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Role of chromosome instability in long term effect of manned-space missions
Adv. Space Res. Vol. 22, No. 4, pp. 591-602, 1998 01998 COSPAR. Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain 0273-l 177/98 $19.00 + 0.00 PII: SO273-1177(98)00082-9
Pergamon
ROLE OF CHROMOSOME INSTABILITY IN LONG TERM EFFECT OF MANNED-SPACE MISSIONS C. Ducray and L. Sabatier Commissariat d L’Energie Atomique, DSVLDRRlaboratory of Radiobiology and Oncology, BP6, F-92265 Fontenay-aux-Roses Cedex, France
ABSTRACT
Astronauts are exposed to heavy ions during space missions and heavy ion induced-chromosome damages have been observed in their lymphocytes. This raises the problem of the consequence of longer space flights. Recent studies show that some alterations can appear many cell generations after the initial radiation exposure as a delayed genomic instability. This delayed instability is characterized by the accumulation of cell alterations leading to cell transformation, delayed cell death and mutations. Chromosome instability was shown in vitro in different model systems (Sabatier et al., 1992; Marder and Morgan, 1993; Kadhim et al., 1994 and Holmberg et al., 1993,1995). All types of radiation used induce a chromosome instability, however, heavy ions cause the most damage. The period of chromosome instability followed by the formation of clones with unbalanced karyotypes seems to be shared by cancer cells. The shortening of telomere sequences leading to the formation of telomere fusions is an important factor in the appearance of this chromosome instability. 01998 COSPAR. Published by Elsevier Science Ltd. INTRODUCTION It has been demonstrated over the years that cancers can be induced by exposure to radiations. Radiation exposure induces a destabilization of the genome. Following exposure to ionizing radiation, some cells die or undergo several normal division cycles before ceasing to divide, whereas surviving cells are defined as cells that continue to proliferate. Irradiation by heavy ions induces multiple chromosomal alterations giving rise eventually, many cell divisions after the exposure, to a transmissible chromosomal instability in the descendants of the surviving irradiated cells. As cancer cells very often present abnormal genomes with aneuploidy and chromosomal rearrangements, chromosome instability could be a crucial event in the occurence of radiation-induced tumors. COSMIC RADIATIONS AND SPACE RESEARCH Cosmic radiations are composed of particles of solar origin (composed of 98% protons and 2% electrons) and predominantly of galactic sources (85% protons, 14% a-particles and 1% heavy ions). Although in small proportion, heavy ions contribute significantly to the dose delivered by cosmic radiations. This implies that it is important to estimate the risks linked to radiations for humans in future long space missions, especially since a single high-LET particle can cause severe damage in most organisms (Reitz et al., 1995). Individual high-LET particles may only traverse a few cells of the body in which they induce considerable damage leading to death or transformation of the target cells. The transformation and carcinogenic potentials of heavy ions are dose and LET-dependent and have more impact than dispersed radiations such as X-rays. The biological dosimetry report performed on 7 astronauts after space missions (Testard et al., 1996) showed different results depending on the duration of the flights. For short-term flights (2-3 weeks), the 597
598
C. Ducray and L. Sabatier
me of damaged metaphases and diceneric yields did not differ from back~rouRd. However, the astronauts who flew for longer periods (6 months) presented chromosomai anomalies in 16%0of the me&phases scored. Two of the astronauts who flew 3 times 6 months also presented serious chromosomal anomalies with 8 to 19 breaks per cell in 10% of their damaged cells. Sabatier et al. (1987) have shown that complex rearrangements involving at least 3 chromosome breaks were induced in lymphocytes irradiated with single particles of neon ions. It was therefore deduced that the cells must have been hit by particles with high LETS.
The question as to whether the potent&I long-tee effects of these high LET radiations are very serious is left u~~swered. It has been shown that many of the aberrant cells will eve~tuaily die since the rate of c~omosome aberration observed decreases at each cell division (Al-Achkar at al. 1988). In vitrcr studies have shown that heavy ion ~diatio~s induce cell lethality at a high rate. Some of the aberrations might also confer a proliferative advantage to the cells and cause cell transfo~ati~n and carcinogenesis. In order to estimate the long-term biological effects of high LET radiation, the behaviour of initial damages has been studied a long period after irradiation and the appearance of & pzovodamages was described as cellular transformation, delayed reproductive death, lethal mutations and radioinduced chromosomal instability. CELLULAR TRANSFORMATION Exposure to X-rays induces a biologic~ change in the su~ivirlg cells which is tmns~tted to the progeny. This enhances the ~robabili~ of the occurence of a second event, tra~sfo~ation. Studies Kennedy and Little in 1980 have shown that the yield of transfo~ed foci increases as a fu~(Ch~g and Little, 199I)ctio~ of X-ray exposure, up to a dose of about 400 Fads. Higher dose exposures give rise to constant yields of ~~sfo~ed foci. Moreover the yield of transformation is inde~~da~t of the number of cells that were initially exposed to X-irradiation. The number of transformed colonies being a~pro~mately constant from one dil~tio~ to another suggests that the cell ~tera~ons that resuit in the formation of a clone of ~~sfo~~d cells is not the immediate, direct consequence of the initial exposure to X-rays. It was suggested that a two step process is responsible for radiation-induced transformation. The exposure to X-rays produces a cellular functional alteration which is transmitted to the progeny during subsequent growth leading to an enhanced probability of the (perhaps mutational) second event expressed as a transformed clone. In fact the initial step determines the frequency of the subsequent rare genetic event (Kennedy et al., 1984). DELAYED ~P~O~U~
DEATH - LETHAL RATIONS
