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Genomic instability and radiation
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2003 J. Radiol. Prot. 23 173 (http://iopscience.iop.org/0952-4746/23/2/304) View the table of contents for this issue, or go to the journal homepage for more
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INSTITUTE OF PHYSICS PUBLISHING
JOURNAL OF RADIOLOGICAL PROTECTION
J. Radiol. Prot. 23 (2003) 173–181
PII: S0952-4746(03)61426-6
REVIEW
Genomic instability and radiation John B Little Harvard School of Public Health, Boston, MA 02115, USA E-mail:
[email protected] Received 24 October 2002, in final form 3 March 2003, accepted for publication 13 March 2003 Published 9 June 2003 Online at stacks.iop.org/JRP/23/R173 Abstract Genomic instability is a hallmark of cancer cells, and is thought to be involved in the process of carcinogenesis. Indeed, a number of rare genetic disorders associated with a predisposition to cancer are characterised by genomic instability occurring in somatic cells. Of particular interest is the observation that transmissible instability can be induced in somatic cells from normal individuals by exposure to ionising radiation, leading to a persistent enhancement in the rate at which mutations and chromosomal aberrations arise in the progeny of the irradiated cells after many generations of replication. If such induced instability is involved in radiation carcinogenesis, it would imply that the initial carcinogenic event may not be a rare mutation occurring in a specific gene or set of genes. Rather, radiation may induce a process of instability in many cells in a population, enhancing the rate at which the multiple gene mutations necessary for the development of cancer may arise in a given cell lineage. Furthermore, radiation could act at any stage in the development of cancer by facilitating the accumulation of the remaining genetic events required to produce a fully malignant tumour. The experimental evidence for such induced instability is reviewed.
1. Introduction The genome is the source of the genetic information in cells which is encoded in the DNA molecules. However, the genome in mammalian cells is constantly challenged by destabilising factors including normal DNA replication and cell division, as well as a host of intracellular and extracellular environmental stresses such as oxidative metabolism, exposure to genotoxic chemical agents and background radiation. Cells have thus developed elaborate systems for maintaining genomic integrity, notably those involved in ensuring the fidelity of DNA replication, the enzymatic repair of endogenous and exogenous DNA damage, and the control of progression through the cell cycle. Although a certain level of genetic instability may be a positive factor in fostering genetic diversity, destabilisation of the genome can lead to an enhanced rate of mutagenesis resulting in alterations in cellular function, including cell death. 0952-4746/03/020173+09$30.00
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Genomic instability is considered a hallmark of the process of carcinogenesis, most human cancers showing loss of genomic integrity. Genomic instability is frequently associated with loss of cell cycle control and alterations in DNA repair processes, but may be manifested by a variety of cellular changes. Notable among these is chromosomal instability. Two types of chromosomal abnormalities can arise in mammalian cells. ‘Unstable’ aberrations are usually lethal to dividing cells and include changes such as dicentrics, ring chromosomes and large deletions. ‘Stable’ aberrations, on the other hand, can be transmitted through many generations of cell replication and include changes such as reciprocal translocations and small deletions. Other manifestations of genomic instability include an increased rate of spontaneous mutations, particularly in microsatellite sequences, and enhanced sensitivity to genotoxic agents. 2. Genetic disorders associated with genomic instability and a predisposition to cancer There are a number of rare genetic disorders characterised by a heritable predisposition to the development of cancer [1, 2]. As the genes from these disorders have now been cloned and characterised, these disorders offer insights into the possible mechanisms of carcinogenesis and the role of genomic instability in this process. Some of these single-gene disorders are listed in table 1, where their cellular and molecular characteristics are described along with the types of cancer to which they are unusually susceptible. As is evident, the gene products associated with all of these disorders are involved in the signalling pathways for DNA replication and repair, as well as cell cycle control, and the disorders are associated with genomic instability. The