistant to IR; middle-size telomere shortening was accelerated, and cells containing the shortest telomeres IRlabile and die on the third day by low-dose IR exposure. These telomeric changes in irradiated cells seemed to be independent of their telomerase activity and their expression levels of telomere-associated proteins, TRF1 and TRF2. Conclusively, the cell fate after IR exposure depends partly on the telomere size of the cell. There exist at least two factors responding to the effects of IR, including the initial damage affecting the stability of chromosomes, thus indicating excessive IR-induced oxidative stress, and late damage of IR-induced chromosome position site deletion, which
Fig. 5 The telomerase activity of endothelial cells after X-irradiation. The left panel shows the telomerase activity after X-irradiation as the relative TPG (Total Product Generated) levels. The TPG is presented as a proportional ratio of a ladder density of a sample to that at day 0. The right panel shows a photograph of representative TRAP assay result for HUVECs after X-irradiation. The materials used for the pc positive control and nc negative control were provided with the kit
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Fig. 6 The expression levels of telomere-associated proteins, TERT, TRF1 and TRF2. Representative (left) and summarized (right) results for the western blot analysis of telomere-associated proteins (TERT, TRF1 and TRF2) after X-irradiation are shown. The relative expression levels were determined compared to that of b-actin, and the level at day 0 was set at 1. Horizontal bars represent standard deviations. n =6
leads to telomere reorganization (these hypotheses are partly supported by the results of previous studies [23, 24]; a 0.5Gy dose of IR not only induced telomeric changes but maintained apoptotic rate, leading to the growth suppression of the normal HUVECs. There was a decreasing tendency among the relatively longer telomeres as well as a significant increase in the shortest telomeres within the two to three days after IR. This implies that two factors may play a role in the damage induced by IR. First, the decreased percentage of length observed in the longer telomeres may have been due to the effects of excessive IR-induced oxidative stress, which is consistent with the findings of previous studies showing that accelerated senescence due to cellular stress is associated with DNA damage, including that induced by ionizing radiation [24–26]. In addition, radiation generates ceramide, which has the capacity to drive cells toward senescence [27]. It is well known that cells with longer telomeres are more vulnerable to oxidative stress than cells with shorter telomeres [28], thus suggesting that the initial damage observed within the first 3 days after IR may result from excessive IRinduced oxidative stress. In the present study, the amount of cell containing shorter telomeres seems to be affected more by IR. Second, short telomeres exhibit enhanced IR-induced lethality in telomerase-defect mice [29], and cells with defects in telomere maintenance are frequently radiosensitive [30]. These findings suggest an inverse relationship between telomere length and chromosomal radiosensitivity [11]. Furthermore, X-rays alter chromosomal organization by affecting telomere stability after irradiation as a delayed effect in X-ray-surviving cells [23], which supports our results showing a persistent apoptotic rate in the late stage, and a relatively obvious change in the shortest telomeres among the irradiated cells over the observation period after
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IR, particularly from the third day to the fourth day; such changes were not observed in other sizes of telomeres. Similar to the results of the percentage analysis, we noted a decreasing tendency in the mean TRF length among the irradiated cells until the third day, whereas the tendency disappeared on the fourth day. This weak increasing tendency in TRF length from third to fourth day may have been due to the proportional decrease in the middle-short range (6.6–4.4 kb) or the shortest range (4.4–2.3 kb) of telomere and the relatively restored percentage of longest telomeres in the irradiated cells on the fourth day after RI. Regarding the initial telomere damage observed in this study, for example, within the first 24 h, we detected a decreasing tendency in the TRF length among the irradiated cells compared to that observed in the controls; this finding is similar to that of a report showing no significant increases in the frequency of telomere abnormalities within 24 h after irradiation [23]. IR is a known cause of telomere dysfunction following telomere instability in normal and radiosensitivity-relative protein-deficient cells [23, 31]. In the present study, there may be a combined IR-induced effect, including initial excessive oxidative stress [25, 26] and late reorganization to telomeres [23, 24, 31]. Our data, therefore, strongly suggest a relationship between telomere length and the duration after IR. However, the cell type, observed period and radiation dose may differ in cellular responses to dysfunction and/or DNA damage. For example, reports on senescence arrest after irradiation are contradictory, with some studies documenting severely shortened telomeres in radiosensitive cells [11, 12]. Telomere length is not a simple marker of the level of chromosomal radiosensitivity, independent of the duration after IR. The present results also imply that the process of wound healing or tumor progression requiring vascular proliferation
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is suppressed by X-ray irradiation even at a low dose especially in the elderly, who bear shorter telomeres in somatic cells in comparison with the young. The present data showed that the shortest telomeres were more sensitive to IR than the other sizes of telomeres based on the telomere percentage analysis. Jones reported that IR induces dysfunction of telomeres, including telomere-negative end fusion, chromatid breaks, and telomere-positive end fusion, with a lack of telomeric signals at most fusion points [32]. The end-to-end fusion of shortened or destabilized telomeres may constitute the mechanism underlying the induction of genomic instability induced by ionizing radiation [23]. In addition, short telomeres may be more prone than long telomeres to structural dysfunction, while long telomeres, but not telomerase, protect cells from the loss of division potential caused by ionizing radiation [8]. Acknowledgments This work was supported, in part, by the National Natural Science Fund (NSFC) (81170329/H2501), the Ministry of Education, Science, and Culture of Japan (#23590885), the 2012 Health and Labour Sciences Research Grants Comprehensive Research on Life-Style Related Diseases including Cardiovascular Diseases and Diabetes Mellitus.
