Radiation carcinogenesis
John B. Little
Department of Cancer Cell Biology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA
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Abstract
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Research on radiation carcinogenesis during the past 2 decades has focused on cellular and molecular mechanisms for the effects of radiation in mammalian cells. This paper will review several of these areas of research, as they may relate specifically to the induction of cancer by ionizing radiation. Knowledge of the critical DNA damage of biologic importance, and how this damage is repaired, will be discussed in relation to its role in the induction of mutations by radiation. The search for the initiating event in radiation carcinogenesis, as well as other genetic events that may be involved, is discussed in terms of the possible role of the activation of oncogenes or tumor suppressor genes and the loss of cell-cycle checkpoints. Finally, evidence will be described indicating that important genetic consequences of radiation may arise in cells that in themselves receive no direct nuclear irradiation. It has been shown that radiation can, by itself, induce a type of genomic instability in cells, which enhances the rate at which mutations and other genetic changes arise in the descendants of the irradiated cell after many generations of replication. Preliminary evidence has been presented that irradiation targeted to the cytoplasm yields a significant increase in the frequency of mutations. Finally, genetic events including the induction of mutations and changes in gene expression may occur in neighboring cells that receive no direct radiation exposure at all. This `bystander effect' involves gap junction mediated cellcell communication, and activation of the p53 damage response pathway. The possible role of these phenomena in radiation carcinogenesis is discussed.
Abbreviations: DSBs, DNA double-strand breaks; [125I]Urd, 125I-labeled iododeoxyuridine; LOH, loss of heterozygosity.
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Introduction
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The carcinogenic potential of ionizing radiation was recognized soon after Roentgen's discovery of X-rays in December, 1895. By 1902, the first radiation-induced cancer was reported arising in an ulcerated area of the skin. Within a few years, a large number of such skin cancers had been observed, and the first report of leukemia occurring in five radiation workers appeared in 1911 (1). Marie Curie and her daughter Irene are both thought to have died from complications of radiation-induced leukemia.
Animal models to study radiation carcinogenesis were developed primarily after World War II, and large-scale tumor-induction studies were carried out in mice and rats over the succeeding 3 decades (2). These studies defined many of the general characteristics of radiation carcinogenesis, and were supported by the emerging findings from various epidemiologic studies in human populations receiving radiation exposure from occupational, medical and accidental sources. Foremost amongst these has been the long-term follow up of the atom bomb survivors from Hiroshima and Nagasaki (3). These studies have shown radiation to be a `universal carcinogen', in that it will induce cancer in most tissues of most species at all ages including the fetus. The cancers induced by radiation are of the same histological types as occur spontaneously, but the distribution of types may differ. Finally, it became evident that radiation carcinogenesis can be modulated by a variety of non-carcinogenic secondary factors.
The universal nature of radiation as a carcinogen relates to a specific characteristic of ionizing radiation that differentiates it from chemical toxic agents or other physical carcinogens, which are usually tissue specific in their action. This is its ability to penetrate cells and to deposit energy within them in a random fashion, unaffected by the usual cellular barriers presented to chemical agents. All cells in the body are thus susceptible to damage by ionizing radiation; the amount of damage will be related to the physical parameters that determine the radiation dose received by the particular cells or tissue. Cellular systems were developed in the 1970s to study the malignant transformation of individual cells in vitro (4,5). The findings in these studies confirmed several of the conclusions derived from the animal studies, notably that radiation is a relatively weak carcinogen and mutagen as compared with certain chemical agents such as the polycyclic hydrocarbons, but that its effects can be modulated significantly by various secondary factors.
Thus, by 1980, the general characteristics of radiation-induced cancer in vivo and of cellular transformation in vitro were well established; in fact, only limited work has proceeded in these areas directly since that time. Rather, research during the past 2 decades has focused on cellular and molecular mechanisms for the effects of radiation in mammalian cells. In fact, ionizing radiation has become a widely used tool for studying cellular and molecular responses to DNA damage, as these serve to enhance our understanding of the carcinogenic process itself.