Some cells from progeny of r~dia&ion~exposedsurviving cells do not continue to proliferate i~de~~itely. This phenomenon has been termed “deiayed reproductive death” {Sinclair, 1964). This ph~no~pic change which can be detected in the surviving population of Chinese hamster ovary cells up to 30-40 genera~ons after X-i~adiation is manifested by a reduced cloning efficiency among the progeny of irradiated cells (Chang and Little, 1991). In 1986 the concept of lethal mutations was introduced (Seymour et al., 1986) to describe the phenomenon of delayed reproductive death. Indeed the authors found that surviving progeny of irradiated cells show a systematic reduced cloning efficiency which is lower than that of the cell population immediately after irradiation. The enhanced death rate which persists for long periods of time (30 generations) after i~adiation, among the progeny of surviving ceils is due to the continuing andlor delayed expression of bthd mutations during the proliferation of these cells. Mutation in some essential genes may be involved in the delayed reproduc~ve death. Heritable defects such as these mu~tio~s with lethal effects and abno~~ ph~not~ic characteristics (lower cell cycle progression, reduced capacity to attach to culturt: dishes, increased number of abortive colonies and enhanced frequencies of giant ce’tls ifi non-homogeneous colonies) occur in some of the surviving progeny post-irradiation. They also appear to be eventually responsible for the late expression of reproductive failure in the descendants. Residual unrepaired damage may be carried by surviving progeny over many mitotic cycles before being expressed, leading to death, although it is improbable. The high frequency of delayed reproductive death among surviving clones (50%) makes it unlikely to be the result of a directly produced mutation in a specific gene or set of genes. It may be induced specifically as a cellular response to DNA DSB (double-strand breaks}. Experiments with X-rays, EMS ~ethyime~anesulfonate) and the restriction endonuclease Hinf I (Chang and Little, 1992) have reinforced
Chromosome fnstabilig and ~aR~~-~~&ce
Missions
599
this hypathesis. In this study, the cloned progeny of CHO cells surviving alkylating, radiation and enzymatic treatment were evaluated for their cloning efficiency. Clones derived from cells suff~ving these treatments showed a reduced cloning efficiency whereas those from UV irradiation, which is not associated with the generation of DSB at the wavelength used, did not, Moreover DSB repair-deficient xrs-S CHO cells did not show delayed reproductive death. The defective cellular processing of the DSB during the repair process seems to be the cause of the decreased reproductive potential. Clones derived from cells surviving X-i~diatio~ showed an increased frequency of spontaneous mutations for up to 100 cellular generations post-irradiation. The phenotype of delayed reproductive death, present in some clones, correlates with genetic instability. The available information on instability around the genome is derived from studies of genes such as TK, HPRT and APRT. Human lymphoblastoid celf lines presenting mutations at the TK gene, induced by X-rays, EMS or arising spontaneously~ were screened for the existence of second-site mutations at ~crosatellite loci (Li et al., 1994). The frequency of mutation at microsateflite loci was significantly higher in the TK- selected cells than in nonselected clones. A h~o~esis on the mechanism of genomic instability could be the action of a mutator factor which can affect more than one (critical) gene at the same time inducing multiple mutations in cells. RADIOINDUCED CHROMOSOMAL INSTABILITY radiation induces chromosome aberrations detected in the first mitosis after the exposure. In the cultures, most of the initial radiation-induced chromosomal damage is unstable. So while death occurs randomly in the descendants of these cells, those with stable chromosome aberrations may survive for many years. Different model systems have been used to study the transmission of radiation-induced chromosome aberrations in ceil clones, most studies being performed on short-term cultures since most induced aberrations, incompatible with cell survival, rapidly disappear in culture. The frequency of the cells carrying dice&c c~omosomes is reduced by half at each genera~on (Al-Acbkar et al., 1988). Thus in few celi generations all the dicentrics directly induced will disappear. Induced chromosome instability refers to the production of de nova aberrations which accumnlate during cell cycles subsequent to ionizing ra~a~on treatment. Holmberg et al. (1993) analysed X-irradiated T lymphocytes stimulated by interleukin 2 at early and late time points, during donal expansion in vifro. They observed two types of radiation-induced chromosomal instability: clonal karyotypic abnormalities characterized by identical chromosomal changes occuring in a minimum of three cells in individual cell clones and sporadic nonclonal chromosome abenations similar to the aberrations observed by Kadhim emal. ( t 992) such as chromatid and chromosome breaks. X-irradiation of human T lymph~~es gives i-&eto two types of clones: homogeneous and heterogeneuus clones. In the first case, the cells show the same c~omosome a~~a~ons which probably originate from one or muitiple events during the first cell divisions after radiation exposure. The other clones classified as heterogeneous contain cells with normal karyotypes and other cells with abnormal karyotypes. The second type of cells might derive from the first by acquiring c~omosome aberrations during growth, since the percentage of cells with normal karyotypes declines with increasing cultivation time. This could be an indication of genomic instability which occurs as a late response to X-irradiation. The authors demonstrate the delayed appetite of sporadic aberrations (Holmberg et al., 1995) prefere~ti~y in irradiated cells with aberrmt karyotypes. This study has shown that a high proportion of T cells with X irradiation-induced chromosame aberrations are able to proliferate giving rise to expanding clones some of which accumulate latent chromosome damage. Marder and Morgan (1993) have used FISH on clonally-derived ceils to insvestigate delayed genomic instability in a hamster-human hybrid cell line exposed to X-rays. Delayed chromosomal instability manifested as multiple rea~angements of human chromosome 4 in an environment of hamster chromosomes_ Small differences were observed between cells exposed twice to X-rays and those exposed only once. A decrease in the frequency of chromosomal rearrangements was observed in cells of colonies surviving two exposures. Chromosome rearrangements seem to contribute to the cells’ inability to survive many generations after the X ray exposure. The mechanism responsible for the radiation induced delayed instability is not known. Several expirations have been suggested such as: a persistence of DNA damage, activation of latent endogenous virus and induction of a mutator phenotype as a response to stress. These last studies indicate that chromosome instabili~ can be induced by X-rays in immo~alized, stimulated cells or in cells after two rounds of subcloning.