References 1. Chiu CP, Harley CB (1997) Replicative senescence and cell immortality: the role of telomeres and telomerase. Proc Soc Exp Biol Med 214:99–106 2. Goytisolo FA, Samper E, Edmonson S, Taccioli GE, Blasco MA (2001) The absence of the dna-dependent protein kinase catalytic subunit in mice results in anaphase bridges and in increased telomeric fusions with normal telomere length and G-strand overhang. Mol Cell Biol 21:3642–3651 3. van Steensel B, Smogorzewska A, de Lange T (1998) TRF2 protects human telomeres from end-to-end fusions. Cell 92:401–413 4. Neuhof D, Ruess A, Wenz F, Weber KJ (2001) Induction of telomerase activity by irradiation in human lymphoblasts. Radiat Res 155:693–697 5. Hyeon Joo O, Hande MP, Lansdorp PM, Natarajan AT (1998) Induction of telomerase activity and chromosome aberrations in human tumour cell lines following X-irradiation. Mutat Res 401:121–131 6. Leteurtre F, Li X, Gluckman E, Carosella ED (1997) Telomerase activity during the cell cycle and in gamma-irradiated hematopoietic cells. Leukemia 11:1681–1689 7. Suzuki K, Mori I, Nakayama Y, Miyakoda M, Kodama S, Watanabe M (2001) Radiation-induced senescence-like growth arrest requires TP53 function but not telomere shortening. Radiat Res 155:248–253 8. Rubio MA, Kim SH, Campisi J (2002) Reversible manipulation of telomerase expression and telomere length. Implications for the ionizing radiation response and replicative senescence of human cells. J Biol Chem 277:28609–28617 9. Hayflick L (1965) The limited in vitro lifetime of human diploid cell strains. Exp Cell Res 37:614–636 10. Jackson SP (2002) Sensing and repairing DNA double-strand breaks. Carcinogenesis 23:687–696 11. McIlrath J, Bouffler SD, Samper E et al (2001) Telomere length abnormalities in mammalian radiosensitive cells. Cancer Res 61:912–915
135 12. Ahmed S, Hodgkin J (2000) MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans. Nature 403:159–164 13. Lanza V, Pretazzoli V, Olivieri G, Pascarella G, Panconesi A, Negri R (2005) Transcriptional response of human umbilical vein endothelial cells to low doses of ionizing radiation. J Radiat Res 46:265–276 14. Harley CB, Futcher AB, Greider CW (1990) Telomeres shorten during ageing of human fibroblasts. Nature 345:458–460 15. Vaziri H, Dragowska W, Allsopp RC, Thomas TE, Harley CB, Lansdorp PM (1994) Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeirc DNA with age. Proc Natl Acad Aci USA 91:9857–9860 16. Cherif H, Tarry JL, Ozanne SE, Hales CN (2003) Ageing and telomeres: a study into organ- and gender-specific telomere shortening. Nucl Acid Res 31:1576–1583 17. de Lange T, Shiue L, Myers RM et al (1990) Structure and variability of human chromosome ends. Mol Cell Biol 10:518–527 18. Nagata T, Takada Y, Ono A et al (2008) Elucidation of the mode of interaction in the UP1-telomerase RNA-telomeric DNA ternary complex which serves to recruit telomerase to telomeric DNA and to enhance the telomerase activity. Nucleic Acids Res 36:6816–6824 19. Sugano M, Tsuchida K, Maeda T, Makino N (2007) siRNA targeting SHP-1 accelerates angiogenesis in a rat model of hindlimb ischemia. Atherosclerosis 191:33–39 20. Robles SJ, Adami GR (1998) Agents that cause DNA double strand breaks lead to p16INK4a enrichment and the premature senescence of normal fibroblasts. Oncogene 16:1113–1123 21. Hemann MT, Strong MA, Hao LY, Greider CW (2001) The shortest telomere, not average telomere length, is critical for cell viability and chromosome stability. Cell 107:67–77 22. von Zglinicki T (2003) Replicative senescence and the art of counting. Exp Gerontol 38:1259–1264 23. Ojima M, Hamano H, Suzuki M, Suzuki K, Kodama S, Watanabe M (2004) Delayed induction of telomere instability in normal human fibroblast cells by ionizing radiation. J Radiat Res 45:105–110 24. Kawanishi S, Oikawa S (2004) Mechanism of telomere shortening by oxidative stress. Ann NY Acad Sci 1019:278–284 25. Przybyszewski WM, Widel M, Rzeszowska-Wolny J (2006) Cardiotoxic consequences of ionizing radiation and anthracyclines. Postepy Hig Med Dosw 60:397–405 26. Little JB, Lauriston S (2006) Taylor lecture: nontargeted effects of radiation: implications for low-dose exposures. Health Phys 91:416–426 27. Ogretmen B, Schady D, Usta J et al (2001) Role of ceramide in mediating the inhibition of telomerase activity in A549 human lung adenocarcinoma cells. J Biol Chem 276:24901–24910 28. Rubio MA, Davalos AR, Campisi J (2004) Telomere length mediates the effects of telomerase on the cellular response to genotoxic stress. Exp Cell Res 298:17–27 29. Wong KK, Chang S, Weiler SR et al (2000) Telomere dysfunction impairs DNA repair and enhances sensitivity to ionizing radiation. Nat Genet 26:85–88 30. Hande P, Slijepcevic P, Silver A, Bouffler S, van Buul P, Bryant P, Lansdorp P (1999) Elongated telomeres in scid mice. Genomics 56:221–223 31. Undarmaa B, Kodama S, Suzuki K, Niwa O, Watanabe M (2004) X-ray-induced telomeric instability in Atm-deficient mouse cells. Biochem Biophys Res Commun 315:51–58 32. Jones KR, Elmore LW, Jackson-Cook C, Demasters G, Povirk LF, Holt SE, Gewirtz DA (2005) p53-Dependent accelerated senescence induced by ionizing radiation in breast tumour cells. Int J Radiat Biol 81:445–458