In this review, I will discuss several of these areas of research carried out over the past 2 decades that may relate specifically to the induction of cancer by ionizing radiation. These areas include the nature of the critical DNA lesions induced by radiation and how these relate to its mutagenic effects, the search for the initiating event in radiation carcinogenesis and of other genetic events that may be involved, and finally some of the emerging evidence to suggest that important genetic consequences of radiation may arise in cells that in themselves received no direct nuclear exposure.
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Critical DNA damage and its relationship to mutagenesis
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DNA lesions of biologic importance
It has long been known that radiation can induce a broad spectrum of DNA lesions including damage to nucleotide bases, cross-linking, and DNA single- and double-strand breaks (DSBs). DSBs were originally assumed to be the critical cytotoxic lesions, whereas base damage particularly thymine glycols were implicated in mutagenesis. It is now accepted, however, that misrepaired DSBs are the principle lesions of importance in the induction of both chromosomal abnormalities and gene mutations (6,7).
Early biological findings to support this conclusion were derived from studies with 125I-labeled iododeoxyuridine ([125I]Urd) incorporated into cellular DNA. 125I is an auger electron emitting radionuclide; each decay releases a shower of 21 very low energy electrons leaving behind a tellurium atom with 21 positive charges which must capture electrons to return to the neutral state. This intense release of energy occurs within a few base pairs of the site of decay in DNA. It was well known that [125I]Urd is highly cytotoxic, but it had been assumed that it killed cells rather than producing viable mutants. In contrast, not only did [125I]Urd prove to be highly effective in inducing malignant transformation (8), but it was also highly mutagenic (9); a single decay within the target gene had a high probability of producing a mutation, whereas the mean lethal dose was 28 decays. The doseresponse curve for the induction of mutations by [125I]Urd is shown in Figure 1
, where it is compared with those for several other types of radiation. [125I]Urd proved to be much more mutagenic than X-rays, even when normalized for cell survival (9). These results suggested that the important mutagenic lesion was an incorrectly repaired DSB.

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Fig. 1. Doseresponse graph for the induction of mutations by [125I]Urd incorporated into DNA as compared with those for several other types of radiation. [125I]Urd was much more mutagenic than X-rays, even when normalized for cell survival. Data are from various experiments reported by Little and coworkers (72).
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Recent track analysis studies of X-ray interactions in DNA have provided evidence for clustered damage which also has a high probability of producing DSBs (6). This appears to result from the fact that radiation interactions are usually associated with the production of several closely associated ion pairs. Certain types of DNA base damage such as 8-hydroxydeoxyguanosine and thymine glycols have significant potential biological importance, but the available data suggest that such base damage probably plays a minor role in radiation mutagenesis (7). Molecular evidence for the importance of DSBs in the biological effects of radiation have also come from studies of mutant cell lines that are defective in the repair of radiation-induced damage (10).
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DNA repair
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Tremendous strides have been made during the past decade in our understanding of DNA repair mechanisms, particularly as regards the isolation and characterization of the genes involved in each pathway. An analysis of the defects associated with the various complementation groups of xeroderma pigmentosum have allowed the cloning and characterization of many of the genes involved in the nucleotide and base excision repair pathways. The phenomenon of transcription-coupled repair, whereby the transcribed strands of expressed genes are more rapidly repaired than the rest of the genome, has been characterized and its biological implications for radiation exposure described (11). Several mismatch repair genes have been identified, and their importance in colonic cancer defined. Finally, genes involved in DSB repair have been characterized; a number of these are homologs of yeast genes.
The genetic analysis of the repair of DSBs indicates that it involves recombinational processes. It had been shown previously that X-irradiation can induce homologous recombination between alleles of a normal gene in situ in human cells, but the yield of recombinants was very low (12). While DSBs can be repaired by homologous recombination in an error-free manner (13), this mechanism is apparently a fairly uncommon one in mammalian cells. Most DSBs appear to be repaired by an illegitimate recombination process that is error-prone and thus likely accounts for many of the potentially mutagenic DNA lesions induced by radiation. This process of non-homologous end rejoining involves the well characterized DNAPK protein complex, the kinase activity being dependent upon the binding of the ku protein to the broken DNA ends (14). Another protein complex which includes hRad50 and hMre11 has also been shown recently to be involved in DSB repair in human cells (15).
