Interleukin 8 exhibits a pro-mitogenic and pro-survival role in radiation induced genomically unstable cells

Radiation induced genomic instability can be perpetuated over time by the transmission of soluble factors. This can occur via cell-to-cell gap junction communication or the secretion/shedding of soluble factors. We have investigated whether our radiation induced chromosomally unstable GM10115 human–hamster hybrid clones secrete factors that can perpetuate the instability phenotype over time. These clones do not have functional gap junctions, but do secrete significant amounts of Interleukin 8 (IL-8) into the culture medium. We then determined whether IL-8 could initiate and or perpetuate genomic instability over time in parental GM10115 cells. Contrary to our hypothesis, IL-8 could induce DNA damage, but was not responsible for the unstable phenotype. Instead it appears that IL-8 secretion provides a pro-survival function in cells that are chromosomally unstable and generally fail to thrive.     

Bystander effects in radiation-induced genomic instability

Exposure of GM10115 hamster-human hybrid cells to X-rays can result in the induction of chromosomal instability in the progeny of surviving cells. This instability manifests as the dynamic production of novel sub-populations of cells with unique cytogenetic rearrangements involving the "marker" human chromosome. We have used the comet assay to investigate whether there was an elevated level of endogenous DNA breaks in chromosomally unstable clones that could provide a source for the chromosomal rearrangements and thus account for the persistent instability observed. Our results indicate no significant difference in comet tail measurement between non-irradiated and radiation-induced chromosomally unstable clones. Using two-color fluorescence in situ hybridization we also investigated whether recombinational events involving the interstitial telomere repeat-like sequences in GM10115 cells were involved at frequencies higher than random processes would otherwise predict. Nine of 11 clones demonstrated a significantly higher than expected involvement of these interstitial telomere repeat-like sequences at the recombination junction between the human and hamster chromosomes. Since elevated levels of endogenous breaks were not detected in unstable clones we propose that epigenetic or bystander effects (BSEs) lead to the activation of recombinational pathways that perpetuate the unstable phenotype. Specifically, we expand upon the hypothesis that radiation induces conditions and/or factors that stimulate the production of reactive oxygen species (ROS). These reactive intermediates then contribute to a chronic pro-oxidant environment that cycles over multiple generations, promoting chromosomal recombination and other phenotypes associated with genomic instability.     

Radiation-induced chromosomal instability and gene expression profiling: searching for clues to initiation and perpetuation

Radiation-induced genomic instability (RIGI) manifests in the progeny of cells surviving ionizing radiation (IR), and can be measured using such endpoints as delayed mutation, micronuclei formation, and chromosomal instability. The frequency of RIGI is relatively high, exceeding the gene mutation rate of IR by orders of magnitude, leading to conjecture that a gene mutation is not the cause of the phenotype. We have started to explore whether differential gene expression patterns are associated with the instability phenotype, in order to shed light on its initiation and perpetuation. Using GM10115 human-hamster hybrid-derived chromosomally stable and radiation-induced unstable clones, gene expression patterns were analyzed using microarray analysis. Two methods were used to find differentially expressed genes, and all candidate genes identified by these methods were under-expressed relative to the chromosomally stable reference sample. Among this set differentially expressed genes identified were two candidates with a relationship to the ubiquitin/proteasome pathway. While follow-up gene expression analyses have confirmed the under-expression of these two genes in some of our chromosomally unstable clones, preliminary functional studies have been unable to demonstrate a link to instability. It is anticipated that as we apply this technology to the study of radiation-induced genomic instability, clues to its onset will be revealed, ultimately contributing to a greater understanding of the mechanisms of radiation carcinogenesis.     

Variation in apoptosis profiles in radiation-induced genomically unstable cell lines

Delayed reproductive cell death or lethal mutations in the survivors of irradiated cells is a well-characterized end point associated with radiation-induced genomic instability. Although the mechanism for this delayed lethality has not been identified, it is thought to be a means of eliminating cells that have sustained extensive damage, thus preventing tissue disruption after radiation exposure. In this study we have tested the hypothesis that delayed reproductive cell death in chromosomally unstable GM10115 clones is due to persistently increased levels of apoptosis. Evidence for differences in apoptosis in two representative genomically unstable clones after irradiation is presented. In addition, one of the unstable clones was found to have abnormal levels of apoptosis after radiation exposure. An understanding of apoptosis in genomically unstable clones may provide insight into the maintenance of genomic instability and the mechanism by which genomically unstable cells evade cell death, potentially contributing to carcinogenesis.     