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Changes in telomere length distribution in low-dose X-ray-irradiated human umbilical vein endothelial cells Jing-Zhi Guan • Wei Ping Guan • Toyoki Maeda Naoki Makino
•
Received: 19 March 2014 / Accepted: 11 July 2014 / Published online: 25 July 2014 Ó Springer Science+Business Media New York 2014
Abstract Ionizing radiation (IR) is known to be a cause of telomere dysfunction in tumor cells; however, very few studies have investigated X-ray-related changes in telomere length and the telomerase activity in normal human cells, such as umbilical vein endothelial cells (HUVECs). The loss of a few hundred base pairs from a shortened telomere has been shown to be important with respect to cellular senescence, although it may not be detected according to traditional mean telomere length [assessed as the terminal restriction fragment (TRF)] analyses. In the present study, a continuous time window from irradiation was selected to examine changes in the telomere length, including the mean TRF length, percentage of the telomere length, telomerase activity, apoptotic rate, and survival rate in HUVECs from the first day to the fourth day after the administration of a 0.5-Gy dose of irradiation. The mean TRF length in the irradiated HUVECs showed shorter telomere length in first 3 days, but they were not statistically significant. On the other hand, according to the percentage analysis of the telomere length, a decreasing
Jing-Zhi Guan, Wei Ping Guan and Toyoki Maeda have authors equally contributed to this article. J.-Z. Guan The 309th Hospital of Chinese People’s Liberation Army, Beijing 100091, China W. P. Guan Nanlou Neurology Department, Chinese PLA general hospital, Beijing 100853, China T. Maeda (&) N. Makino The Division of Cardiovascular, Respiratory and Geriatric Medicine, Kyushu University Beppu Hospital, Beppu, Oita 874-0838, Japan e-mail: [email protected]
tendency was noted in the longer telomere lengths (9.4–4.4 kb), with a significant increase in the shortest telomeres (4.4–2.3 kb) among the irradiated cells versus the controls from the first day to the third after irradiation; no significant differences were noted on the fourth day. These results suggest that the shortest telomeres are sensitive to the late stage of radiation damage. The proliferation of irradiated cells was suppressed after IR in contrast to the non-irradiated cells. The apoptotic rate was elevated initially both in IR- and non-IR-cells, but that of IR-cells was maintained at an elevated level thereafter in contrast to that of non-IR-cells decreasing promptly. Therefore, a 0.5Gy dose of IR induces persistent apoptosis leading to an apparent growth arrest of the normal HUVECs. Keywords Telomere length distribution Telomerase Vascular endothelial cell X-ray Ionizing irradiation
Introduction Human cells undergo senescence growth arrest, when their telomeres reach a critical length [1]. Telomere instability causes chromosome fusion [2, 3], which may be one mechanism of radiation-induced genomic instability. Accelerated senescence is also associated with telomere shortening, telomere dysfunction, and/or alterations in the telomerase activity [4–8]. Endothelial cells play a key role in the inflammatory response. The modulation of apoptosis by ionizing radiation in endothelial cells is very important with respect to anticancer radiotherapeutic protocols. Previous studies have demonstrated that the replication of normal somatic cells is finite and that, after a critical number of cell divisions, the cells reach a state in which further division