In sum, the biologically important DNA lesion in irradiated cells appears to be the DSB. DSBs are repaired primarily by a non-homologous end joining process which is error-prone; multiple DSBs in a cell may thus facilitate the production of chromosomal rearrangements and other large-scale changes as occur frequently in irradiated cells. What are the implications of these observations for the induction of mutations? This is an important question as the initiating event in radiation carcinogenesis is thought to be a mutational event in a target gene.
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Radiation-induced mutations
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Although the mutagenic potential of radiation was first described by Muller in 1927, it is only within the past 2 decades that information has been gained concerning the molecular structure of mutations in mammalian cells. Earlier studies with the hemizygous X-linked HPRT gene showed that radiation could induce both point mutations and deletions, the latter sometimes including the entire gene (16). However, the size of the deletions at a hemizygous locus that can give rise to a viable cell is limited, owing to the presence nearby of essential genes, and mutations cannot be induced by homologous recombinational processes. When a system was developed to study mutational events at an autosomal locus where both copies of the gene were present, large-scale events involving loss of heterozygosity (LOH) were found to be the most frequent ones (17). This LOH frequently extended to include other genes on the chromosome both proximal and distal to the target gene (18). Although LOH was often the result of a simple deletion, it was shown at least in some cases to be the result of recombinational processes (18,19).
Thus, the predominant molecularstructural changes associated with radiation-induced mutations are large-scale events including LOH of the gene that may extend to other loci on the chromosome. These large-scale changes may include deletions, chromosomal rearrangements or recombinational processes; such mutational changes are of the type one would expect to arise as a consequence of DSBs. These results thus suggest that the initiating event in radiation carcinogenesis would be more likely to involve inactivation of a tumor suppressor gene by LOH rather than the activation of a proto-oncogene such as RAS.
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Search for the initiating event in radiation carcinogenesis
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Oncogenes and tumor supressor genes
The search for genetic changes associated specifically with radiation exposure has proven very disappointing. There is no evidence of site specificity for mutations induced by radiation. The spectrum of molecular-structural changes associated with direct radiation-induced mutations differs markedly from that for spontaneous mutations; in the latter case, point mutations predominate. This is shown graphically for the X-linked HPRT gene in Figure 2
. However, there appears to be no site specificity for DNA breakpoints that lead to deletions and, similarly, sequence analyses of radiation-induced point mutations have generally shown no site specificity (20). Furthermore, no genetic alterations unique to radiation have as yet been found in radiation-induced tumors.
It is well known that radiation can induce a wide variety of stable chromosomal aberrations including deletions and reciprocal translocations. It is tempting to speculate that they may play a more fundamental role in the process of radiation carcinogenesis. Cancer cells are typically aneuploid and contain multiple chromosomal changes; in some cases, specific chromosomal abnormalities have been associated with specific tumor types such as some leukemias. However, these abnormalities occur in both radiation-induced and naturally occurring leukemias. While radiation-induced cancers show multiple unbalanced chromosomal rearrangements, few show specific translocations or deletions as would be associated with the activation of known oncogenes or tumor suppressor genes (21).
It is known that unbalanced translocations can lead to deletions. As certain cancers that are known to be induced by radiation (such as some types of leukemia and sarcoma) are specifically those in which deletions occur, it has been proposed that the most likely mutational event in the initiation of radiation carcinogenesis involves LOH of a tumor suppressor gene (21). One specific example of this phenomenon is the RB tumor suppressor gene located on chromosome 13q14. The hypersensitivity of retinoblastoma patients to the induction of secondary cancers, primarily osteosarcomas in the irradiated field following radiotherapy, is presumably the result of radiation-induced LOH of the RB gene (22). However, RB is a rare disorder and there are few other examples from animal or human carcinogenesis to support the attractive hypothesis that the induction of LOH at tumor suppressor loci is a general phenomenon in the initiation of radiation carcinogenesis. If it were the case, there must be multiple as yet unrecognized genes involved. Although knockout mice heterozygous for the p53 tumor suppressor gene are more susceptible to radiation-induced tumors, evidence has been presented to suggest that LOH at this locus may not be the initiating event (23). It is of interest in this context that the expression of p53 mutations also appears to occur late in the process of radiation-induced malignant transformation, during the growth of the visible transformed foci (23a), an observation consistent with the findings in some human tumors (24).