Ionizing radiation induces delayed hyperrecombination in Mammalian cells

Exposure to ionizing radiation can result in delayed effects that can be detected in the progeny of an irradiated cell multiple generations after the initial exposure. These effects are described under the rubric of radiation-induced genomic instability and encompass multiple genotoxic endpoints. We have developed a green fluorescence protein (GFP)-based assay and demonstrated that ionizing radiation induces genomic instability in human RKO-derived cells and in human hamster hybrid GM10115 cells, manifested as increased homologous recombination (HR). Up to 10% of cells cultured after irradiation produce mixed GFP(+/-) colonies indicative of delayed HR or, in the case of RKO-derived cells, mutation and deletion. Consistent with prior studies, delayed chromosomal instability correlated with delayed reproductive cell death. In contrast, cells displaying delayed HR showed no evidence of delayed reproductive cell death, and there was no correlation between delayed chromosomal instability and delayed HR, indicating that these forms of genome instability arise by distinct mechanisms. Because delayed hyperrecombination can be induced at doses of ionizing radiation that are not associated with significantly reduced cell viability, these data may have important implications for assessment of radiation risk and understanding the mechanisms of radiation carcinogenesis.     

Intrachromosomal changes and genomic instability in site-specific microbeam-irradiated and bystander human-hamster hybrid cells

Exposure to ionizing radiation may induce a heritable genomic instability phenotype that results in a persisting and enhanced genetic and functional change among the progeny of irradiated cells. Since radiation-induced bystander effects have been demonstrated with a variety of biological end points under both in vitro and in vivo conditions, this raises the question whether cytoplasmic irradiation or the radiation-induced bystander effect can also lead to delayed genomic instability. In the present study, we used the Radiological Research Accelerator Facility charged-particle microbeam for precise nuclear or cytoplasmic irradiation. The progeny of irradiated and the bystander human hamster hybrid (A(L)) cells were analyzed using multicolor banding (mBAND) to examine persistent chromosomal changes. Our results showed that the numbers of metaphase cells involving changes of human chromosome 11 (including rearrangement, deletion and duplication) were significantly higher than that of the control in the progeny of both nuclear and cytoplasmic targeted cells. These chromosomal changes could also be detected among the progeny of bystander cells. mBAND analyses of clonal isolates from nuclear and cytoplasm irradiations as well as the bystander cell group showed that chromosomal unstable clones were generated. Analyses of clonal stability after long-term culture indicated no significant change in the number of unstable clones for the duration of culture in each irradiated group. These results suggest that genomic instability that is manifested after ionizing radiation exposure is not dependent on direct damage to the cell nucleus.     

Transmission of genomic instability from a single irradiated human chromosome to the progeny of unirradiated cells

Ionizing radiation can induce chromosome instability that is transmitted over many generations after irradiation in the progeny of surviving cells, but it remains unclear why this instability can be transmitted to the progeny. To acquire knowledge about the transmissible nature of genomic instability, we transferred an irradiated human chromosome into unirradiated mouse recipient cells by microcell fusion and examined the stability of the transferred human chromosome in the microcell hybrids. The transferred chromosome was stable in all six microcell hybrids in which an unirradiated human chromosome had been introduced. In contrast, the transferred chromosome was unstable in four out of five microcell hybrids in which an irradiated human chromosome had been introduced. The aberrations included changes in the irradiated chromosome itself and rearrangements with recipient mouse chromosomes. Thus the present study demonstrates that genomic instability can be transmitted to the progeny of unirradiated cells by a chromosome exposed to ionizing radiation, implying that the instability is caused by the irradiated chromosome itself and also that the instability is induced by the nontargeted effect of radiation.     

