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Radiation-induced genomic instability triggered by telomere dysfunction
International Congress Series 1258 (2003) 255 – 259
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Radiation-induced genomic instability triggered by telomere dysfunction Seiji Kodama a,*, Ayumi Urushibara a, Barkhaa Undarmaa a, Naoki Mukaida a, Keiji Suzuki a, Mitsuo Oshimura b, Masami Watanabe a a
Division of Radiation Biology, Nagasaki University Graduate School of Biomedical Sciences, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan b Department of Molecular and Cell genetics, School of Life Science, Faculty of Medicine, Tottori University, Yonago 638-8503, Japan
Abstract. To examine the possibility that an irradiated chromosome has the potential to promote genome rearrangement, we investigated the stability of irradiated human chromosomes in unirradiated mouse cells using a chromosome transfer technique. The stability of the irradiated human chromosome was analyzed by fluorescence in situ hybridization (FISH) using a probe specific for the human chromosome. The chromosome analysis revealed that the irradiated human chromosome was rearranged after chromosome transfer in two out of four microcell hybrids examined. This result suggests that the irradiated chromosome per se is unstable, even under the unirradiated environment. To determine the trigger for the delayed chromosome instability by radiation, we analyzed chromosome aberrations by telomere FISH technique, and found that the irradiated chromosome showed frequent end-to-end fusions with positive telomere signals at a fusion point. This suggested that telomeres do not function in preventing fusions, in spite of the presence of telomere sequences. These results indicate that radiation promotes telomere dysfunction and that this dysfunction accelerates genomic instability. Thus, we propose that an irradiated chromosome has an elevated potential to rearrange the chromosome in cis- and also in trans-action, and that this chromosomal instability can be triggered by telomere dysfunction. D 2003 Elsevier B.V. All rights reserved. Keywords: Radiation; Genomic instability; Chromosome transfer; Chromosome aberration; Telomere dysfunction
1. Introduction There is accumulated evidence that radiation induces genomic instability, such as delayed chromosomal instability in the progeny of irradiated cells [1 – 3], and that this instability might contribute to subsequent genome rearrangement toward development of
* Corresponding author. Tel.: +81-95-819-2460; fax: +81-95-819-2460. E-mail address: [email protected] (S. Kodama). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0531-5131(03)01203-2
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cancers [4]. Radiation-induced genomic instability is characterized by a high frequency, an untargeted induction, and a transmissible nature over many cell divisions postirradiation [5], suggesting that a single mutation of the specific genes might not fully explain this phenomenon. Although the initial event that leads to genomic instability is still unknown, there is evidence to suggest that some residual DNA damage remains somewhere in the irradiated genome and initiates genomic instability. For example, Niwa and Kominami [6] have demonstrated that 10 to 100 times higher mutation than expected was induced at a hypervariable minisatellite locus of F1 mice by irradiation at the postmeiotic spermatozoa stage, implying that the initiation of genomic instability was retained in the irradiated sperm genome. Of particular interest is that the mutant frequency increased also at the maternally derived allele when the male parents were irradiated at the spermatozoa stage. These results suggest that introduction of DNA damage by the irradiated sperm triggers genomic instability, and this instability induces untargeted mutation in cis at paternal allele and in trans at maternal allele. In somatic cells, however, there is no direct evidence to show that radiationinduced genomic instability is mediated by an irradiated chromosome per se. The former study using cell hybrids between irradiated cells and unirradiated cells demonstrated that genomic instability showed a dominant trait [7]. This result, however, was not clear to show what components in the irradiated cells are crucial to induce genomic instability. In the present study, we focused on the mechanism of radiation-induced, delayed chromosomal instability using a chromosome transfer technique. By this experimental system, we investigated the stability of an irradiated chromosome in unirradiated recipient cells, and examined the hypothesis that the irradiated chromosome per se is unstable, and has the potential to promote genomic instability. 