While the involvement of various oncogenes in experimental and human carcinogenesis is well established, no data have emerged from animal models to suggest a general role for oncogene activation in radiation-induced cancer. Activation of members of the RAS family has been reported in a small fraction of certain mouse lymphomas, and amplification and rearrangement of C-MYC was found in a small percentage of radiation-induced murine osteosarcomas. To my knowledge, however, these scattered early results have not proven to be a consistent finding, though it is clear that the pattern of oncogene activation differs significantly for carcinogenesis induced by chemical carcinogens as compared with radiation. There is some evidence to suggest that radiation may induce papillary thyroid carcinomas in children as a result of activation of the ret oncogene (25). This, however, is a rather special case.
Studies of oncogene and tumor suppressor gene activation in radiation transformation in vitro have been equally disappointing. Amplification and/or overexpression of MDM2 was found in some X-ray transformed foci, while a few others expressed mutant p53 (26). Such findings have led to the hypothesis that there may be multiple pathways for the development of transformation, some involving as yet unidentified oncogenes or tumor suppressor genes (26). This conclusion is perhaps consistent with the seemingly random, non-specific nature of radiation-induced DNA damage and mutations.
Cell-cycle checkpoints
The existence of cell-cycle checkpoints was first demonstrated by a number of radiobiologists in the 1960s. Most prominent among these was a radiation-induced reversible G2/M delay observed in essentially all types of mammalian cells, and a G1/S delay observed primarily in normal diploid cells. As most of these studies were carried out with either transformed rodent or human tumor cell lines, this early research focused on the G2 checkpoint. However, the G2 delay occurs in all cell types both normal and malignant, and can be abrogated only under a few very specific conditions. Thus, research has focused primarily on loss of the G1 checkpoint as a possible initiating event in radiation carcinogenesis.
It was not described until the mid-1980s that, following irradiation of normal human diploid fibroblast cultures, many of the cells were irreversibly arrested at the G1/S border and appeared to enter a senescent-like state (27). When the cells were held in confluence for several hours after irradiation to allow for the repair of potentially lethal radiation damage, the fraction of cells arrested in G1 was reduced in a dose-dependent manner (27). These findings led to the hypothesis that such an irreversible G1 arrest may serve as a mechanism for the elimination of heavily damaged and thus potentially mutated cells from the irradiated population, representing a parallel mechanism to that proposed for cell types such as those of lymphoid or hematopoietic origin that readily undergo apoptotic cell death as a consequence of radiation exposure. The occurrence of this irreversible G1 arrest as well as the reversible G1 delay that occurs in the remaining cells was subsequently shown to be dependent upon wild-type p53 protein expression, owing to activation of its downstream effector p21waf1 (28,29). An association of p53 with a G1 checkpoint had been shown earlier (30); loss of p53 not only eliminated normal checkpoint control but also enhanced the occurrence of apoptotic cell death (30).
It was originally proposed that the loss of the G1 checkpoint was responsible for the genetic instability that occurs in irradiated cells lacking normal p53 function (31). This loss of G1 checkpoint control might thus represent an early event in radiation carcinogenesis. It was hypothesized that cells with extensive genetic damage would now progress through the cell cycle and continue proliferating, rather than undergoing apoptotic cell death or arresting in G1 and becoming senescent (31). This hypothesis remains controversial, however, with G1 arrest appearing to be only one aspect of a complex cellular response to DNA damage (32). Cells that lose wild-type p53 function do become genetically unstable, as manifested, for example, by a marked enhancement in recombinational activity involving non-homologous end rejoining (33), and transgenic mice lacking p53 function are highly susceptible to radiation-induced cancer (34). However, it appears more likely that this is related to the loss of normal p53 function rather than a result specifically of loss of the G1 checkpoint (32). Interestingly, however, the G1 checkpoint is significantly reduced in a wide variety of human tumor cells, irrespective of p53 function (35), as has been observed in radiation-transformed clones of mouse 10T
cells (36).