ATM protein is an important sensor of DNA damage which activates by phosphorylation a constellation of downstream proteins involved in DNA repair and cell cycle control. Cells homozygous for mutations in the ATM gene are extremely sensitive to the cytotoxic effects of ionising radiation. NBS, BRCA1 and BRCA2 code for proteins involved in DNA repair, specifically the recombinational repair pathways. Bloom’s syndrome gene (BS) codes for a helicase which is important for DNA replication as well as being involved in repair; this disorder is of interest as it is characterised by a predisposition to a wide variety of types of cancer. Recent evidence links the seven cloned FA genes with BRCA1, ATM and NB51 in cell cycle checkpoint and DNA repair pathways [3]. Hereditary non-polyposis colon cancer is associated with a defect in mismatch repair of DNA base damage, and is characterised specifically by microsatellite instability. Although these are rare disorders which affect only a small fraction of the population, they provide an important insight into the mechanisms for genomic instability and its association with cancer. As noted earlier, the mammalian cell has developed complex machinery for the faithful replication of its DNA prior to cell division, as well as for the repair of both endogenous and exogenous DNA damage. It is thus of interest that the genes responsible for all of these genetic disorders are involved in pathways that ensure the fidelity of DNA replication and repair. Inactivation of genes involved in the non-homologous end joining DNA repair pathway in transgenic mice has been associated specifically with the induction of chromosomal instability [4]. Mutations in genes in this recombinational repair pathway are involved in many of the disorders in table 1. 3. p53 and genomic instability One pathway of importance in maintaining genomic integrity involves the p53 tumour suppressor gene, a transcriptional regulator that plays a central role in the cellular response to
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Table 1. Some genetic disorders characterised by genomic instability and a predisposition to cancer. Clinical disorder Ataxia telangiectasia
Gene
Function
ATM
DNA damage sensor
Major cellular abnormalities
Chromosomal instability Radiosensitivity Cell cycle abnormalities Nijmegen breakage NBS1 Recombinational DNA Chromosomal instability syndrome repair Radiosensitivity Cell cycle abnormalities Bloom’s BS Helicase Chromosomal instability syndrome (DNA replication) Elevated sister chromatid exchanges DNA damage Chromosomal instability Fanconi’s anaemia FAa sensing and repair Sensitivity to DNA crosslinking agents Familial breast BRCA1 Recombinational DNA Chromosomal instability cancer BRCA2 repair Radiosensitivity Hereditary nonMMRb Mismatch DNA repair Microsatellite instability polyposis colon Mutational instability cancer a b
Cancer types Primarily leukaemia and lymphoma Some solid tumours Lymphoma and leukaemia
Multiple cancers of all types
Leukaemia and solid tumours
Breast and ovarian cancer Colon and certain other solid tumours
There are seven interacting FA genes. Mismatch repair. There several different MMR genes, inactivation of any one of which will give rise to the disorder.
DNA damage. Mutations in p53 in humans are associated with the Li–Fraumeni syndrome, a rare genetic disorder associated with a predisposition to multiple malignancies. p53 functions primarily in cell cycle control; while control of the G1 /S checkpoint is an important function of p53, it also acts at the S and G2 phases of the cycle. These checkpoints or delays in cell cycle progression allow the cell time to repair DNA damage prior to DNA replication or mitosis. Thus, irradiated cells that have lost p53 function enter and proceed through the cell cycle unchecked; such cells develop chromosomal instability. p53 also plays a role in the apoptotic response by which heavily damaged cells are thought to be removed from the population by undergoing apoptotic cell death. In cells such as human diploid fibroblasts which do not undergo apoptosis, heavily damaged cells may experience a p53-dependent, irreversible G1 arrest, thus effectively removing them from a proliferating population. Neither of these processes occurs in cells in which the p53 pathway is inactivated; thus, heavily damaged cells may survive and contribute further to genomic instability. Interestingly, p53 deficiency by itself may be insufficient for the induction of genomic instability [5–7]. Rather, instability is generally observed in cells that have been exposed to genotoxic or clastogenic agents, or by activation of certain oncogenes [8, 9]. However, the organism is constantly subjected to endogenous and exogenous environmental stresses that may serve to induce instability in p53-deficient cells. In this light, it is of interest that an increased susceptibility to spontaneous or radiation-induced cancer has been reported in certain strains of transgenic mice either homozygous or heterozygous for p53 mutations [10, 11], consistent with the elevated incidence of cancer occurring in Li–Fraumeni syndrome patients, a human disorder characterised by p53 mutations. 4. Genomic instability and the development of cancer Evidence is growing to support the role of instability in carcinogenesis. Almost all human tumours show such instability manifested as multiple unbalanced chromosomal