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cannot occur, termed ‘‘replicative senescence,’’ as DNA replication during mitosis is incomplete, thus resulting in a loss of terminal 50–200 bp in telomeres per cell division [9]. Ionizing radiation causes DNA damage in the form of single- and double-strand breaks [10]. Reports of senescence arrest after irradiation are contradictory, with some authors suggesting induction of the telomerase activity [4– 6], others reporting a senescence expression without net telomere shortening [7], and still others demonstrating a reduction in the telomere activity [8]. Radiosensitive cells exhibit severely shortened telomeres [11, 12]. Additionally, IR is widely used in anticancer therapy. Therefore, the relationship between telomere stability and the telomerase activity in normal HUVECs is important. This background led us to investigate the response of IR-related arrest of senescence in the telomeres of normal cells and the side effects of IR anticancer therapy in order to determine, whether there is a relationship between telomere length and radiosensitivity in normal human endothelial cells. A previous study reported that HUVECs display high sensitivity to ionizing radiation and that a high number of genes with very important functions in endothelial cells demonstrate the highest level of upregulation at intermediate doses [13]. Therefore, in this study, we evaluated changes in the telomere length and telomerase activity in HUVECs following X-ray treatment at an intermediate dose of 0.5 Gy.
Methods Cell culture and X-irradiation For the experiment, triplicate independent samples were obtained. HUVECs were seeded at 3 9 105 cells/ml and precultured until approximately 80 % of confluence in a 5 % CO2 atmosphere at 37 °C in complete endothelial cell basal medium-2 (EGM-2) (containing growth factors and enriched with 2 % fetal calf serum) (Cambrex Bio Science Walkersville, Walkersville, MD, USA). At that point, the cells in the culture dishes were irradiated with X-ray at a dose of 0.5 Gy. The X-rays were delivered from an X-ray generator ((SOF-TEX M-150WE, Japan). The cells in the culture dishes were placed on an irradiation stage 30 cm from the radiation source, and the corresponding rate of administration was 2 Gy/min. The cells were then incubated for four days after which they were collected. The culture medium was changed every 24 h (2 ml/50 cm2) and then removed, and the cells were washed with Ca2?free phosphate-buffered saline (PBS-). Subsequently, the cells were immersed in 3 ml of 0.1 % trypsin for less than 2 min, after which 3 ml of medium was added to the dish,
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and the cells were collected via pipetting. The HUVECs were maintained under subconfluent conditions at all times. The number of viable cells determined based on the exclusion of trypan blue was calculated on days 0, 1, 2, 3, and 4 after IR exposure. All experiments were performed using cells of passage 4. Telomere detection Telomere detection was performed as previously described [14–16]. Briefly, Aliquots of DNA (1 lg) were digested at 37 °C with 3 U Msp I for 2 h. The digests (20 ll) were electrophoresed and transferred to a positively charged nylon membrane (Roche Diagnostics, Mannheim, Germany) by the conventional Southern blotting method. The blotted membranes were hybridized to a 500-bp long (TTAGGG)n (n = 80–90) digoxigenin (dig)-labeled telomere probe. The membrane was then incubated with antidigoxigenin-AP-specific antibody. The telomere probe was visualized by CSPD (provided with the kit). The membrane was then exposed to Fuji XR film with an intensifying screen. The smears of the autoradiogram were captured on an Image Master, and the telomere length was assessed quantitatively. Terminal restriction fragment (TRF) analysis The mean TRF was estimated using the formula R(ODibackground)/R(ODi-background/Li) [17], where ODi is the chemiluminescent signal and Li is the length of the TRF fragment at position i. In brief, the intensity (photo-stimulated luminescence: PSL) was quantified as follows: each telomeric sample was divided into grid squares as follows according to the molecular size ranges: 23.1 9.4, 9.4 6.6, 6.6 4.4, and 4.4 2.3 kb (Fig. 1a). The percent of PSL in each molecular weight range was measured (%PSL = intensity of a defined region-background 9 100/total lane intensity-background). Senescence-associated b-galactosidase (SA-b-Gal) expression The cells were washed in PBS, fixed for 10 min at room temperature in 2 % formaldehyde/0.2 % glutaraldehyde, rinsed in PBS, and then incubated at 37 °C (no CO2) with fresh SA-b-Gal staining solution. The staining solution consisted of 1 mg X-gal, per ml solution, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, 150 mM NaCl, and 40 mM citric acid and sodium phosphate at pH 6.0. This solution was left on the cells for 12 h to achieve the maximum staining. Hundred cells were scored from each well (plate) using a light microscope.
