Cell Killing

Radiation can kill cells by two distinct mechanisms. The first is apoptosis, also called programmed cell death or interphase death.^2–4 Cells undergoing apoptosis as an immediate consequence of radiation damage usually die in interphase within a few hours of irradiation, irrespective of and without intervening mitosis. They share distinct morphologic changes, including loss of normal nuclear structure and degradation of DNA that can be demonstrated by a classical pattern of “laddering” on DNA blots. It has long been known that apoptotic cell death can be induced by exposure to relatively low doses of radiation in a few cell types including small lymphocytes, type A spermatogonia and oocytes.² However, apoptosis may also be a significant cause of death in a broader variety of cell types exposed to higher radiation doses, particularly those of hematopoietic or lymphoid origin as well as some tumor cells.

Loss of apoptotic control is thought to be an important factor in tumor development. Early evidence suggested that radiation-induced apoptosis was dependent upon the functional activity of the p53 gene, but it soon became evident that p53-independent pathways may also be involved, such as that mediated by the Bcl-2/BAX family,⁵ all of which converge on the activation of proteases called caspases.⁶ It has been proposed that p53-dependent apoptosis may involve the transcriptional induction of redox-related genes with the formation of reactive oxygen species, leading to cell death by oxidative stress.⁷ Although DNA damage is thought to be important in triggering the apoptotic response,^{8, 9} some studies suggest a role for membrane damage and signaling pathways outside the nucleus that involve tyrosine kinases, especially ceramide.¹⁰ In any case, apoptosis may serve as a protective mechanism for the elimination of heavily damaged and thus potentially mutated cells from an irradiated population.

Another potential mechanism for removing heavily damaged cells from an irradiated population is the induction of an irreversible G₁/S arrest.¹¹ This is observed, for example, following irradiation of human diploid fibroblasts which are very resistant to apoptosis. The fraction of cells irreversibly blocked in G₁/S is reduced if the cells are allowed to repair potentially lethal damage prior to assaying for survival.¹¹ This arrest is p53 and ATM dependent^12–14; no arrest occurs following irradiation of ATM homozygotic cells,¹¹ nor of p53^-/- cells from patients with the Li Fraumeni syndrome.¹² The activation of the p53/p21 pathway by radiation damage in some human tumor cells also suppresses the progression of G₁ cells into the DNA synthetic (S) phase of the cell cycle, as well as enhancing apoptotic cell death.^{15, 16} Interestingly, overexpression of the C-MYC oncogene can attenuate this effect in irradiated cells.¹⁷ It has been hypothesized that the absence of a G₁ arrest is responsible for the genetic instability that occurs in irradiated cells lacking normal p53 function; cells with extensive genetic damage will progress through the cell cycle and continue proliferation rather than becoming arrested in G₁ and undergoing apoptotic cell death or becoming senescent. However, the G₁ arrest is only one aspect of a complex cellular response to DNA damage.

The second mechanism for cell killing is radiation-induced reproductive failure. Radiation in sufficient doses can inhibit mitosis, that is, the cell's ability to divide and proliferate indefinitely. The inhibition of cellular proliferation is the mechanism by which radiation kills most mammalian cells. The nature and kinetics of the cytotoxic effects of radiation have been reviewed elsewhere,^{2, 12} and they are discussed in Chapter 39, particularly as they relate to tumor cells and radiation oncology. As radiation kills cells by inhibiting their ability to divide, its effects in human beings occur primarily in tissues with high cell turnover or renewal rates characterized by a large amount of proliferative activity. These include tissues such as the bone marrow and the mucosal lining of the stomach and small intestine. Symptoms of acute exposure to whole-body irradiation in human beings are usually observed only following doses of 150 cGy or greater, whereas significant cell killing in vitro can be detected with doses as low as 10 cGy.

Another important somatic effect related to cell killing arises from irradiation of the developing embryo and fetus.^{18, 19} Whereas irradiation of experimental animals with doses in the order of 200 to 400 cGy during the first trimester of pregnancy has led to a variety of congenital anomalies in the offspring, no such effects were found in large populations of mice exposed to doses below 25 cGy.¹⁸ Moreover, no increase in the frequency of congenital anomalies has been observed in human beings, even following relatively high radiation doses.