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aberrations [12]. It has been hypothesised that as many as six to eight separate genetic events may be required to convert a normal cell into one with a fully malignant phenotype; a number of the specific events involved have been identified for certain types of colon cancer where they are associated with increasing levels of malignant change [13]. The perplexing question, however, is how a single cell lineage can accumulate this number of independent mutations within the lifespan of the individual, if each of these mutations arise independently and at a frequency of approximately 10−5 . It has thus been tempting to speculate that cells developing mutator phenotypes may be an important mechanism in the initiation and/or progression of cancer [14]. The association of cancer predisposition with genomic instability resulting from mutations in genes controlling DNA replication and repair support this hypothesis, and the observation that mismatch repair defects are associated with microsatellite instability and the development of certain types of cancer represents an appropriate model. However, cells from most common types of human tumours do not show a mutator phenotype nor known defects in DNA repair capacity. Thus, the discovery that exposure to ionising radiation can in itself induce transmissible genomic instability in a large fraction of the irradiated cell population has generated considerable interest. This instability is characterised by hypermutability, chromosomal instability and other genetic changes occurring in the progeny of the irradiated cell, which persist for many generations of cell replication [15, 16]. 5. Radiation-induced genomic instability It has long been thought that the initial genetic events induced by radiation in mammalian cells occur as a direct consequence of DNA damage that is not correctly restored by metabolic repair processes [17]. Genetic changes such as mutations and chromosomal aberrations would thus arise in the irradiated cell at the site of DNA damage. It is now evident, however, that exposure of cell populations to ionising radiation may also produce non-targeted effects; that is, the genetic consequences of radiation may arise in cells that in themselves receive no direct radiation exposure. There is now convincing evidence that radiation may induce a process of genomic instability in individual cells that is transmitted to their progeny, leading to a persistent enhancement in the rate at which genetic changes arise in the descendants of the irradiated cell after many generations of replication [18, 19]. This is a non-targeted effect of radiation in that the ultimate genetic damage arises in the descendants of the irradiated cell; that is, in cells that in themselves have received no direct radiation exposure. Radiation has in essence enhanced the frequency with which genetic changes arise spontaneously in the cell population derived from the irradiated cell. Typically, this phenomenon has been studied by examining the occurrence of such genetic effects in clonal populations derived from single cells surviving radiation exposure [20]. Early evidence for the existence of such a phenomenon was derived from an examination of the kinetics of radiation-induced malignant transformation of cells in vitro [21]. These results suggested that transformed foci did not arise from a single, radiation-damaged cell. Rather, radiation appeared to induce a type of instability in 20–30% of the irradiated cell population; this instability enhanced the probability of the occurrence of a second, neoplastic transforming event. This finding was in contradistinction to classic theories of carcinogenesis in which the initiating event is thought to be rare and likely mutagenic in nature. This second event was a rare one, however, occurring with a frequency of approximately 10−6 , and involved the actual transformation of one or more of the progeny of the original irradiated cells after may rounds of cell division. This second, transforming, event occurred with the constant frequency per