2. Materials and methods Mouse A9 cells containing a human chromosome 4 or 11 were used as chromosome donor cells and mouse m5S cells, which retained near-diploid karyotype, were used as chromosome recipient cells. The donor cells were irradiated with 6 or 15 Gy of X-rays, and then a human chromosome 4 or 11 was introduced into the unirradiated m5S cells by microcell-mediated chromosome transfer as previously described [8]. The cells were cultured in a-MEM medium (Life Technologies, Gaithersburg, USA) with 10% FBS (Trace Scientific, Melbourne, Australia), penicillin (100 U/ml), and streptomycin (100 Ag/ ml). The microcell hybrids were cultured in the a-MEM medium supplemented with 3 Ag/ ml blasticidin-S-hydrochloride (Funakoshi, Tokyo, Japan). The stability of the irradiated human chromosome, in the microcell hybrids over 20 population doublings postirradiation, was examined by fluorescence in situ hybridization (FISH) using a probe specific for a human chromosome 4 or 11. The existence of telomere sequences at the chromosome termini was examined by telomere FISH using a peptide nucleic acid (PNA) probe, (CCCTAA)3, as previously reported [9]. Phosphorylated histone H2AX was detected by fluorescent immunostaining using the monoclonal antibody against histone H2AX phosphorylated at Ser139.
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3. Results We isolated independently four microcell hybrids introduced with unirradiated human chromosomes 4 and 11, and also four hybrids transferred with the same set of the irradiated chromosomes. To examine the possibility that the irradiated chromosome has a potential to promote genome rearrangement, we analyzed the transferred human chromosome by FISH. The unirradiated human chromosomes transferred into the recipient mouse cells were relatively stable under the present experimental condition, showing that the fraction of cells having rearranged human chromosomes was less than 7%. In contrast, two microcell hybrids introduced with the irradiated (6 and 15 Gy of X-rays) human chromosomes showed the higher fraction of cells (45 – 72%) having rearranged human chromosomes than those with unirradiated chromosomes after chromosome transfer, suggesting that the irradiated chromosome per se is of an unstable nature. To examine the possibility that some radiation damage remains in the irradiated chromosome, we investigated focus formation of phosphorylated histone H2AX (gH2AX) that might be responsible for residual radiation damage in the microcell hybrids. In two microcell hybrids, introduced with unirradiated human chromosome 11, the average number of foci per cell was 2.0. In a microcell hybrid, 6X11-11, of which human chromosome 11 was exposed to 6 Gy of X-rays and highly unstable (the unstable fraction was 72%), the number of foci per cells was 1.9. Thus, we failed to show the difference in numbers of foci of g-H2AX between microcell hybrids with the unirradiated chromosome and those with the irradiated chromosome, implying that the residual radiation damage could not be visualized, possibly due to technical limitation in the present condition. To determine the trigger for delayed chromosomal instability by radiation observed in the microcell hybrids, we investigated the integrity of telomere function of the irradiated human chromosome by telomere FISH technique. We found that the major aberrations observed in 6X11-11 cells, such as a ring of human chromosome 11 and a translocation between a human chromosome 11 and a mouse chromosome, were formed by end-toend fusions in spite of the presence of telomere sequences at a fusion point. This result implies that irradiation compromises telomere function in protecting end-to-end fusions, suggesting that telomeres are a target in radiation-induced delayed chromosomal instability. 