Altogether, however, these results suggest that the loss of normal checkpoint control in itself is not likely to be a principle initiating event in radiation carcinogenesis. This is consistent with the lack of evidence for the specific involvement of p53 mutations in malignant transformation or radiation-induced tumors. Indeed, when p53 mutations do occur, they appear to be a late event in these processes.
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Evidence that the initial genetic event may not be a direct consequence of nuclear irradiation
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It has been widely assumed that the initial genetic events induced by radiation in mammalian cells occur as a direct result of DNA damage that is not correctly restored by metabolic repair processes. As a consequence, genetic changes such as mutations and chromosomal aberrations would arise in the irradiated cell at the site of DNA damage. There is now increasing evidence, however, that exposure of cell populations to ionizing radiation may also produce non-targeted effects; that is, important genetic consequences of radiation may arise in cells that in themselves received no direct nuclear exposure. Current research in three of these areas is described below.
Radiation-induced genomic instability
In 1980, the results of a series of experiments designed to examine in detail the kinetics of radiation transformation of mouse 10T
cells in vitro yielded a rather surprising result (37). It appeared that transformation involved at least two distinct events. The first was a frequent event which involved a large fraction (2030%) of the irradiated cell population, and enhanced the probability of the occurrence of the second event. The second is a rare event occurring with a frequency of approximately 106 and involves the actual transformation of one or more of the progeny of the original irradiated cell after many rounds of cell division. The second event occurred with a constant frequency per cell per generation during the growth of the irradiated population to confluence, and has the characteristics of a mutagenic event (38). Based on these findings, it was hypothesized that radiation induced a type of transmissible genetic instability in a large fraction of cells in the population; this instability had the effect of enhancing the rate at which transformants arose spontaneously in the progeny population. Evidence was presented that this unstable phenotype persisted for at least 12 and perhaps as many as 25 population doublings after radiation exposure. Data derived from certain experimental animal systems were consistent with this hypothesis.
This hypothesis, which is described schematically in Figure 3
, has now been confirmed in a number of different experimental systems for various endpoints including mutagenesis, chromosomal aberrations and delayed cell death (39,40). In studies of mutagenesis, ~10% of clonal populations derived from single cells surviving radiation exposure showed a persistent increase in the rate in which new mutations arose (41,42). This increased mutation rate persisted for at least 30 generations post-irradiation. As is shown in Figure 2
, the molecular spectrum of these late arising mutants differed markedly from those of direct X-ray induced mutations; in reality, they were very similar to those of spontaneously arising mutants in that the majority were point mutations (42,43). This finding is consistent with the hypothesis that this transmissible instability had the effect of enhancing the rate at which spontaneous mutations arose in the descendants of the irradiated cells. Additional evidence suggesting that a subpopulation of genetically unstable cells may arise in irradiated populations came from the observation that an enhanced frequency of minisatellite mutations occurred in irradiated cells selected for mutation at the TK locus (44).

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Fig. 3. Schematic representation of radiation-induced genomic instability (C and D). Open circles represent normal wild-type cells, while closed circles represent mutated cells. (B) Example of a cell directly mutated by radiation exposure; the mutation is transmitted to all of its progeny. However, most of the cells in the irradiated population will retain the wild-type phenotype (A). (C and D) Examples of mutations arising as a result of radiation-induced genomic instability. The irradiated cell and its immediate progeny are wild-type, but the frequency with which mutations arise amongst the more distant descendants of the irradiated cell is elevated.
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A higher frequency of non-clonal chromosomal aberrations was first reported in clonal descendants of mouse hematopoietic stem cells 1214 generations after
-irradiation (45). The induction of transmissible chromosomal instability by radiation has since been shown to occur in a number of cellular systems (4649). Transmission of such chromosomal instability has also been demonstrated in vivo (50,51). Indeed, instability may be enhanced by passaging cells in vivo (52). A persistently increased rate of cell death has also been found to occur in cell populations many generations after irradiation (53,54).