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horseradish peroxidase-conjugated antibodies (Chemicon) and the ECL detection system as previously described [19]. Statistical analysis Assays were repeated three times and analyzed statistically. Intergroup comparisons were performed using an independent samples t test and one-way ANOVA. Paired samples were compared using the paired t test. Significance was defined as p values of B0.05. Group data are expressed as mean ± standard deviation. Statistical analyses were performed using the SPSS 10.0 software package (SPSS, Chicago, IL).
Fig. 1 The change of the mean telomere lengths (kb) of HUVECs after 0.5 Gy X-irradiation. Pale and dark gray columns depict controls and irradiated subjects, respectively. Asterisk means p value \0.05 in statistical significance of the comparison between control and irradiated condition
Telomerase activity Telomerase activity was examined by means of a modified telomerase repeat amplification protocol (TRAP) method [18] with TeloChaser (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. Briefly, the substrate oligonucleotide is added to 0.5 mg protein extract. If telomerase is present and active, telomeric repeats (GGTTAG) are added to the 30 end of the oligonucleotide. After amplification, the PCR products were resolved on a 12 % polyacrylamide gel, stained with ethidium bromide, and detected using a FLA 5000 system (Fuji Film, Tokyo, Japan). The intensities of the bands were quantified with Image J (NIH). According to the manufacturer’s instructions, the telomerase activities were calculated and presented as Total Product Generated (TPG). Western blot and other analyses Cells from a dish were homogenized with 100 ll lysis buffer (100 mM Tris pH 6.8; 4 % SDS; 20 % glycerol containing the protease inhibitor M phenylmethanesulfonyl fluoride, 0.1 mM; leupeptin, 0.1 ll; and aprotinin, 0.1 ll). Gel electrophoresis was used to separate 10 lg protein on a 10 % SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose membranes (162–0112, Bio-Rad Labo-ratories, Hercules, California) blocked with 5 % dry milk or blocking solution for Western blot (Roche). Membranes were blocked and incubated with antibodies against telomerase reverse transcriptase (TERT) (Rockland), TRF1 (Imgenex), TRF2 (Cell Signaling), or b-actin (Santa Cruz Biotechnology). Detection was performed with secondary
Results Effects of IR on the average telomere length in the HUVECs The telomere length, as assessed according to the TRF length, was measured in the HUVECs (Fig. 1). There was a tendency toward shortening of the TRF length in the IRcells compared to that observed in the control cells (Control vs. IR, 6.8 ± 0.2 vs. 6.1 ± 2.1, 7.2 ± 0.7 vs. 6.3 ± 0.8, and 7.1 ± 1.0 vs. 6.5 ± 1.4 kb) on the first through the third day after IR, followed by the fourth day when the tendency seemed to disappear (6.9 ± 0.6 vs. 6.9 ± 0.4 kb), although none of their difference was statistically significant (Fig. 1). Effects of IR on the percentage of telomere length in the HUVECs The result of the percentage analysis of the telomere length is shown in Fig. 2. No significant change of telomere length distribution was observed during 4 days in control cell. There was a slight decreasing tendency in the longest telomere length (23.1–9.4 kb) of IR-cells in the first 3 days. In the middle ranges (9.4–6.6 and 6.6–4.4 kb), the peak was observed on the third day, and the lowest percentage was observed on the fourth day. On the other hand, the shortest range (4.4–2.3 kb) increased on 1st and 2nd day, followed by the lowest on the third day, and its percentage re-increased on the fourth day (Fig. 2). Relationship between the survival rate and duration after IR The number of cells in the mass cultures was followed over 4 days. The non-irradiated cells grew exponentially, whereas the irradiated cells showed only minimal cell
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Fig. 2 The change of the telomere length distributions of HUVECs after 0.5 Gy X-irradiation. Pale and dark gray columns depict controls and irradiated subjects, respectively. Asterisk means p value \0.05 in statistical significance of the comparison between control
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and irradiated condition. The telomere length ranges are as follows: a 23.1–9.4 kb, b 9.4–6.6 kb, c 6.6–4.4 kb, and d 4.4–2.3 kb. Asterisk means a significant difference between IR-cells and control (p \ 0.05)
The apoptotic rate after IR The percentage of apoptotic cells was detected by SA-b– Gal staining (Fig. 4). The apoptotic rate (%) were as follows; Non-IR versus IR, 0.0 ± 0.0 versus 0.0 ± 0.0 on day 0, 2.4 ± 0.1 versus 2.2 ± 0.5 on day 1, 1.0 ± 0.6 versus 2.4 ± 0.1 on day 2.5 (p \ 0.05), and 0.0 ± 0.0 versus 2.5 ± 0.7 on day 4 (p \ 0.02); Both IR-cells and non-IRcells showed a similar increase of apoptotic rate (*2.5 %) on day 1. Thereafter, they showed different ways. IR-cells maintained the elevated level of apoptotic rate in contrast to non-IR-cells, in which it decreased to 0 % on day 4. Fig. 3 The cell numbers of HUVECs after 0.5 Gy X-irradiation. Viable (trypan blue-non- stained) cell numbers per dish are plotted
proliferation over this period (Fig. 3). These results indicate that a 0.5-Gy dose of IR induced growth arrest in the normal HUVECs, similar to the findings of previous studies [20].