600
C. Ductay and L. Sabatier
In order to follow the transmission of radioinduced chromosome damage in normal ceils, Kadhim et al. (1992) studied the effects of low dose a-particle irradiation from plutonium-238 on clonal descendants of murine haematopoietic stem cells. Alpha-particles induced lesions in the stem cells which result in a diversity of chromosomal aberrations in their progeny many cell divisions later. This radiation exposure produced a high frequency of nonclonal chromosome aberrations in the individual colonies, derived from the surviving stem cells. These abnormalities are characterized mostly by chromatid rather than chromosome aberrations, with no evidence of any preferential site of breakage. This nonclonality in the aberrations observed might be due to de nuv~ occurence of transmissible instability in the clonogenic cells surviving radiation. Similar results were observed for a-particle radiation of human bone marrow cells (Kadhim et al., 1994). However, the results demonstrate interindividu~ v~iations: the stem cells of two individu~s showed c~omosom~ instabiIity when those of two other donors seemed resistant to instability. Moreover, the results were totally different when the cells, murine or human, were exposed to X-rays rather than a-particles. The aberrations were clonal and for the most part, chromosome aberrations. In this model they failed to observe any chromosomal instability after X-ray irradiation. These studies demonstrate that cells which survive a-particle exposure may show a delayed chromosomal instability since they continue to develop chromosomal aberrations at an increased frequency for many cell cycles after the irradiation. The delayed chromosome instability was also observed on human dermal fibroblasts exposed to heavy ions, neon, argon and lead but not after y rays.(Sabatier and Dutrillaux, 1992). The lesions created by these heavy ions are more complex than those induced by X-rays. Their complexity is pro~~on~ to the fluence of the particles (Sabatier et al., 1987). The authors analysed the karyotypes of the irradiated cells at each passage after the exposure and found that after a short period of heavy chromosomal aberrations directly induced by the irradiation, almost all karyotypes of the surviving cells bore stable rearrangements (translocations, inversions) or presented normal karyotypes. These cells acquire de nova transmissible chromosome instability which, several cell generations after exposure, persist from the 15th to the 25th passage. It was characterized by a high proportion of dicentrics, resulting from end-to-end fusions (Martins et al., 1993, Sabatier and Dutrillaux, 1992). Contrary to the results observed by Kadhim, the location of the corresponding breakpoints were specific for the donor analysed. Specific chromosome structures are involved: telomeric regions of, in decreasing order of frequency, chromosome 13, 16, the short arm of chromosome 1 and other chromosomes. This non-random chromosome instability is a late consequence of radiation by high LET particles. This period of chromosome instability was foflowed by the formation of clones with unb~anced karyotypes, such as loss of chromosome 13. These clones progressively invade all the culture. Cell cultures present an increased lifespan (20%). Another hypothesis on the mechanism of genomic instability could be the bypass of the senescence process. Thus irradiation would induce the early emergence of a naturally occuring phenomenon: chromosome instability during senescence. CHROMOSOME INSTABILITY AND CARCINOGENESIS Genomic instability occurs after many generations in the progeny of irradiated cells. The chromosome instability induced by heavy ions leads to multiple genetic alterations and preferentially affects some chromosome structures, particularly telomeres by end-to-end fusions, leading to specific chromosomal imbalances. This instability, specific to certain chromosomes, could differ from one individual to another. The specificity of the chromosomes involved in senescence is a characteristic of each donor (Benn, 1976). Moreover, Sabatier observed that, for one donor, the chromosomal instability seems to be the same independent of the stress (senescence, heavy ion i~adiation, ~ansfo~ation) (Sabatier et al., 1995; data not shown). This period of instability gives rise to the appearance of unbalanced karyotypes. The chromosome imbalances which confer a proliferative advantage to the cells will be selected. These cells then progressively invade the culture. This implies the emergence of unbalanced clones in the progeny of irradiated cells. The chromosome instability followed by the formation of aneuploid clones appears to be shared by cancer ceils. In fact it could represent a key step in the carcinogenesis process. Human solid tumours are characterized by specific chromosome imbalances. Indeed these tumours not only contain structural changes but also numerical changes (gain or loss of whole chromosomes). In colorectal cancers, for example, karyotypes may either remain normal (7% of the cases), or may principally be characterized
Chromosome In~abiiity and Many-Space
Missions
601
by unbalanced clonal rearrangements: gains in 25% of the cases and losses and/or endoreduplication in the rest of the cases (Muleris et al., 1991). It could be generalized that, depending on the first genetic alterations, the selective pressure will determine the chromosome pattern of the tumour.