Recent epidemiologic studies on the atom bomb survivors of Hiroshima and Nagasaki have focused on mental retardation and other measures of intelligence such as test scores and school performance.¹⁹ These are presumably more sensitive indicators of radiation effects owing to cell depletion amongst the neuroblasts during development. Neuroblasts comprise by far the largest population of cells in the early fetus and continue proliferating until the fifth or sixth month of pregnancy. The number of children with such disorders in the atom bomb survivor study is small, and the mean values for all end points are not significantly different from those in controls for the dose groups below 50 to 100 cGy. The Committee on the Biologic Effects of Ionizing Radiations of the National Research Council (BEIR V Committee)²⁰ concluded that for mental retardation, the best documented of the developmental abnormalities, the prevalence appeared to increase with dose in a linear manner for individuals irradiated between 8 and 15 weeks, the most sensitive time period after conception. However, the data do not exclude a threshold in the range of 20 to 40 cGy and, indeed, best fit a threshold dose-response relationship with a lower bound of 12 to 20 cGy.²⁰ On the assumption of a linear, nonthreshold relationship, however, the magnitude of the risk would be approximately a 4% chance of occurrence per 10 cGy for exposure at 8 to 15 weeks of gestational age, with less risk occurring for exposure at other ages.

Mutagenesis

The mutagenic effects of ionizing radiation were first described by Herman Muller, in 1927, in his classic experiments with the fruit fly Drosophila. Subsequent experiments showed the dose-response relationship for such mutations to be a linear function of exposure over a wide range of radiation doses from as low as 10 to as high as 1,000 cGy. Studies of the induction of single-gene mutations in human cells have been limited to several genetic loci. The results of most of these studies also suggest that the induction of mutations in human cells is a linear function of dose with doses as low as 10 cGy, and perhaps as low as 1 cGy, and that the dose-rate effect appears to be relatively small.^{21, 22} DNA structural analyses show that the majority of radiation-induced mutations in human cells result from large-scale genetic events involving loss of the entire active gene and often extending to other loci on the same chromosome.²³

The major potential consequence of radiation-induced mutations in human populations is heritable genetic effects resulting from mutations induced in germinal cells. Such effects have been examined in several different animal systems.^{20, 24} For high dose-rate exposure, the induced mutation rate per gamete generally falls in the range of 10^-4 to 10^-5 per cGy. The rates per locus are in the range of 10^-7 to 10^-8 per cGy. Protraction of exposure appears to decrease the mutation rate in rodent systems by a factor of 2 or greater. When all of the experimental data for the various genetic end points are considered, the genetic doubling dose (radiation dose necessary to double the spontaneous mutation rate) for low dose-rate exposure appears to be in the range of 100 cGy. Although significant heritable genetic effects of radiation have not yet been demonstrated in human populations, a doubling dose of 100 cGy is not inconsistent with the absence of a statistically significant increase in hereditary disease among the children of atom bomb survivors.²⁵ Indeed, 100 cGy represents approximately the lower 95% confidence interval (CI) for the human doubling dose calculated from the atom bomb survivor data.²⁰

Chromosomal Aberrations

Radiation can induce two types of chromosomal aberrations in mammalian cells. The first have been termed “unstable” aberrations in that they are usually lethal to dividing cells. They include such changes as dicentrics, ring chromosomes, large deletions, and fragments. These types of aberrations do not allow the equal distribution of genetic material into daughter cells; in many cases, the frequency of such aberrations correlates well with the cytotoxic effects of radiation.

The second type has been termed “stable” aberrations. These include changes such as small deletions, reciprocal translocations, and aneuploidy—changes that do not preclude the cell from dividing and proliferating. Figure 19-2 shows a karyotype of a human cell showing a stable aberration. Radiation-induced reciprocal translocations such as have occurred in this cell may be passed on through many generations of cell replication and emerge in clonal cell populations.^{26, 27}

Figure 19-2

Karyotype of normal human diploid fibroblast showing stable chromosomal rearrangement (1:16 translocation) induced by radiation. The irradiated cells were serially subcultivated for 3 months (approximately 20 cell generations) before this cell was analyzed. (more...)