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Effects of IR on the telomerase activity in the HUVECs We examined the effects of IR on the telomerase activity in the HUVECs. The cells were cultured for 4 days after IR, and the pellets were analyzed every day after IR using a TRAP assay. There were no significant differences between the groups (Fig. 5).
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Expression of telomere-associated proteins The protein expression levels of TERT, TRF1, and TRF2 were analyzed (Fig. 6). They showed no significant change after irradiation from day 0 to day 4.
Discussion Ionizing radiation does not induce significant overall telomere shortening in senescence cells, although it is possible that a single shortened telomere may be responsible for the senescence response [16, 21, 22]. However, it should be noted that the TRF (telomere length) assay evaluates only the average telomere length. Therefore, in the present study, we analyzed the effects of 0.5 Gy of X-rays on changes in the
Fig. 4 The ratio of senescence-associated b-galactosidase (SA-bGal) staining of HUVECs cultured after X-irradiation. The graph shows the percentages of IR-cells and non-IR-cells that were stained
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telomere length, including the mean TRF length, percentage of the telomere length, telomerase activity, the expression of telomere-associated proteins (TRF1 and TRF2), apoptotic rate, and survival rate in HUVECs. We found that the mean TRF length in the irradiated HUVECs exhibited no significant change, but a relatively decreasing tendency in the first 3 days. The changes of TL distribution after IR in this study suggested that cells containing the longest range of telomere were relatively conserved. It was also suggested that middlesized telomeres were shortened and entered the shortest range, and cells with the shortest telomeres increased for 2 days but some of them entered senescence stage and died on the third day, followed by the fourth day when the shortest range increased by shortening of middle-sized telomeres from the 3rd to 4th day, from the observation that middlesized range increased the best on the third day and sharply decreased on the fourth day, and the shortest range decreased on the third day and restored on the fourth day. Cell growth was suppressed from the third day after IR. In this period, the apoptotic rate of IR-cell population was maintained at a constantly elevated rate in contrast to non-IR-cell population. These observations characterized three size ranges of telomere. Cells containing the longest telomeres were relatively resistant to IR; middle-size telomere shortening was accelerated, and cells containing the shortest telomeres IRlabile and die on the third day by low-dose IR exposure. These telomeric changes in irradiated cells seemed to be independent of their telomerase activity and their expression levels of telomere-associated proteins, TRF1 and TRF2. Conclusively, the cell fate after IR exposure depends partly on the telomere size of the cell. There exist at least two factors responding to the effects of IR, including the initial damage affecting the stability of chromosomes, thus indicating excessive IR-induced oxidative stress, and late damage of IR-induced chromosome position site deletion, which
Fig. 5 The telomerase activity of endothelial cells after X-irradiation. The left panel shows the telomerase activity after X-irradiation as the relative TPG (Total Product Generated) levels. The TPG is presented as a proportional ratio of a ladder density of a sample to that at day 0. The right panel shows a photograph of representative TRAP assay result for HUVECs after X-irradiation. The materials used for the pc positive control and nc negative control were provided with the kit
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Fig. 6 The expression levels of telomere-associated proteins, TERT, TRF1 and TRF2. Representative (left) and summarized (right) results for the western blot analysis of telomere-associated proteins (TERT, TRF1 and TRF2) after X-irradiation are shown. The relative expression levels were determined compared to that of b-actin, and the level at day 0 was set at 1. Horizontal bars represent standard deviations. n =6