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cell per generation, and had the characteristics of a mutagenic event [22]. Thus, neoplastically transformed foci did not appear to arise from the original irradiated cell but rather from one or more of its progeny. This phenomenon has subsequently been demonstrated in a number of experimental systems for various genetic endpoints [18–20, 23]. In terms of mutagenesis, approximately 10% of clonal populations derived from single cells surviving radiation exposure showed a significant elevation in the frequency of spontaneously arising mutations as compared with clonal populations derived from non-irradiated cells [15, 24]. This increased mutation rate persisted for approximately 30 generations post-irradiation then gradually subsided. The molecular structural spectrum of these late-arising mutants resembles those of spontaneous mutations in that the majority of them are point mutations [24, 25] whereas direct x-ray-induced mutations involve primarily deletions. This phenomenon was associated with the development of chromosomal instability. An enhancement of both minisatellite [26] and microsatellite [27] instability has been observed in the progeny of irradiated cells selected for mutations at the thymidine kinase locus. A heritable reduction in cloning efficiency and an increased frequency of apoptotic cell death have also been reported among the progeny of irradiated cells [15, 28– 30]. This may represent another mechanism for the elimination of genetically damaged and therefore potentially cancer forming cells such as was described above for p53 expression. An enhanced frequency of non-clonal chromosomal aberrations was first reported in clonal descendants of mouse haematopoietic stem cells 12 to 14 generations after exposure to alpha radiation [16]. Persistent radiation-induced chromosomal instability has since been demonstrated in a number of other cellular systems, and transmission of such chromosomal instability has also been shown to occur in vivo [31–33]. Susceptibility to radiation-induced chromosomal instability in mice differed significantly among different strains [34, 35]. Of particular importance in terms of radiation carcinogenesis are the emerging observations suggesting that this phenomenon not only occurs in vivo, but that it may be related to the induction of cancer. The transmission of chromosomal instability in vivo has been demonstrated in several distinct experimental models [31–33], and evidence has been presented to suggest that instability induced in x-irradiated mouse haematopoietic stem cells may be related to the occurrence of the non-specific genetic damage found in radiation-induced leukaemias in these mice [36]. Of particular interest, however, is the finding that sensitivity to mammary tumour induction in mice was not only strain specific, but correlated with the strain specificity for the induction of chromosomal instability in mammary epithelial cells irradiated in vivo [33]. These were related to reduced expression of the DNA repair enzyme DNA-PKcs, an important component of the non-homologous end joining pathway of DNA repair [37]. Thus, this mouse model relates radiation-induced genomic instability to a defect in DNA repair and associates it with the enhanced susceptibility to radiation-induced cancer. Another non-targeted effect of radiation of current interest has been termed the bystander effect [17, 38]. When a small fraction of cells in a population are exposed to radiation, damage signals may be transmitted to neighbouring non-irradiated cells leading to genetic changes including mutations and chromosomal aberrations in these ‘bystander’ cells. These signals may be transmitted through the culture medium [38], or by way of gap-junction mediated intercellular communication [39]. Both radiation-induced genomic instability [29, 40] and the bystander effect [41] have been associated with enhanced oxidative stress, and evidence for the involvement of the non-homologous end joining DNA repair pathway in the bystander effect has been reported recently [42]. Thus, a convergence between the two phenomena may be emerging. The molecular mechanisms involved in radiation-induced genomic instability remain elusive, both in terms of how the process is initiated and how it is maintained. It would not