4. Discussion Recent studies clearly demonstrated that the descendants of irradiated cells show a variety of delayed effects, including reproductive cell death, giant cell formation, chromosome aberration, and gene mutation [2,3,5]. In the present study, we adopted the chromosome transfer technique to know the mechanism of radiation-induced delayed chromosomal instability by examining the stability of the irradiated chromosome in unirradiated recipient cells. Our results indicated that the irradiated chromosome, per se, had an unstable nature and a potency to interact with other chromosomes even under the unirradiated environment in two out of four microcell hybrids. The chromosome analysis using telomere FISH revealed that the unstable chromosome showed frequent end-to-end fusions with positive telomere signals
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at a fusion point, suggesting that telomere dysfunction triggers the instability of the irradiated chromosome. Thus, the present study suggests that radiation promotes telomere dysfunction, and this dysfunction accelerates genomic instability. However, in the remaining two-microcell hybrids, the fraction of cells containing rearranged human chromosomes after chromosome transfer was similar (7– 8%) to that of cells with unirradiated human chromosomes. Thus, not all irradiated chromosomes are destabilized by radiation. Rather, a certain fraction of irradiated chromosomes gives rise to instability, even many cell divisions postirradiation. It should be noticed that our results clearly indicate that a main cause of the instability exists in the irradiated chromosome but not in the environment of recipient cells, although we cannot exclude the possibility that an extent of its manifestation is affected by the environment. In the present study, we focused on telomeric instability as one of the causal roles in inducing chromosomal instability. Telomere consists of a specific repetitious sequence of the hexamer, TTAGGG, which ends in a 3V-single strand overhang [10,11]. This telomeric overhang has been implicated as a critical component of telomere structure that is required for proper telomere function [12]. The 3V-telomeric overhang invades upstream duplex telomeric repeats to form a large duplex loop, and this loop, combined with several telomere-associated proteins, forms a nucleoprotein structure designated as a T-loop [13]. This structure may distinguish telomeres from the other DNA ends that would be a target for the repair enzymes of DNA doublestrand breaks. It has recently been demonstrated that erosion of the 3V-telomeric overhang, of probably less than 100 nucleotides, rather than erosion of overall telomere length, is potentially crucial for disruption of the T-loop structure, resulting in destabilization of telomere function [14]. From this point of view, a step of DNA end processing after DNA replication is a matter of interest. Recent reports have demonstrated that the ends of leading and lagging strands must be differentially processed to form a 3V-overhang for stabilization of the telomere structure postreplication [15]. A defect in TRF-2 function enhances the destabilization of telomeres, especially in the leading strand. If radiation indirectly affects this postreplication process of the leading strand and promotes immature formation of the 3V-overhang, telomeres might become unstable, resulting in the formation of end-to-end fusions. Finally, based on the present results, we propose that an irradiated chromosome has an elevated potential to rearrange the chromosome in cis- and also in trans-action, and that radiation-induced delayed chromosomal instability can be triggered by telomere dysfunction. Acknowledgements The authors thank Prof. Takeki Tsutsui, The Nippon Dental University, School of Dentistry, for kindly providing a PNA telomere probe. This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a Grant from the Health Research Foundation, Kyoto, Japan.