leads to telomere reorganization (these hypotheses are partly supported by the results of previous studies [23, 24]; a 0.5Gy dose of IR not only induced telomeric changes but maintained apoptotic rate, leading to the growth suppression of the normal HUVECs. There was a decreasing tendency among the relatively longer telomeres as well as a significant increase in the shortest telomeres within the two to three days after IR. This implies that two factors may play a role in the damage induced by IR. First, the decreased percentage of length observed in the longer telomeres may have been due to the effects of excessive IR-induced oxidative stress, which is consistent with the findings of previous studies showing that accelerated senescence due to cellular stress is associated with DNA damage, including that induced by ionizing radiation [24–26]. In addition, radiation generates ceramide, which has the capacity to drive cells toward senescence [27]. It is well known that cells with longer telomeres are more vulnerable to oxidative stress than cells with shorter telomeres [28], thus suggesting that the initial damage observed within the first 3 days after IR may result from excessive IRinduced oxidative stress. In the present study, the amount of cell containing shorter telomeres seems to be affected more by IR. Second, short telomeres exhibit enhanced IR-induced lethality in telomerase-defect mice [29], and cells with defects in telomere maintenance are frequently radiosensitive [30]. These findings suggest an inverse relationship between telomere length and chromosomal radiosensitivity [11]. Furthermore, X-rays alter chromosomal organization by affecting telomere stability after irradiation as a delayed effect in X-ray-surviving cells [23], which supports our results showing a persistent apoptotic rate in the late stage, and a relatively obvious change in the shortest telomeres among the irradiated cells over the observation period after
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IR, particularly from the third day to the fourth day; such changes were not observed in other sizes of telomeres. Similar to the results of the percentage analysis, we noted a decreasing tendency in the mean TRF length among the irradiated cells until the third day, whereas the tendency disappeared on the fourth day. This weak increasing tendency in TRF length from third to fourth day may have been due to the proportional decrease in the middle-short range (6.6–4.4 kb) or the shortest range (4.4–2.3 kb) of telomere and the relatively restored percentage of longest telomeres in the irradiated cells on the fourth day after RI. Regarding the initial telomere damage observed in this study, for example, within the first 24 h, we detected a decreasing tendency in the TRF length among the irradiated cells compared to that observed in the controls; this finding is similar to that of a report showing no significant increases in the frequency of telomere abnormalities within 24 h after irradiation [23]. IR is a known cause of telomere dysfunction following telomere instability in normal and radiosensitivity-relative protein-deficient cells [23, 31]. In the present study, there may be a combined IR-induced effect, including initial excessive oxidative stress [25, 26] and late reorganization to telomeres [23, 24, 31]. Our data, therefore, strongly suggest a relationship between telomere length and the duration after IR. However, the cell type, observed period and radiation dose may differ in cellular responses to dysfunction and/or DNA damage. For example, reports on senescence arrest after irradiation are contradictory, with some studies documenting severely shortened telomeres in radiosensitive cells [11, 12]. Telomere length is not a simple marker of the level of chromosomal radiosensitivity, independent of the duration after IR. The present results also imply that the process of wound healing or tumor progression requiring vascular proliferation
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is suppressed by X-ray irradiation even at a low dose especially in the elderly, who bear shorter telomeres in somatic cells in comparison with the young. The present data showed that the shortest telomeres were more sensitive to IR than the other sizes of telomeres based on the telomere percentage analysis. Jones reported that IR induces dysfunction of telomeres, including telomere-negative end fusion, chromatid breaks, and telomere-positive end fusion, with a lack of telomeric signals at most fusion points [32]. The end-to-end fusion of shortened or destabilized telomeres may constitute the mechanism underlying the induction of genomic instability induced by ionizing radiation [23]. In addition, short telomeres may be more prone than long telomeres to structural dysfunction, while long telomeres, but not telomerase, protect cells from the loss of division potential caused by ionizing radiation [8]. Acknowledgments This work was supported, in part, by the National Natural Science Fund (NSFC) (81170329/H2501), the Ministry of Education, Science, and Culture of Japan (#23590885), the 2012 Health and Labour Sciences Research Grants Comprehensive Research on Life-Style Related Diseases including Cardiovascular Diseases and Diabetes Mellitus.