It is well known that such deletions and translocations can result in gene mutations. It is tempting to speculate that they may play a more fundamental role in the process of radiation carcinogenesis. Typically, cancer cells are aneuploid and contain multiple stable chromosomal aberrations. In a number of cases, specific chromosomal abnormalities are associated with specific tumor types. In some instances, such as the chromosome 8:14 translocation in Burkitt lymphoma, the chromosomal change results in the activation of a specific oncogene. In others, such as the chromosome 13q14 deletion found in retinoblastoma (RB), tumor development has been ascribed to loss or inactivation of the RB tumor-suppressor gene. Although radiation-induced cancers show multiple unbalanced chromosomal rearrangements, few show such specific translocations as would be associated with the activation of specific oncogenes or known tumor-suppressor genes.²⁸

Neoplastic Transformation In Vitro

An important cellular effect of radiation is neoplastic transformation, or the conversion of a normal cell to one with the phenotype of a cancer cell, including the ability to form an invasive, malignant tumor upon re-injection into syngeneic hosts. Most human cancers have been shown to be clonal in origin. That is, all of the cells within a tumor are descendants of a single cell that has undergone the process of neoplastic transformation. The transformation of one or more normal cells in a tissue in vivo is presumed to represent the earliest step in the overall process of carcinogenesis. Whether or not such a transformed cell can successfully give rise to an invasive, malignant tumor depends upon a number of tissue and systemic factors. Although a number of different in vitro transformation systems involving various species and cell types are under investigation, those that generate reliable quantitative data have been restricted to rodent cells, and in none of these is the entire process of malignant transformation measured.^{29, 30} Rather, surrogate features of transformation are assayed such as changes in colony morphology, focus formation, or growth under anchorage-independent conditions.

Studies of cellular and animal models for radiation carcinogenesis indicate that it is a progressive, multistep process by which normal cells acquire the various phenotypic characteristics of cancer cells.³¹ There appear to be three major independent stages in the malignant transformation of cells in vitro: the development of morphologic changes; cellular immortality; and tumorigenicity.²⁹ Morphologic changes are many and varied, including the development of abnormalities in cytology, growth pattern and the control of cell proliferation. Immortalization occurs frequently in rodent cells but extremely rarely in human cells, either spontaneously or as a result of treatment with radiation or chemical carcinogens. It can be induced, however, by transfection of human diploid cells with certain oncogenes and/or genes associated with tumor viruses such as the SV40 T antigen or the E6/E7 genes of human papillomavirus 16, and has been associated with the production of telomerase. Immortalization may thus be an important rate-limiting step in human cell transformation and perhaps in human carcinogenesis in vivo.²⁹ Tumorigenicity also appears to be an independent phenotype that generally occurs only in previously immortalized cells. A subpopulation of such immortal cells may undergo additional genomic rearrangements that give them a selective growth advantage in vivo perhaps related to factors present in the host animal.³²

As compared with many chemical agents, ionizing radiation is not a potent inducer of transformation. Polycyclic hydrocarbons, for example, can induce much higher frequencies of transformation at doses which produce very little cell killing. Incubation of cells with various agents during the 4- to 6-week postirradiation expression period for transformation, however, can markedly modify the ultimate yield of transformed cells.³³ For example, the phorbol ester compound 12-0-tetradecanoyl-phorbol-13-acetate (TPA) acts as a potent promoter of x-ray transformation, if applied repeatedly beginning either immediately after irradiation or several weeks later. Indeed, these in vitro findings offered the first evidence that the phenomenon of tumor promotion was a general one and not simply limited to mouse skin. A number of different classes of agents applied by a similar experimental protocol can suppress transformation.³⁴ These classes include selenium, retinoids, carotenoids, and ascorbic acid, as well as certain protease inhibitors that have shown promise as chemopreventive agents in vivo.^{35, 36} Transformation can also be modulated by hormones, growth factors, and antiinflammatory agents.³⁴

It has thus become evident that a number of noncarcinogenic secondary factors can markedly modulate the frequency of radiation-induced transformation. As transformation can be markedly enhanced, suppressed, or completely inhibited by such factors, they may become the controlling ones in the overall process of transformation of cells exposed to radiation. In many cases, the effects of such agents in vitro have been predictive of those observed in experimental animal systems. It therefore seems likely that secondary factors may be of importance in human radiation carcinogenesis, although there are few epidemiologic data to support this contention.

Radiation-Induced Genomic Instability

This term refers to a phenomenon observed in a number of different cellular systems whereby radiation exposure appears to induce a type of transmissible genetic 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. The occurrence of such a process could enhance the probability that a single cell lineage would acquire the multiple sequential and interacting gene mutations necessary to convert a normal cell to a full malignant cell. It would also imply that radiation could act at any point in this carcinogenic process. This phenomenon has been termed a nontargeted effect of radiation, as genetic damage occurs in cells that in themselves received no direct radiation exposure. The end points studied include malignant transformation, specific gene mutations, and chromosomal aberrations. Typically, this phenomenon is studied by examining the occurrence of such genetic effects in clonal populations derived from single cells surviving radiation exposure.³⁶ Figure 19-3 shows this schematically.