It is thus well established that radiation by itself can induce a type of transmissible instability in cells that leads to an enhanced probability that multiple genetic events may arise in the surviving cell population, after many generations of replication. The precise mechanisms associated with this phenomenon, however, remain to be elucidated. These include how it is initiated and how it is maintained. Recent evidence suggests that DNA is at least one of the critical targets in the initiation of such genomic instability (55), and that oxidative stress resulting from enhancement of a p53-independent apoptotic process may contribute to the maintenance of the instability phenotype (56). It has been hypothesized that various closely regulated cellular processes may be disrupted by radiation leading to a state of chaos that perturbs the normal regulatory and signaling pathways thus disrupting cellular homeostasis, a state from which the cell never completely recovers (55). Thus, though the nucleus may indeed be the target for the induction of instability in the irradiated cell, the initial genetic consequence of radiation may occur in one of the descendants of the irradiated cell at a remote point in time.
Although the importance of such induced instability to the early events in radiation carcinogenesis in vivo remains unknown, the phenomenon is now well documented in a wide variety of in vitro systems, and certainly would change the way we think about the early events in the initiation of carcinogenesis. It is tempting to speculate, for example, that such instability would enhance the probability of the occurrence of the multiple genetic events in a single cell lineage that may be required for the development of cancer. Interestingly, the concept of induced instability as an initial step in carcinogenesis is consistent with emerging findings from epidemiologic studies in human populations which suggests that some types of radiation-induced cancer may follow a relative risk model; that is, a given dose of radiation enhances the rate at which cancer develops normally at all times post-irradiation rather than inducing a specific cohort of new tumors.
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Induction of mutations by cytoplasmic irradiation
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It has long been considered that the nucleus is the target for the important biological effects of radiation, and that the consequent genetic alterations arise from radiation-induced DNA damage. Indeed, a number of earlier studies in several biological systems indicated that nuclear irradiation was critical for its cytotoxic effects. The development of precision microbeam irradiators has now permitted the study of the effects of radiation localized to specific parts of the cell. The
-particle irradiator developed by scientists at Columbia University allows the delivery of a specific number of
-particles to the cytoplasm of individual cells without concomitant exposure to the nucleus. Utilizing this source, Wu et al. (57) have shown that cytoplasmic irradiation with low fluences of
-particles can induce a significant frequency of mutations in mammalian cells. The results of these experiments are summarized in Figure 4
. As was expected on the basis of earlier reports, cytoplasmic irradiation had relatively little effect on cell survival (Figure 4A
). However, a single traversal by an
-particle approximately doubled the spontaneous mutation frequency, whereas a 23-fold enhancement in the mutation frequency was observed with up to four particle traversals per cell. No increase in the frequency of mutations was observed with higher particle fluences.

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Fig. 4. Cell survival and induced frequency of mutations in AL cells in which the cytoplasm was irradiated with an exact number of -particles from a precision microbeam irradiator. The background mutation frequency was 43 ± 15 mutants per 105 survivors. Data from Wu et al. (57).
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These results differ from those of an earlier study involving nuclear irradiation (58). For nuclear traversals, mutation frequencies were 23-fold higher than for the same number of cytoplasmic traversals, and the frequency continued to rise with fluences up to eight or more particles per cell. Of particular interest was the observation that the spectrum of molecularstructural changes differed markedly between the two types of irradiation. Direct nuclear hits yielded primarily large-scale changes such as described previously for X-rays (Figure 2
). Cytoplasmic radiation, on the other hand, yielded primarily point mutations with the spectrum resembling that of spontaneously arising mutants (57). This observation is reminiscent of that for late-arising mutations associated with genomic instability (Figure 2
). It is of interest that both phenomena have been hypothesized to involve an enhanced production of reactive oxygen species (56,57).
Although the results of this study are preliminary and need to be reproduced, they are very provocative in that they suggest that direct nuclear radiation is not required for the production of important genetic effects.
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Bystander effects in irradiated cell populations
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The bystander effect in irradiated cell populations goes one step further; that is, it implies that genetic alterations can occur in cells that receive no direct radiation exposure at all. Rather, damage signals are transmitted to these cells from neighboring irradiated cells in the population. Indeed, evidence is now accumulating to support this hypothesis that important biological effects may occur in cells that receive no direct radiation exposure (59). If this phenomenon occurs in vivo, it could be of considerable importance as regards the carcinogenic effects of very low doses of densely ionizing radiation such as
-particles from radon. Only a small fraction of a person's bronchial epithelial cells, the presumed target for lung cancer, will actually be hit by an
-particle from residential radon exposure during the person's lifetime.