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appear to be the result of a mutation occurring in a mutator gene or a family of mutator genes, as instability is induced in a large fraction of the cell population (10–20%) by quite low radiation doses. Upregulation of oxidative metabolism has been associated with the phenomenon, and it has been proposed that oxidative stress perhaps consequent to enhanced p53-independent apoptosis may contribute to the perpetuation of the instability phenotype [29]. Finally, it has been proposed that telomeric sequences may play a role in transmissible genomic instability [43] with unstable telomeric structures undergoing successive bridge-breakage–fusion cycles perpetuating the instability phenotype. 6. Conclusions Genomic instability is a hallmark of cancer cells, and is presumably involved in the process of carcinogenesis. The fact that it can be induced by exposure to genotoxic agents such as ionising radiation, as well as certain non-genotoxic stress exposures [44], is provocative. It could imply that the initial event in radiation carcinogenesis is not a mutation occurring in a specific gene or set of genes in an occasional cell, which subsequently gains a selective growth advantage. Rather, radiation may induce a process of instability in many cells in the irradiated population, which could enhance the rate at which multiple mutations would occur in a given cell lineage. Radiation could thus act at any stage in the development of the tumour; radiation exposure would facilitate the accumulation of the remaining genetic events required to produce a fully malignant tumour. Indeed, the studies of mutational instability in vitro are consistent with radiation enhancing the rate at which spontaneous mutations arise in the descendants of the irradiated cell. The extent to which this phenomenon may be of importance in the induction of human cancer by ionising radiation remains, however, to be determined. Emerging evidence with animal models suggests that it does occur in vivo. It could have significant impact on the shape of the dose response curve for the induction of cancer by low radiation doses. In another light, it would offer a mechanism for transgenerational effects of irradiation which have now been shown to occur in several experimental animal models. Acknowledgments The research described from my laboratory was supported by grants from the US Department of Energy and the National Institutes of Health. R´esum´e L’instabilit´e g´enomique est le poinson des cellules canc´ereuses; on pense qu’elle est impliqu´ee dans la carcinog´en`ese. En effet, un certain nombre de d´esordres g´en´etiques rares, associ´es a` une pr´edisposition au cancer, sont caract´eris´es par une instabilit´e g´enomique, apparaissant dans leurs cellules somatiques. Une observation est particuli`erement int´eressante: l’instabilit´e transmissible peut eˆ tre induite dans les cellules somatiques venant d’individus normaux, par une exposition a` un rayonnement ionisant, conduisant a` une acc´el´eration persistante de la vitesse avec laquelle des mutations et des aberrations chromosomiques surviennent dans les descendants des cellules irradi´ees, apr`es plusieurs g´en´erations de r´eplication. Si la carcinog´en`ese par rayonnement produit une telle instabilit´e induite, cela implique que l’´ev´enement carcinog´enique initial peut ne pas eˆ tre une mutation rare se produisant dans un g`ene, ou une s´erie de g`enes sp´ecifiques. Pour mieux dire, le rayonnement peut induire
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un processus d’instabilit´e dans de nombreuses cellules d’une population; il augmente ainsi la vitesse avec laquelle peuvent apparaˆıtre, dans un lignage donn´e de cellules, les mutations multiples des g`enes n´ecessaires pour d´evelopper un cancer. Par ailleurs, le rayonnement pourrait agir a` n’importe quel stade du d´eveloppement d’un cancer, en facilitant l’accumulation des e´ v`enements g´en´etiques permanents, n´ecessaires pour produire une tumeur totalement maligne. On passe en revue les preuves exp´erimentales d’une telle instabilit´e induite. Zusammenfassung Genomische Instabilit¨at ist ein Kennzeichen von Krebszellen und man glaubt, dass sie am Prozess der Karzinogenese beteiligt ist. Tats¨achlich sind eine Reihe seltener genetischer St¨orungen, die mit einer Anf¨alligkeit f¨ur Krebs in Verbindung gebracht werden, gekennzeichnet durch genomische Instabilit¨at, die in ihren K¨orperzellen auftritt. Von besonderem Interesse sind die Beobachtungen, dass u¨ bertragbare Instabilit¨at in den K¨orperzellen normaler Personen durch Bestrahlung mit ionisierende Strahlung ausgel¨ost werden kann; dies f¨uhrt zu einer zunehmenden Erh¨ohung der Geschwindigkeit, mit der Mutationen und Chromosomenaberrationen in der Nachkommenschaft der bestrahlten Zellen nach vielen Generationen der Replikation entstehen. Ist eine derart induzierte Instabilit¨at an der StrahlenKarzinogenese beteiligt, so w¨urde dies bedeuten, dass das urspr¨ungliche karzinogene Ereignis m¨oglicherweise keine seltene Mutation ist, die in einem spezifischen Gen oder einem Gensatz vorkommt. Stattdessen k¨onnte die Strahlung einen Prozess der Instabilit¨at in vielen Zellen in einer Population ausl¨osen und dadurch die Geschwindigkeit erh¨ohen, mit der multiple Genmutationen, die f¨ur die Entwicklung von Krebs erforderlich sind, in einer bestimmten Zellabstammung entstehen. Außerdem