References 1. Chiu CP, Harley CB (1997) Replicative senescence and cell immortality: the role of telomeres and telomerase. Proc Soc Exp Biol Med 214:99–106 2. Goytisolo FA, Samper E, Edmonson S, Taccioli GE, Blasco MA (2001) The absence of the dna-dependent protein kinase catalytic subunit in mice results in anaphase bridges and in increased telomeric fusions with normal telomere length and G-strand overhang. Mol Cell Biol 21:3642–3651 3. van Steensel B, Smogorzewska A, de Lange T (1998) TRF2 protects human telomeres from end-to-end fusions. Cell 92:401–413 4. Neuhof D, Ruess A, Wenz F, Weber KJ (2001) Induction of telomerase activity by irradiation in human lymphoblasts. Radiat Res 155:693–697 5. Hyeon Joo O, Hande MP, Lansdorp PM, Natarajan AT (1998) Induction of telomerase activity and chromosome aberrations in human tumour cell lines following X-irradiation. Mutat Res 401:121–131 6. Leteurtre F, Li X, Gluckman E, Carosella ED (1997) Telomerase activity during the cell cycle and in gamma-irradiated hematopoietic cells. Leukemia 11:1681–1689 7. Suzuki K, Mori I, Nakayama Y, Miyakoda M, Kodama S, Watanabe M (2001) Radiation-induced senescence-like growth arrest requires TP53 function but not telomere shortening. Radiat Res 155:248–253 8. Rubio MA, Kim SH, Campisi J (2002) Reversible manipulation of telomerase expression and telomere length. Implications for the ionizing radiation response and replicative senescence of human cells. J Biol Chem 277:28609–28617 9. Hayflick L (1965) The limited in vitro lifetime of human diploid cell strains. Exp Cell Res 37:614–636 10. Jackson SP (2002) Sensing and repairing DNA double-strand breaks. Carcinogenesis 23:687–696 11. McIlrath J, Bouffler SD, Samper E et al (2001) Telomere length abnormalities in mammalian radiosensitive cells. Cancer Res 61:912–915
135 12. Ahmed S, Hodgkin J (2000) MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans. Nature 403:159–164 13. Lanza V, Pretazzoli V, Olivieri G, Pascarella G, Panconesi A, Negri R (2005) Transcriptional response of human umbilical vein endothelial cells to low doses of ionizing radiation. J Radiat Res 46:265–276 14. Harley CB, Futcher AB, Greider CW (1990) Telomeres shorten during ageing of human fibroblasts. Nature 345:458–460 15. Vaziri H, Dragowska W, Allsopp RC, Thomas TE, Harley CB, Lansdorp PM (1994) Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeirc DNA with age. Proc Natl Acad Aci USA 91:9857–9860 16. Cherif H, Tarry JL, Ozanne SE, Hales CN (2003) Ageing and telomeres: a study into organ- and gender-specific telomere shortening. Nucl Acid Res 31:1576–1583 17. de Lange T, Shiue L, Myers RM et al (1990) Structure and variability of human chromosome ends. Mol Cell Biol 10:518–527 18. Nagata T, Takada Y, Ono A et al (2008) Elucidation of the mode of interaction in the UP1-telomerase RNA-telomeric DNA ternary complex which serves to recruit telomerase to telomeric DNA and to enhance the telomerase activity. Nucleic Acids Res 36:6816–6824 19. Sugano M, Tsuchida K, Maeda T, Makino N (2007) siRNA targeting SHP-1 accelerates angiogenesis in a rat model of hindlimb ischemia. Atherosclerosis 191:33–39 20. Robles SJ, Adami GR (1998) Agents that cause DNA double strand breaks lead to p16INK4a enrichment and the premature senescence of normal fibroblasts. Oncogene 16:1113–1123 21. Hemann MT, Strong MA, Hao LY, Greider CW (2001) The shortest telomere, not average telomere length, is critical for cell viability and chromosome stability. Cell 107:67–77 22. von Zglinicki T (2003) Replicative senescence and the art of counting. Exp Gerontol 38:1259–1264 23. Ojima M, Hamano H, Suzuki M, Suzuki K, Kodama S, Watanabe M (2004) Delayed induction of telomere instability in normal human fibroblast cells by ionizing radiation. J Radiat Res 45:105–110 24. Kawanishi S, Oikawa S (2004) Mechanism of telomere shortening by oxidative stress. Ann NY Acad Sci 1019:278–284 25. Przybyszewski WM, Widel M, Rzeszowska-Wolny J (2006) Cardiotoxic consequences of ionizing radiation and anthracyclines. Postepy Hig Med Dosw 60:397–405 26. Little JB, Lauriston S (2006) Taylor lecture: nontargeted effects of radiation: implications for low-dose exposures. Health Phys 91:416–426 27. Ogretmen B, Schady D, Usta J et al (2001) Role of ceramide in mediating the inhibition of telomerase activity in A549 human lung adenocarcinoma cells. J Biol Chem 276:24901–24910 28. Rubio MA, Davalos AR, Campisi J (2004) Telomere length mediates the effects of telomerase on the cellular response to genotoxic stress. Exp Cell Res 298:17–27 29. Wong KK, Chang S, Weiler SR et al (2000) Telomere dysfunction impairs DNA repair and enhances sensitivity to ionizing radiation. Nat Genet 26:85–88 30. Hande P, Slijepcevic P, Silver A, Bouffler S, van Buul P, Bryant P, Lansdorp P (1999) Elongated telomeres in scid mice. Genomics 56:221–223 31. Undarmaa B, Kodama S, Suzuki K, Niwa O, Watanabe M (2004) X-ray-induced telomeric instability in Atm-deficient mouse cells. Biochem Biophys Res Commun 315:51–58 32. Jones KR, Elmore LW, Jackson-Cook C, Demasters G, Povirk LF, Holt SE, Gewirtz DA (2005) p53-Dependent accelerated senescence induced by ionizing radiation in breast tumour cells. Int J Radiat Biol 81:445–458
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