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References [1] M.A. Kadhim, D.A. Macdonald, D.T. Goodhead, S.A. Lorimore, S.J. Marsden, E.G. Wright, Transmission of chromosomal instability after plutonium a-particle irradiation, Nature 355 (1992) 738 – 740. [2] K. Roy, S. Kodama, K. Suzuki, M. Watanabe, Delayed cell death, giant cell formation and chromosome instability induced by X-irradiation in human embryo cells, J. Radiat. Res. 40 (1999) 311 – 322. [3] K. Roy, S. Kodama, K. Suzuki, K. Fukase, M. Watanabe, Hypoxia relieves X-ray-induced delayed effects in normal human embryo cells, Radiat. Res. 154 (2000) 659 – 666. [4] R.S. Maser, R.A. DePinho, Connecting chromosomes, crisis, and cancer, Science 297 (2002) 565 – 659. [5] J.B. Little, Radiation-induced genomic instability, Int. J. Radiat. Biol. 74 (6) (1998) 663 – 671. [6] O. Niwa, R. Kominami, Untargeted mutation of the maternally derived mouse hypervariable minisatellite allele in F1 mice born to irradiated spermatozoa, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 1705 – 1710. [7] W.P. Chang, J.B. Little, Delayed reproductive death as a dominant phenotype in cell clones surviving X-irradiation, Carcinogenesis 13 (1992) 923 – 928. [8] S. Kodama, G. Kashino, K. Suzuki, T. Takatsuji, Y. Okumura, M. Oshimura, M. Watanabe, J.C. Barrett, Failure to complement abnormal phenotypes of simian virus 40-transformed Werner syndrome cells by introduction of a normal human chromosome 8, Cancer Res. 58 (1998) 5188 – 5195. [9] P.M. Lansdorp, N.P. Verwoerd, F.M. van de Rijke, V. Dragowska, M.-T. Little, R.W. Dirks, A.K. Raap, H.J. Tanke, Heterogeneity in telomere length of human chromosomes, Hum. Mol. Genet. 5 (1996) 685 – 691. [10] R.K. Moyzis, J.M. Buckingham, L.S. Cram, M. Dani, L.L. Deaven, M.D. Jones, J. Meyne, R.L. Ratliff, J.-R. Wu, A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 6622 – 6626. [11] V.L. Makarov, Y. Hirose, J.P. Langmore, Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening, Cell 88 (1997) 657 – 666. [12] E.R. Henderson, E.H. Blackburn, An overhanging 3V terminus is a conserved feature of telomeres, Mol. Cell. Biol. 9 (1989) 345 – 348. [13] J.D. Griffith, L. Comeau, S. Rosenfield, R.M. Stansel, A. Bianchi, H. Moss, T. de Lange, Mammalian telomeres end in a large duplex loop, Cell 97 (1999) 503 – 514. [14] S.A. Stewart, I. Ben-Porath, V.J. Carey, B.F. O’Connor, W.C. Hahn, R.A. Weinberg, Erosion of the telomeric single-strand overhang at replicative senescence, Nat. Genet. 33 (2003) 492 – 496. [15] S.M. Bailey, M.N. Cornforth, A. Kurimasa, D.J. Chen, E.H. Goodwin, Strand-specific postreplicative processing of mammalian telomeres, Science 293 (2001) 2462 – 2465.
www.ics-elsevier.com
Radiation-induced genomic instability triggered by telomere dysfunction Seiji Kodama a,*, Ayumi Urushibara a, Barkhaa Undarmaa a, Naoki Mukaida a, Keiji Suzuki a, Mitsuo Oshimura b, Masami Watanabe a a
Division of Radiation Biology, Nagasaki University Graduate School of Biomedical Sciences, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan b Department of Molecular and Cell genetics, School of Life Science, Faculty of Medicine, Tottori University, Yonago 638-8503, Japan
Abstract. To examine the possibility that an irradiated chromosome has the potential to promote genome rearrangement, we investigated the stability of irradiated human chromosomes in unirradiated mouse cells using a chromosome transfer technique. The stability of the irradiated human chromosome was analyzed by fluorescence in situ hybridization (FISH) using a probe specific for the human chromosome. The chromosome analysis revealed that the irradiated human chromosome was rearranged after chromosome transfer in two out of four microcell hybrids examined. This result suggests that the irradiated chromosome per se is unstable, even under the unirradiated environment. To determine the trigger for the delayed chromosome instability by radiation, we analyzed chromosome aberrations by telomere FISH technique, and found that the irradiated chromosome showed frequent end-to-end fusions with positive telomere signals at a fusion point. This suggested that telomeres do not function in preventing fusions, in spite of the presence of telomere sequences. These results indicate that radiation promotes telomere dysfunction and that this dysfunction accelerates genomic instability. Thus, we propose that an irradiated chromosome has an elevated potential to rearrange the chromosome in cis- and also in trans-action, and that this chromosomal instability can be triggered by telomere dysfunction. D 2003 Elsevier B.V. All rights reserved. Keywords: Radiation; Genomic instability; Chromosome transfer; Chromosome aberration; Telomere dysfunction