k¨onnte die Strahlung in jedem Stadium in der Entwicklung von Krebs wirksam werden, indem sie die Akkumulation der restlichen genetischen Ereignisse erleichtert, die erforderlich sind, um einen vollst¨andig b¨osartigen Tumor zu erzeugen. Die experimentellen Beweise f¨ur derart induzierte Instabilit¨at werden untersucht. References [1] Fearon E R 1997 Human cancer syndromes: clues to the origin and nature of cancer Science 278 1043–50 [2] Sankaranarayanan K and Chakraborty R 2001 Impact of cancer predisposition and radiosensitivity on the population risk of radiation-induced cancers Radiat. Res. 156 648–56 [3] D’Andrea A D and Grompe M 2003 The Fanconi anaemia/BRCA pathway Nat. Rev. Cancer 3 23–43 [4] d’Adda di Fagagna F, Hande M P, Tong W M, Roth D, Lansdorp P M, Wang Z Q and Jackson S P 2001 Effects of DNA nonhomologous end-joining factors on telomere length and chromosomal stability in mammalian cells Curr. Biol. 11 1192–6 [5] Lu X, Magrane G, Yin C, Louis D N, Gray J and Van Dyke T 2001 Selective inactivation of p53 facilitates mouse epithelial tumor progression without chromosomal instability Mol. Cell. Biol. 21 6017–30 [6] Van Gent D C, Hoeijmakers J H and Kanaar R 2002 Chromosomal stability and the DNA double-stranded break connection Nat. Rev. Genet. 2 196–206 [7] Bunz F, Fauth C, Speicher M R, Dutriaux A, Sedivy J M, Kinzler K W, Vogelstein B and Lengauer C 2002 Targeted inactivation of p53 in human cells does not result in aneuploidy Cancer Res. 62 1129–33 [8] Paulson T G, Almasan A, Brody L L and Wahl G M 1998 Gene amplification in a p53-deficient cell line requires cell cycle progression under conditions that generate DNA breakage Mol. Cell. Biol. 18 3089–100 [9] Vafa O, Wade M, Kern S, Beeche M, Pandita T K, Hampton G M and Wahl G M 2002 c-Myc can induce DNA damage, increase reactive oxygen species, and mitigate p53 function: a mechanism for oncogene-induced genetic instability Mol. Cell. 9 1031–44 [10] Kemp C J, Wheldon T and Balmain A 1994 p53-deficient mice are extremely susceptible to radiation-induced tumorigenesis Nat. Genet. 8 66–9
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[11] Donehower L A, Harvey M, Slagle B L, McArthur M J, Montgomery C A, Butel J S and Bradley A 1992 Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours Nature 356 215–21 [12] Dutrillaux B 1997 Ionizing radiation induced malignancies in man Radioprotection 32 C431–40 [13] Vogelstein B and Kinzler K W 1993 The multistep nature of cancer Trends Genet. 9 138–41 [14] Loeb L A 2001 A mutator phenotype in cancer Cancer Res. 61 3230–9 [15] Chang W P and Little J B 1992 Persistently elevated frequency of spontaneous mutations in progeny of CHO clones surviving X-irradiation: association with delayed reproductive death phenotype Mutat. Res. 270 191–9 [16] Kadhim M A, Macdonald D A, Goodhead D T, Lorimore S A, Marsden S J and Wright E G 1996 Transmission of chromosomal instability after plutonium alpha-particle radiation Radiat. 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Carcinog. 32 118–27 [24] Little J B, Nagasawa H, Pfenning T and Vetrovs H 1997 Radiation-induced genomic instability: delayed mutagenic and cytogenetic effects of x-rays and alpha particles Radiat. Res. 148 299–307 [25] Grosovsky A J, Parks K K, Giver C R and Nelson S L 1996 Clonal analysis of delayed karyotypic abnormalities and gene mutations in radiation-induced genetic instability Mol. Cell. Biol. 16 6252–62 [26] Li C Y, Yandell D W and Little J B 1992 Evidence for coincident mutations in human lymphoblast clones selection for functional loss of a thymidine kinase gene Mol. Carcinog. 5 270–7 [27] Romney C A, Paulauskis J D and Little J B 2001 X-ray induction of microsatellite instability at autosomal loci in human lymphoblastoid WTK1 cells Mutat. Res. 478 97–106 [28] Seymour C B, Mothersill C and Alper T 1986 High yields of lethal mutations in somatic mammalian cells that survive ionizing radiation Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. 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[41] Azzam E I, de Toledo S M, Spitz D R and Little J B 2002 Oxidative metabolism modulates signal transduction and micronucleus formation in bystander cells from alpha-particle-irradiated normal human fibroblast cultures Cancer Res. 62 5436–42 [42] Little J B, Nagasawa H, Li G C and Chen D J 2003 Involvement of the nonhomologous end joining DNA repair pathway in the bystander effect for chromosomal aberrations Radiat. Res. 159 262–7 [43] Bouffler S D, Blasco M A, Cox R and Smith P J 2001 Telomeric sequences, radiation sensitivity and genomic instability Int. J. Radiat. Biol. 77 995–1005 [44] Li C Y, Little J B, Hu K, Zhang W, Zhang L, Dewhirst M W and Huang Q 2001 Persistent genetic instability in cancer cells induced by non-DNA-damaging stress exposures Cancer Res. 61 428–32