Chromosome instability characterized by end-to-end fusions is maintained during many cell generations without inducing ceil death. The transmission of this instability could result from tefomere associations followed by fusion-breakage-fusion cycles leading to the appearance of chromosome imbalances. The rea~angements which confer a proliferative advantage to the cells are selected and progressively invade the culture. Chromosomal instability induced by telomere sho~ening may be an early step in the conversion of a normal diploid cell into an aneupioid cell, as observed in many cancer cells. In somatic cells, telomerase, a ~bonucleoprotein which can replicate telomere ends, is not active (Counter er at., 1992; Greider, 1990). Without telomerase activity, telomeres progressively shorten at each cell division. This shortening of telomeres eventually leads to a destabilization of the chromosomes for which the telomeres have reached a critically small size thus leading to end-to-end fusions, contributing to genome instability. The hypothesis by which the chromosomes with the shortest telomeres become unstable first was tested by inducing a high level of chromosome instability with SV40 transfection. Indeed SV40 large T antigen enables cells to progress beyond senescence. Cells continue to divide, losing more telomeric DNA, therefore con~buting to more severe chromosome damage, until they face crisis which is not circumvented by viral ~nsfo~ation. Progression beyond crisis occurs at low frequency and seems to require telomerase activation which is needed for telomeres to be stably m~nt~ned, In order for cells to become i~o~al, it is necessary for telomeres to be maintained or lengthened. This will prevent further destabilization of chromosomes in the i~o~alized clones and imply a great decrease of the high level of genetic instability. This instability will not however disappear since 20% of the overall alterations still remain. Although some immortal cell lines have been described with no telomerase activity (Murnane et al., 1994; Bryan et al., 1995), usually, during the transformation process, telomeres decrease in size in cells when no telomerase is detectable, resulting in c~omosome fusions. Telomere associations, followed by several rounds of fusionbre~age-fusion cycles, allow the transmission of chromosome instability, leading to the selection of chromosome imbalances. In summary, ionizing radiation induce a transmissible chromosomal instability in the progeny of exposed cells many generations after the initial radiation. This delayed instability can be the result of X rays but densely ionizing radiation seems more efficient. The transmission of chromosome instability which p~ferenti~y affects s~cmres such as telomeres when telomerase activity is not detected is due to telomere fusions occuring when a critical telomere length is reached. Chromosome i~tabili~ induced by radiation seems to be a property shared by cancer cells. This raises the question as to whether high LET radiation has a deleterious long-term effect on astronauts after long space missions. ACKNOWLEDGMENTS This work was supported by the CNES 95/421 and the ministry ACCSV8. REFEZBNCES Al-A&car, W., L. Sabatier and B. Dutrilfaux, Transmission of ra~a~on-induced re~ngemen~ through cell divisions, ~~f~f~~~ Researcia, f98, pp. 191-198 (1988). Benn, P. A., Specific chromosome abe~ation~ in senescent ~brobl~t cell lines derived from human embryos, A~er~cun Journal of Human Genetics, 28, pp. 465-473 (1976). Bryan, T. M., A. Englezou, J. Gupta, S, Bacchetti and R. R. Reddel, Telomere elongation in immortal human cells without detectable telomerase activity, The EMBU Journal, 14, pp. 4240-4248 (1995). Chang, W. P. and J. B. Little, Delayed reproductive death in X-irradiated Chinese hamster ovary cells, i~fe~a~io~ai Journal of Radiation Bioology,60, pp. 483-496 (1991). Chang, W. P. and J. B. Little, Evidence that DNA double-stand breaks initiate the pheno~pe of delayed reproductive death in Chinese hamster ovary cells, Radiation Reseurch, 131, pp. 53-59 (1992).
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Counter, C. M., A. A. Avilion, C. E. LeFeuvre, N. G. Stewart, C. W. Greider, C. B. Harley and S. Bacchetti, Telomere shortening associated with chromosome instability is arrested in immortai cells which express telomerase activity, The EMBO Journal, 11,pp. 1921-1929 (1992). Greider, C. W., Telomeres, telomerase and senescence, Bi~~ssuys, 12, pp. 363-369 (1990). Holmberg, K., A. E. Meijer, G. Auer and B. Lambert, Delayed chromosomal instability in human Tlymphocyte clones exposed to ionising radiation, International Journal of Radiation Biology, 68, pp. 245-255 (1995). Kadhim, M. A., S. A. Lorimore, M. D. Hepburn, D. T. Goodhead, V. J. Buckle and E. G. Wright, aparticle-induced chromosomal instability in human bone marrow cells, The Lancet, 344, pp. 987-988 (1994). Kennedy, A. B., J. Cairns and J. B. Little, Timing of the steps in transformation of C3H lOT112cells by x-i~adiation, ~uf~~~~,307, pp. 85-86 (1984). Kennedy, A. B., M. Fox, G. Murphy and J. B. Little, Relationship between x-ray exposure and malignant transformation in C3H 10T 112cells, Proceedings of the National Academy of Science USA, 77, pp. 7262-7266 (1980). Li, C. Y., D. W. Yandell and J. B. Little, Elevated frequency of microsatellite mutations in TK6 human lymphoblast clones selected for mutations at the thymidine kinase locus, Molecular and Cellular Biology, 14, pp. 4373-4379 (1994). Martins, M., L. Sabatier, M. Rico& A. Pinton and B. Dutrillaux, Specific chromosome instability induced by heavy ions: a