1. Introduction There is accumulated evidence that radiation induces genomic instability, such as delayed chromosomal instability in the progeny of irradiated cells [1 – 3], and that this instability might contribute to subsequent genome rearrangement toward development of
* Corresponding author. Tel.: +81-95-819-2460; fax: +81-95-819-2460. E-mail address: [email protected] (S. Kodama). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0531-5131(03)01203-2
256
S. Kodama et al. / International Congress Series 1258 (2003) 255–259
cancers [4]. Radiation-induced genomic instability is characterized by a high frequency, an untargeted induction, and a transmissible nature over many cell divisions postirradiation [5], suggesting that a single mutation of the specific genes might not fully explain this phenomenon. Although the initial event that leads to genomic instability is still unknown, there is evidence to suggest that some residual DNA damage remains somewhere in the irradiated genome and initiates genomic instability. For example, Niwa and Kominami [6] have demonstrated that 10 to 100 times higher mutation than expected was induced at a hypervariable minisatellite locus of F1 mice by irradiation at the postmeiotic spermatozoa stage, implying that the initiation of genomic instability was retained in the irradiated sperm genome. Of particular interest is that the mutant frequency increased also at the maternally derived allele when the male parents were irradiated at the spermatozoa stage. These results suggest that introduction of DNA damage by the irradiated sperm triggers genomic instability, and this instability induces untargeted mutation in cis at paternal allele and in trans at maternal allele. In somatic cells, however, there is no direct evidence to show that radiationinduced genomic instability is mediated by an irradiated chromosome per se. The former study using cell hybrids between irradiated cells and unirradiated cells demonstrated that genomic instability showed a dominant trait [7]. This result, however, was not clear to show what components in the irradiated cells are crucial to induce genomic instability. In the present study, we focused on the mechanism of radiation-induced, delayed chromosomal instability using a chromosome transfer technique. By this experimental system, we investigated the stability of an irradiated chromosome in unirradiated recipient cells, and examined the hypothesis that the irradiated chromosome per se is unstable, and has the potential to promote genomic instability. 2. Materials and methods Mouse A9 cells containing a human chromosome 4 or 11 were used as chromosome donor cells and mouse m5S cells, which retained near-diploid karyotype, were used as chromosome recipient cells. The donor cells were irradiated with 6 or 15 Gy of X-rays, and then a human chromosome 4 or 11 was introduced into the unirradiated m5S cells by microcell-mediated chromosome transfer as previously described [8]. The cells were cultured in a-MEM medium (Life Technologies, Gaithersburg, USA) with 10% FBS (Trace Scientific, Melbourne, Australia), penicillin (100 U/ml), and streptomycin (100 Ag/ ml). The microcell hybrids were cultured in the a-MEM medium supplemented with 3 Ag/ ml blasticidin-S-hydrochloride (Funakoshi, Tokyo, Japan). The stability of the irradiated human chromosome, in the microcell hybrids over 20 population doublings postirradiation, was examined by fluorescence in situ hybridization (FISH) using a probe specific for a human chromosome 4 or 11. The existence of telomere sequences at the chromosome termini was examined by telomere FISH using a peptide nucleic acid (PNA) probe, (CCCTAA)3, as previously reported [9]. Phosphorylated histone H2AX was detected by fluorescent immunostaining using the monoclonal antibody against histone H2AX phosphorylated at Ser139.