Figure 19-3

Schematic of radiation-induced mutagenesis. Open circles represent normal wild-type cells, while closed circles represent mutated cells. B is an example of a cell directly mutated by radiation exposure; the mutation is transmitted to all of its progeny. (more...)

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.^{37, 38} 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% to 30% of the irradiated cell population; this instability enhanced the probability of the occurrence of a second, neoplastic transforming event. This finding is in contradistinction to the 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 the frequency of approximately 10^-6, and involved in the actual transformation of one or more of the progeny of the original irradiated cells after many rounds of cell division. This second transforming event occurred with the constant frequency per cell per generation, and had the characteristics of a mutagenic event.³⁹ 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 was subsequently demonstrated in a number of experiment systems for various genetic end points.^40–43 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 nonirradiated cells.^{44, 45} This increased mutation rate persisted for approximately 30 to 50 generations postirradiation. The molecular structural spectrum of these late-arising mutants resembles those of spontaneous mutations in that the majority of them are point mutations,^{45, 46} whereas direct x-ray-induced mutations involve primarily deletions. An enhancement of both minisatellite⁴⁷ and microsatellite⁴⁸ instability has also been observed in the progeny of irradiated cells selected for mutations at the thymidine kinase locus.

An enhanced frequency of nonclonal chromosomal aberrations was first reported in clonal descendants of mouse hematopoietic stem cells 12 to 14 generations after exposure to alpha radiation.⁴⁹ Persistent radiation-induced chromosomal instability has since been shown to occur in a number of other cellular systems.^50–53 Transmission of such chromosomal instability has also been shown to occur in vivo,^{54, 55} but susceptibility to radiation-induced chromosomal instability differed significantly among different strains of mice.^{56, 57} Finally, a persistent increase in the rate of cell death has been shown to occur in cell populations many generations after radiation exposure.^58–60 Delayed reproductive failure has been linked to chromosomal instability⁶¹ and malignant transformation,^{62, 63} and evidence presented to suggest that DNA is at least one of the critical targets in the initiation of this phenomenon.⁶⁴ It has been proposed that oxidative stress perhaps consequent to enhanced, p53-independent apoptosis may contribute to the perpetuation of the instability phenotype in these populations.^{61, 63, 65}

Of importance in terms of radiation carcinogenesis are the emerging observations indicating that this phenomenon occurs in vivo and may be related to the induction of cancer. The transmission of radiation-induced chromosomal instability in vivo has been demonstrated in several distinct experimental models,^{54, 55, 66} and evidence presented to suggest that instability induced in X-irradiated mouse hematopoietic stem cells may be related to the occurrence of the nonspecific genetic damage found in radiation-induced leukemias in these mice.⁶⁷ Sensitivity to mammary tumor induction was found not only to be strain specific but to correlate with the strain specificity for the induction of chromosomal instability in mammary epithelial cells irradiated in vivo.⁶⁶ These were related to reduced expression of the DNA repair enzymes DNA-PKcs⁶⁸ and a high frequency of telomere fusions.⁶⁹ This mouse model thus relates radiation-induced genomic instability to a defect in DNA repair and associates it with an enhanced susceptibility to radiation-induced cancer.

It is thus well established that radiation can induce a type of instability in cells that enhances the probability of the occurrence of multiple genetic events in surviving cell populations, sometimes after many generations of replication. Thus, rather than inducing an “initiating” mutation, radiation may play a more general role in the process of carcinogenesis. If this is the case, the initiating event would not be directly related to the tumor itself, but one that enhances the probability that the required mutations would arise in a given cell lineage. Radiation could thus act at any state in tumor development. However, the precise mechanisms for this phenomenon including how it is initiated and maintained remain to be elucidated. Various tightly regulated cellular processes may be disrupted by radiation, leading to a chaotic state that perturbs the normal regulatory and signaling pathways, thus disrupting cellular homeostasis, a state from which the cell never completely recovers.⁶⁴ It is tempting to speculate that the various factors known to modulate malignant transformation in vitro may act on this process. Interestingly, this concept is consistent with the emerging findings in human populations which suggest that some types of radiation-induced cancer may follow a relative risk model (see below); that is, a given dose of radiation increases the rate of occurrence of cancer at all follow-up times rather than inducing a specific cohort of new tumors.