step towards transfo~ation of human fibroblasts?, mutation Research, 285, pp. 229237 (1993). Mule& M., R.J. Salmon and B. Dutrillaux, Cytogenetics of colorectal adenocarcinomas, Cancer Genetics and Cytogenekics, 46, pp. 143- 156 (199 1). Mumane, P.M., L. Sabatier, B.A. Marder and W.F. Morgan, Telomere dynamics in an immortal human cell line, The EMBO Journal, 13, pp. 4953-4962 (1994). Reitz, G., G. Homeck, R. Facius and M. Schafer, Results of space experiments, Radiation and Environmental Biophysics, 34, pp. 139- 144 (1995). Sabatier, L., W. Al Achkar, F. Hoffshir, C. Luccioni and B. Dutrillaux, Qualitative study of chromosomai lesions induced by neurons and neon ions in human lymphocytes at GOphase, ~ufario~ Research, 178, pp. 91-97 (1987). Sabatier, L. and B. Dutrillaux, Chromosomal instability, Nature, 357, pp. 548 (1992). Sabatier L., J. Lebeau, J.P. Pommier and B. Dutrillaux, Chromosome instability and telomeric alteration detected in irradiated human fibroblasts, Proceedings of the Tenth International Congress of Radiation Research, ed. Hagen U., D. Harder, H. Jung and C. Streffer, 2, pp. 509-512, Wtirzburg, Germany (1995). Seymour, C. B., C. Mothersill and T. Alper, High yieids of lethal mutations in somatic mammalian cells that survive ionising radiation, ~nte~a~ionaZ Jou~a~ of Radiation Biology, 50, pp. 167-179 (1986). Sinclair, W.K., X-ray induced heritable damage (small colony fo~ation) in cultured rn~~i~ cells, Radiation Research, 21, pp. 584-611 (19641. Testard, I., M. Ricoul, F. Hoffschir, A. Fluty-Herard, B. Dutrillaux, B. Fedorenko, V. Gerasimenko and L. Sabatier, Radiation-induced chromosome damage in astronauts’ lymphocytes, Znternational Journal of Radiation Biology, 70, pp. 403-411 (1996).
Pergamon
ROLE OF CHROMOSOME INSTABILITY IN LONG TERM EFFECT OF MANNED-SPACE MISSIONS C. Ducray and L. Sabatier Commissariat d L’Energie Atomique, DSVLDRRlaboratory of Radiobiology and Oncology, BP6, F-92265 Fontenay-aux-Roses Cedex, France
ABSTRACT
Astronauts are exposed to heavy ions during space missions and heavy ion induced-chromosome damages have been observed in their lymphocytes. This raises the problem of the consequence of longer space flights. Recent studies show that some alterations can appear many cell generations after the initial radiation exposure as a delayed genomic instability. This delayed instability is characterized by the accumulation of cell alterations leading to cell transformation, delayed cell death and mutations. Chromosome instability was shown in vitro in different model systems (Sabatier et al., 1992; Marder and Morgan, 1993; Kadhim et al., 1994 and Holmberg et al., 1993,1995). All types of radiation used induce a chromosome instability, however, heavy ions cause the most damage. The period of chromosome instability followed by the formation of clones with unbalanced karyotypes seems to be shared by cancer cells. The shortening of telomere sequences leading to the formation of telomere fusions is an important factor in the appearance of this chromosome instability. 01998 COSPAR. Published by Elsevier Science Ltd. INTRODUCTION It has been demonstrated over the years that cancers can be induced by exposure to radiations. Radiation exposure induces a destabilization of the genome. Following exposure to ionizing radiation, some cells die or undergo several normal division cycles before ceasing to divide, whereas surviving cells are defined as cells that continue to proliferate. Irradiation by heavy ions induces multiple chromosomal alterations giving rise eventually, many cell divisions after the exposure, to a transmissible chromosomal instability in the descendants of the surviving irradiated cells. As cancer cells very often present abnormal genomes with aneuploidy and chromosomal rearrangements, chromosome instability could be a crucial event in the occurence of radiation-induced tumors. COSMIC RADIATIONS AND SPACE RESEARCH Cosmic radiations are composed of particles of solar origin (composed of 98% protons and 2% electrons) and predominantly of galactic sources (85% protons, 14% a-particles and 1% heavy ions). Although in small proportion, heavy ions contribute significantly to the dose delivered by cosmic radiations. This implies that it is important to estimate the risks linked to radiations for humans in future long space missions, especially since a single high-LET particle can cause severe damage in most organisms (Reitz et al., 1995). Individual high-LET particles may only traverse a few cells of the body in which they induce considerable damage leading to death or transformation of the target cells. The transformation and carcinogenic potentials of heavy ions are dose and LET-dependent and have more impact than dispersed radiations such as X-rays. The biological dosimetry report performed on 7 astronauts after space missions (Testard et al., 1996) showed different results depending on the duration of the flights. For short-term flights (2-3 weeks), the 597
598
C. Ducray and L. Sabatier
me of damaged metaphases and diceneric yields did not differ from back~rouRd. However, the astronauts who flew for longer periods (6 months) presented chromosomai anomalies in 16%0of the me&phases scored. Two of the astronauts who flew 3 times 6 months also presented serious chromosomal anomalies with 8 to 19 breaks per cell in 10% of their damaged cells. Sabatier et al. (1987) have shown that complex rearrangements involving at least 3 chromosome breaks were induced in lymphocytes irradiated with single particles of neon ions. It was therefore deduced that the cells must have been hit by particles with high LETS.