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3. Results We isolated independently four microcell hybrids introduced with unirradiated human chromosomes 4 and 11, and also four hybrids transferred with the same set of the irradiated chromosomes. To examine the possibility that the irradiated chromosome has a potential to promote genome rearrangement, we analyzed the transferred human chromosome by FISH. The unirradiated human chromosomes transferred into the recipient mouse cells were relatively stable under the present experimental condition, showing that the fraction of cells having rearranged human chromosomes was less than 7%. In contrast, two microcell hybrids introduced with the irradiated (6 and 15 Gy of X-rays) human chromosomes showed the higher fraction of cells (45 – 72%) having rearranged human chromosomes than those with unirradiated chromosomes after chromosome transfer, suggesting that the irradiated chromosome per se is of an unstable nature. To examine the possibility that some radiation damage remains in the irradiated chromosome, we investigated focus formation of phosphorylated histone H2AX (gH2AX) that might be responsible for residual radiation damage in the microcell hybrids. In two microcell hybrids, introduced with unirradiated human chromosome 11, the average number of foci per cell was 2.0. In a microcell hybrid, 6X11-11, of which human chromosome 11 was exposed to 6 Gy of X-rays and highly unstable (the unstable fraction was 72%), the number of foci per cells was 1.9. Thus, we failed to show the difference in numbers of foci of g-H2AX between microcell hybrids with the unirradiated chromosome and those with the irradiated chromosome, implying that the residual radiation damage could not be visualized, possibly due to technical limitation in the present condition. To determine the trigger for delayed chromosomal instability by radiation observed in the microcell hybrids, we investigated the integrity of telomere function of the irradiated human chromosome by telomere FISH technique. We found that the major aberrations observed in 6X11-11 cells, such as a ring of human chromosome 11 and a translocation between a human chromosome 11 and a mouse chromosome, were formed by end-toend fusions in spite of the presence of telomere sequences at a fusion point. This result implies that irradiation compromises telomere function in protecting end-to-end fusions, suggesting that telomeres are a target in radiation-induced delayed chromosomal instability. 4. Discussion Recent studies clearly demonstrated that the descendants of irradiated cells show a variety of delayed effects, including reproductive cell death, giant cell formation, chromosome aberration, and gene mutation [2,3,5]. In the present study, we adopted the chromosome transfer technique to know the mechanism of radiation-induced delayed chromosomal instability by examining the stability of the irradiated chromosome in unirradiated recipient cells. Our results indicated that the irradiated chromosome, per se, had an unstable nature and a potency to interact with other chromosomes even under the unirradiated environment in two out of four microcell hybrids. The chromosome analysis using telomere FISH revealed that the unstable chromosome showed frequent end-to-end fusions with positive telomere signals
258
S. Kodama et al. / International Congress Series 1258 (2003) 255–259
at a fusion point, suggesting that telomere dysfunction triggers the instability of the irradiated chromosome. Thus, the present study suggests that radiation promotes telomere dysfunction, and this dysfunction accelerates genomic instability. However, in the remaining two-microcell hybrids, the fraction of cells containing rearranged human chromosomes after chromosome transfer was similar (7– 8%) to that of cells with unirradiated human chromosomes. Thus, not all irradiated chromosomes are destabilized by radiation. Rather, a certain fraction of irradiated chromosomes gives rise to instability, even many cell divisions postirradiation. It should be noticed that our results clearly indicate that a main cause of the instability exists in the irradiated chromosome but not in the environment of recipient cells, although we cannot exclude the possibility that an extent of its manifestation is affected by the environment. In the present study, we focused on telomeric instability as one of the causal roles in inducing chromosomal instability. Telomere consists of a specific repetitious sequence of the hexamer, TTAGGG, which ends in a 3V-single strand overhang [10,11]. This telomeric overhang has been implicated as a critical component of telomere structure that is required for proper telomere function [12]. The 3V-telomeric overhang invades upstream duplex telomeric repeats to form a large duplex loop, and this loop, combined with several telomere-associated proteins, forms a nucleoprotein structure designated as a T-loop [13]. This structure may distinguish telomeres from the other DNA ends that would be a target for the repair enzymes of DNA doublestrand breaks. It has recently been demonstrated that erosion of the 3V-telomeric overhang, of probably less than 100 nucleotides, rather than erosion of overall telomere length, is potentially crucial for disruption of the T-loop structure, resulting in destabilization of telomere function [14]. From this point of view, a step of DNA end processing after DNA replication is a matter of interest. Recent reports have demonstrated that the ends of leading and lagging strands must be differentially processed to form a 3V-overhang for stabilization of the telomere structure postreplication [15]. A defect in TRF-2 function enhances the destabilization of telomeres, especially in the leading strand. If radiation indirectly affects this postreplication process of the leading strand and promotes immature formation of the 3V-overhang, telomeres might become unstable, resulting in the formation of end-to-end fusions. Finally, based on the present results, we propose that an irradiated chromosome has an elevated potential to rearrange the chromosome in cis- and also in trans-action, and that radiation-induced delayed chromosomal instability can be triggered by telomere dysfunction. Acknowledgements The authors thank Prof. Takeki Tsutsui, The Nippon Dental University, School of Dentistry, for kindly providing a PNA telomere probe. This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a Grant from the Health Research Foundation, Kyoto, Japan.