Bystander Effects in Irradiated Cell Populations

It has long been thought that the cell nucleus is the target for the important biologic effects of radiation; these effects occur in the irradiated cell as a direct result of DNA damage that has not been correctly restored by enzymatic repair processes. Such a direct mutational event in a critical gene has been hypothesized to represent the first step in radiation carcinogenesis. However, recent evidence shows that targeted cytoplasmic radiation is significantly mutagenic.⁷⁰ Moreover, evidence is accumulating that damage signals may be transmitted from irradiated to nonirradiated cells in the population, leading to the occurrence of biologic effects in cells that received no direct radiation exposure.⁷¹ This phenomenon has been termed the “bystander” effect of radiation; it could be of considerable importance in the carcinogenic effects of very-low doses of densely ionizing radiation such as alpha particles released by radon. Only a small fraction of a person's bronchial epithelial cells, the presumed target for lung cancer, will actually be hit by an alpha particle from residential radon exposure during an exposed person's lifetime.

The experimental model used to study this effect has generally involved the exposure of monolayer cultures of cells to very-low fluences of alpha particles, fluences whereby a very small fraction of the cell population will actually be hit by a particle. In the initial study, an enhanced frequency of sister chromatid exchanges (SCE) was observed in 20% to 40% of cells exposed to fluences by which only 1/1,000 to 1/100 cells were traversed by an alpha particle.⁷² This finding was later confirmed,⁷³ and evidence presented that it involves the secretion of cytokines or other factors by irradiated cells that leads to an upregulation of oxidative metabolism in bystander cells.^{74, 75} There is also evidence that incubation with conditioned medium from irradiated cells has cytotoxic effects on nonirradiated cells, which may be related to the release of a factor(s) into the medium, including reactive oxygen species.^{76, 77} These findings are reminiscent of the reports that clastogenic activity can be isolated from the plasma of radiation-exposed people.⁷⁸

Of particular note is the observation that an enhanced frequency of specific gene mutations occurs in bystander cells in populations exposed to very-low fluences of alpha particles.^{79, 80} As a result, the induced mutation frequency per alpha particle track increases at low fluences where bystander as well as directly irradiated cells are at risk for the induction of mutations. This leads to hyperlinearity of the dose-response curve in the low-dose region.

Changes in gene expression also occur in bystander cells in monolayer cultures; the expression levels of p53, p21^Waf1, CDC2, cyclin-B1, and rad51 were significantly modulated in nonirradiated cells in confluent human diploid cell populations exposed to very-low fluences of alpha particles.⁷¹ As seen in Figure 19-4, clusters of cells showed enhanced expression of p21^Waf1 as determined by in situ immunofluorescence staining techniques, although only approximately 1% to 2% of the cell nuclei were actually traversed by an alpha particle. This phenomenon involved cell-to-cell communication via gap junctions.⁸¹ Evidence that the upregulation of the p53 signaling pathway in bystander cells is a consequence of DNA damage is supported by the observation that p53 was phosphorylated on serine 15.⁸¹ Interestingly, however, DNA damage in bystander cells appears to differ from that occurring in directly irradiated cells; whereas the mutations induced in directly irradiated cells were primarily partial and total gene deletions, more than 90% of those arising in bystander cells were point mutations.⁸² This would be consistent with the evidence that oxidative metabolism is upregulated in bystander cells,^{74, 83} and has led to the hypothesis that the point mutations are a result of oxidative base damage occurring in bystander cells.⁸² The activation of MAP K proteins and their downstream effectors in bystander cells⁸³ is of particular interest in terms of the preliminary evidence that membrane signaling is involved in the bystander effect in monolayer cultures.⁸⁴

Figure 19-4

Bystander effect of radiation. The expression of p21^Waf1 was determined in individual cells in monolayer cultures of human diploid fibroblasts by in situ immunofluorescence. In cultures exposed to 0.3 cGy (< 1 to 2% of nuclei traversed by an alpha (more...)

In sum, the results of these studies of bystander effects indicate clearly that damage signals can be transmitted from irradiated to nonirradiated cells. In confluent monolayer cultures, this phenomenon involves gap junction mediated cell-to-cell communication, and appears to involve both the induction of reactive oxygen species and the activation of extranuclear signal transduction pathways. Some evidence suggests that regulation of the p53 damage-response pathway may be central to this phenomenon. These findings could potentially be of considerable significance in terms of residential radon exposure where the mutagenic and carcinogenic effect could be greater than that predicted on the basis only of those bronchial epithelial cells actually traversed by an alpha particle.