The question as to whether the potent&I long-tee effects of these high LET radiations are very serious is left u~~swered. It has been shown that many of the aberrant cells will eve~tuaily die since the rate of c~omosome aberration observed decreases at each cell division (Al-Achkar at al. 1988). In vitrcr studies have shown that heavy ion ~diatio~s induce cell lethality at a high rate. Some of the aberrations might also confer a proliferative advantage to the cells and cause cell transfo~ati~n and carcinogenesis. In order to estimate the long-term biological effects of high LET radiation, the behaviour of initial damages has been studied a long period after irradiation and the appearance of & pzovodamages was described as cellular transformation, delayed reproductive death, lethal mutations and radioinduced chromosomal instability. CELLULAR TRANSFORMATION Exposure to X-rays induces a biologic~ change in the su~ivirlg cells which is tmns~tted to the progeny. This enhances the ~robabili~ of the occurence of a second event, tra~sfo~ation. Studies Kennedy and Little in 1980 have shown that the yield of transfo~ed foci increases as a fu~(Ch~g and Little, 199I)ctio~ of X-ray exposure, up to a dose of about 400 Fads. Higher dose exposures give rise to constant yields of ~~sfo~ed foci. Moreover the yield of transformation is inde~~da~t of the number of cells that were initially exposed to X-irradiation. The number of transformed colonies being a~pro~mately constant from one dil~tio~ to another suggests that the cell ~tera~ons that resuit in the formation of a clone of ~~sfo~~d cells is not the immediate, direct consequence of the initial exposure to X-rays. It was suggested that a two step process is responsible for radiation-induced transformation. The exposure to X-rays produces a cellular functional alteration which is transmitted to the progeny during subsequent growth leading to an enhanced probability of the (perhaps mutational) second event expressed as a transformed clone. In fact the initial step determines the frequency of the subsequent rare genetic event (Kennedy et al., 1984). DELAYED ~P~O~U~
DEATH - LETHAL RATIONS
Some cells from progeny of r~dia&ion~exposedsurviving cells do not continue to proliferate i~de~~itely. This phenomenon has been termed “deiayed reproductive death” {Sinclair, 1964). This ph~no~pic change which can be detected in the surviving population of Chinese hamster ovary cells up to 30-40 genera~ons after X-i~adiation is manifested by a reduced cloning efficiency among the progeny of irradiated cells (Chang and Little, 1991). In 1986 the concept of lethal mutations was introduced (Seymour et al., 1986) to describe the phenomenon of delayed reproductive death. Indeed the authors found that surviving progeny of irradiated cells show a systematic reduced cloning efficiency which is lower than that of the cell population immediately after irradiation. The enhanced death rate which persists for long periods of time (30 generations) after i~adiation, among the progeny of surviving ceils is due to the continuing andlor delayed expression of bthd mutations during the proliferation of these cells. Mutation in some essential genes may be involved in the delayed reproduc~ve death. Heritable defects such as these mu~tio~s with lethal effects and abno~~ ph~not~ic characteristics (lower cell cycle progression, reduced capacity to attach to culturt: dishes, increased number of abortive colonies and enhanced frequencies of giant ce’tls ifi non-homogeneous colonies) occur in some of the surviving progeny post-irradiation. They also appear to be eventually responsible for the late expression of reproductive failure in the descendants. Residual unrepaired damage may be carried by surviving progeny over many mitotic cycles before being expressed, leading to death, although it is improbable. The high frequency of delayed reproductive death among surviving clones (50%) makes it unlikely to be the result of a directly produced mutation in a specific gene or set of genes. It may be induced specifically as a cellular response to DNA DSB (double-strand breaks}. Experiments with X-rays, EMS ~ethyime~anesulfonate) and the restriction endonuclease Hinf I (Chang and Little, 1992) have reinforced
Chromosome fnstabilig and ~aR~~-~~&ce
Missions
599
this hypathesis. In this study, the cloned progeny of CHO cells surviving alkylating, radiation and enzymatic treatment were evaluated for their cloning efficiency. Clones derived from cells suff~ving these treatments showed a reduced cloning efficiency whereas those from UV irradiation, which is not associated with the generation of DSB at the wavelength used, did not, Moreover DSB repair-deficient xrs-S CHO cells did not show delayed reproductive death. The defective cellular processing of the DSB during the repair process seems to be the cause of the decreased reproductive potential. Clones derived from cells surviving X-i~diatio~ showed an increased frequency of spontaneous mutations for up to 100 cellular generations post-irradiation. The phenotype of delayed reproductive death, present in some clones, correlates with genetic instability. The available information on instability around the genome is derived from studies of genes such as TK, HPRT and APRT. Human lymphoblastoid celf lines presenting mutations at the TK gene, induced by X-rays, EMS or arising spontaneously~ were screened for the existence of second-site mutations at ~crosatellite loci (Li et al., 1994). The frequency of mutation at microsateflite loci was significantly higher in the TK- selected cells than in nonselected clones. A h~o~esis on the mechanism of genomic instability could be the action of a mutator factor which can affect more than one (critical) gene at the same time inducing multiple mutations in cells. RADIOINDUCED CHROMOSOMAL INSTABILITY radiation induces chromosome aberrations detected in the first mitosis after the exposure. In the cultures, most of the initial radiation-induced chromosomal damage is unstable. So while death occurs randomly in the descendants of these cells, those with stable chromosome aberrations may survive for many years. Different model systems have been used to study the transmission of radiation-induced chromosome aberrations in ceil clones, most studies being performed on short-term cultures