S. Kodama et al. / International Congress Series 1258 (2003) 255–259
259
References [1] M.A. Kadhim, D.A. Macdonald, D.T. Goodhead, S.A. Lorimore, S.J. Marsden, E.G. Wright, Transmission of chromosomal instability after plutonium a-particle irradiation, Nature 355 (1992) 738 – 740. [2] K. Roy, S. Kodama, K. Suzuki, M. Watanabe, Delayed cell death, giant cell formation and chromosome instability induced by X-irradiation in human embryo cells, J. Radiat. Res. 40 (1999) 311 – 322. [3] K. Roy, S. Kodama, K. Suzuki, K. Fukase, M. Watanabe, Hypoxia relieves X-ray-induced delayed effects in normal human embryo cells, Radiat. Res. 154 (2000) 659 – 666. [4] R.S. Maser, R.A. DePinho, Connecting chromosomes, crisis, and cancer, Science 297 (2002) 565 – 659. [5] J.B. Little, Radiation-induced genomic instability, Int. J. Radiat. Biol. 74 (6) (1998) 663 – 671. [6] O. Niwa, R. Kominami, Untargeted mutation of the maternally derived mouse hypervariable minisatellite allele in F1 mice born to irradiated spermatozoa, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 1705 – 1710. [7] W.P. Chang, J.B. Little, Delayed reproductive death as a dominant phenotype in cell clones surviving X-irradiation, Carcinogenesis 13 (1992) 923 – 928. [8] S. Kodama, G. Kashino, K. Suzuki, T. Takatsuji, Y. Okumura, M. Oshimura, M. Watanabe, J.C. Barrett, Failure to complement abnormal phenotypes of simian virus 40-transformed Werner syndrome cells by introduction of a normal human chromosome 8, Cancer Res. 58 (1998) 5188 – 5195. [9] P.M. Lansdorp, N.P. Verwoerd, F.M. van de Rijke, V. Dragowska, M.-T. Little, R.W. Dirks, A.K. Raap, H.J. Tanke, Heterogeneity in telomere length of human chromosomes, Hum. Mol. Genet. 5 (1996) 685 – 691. [10] R.K. Moyzis, J.M. Buckingham, L.S. Cram, M. Dani, L.L. Deaven, M.D. Jones, J. Meyne, R.L. Ratliff, J.-R. Wu, A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 6622 – 6626. [11] V.L. Makarov, Y. Hirose, J.P. Langmore, Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening, Cell 88 (1997) 657 – 666. [12] E.R. Henderson, E.H. Blackburn, An overhanging 3V terminus is a conserved feature of telomeres, Mol. Cell. Biol. 9 (1989) 345 – 348. [13] J.D. Griffith, L. Comeau, S. Rosenfield, R.M. Stansel, A. Bianchi, H. Moss, T. de Lange, Mammalian telomeres end in a large duplex loop, Cell 97 (1999) 503 – 514. [14] S.A. Stewart, I. Ben-Porath, V.J. Carey, B.F. O’Connor, W.C. Hahn, R.A. Weinberg, Erosion of the telomeric single-strand overhang at replicative senescence, Nat. Genet. 33 (2003) 492 – 496. [15] S.M. Bailey, M.N. Cornforth, A. Kurimasa, D.J. Chen, E.H. Goodwin, Strand-specific postreplicative processing of mammalian telomeres, Science 293 (2001) 2462 – 2465.