The experimental model employed to study the bystander effect has generally involved the exposure of monolayer cultures to very low fluences of
-particles, such that only a very small fraction of the cell population will actually be hit by a particle. In the initial report of this phenomenon, an enhanced frequency of sister chromatid exchanges (SCE) was observed in 3050% of the cells exposed to fluences where only 0.11% of the cells were actually traversed by an
-particle (60). These results indicated that the target for genetic damage by
-particles is much larger than the nucleus or in fact than the entire cell itself. This finding was later confirmed (61), and evidence presented that it involves the secretion by irradiated cells of cytokines or other factors that leads to enhanced production of reactive oxygen species in bystander cells (6264). There is also evidence that cytotoxic effects in bystander cells may be related to the release of a factor into the medium (65,66). These findings are reminiscent of reports that clastogenic activity can be isolated from the plasma of radiation-exposed individuals (67). Microbeam studies of cells individually irradiated with 3He particles have provided evidence that more micronucleated and apoptotic cells arise in the population than expected on the basis of direct hits, providing further evidence for interactions between hit and non-hit cells (59). We have also found evidence for an increased occurrence of specific gene mutations in bystander cells under conditions similar to those described above for SCE (68). Finally, it has been reported that chromosomal instability may be transmitted to the clonal descendants of unirradiated bystander cells following
-irradiation of murine bone marrow (69).
Changes in gene expression also occur in bystander cells (70). The expression levels of p53, p21, cyclin B1, rad51 and CDC2 were significantly modulated in non-irradiated cells in confluent human diploid cell populations exposed to very low fluences of
-particles. The changes in expression levels were determined both by western blotting and in situ immunofluorescence techniques. An example of one of the latter studies is shown in Figure 5
in which an antibody to p21waf1 (a downstream effector in the p53 damage signaling pathway) was utilized to examine the expression of this gene product in individual cells by confocal fluoresence microscopy. The cultures were irradiated with 0.3 cGy, an
-particle fluence by which ~12% of the cells in the population should be hit. As can be seen in Figure 5
, increased expression of p21waf1 was observed in clusters of cells in the monolayer, whereas only occasional individual cells should be labeled if the effect occurred only in those nuclei actually traversed by an
-particle.

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Fig. 5. Bystander effect in confluent cultures of normal human diploid fibroblasts, as examined by in situ immunofluoresence detection of p21waf1 by a secondary antibody conjugated to FITC. The panel on the left represents control, non-irradiated cultures, whereas the two panels on the right are from cultures irradiated with a 0.3 cGy. Focal areas were observed in which up to 50% of the cells showed enhanced expression of p21, whereas only 12% of the nuclei were actually traversed by an -particle.
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This phenomenon has been shown to involve cellcell communication via gap junctions (70). Thus, the p53-mediated DNA damage response pathway appears to be induced in bystander as well as irradiated cells; it has been hypothesized that activation of this signal transduction pathway in bystander cells could lead to genetic changes in these cells through reduced replication fidelity or increased recombinational activity (71). Overall, however, these results suggest that similar signaling pathways are induced in bystander as occurs in irradiated cells. Thus, biological effects in cell populations may not be restricted to the response of individual cells to the DNA damage they receive.
The three phenomena described in this section have been observed primarily in in vitro cell culture systems. There is no reason to believe, however, that the mutagenic effects of cytoplasmic radiation or the biological effects mediated by gap junction communication in bystander cells would not also occur in cell populations in vivo. Preliminary evidence has already been presented for the transmission of radiation-induced genomic instability in experimental animal systems in vivo (50,51). The importance of these phenomena in radiation carcinogenesis remains, of course, to be elucidated. These results, however, may change our thinking about the early events in this process, and particularly about the critical targets for genetic and carcinogenic damage by radiation.
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Notes
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Email: jlittle{at}hsph.harvard.edu
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Received August 16, 1999;
accepted September 20, 1999.