since most induced aberrations, incompatible with cell survival, rapidly disappear in culture. The frequency of the cells carrying dice&c c~omosomes is reduced by half at each genera~on (Al-Acbkar et al., 1988). Thus in few celi generations all the dicentrics directly induced will disappear. Induced chromosome instability refers to the production of de nova aberrations which accumnlate during cell cycles subsequent to ionizing ra~a~on treatment. Holmberg et al. (1993) analysed X-irradiated T lymphocytes stimulated by interleukin 2 at early and late time points, during donal expansion in vifro. They observed two types of radiation-induced chromosomal instability: clonal karyotypic abnormalities characterized by identical chromosomal changes occuring in a minimum of three cells in individual cell clones and sporadic nonclonal chromosome abenations similar to the aberrations observed by Kadhim emal. ( t 992) such as chromatid and chromosome breaks. X-irradiation of human T lymph~~es gives i-&eto two types of clones: homogeneous and heterogeneuus clones. In the first case, the cells show the same c~omosome a~~a~ons which probably originate from one or muitiple events during the first cell divisions after radiation exposure. The other clones classified as heterogeneous contain cells with normal karyotypes and other cells with abnormal karyotypes. The second type of cells might derive from the first by acquiring c~omosome aberrations during growth, since the percentage of cells with normal karyotypes declines with increasing cultivation time. This could be an indication of genomic instability which occurs as a late response to X-irradiation. The authors demonstrate the delayed appetite of sporadic aberrations (Holmberg et al., 1995) prefere~ti~y in irradiated cells with aberrmt karyotypes. This study has shown that a high proportion of T cells with X irradiation-induced chromosame aberrations are able to proliferate giving rise to expanding clones some of which accumulate latent chromosome damage. Marder and Morgan (1993) have used FISH on clonally-derived ceils to insvestigate delayed genomic instability in a hamster-human hybrid cell line exposed to X-rays. Delayed chromosomal instability manifested as multiple rea~angements of human chromosome 4 in an environment of hamster chromosomes_ Small differences were observed between cells exposed twice to X-rays and those exposed only once. A decrease in the frequency of chromosomal rearrangements was observed in cells of colonies surviving two exposures. Chromosome rearrangements seem to contribute to the cells’ inability to survive many generations after the X ray exposure. The mechanism responsible for the radiation induced delayed instability is not known. Several expirations have been suggested such as: a persistence of DNA damage, activation of latent endogenous virus and induction of a mutator phenotype as a response to stress. These last studies indicate that chromosome instabili~ can be induced by X-rays in immo~alized, stimulated cells or in cells after two rounds of subcloning.
600
C. Ductay and L. Sabatier
In order to follow the transmission of radioinduced chromosome damage in normal ceils, Kadhim et al. (1992) studied the effects of low dose a-particle irradiation from plutonium-238 on clonal descendants of murine haematopoietic stem cells. Alpha-particles induced lesions in the stem cells which result in a diversity of chromosomal aberrations in their progeny many cell divisions later. This radiation exposure produced a high frequency of nonclonal chromosome aberrations in the individual colonies, derived from the surviving stem cells. These abnormalities are characterized mostly by chromatid rather than chromosome aberrations, with no evidence of any preferential site of breakage. This nonclonality in the aberrations observed might be due to de nuv~ occurence of transmissible instability in the clonogenic cells surviving radiation. Similar results were observed for a-particle radiation of human bone marrow cells (Kadhim et al., 1994). However, the results demonstrate interindividu~ v~iations: the stem cells of two individu~s showed c~omosom~ instabiIity when those of two other donors seemed resistant to instability. Moreover, the results were totally different when the cells, murine or human, were exposed to X-rays rather than a-particles. The aberrations were clonal and for the most part, chromosome aberrations. In this model they failed to observe any chromosomal instability after X-ray irradiation. These studies demonstrate that cells which survive a-particle exposure may show a delayed chromosomal instability since they continue to develop chromosomal aberrations at an increased frequency for many cell cycles after the irradiation. The delayed chromosome instability was also observed on human dermal fibroblasts exposed to heavy ions, neon, argon and lead but not after y rays.(Sabatier and Dutrillaux, 1992). The lesions created by these heavy ions are more complex than those induced by X-rays. Their complexity is pro~~on~ to the fluence of the particles (Sabatier et al., 1987). The authors analysed the karyotypes of the irradiated cells at each passage after the exposure and found that after a short period of heavy chromosomal aberrations directly induced by the irradiation, almost all karyotypes of the surviving cells bore stable rearrangements (translocations, inversions) or presented normal karyotypes. These cells acquire de nova transmissible chromosome instability which, several cell generations after exposure, persist from the 15th to the 25th passage. It was characterized by a high proportion of dicentrics, resulting from end-to-end fusions (Martins et al., 1993, Sabatier and Dutrillaux, 1992). Contrary to the results observed by Kadhim, the location of the corresponding breakpoints were specific for the donor analysed. Specific chromosome structures are involved: telomeric regions of, in decreasing order of frequency, chromosome 13, 16, the short arm of chromosome 1 and other chromosomes. This non-random chromosome instability is a late consequence of radiation by high LET particles. This period of chromosome instability was foflowed by the formation of clones with unb~anced karyotypes, such as loss of chromosome 13. These clones progressively invade all the culture. Cell cultures present an increased lifespan (20%). Another hypothesis on the mechanism of genomic instability could be the bypass of the senescence process. Thus irradiation would induce the early emergence of a naturally occuring phenomenon: chromosome instability during senescence. CHROMOSOME INSTABILITY AND CARCINOGENESIS Genomic instability occurs after many generations in the progeny of irradiated cells. The chromosome instability induced by heavy ions leads to multiple genetic alterations and preferentially affects some chromosome structures, particularly telomeres by end-to-end fusions, leading to specific chromosomal imbalances. This instability, specific to certain chromosomes, could differ from one individual to another. The specificity of the chromosomes involved in senescence is a characteristic of each donor (Benn, 1976). Moreover, Sabatier observed that, for one donor, the chromosomal instability seems to be the same independent of the stress (senescence, heavy ion i~adiation, ~ansfo~ation) (Sabatier et al., 1995; data not shown). This period of instability gives rise to the appearance of unbalanced karyotypes. The chromosome imbalances which confer a proliferative advantage to the cells will be selected. These cells then progressively invade the culture. This implies the emergence of unbalanced clones in the progeny of irradiated cells. The chromosome instability followed by the formation of aneuploid clones appears to be shared by cancer ceils. In fact it could represent a key step in the carcinogenesis process. Human solid tumours are characterized by specific chromosome imbalances. Indeed these tumours not only contain structural changes but also numerical changes (gain or loss of whole chromosomes). In colorectal cancers, for example, karyotypes may either remain normal (7% of the cases), or may principally be characterized
Chromosome In~abiiity and Many-Space
Missions
601
by unbalanced clonal rearrangements: gains in 25% of the cases and losses and/or endoreduplication in the rest of the cases (Muleris et al., 1991). It could be generalized that, depending on the first genetic alterations, the selective pressure will determine the chromosome pattern of the tumour.
Chromosome instability characterized by end-to-end fusions is maintained during many cell generations without inducing ceil death. The transmission of this instability could result from tefomere associations followed by fusion-breakage-fusion cycles leading to the appearance of chromosome imbalances. The rea~angements which confer a proliferative advantage to the cells are selected and progressively invade the culture. Chromosomal instability induced by telomere sho~ening may be an early step in the conversion of a normal diploid cell into an aneupioid cell, as observed in many cancer cells. In somatic cells, telomerase, a ~bonucleoprotein which can replicate telomere ends, is not active (Counter er at., 1992; Greider, 1990). Without telomerase activity, telomeres progressively shorten at each cell division. This shortening of telomeres eventually leads to a destabilization of the chromosomes for which the telomeres have reached a critically small size thus leading to end-to-end fusions, contributing to genome instability. The hypothesis by which the chromosomes with the shortest telomeres become unstable first was tested by inducing a high level of chromosome instability with SV40 transfection. Indeed SV40 large T antigen enables cells to progress beyond senescence. Cells continue to divide, losing more telomeric DNA, therefore con~buting to more severe chromosome damage, until they face crisis which is not circumvented by viral ~nsfo~ation. Progression beyond crisis occurs at low frequency and seems to require telomerase activation which is needed for telomeres to be stably m~nt~ned, In order for cells to become i~o~al, it is necessary for telomeres to be maintained or lengthened. This will prevent further destabilization of chromosomes in the i~o~alized clones and imply a great decrease of the high level of genetic instability. This instability will not however disappear since 20% of the overall alterations still remain. Although some immortal cell lines have been described with no telomerase activity (Murnane et al., 1994; Bryan et al., 1995), usually, during the transformation process, telomeres decrease in size in cells when no telomerase is detectable, resulting in c~omosome fusions. Telomere associations, followed by several rounds of fusionbre~age-fusion cycles, allow the transmission of chromosome instability, leading to the selection of chromosome imbalances. In summary, ionizing radiation induce a transmissible chromosomal instability in the progeny of exposed cells many generations after the initial radiation. This delayed instability can be the result of X rays but densely ionizing radiation seems more efficient. The transmission of chromosome instability which p~ferenti~y affects s~cmres such as telomeres when telomerase activity is not detected is due to telomere fusions occuring when a critical telomere length is reached. Chromosome i~tabili~ induced by radiation seems to be a property shared by cancer cells. This raises the question as to whether high LET radiation has a deleterious long-term effect on astronauts after long space missions. ACKNOWLEDGMENTS This work was supported by the CNES 95/421 and the ministry ACCSV8. REFEZBNCES Al-A&car, W., L. Sabatier and B. Dutrilfaux, Transmission of ra~a~on-induced re~ngemen~ through cell divisions, ~~f~f~~~ Researcia, f98, pp. 191-198 (1988). Benn, P. A., Specific chromosome abe~ation~ in senescent ~brobl~t cell lines derived from human embryos, A~er~cun Journal of Human Genetics, 28, pp. 465-473 (1976). Bryan, T. M., A. Englezou, J. Gupta, S, Bacchetti and R. R. Reddel, Telomere elongation in immortal human cells without detectable telomerase activity, The EMBU Journal, 14, pp. 4240-4248 (1995). Chang, W. P. and J. B. Little, Delayed reproductive death in X-irradiated Chinese hamster ovary cells, i~fe~a~io~ai Journal of Radiation Bioology,60, pp. 483-496 (1991). Chang, W. P. and J. B. Little, Evidence that DNA double-stand breaks initiate the pheno~pe of delayed reproductive death in Chinese hamster ovary cells, Radiation Reseurch, 131, pp. 53-59 (1992).
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