Lack of consensus gene expression changes associated with radiation-induced chromosomal instability

The relatively high frequency with which ionizing radiation induces genomic instability suggests that a gene mutation occurring after irradiation is an unlikely cause of the phenotype. To search for mechanism(s) of initiation and perpetuation of this instability phenotype, gene expression profiles of clones exhibiting delayed chromosomal instability were analyzed. Microarray analysis using two pools of isogenic radiation-induced chromosomally unstable clones compared to an irradiated but chromosomally stable clone uncovered a set of 68 differentially expressed genes using two methods of analysis. Unexpectedly, all 68 genes were under-expressed relative to the chromosomally stable reference clone. Further analysis of the candidates placed the differentially expressed genes into pathways implicating differential MAP kinase signaling, ubiquitin/proteasome function, DNA repair, cell cycle control, lipid signaling, nucleotide metabolism, and other potentially disrupted pathways. Validation studies using northern and western blotting, and functional assays concluded that although differences in some of these pathways exist, no single gene or molecular pathway was found to be differentially regulated in all of the chromosomally unstable clones tested. Inferred from these data is that there are multiple potential molecular pathways and/or events that maintain the unstable phenotype, and no single expression pattern is linked to instability in the unstable clones analyzed.     

Genetic and Epigenetic Changes in Chromosomally Stable and Unstable Progeny of Irradiated Cells

Radiation induced genomic instability is a well-studied phenomenon, the underlying mechanisms of which are poorly understood. Persistent oxidative stress, mitochondrial dysfunction, elevated cytokine levels and epigenetic changes are among the mechanisms invoked in the perpetuation of the phenotype. To determine whether epigenetic aberrations affect genomic instability we measured DNA methylation, mRNA and microRNA (miR) levels in well characterized chromosomally stable and unstable clonally expanded single cell survivors of irradiation. While no changes in DNA methylation were observed for the gene promoters evaluated, increased LINE-1 methylation was observed for two unstable clones (LS12 and CS9) and decreased Alu element methylation was observed for the other two unstable clones (115 and Fe5.0–8). These relationships also manifested for mRNA and miR expression. mRNA identified for the LS12 and CS9 clones were most similar to each other (261 mRNA), while the 115 and Fe5.0–8 clones were more similar to each other, and surprisingly also similar to the two stable clones, 114 and 118 (286 mRNA among these four clones). Pathway analysis showed enrichment for pathways involved in mitochondrial function and cellular redox, themes routinely invoked in genomic instability. The commonalities between the two subgroups of clones were also observed for miR. The number of miR for which anti-correlated mRNA were identified suggests that these miR exert functional effects in each clone. The results demonstrate significant genetic and epigenetic changes in unstable cells, but similar changes are almost as equally common in chromosomally stable cells. Possible conclusions might be that the chromosomally stable clones have some other form of instability, or that some of the observed changes represent a sort of radiation signature and that other changes are related to genomic instability. Irrespective, these findings again suggest that a spectrum of changes both drive genomic instability and permit unstable cells to persist and proliferate.     

Quantitative proteomic analysis of mitochondrial proteins reveals prosurvival mechanisms in the perpetuation of radiation-induced genomic instability

Radiation-induced genomic instability is a well-studied phenomenon that is measured as mitotically heritable genetic alterations observed in the progeny of an irradiated cell. The mechanisms that perpetuate this instability are unclear; however, a role for chronic oxidative stress has consistently been demonstrated. In the chromosomally unstable LS12 cell line, oxidative stress and genomic instability were correlated with mitochondrial dysfunction. To clarify this mitochondrial dysfunction and gain insight into the mechanisms underlying radiation-induced genomic instability we have evaluated the mitochondrial subproteome and performed quantitative mass spectrometry analysis of LS12 cells. Of 98 quantified mitochondrial proteins, 17 met criteria for fold changes and reproducibility; and 11 were statistically significant in comparison with the stable parental GM10115 cell line. Previous observations implicated defects in the electron transport chain (ETC) in the LS12 cell mitochondrial dysfunction. Proteomic analysis supports these observations, demonstrating significantly reduced levels of mitochondrial cytochrome c, the intermediary between complexes III and IV of the ETC. Results also suggest that LS12 cells compensate for ETC dysfunction and oxidative stress through increased levels of tricarboxylic acid cycle enzymes and upregulation of proteins that protect against oxidative stress and apoptosis. More than one cellular defect is likely to contribute to the genomic instability phenotype, and evaluation of gene and microRNA expression suggests that epigenetics play a role in the phenotype. These data suggest that LS12 cells have adapted mechanisms that allow survival under suboptimal conditions of oxidative stress and compromised mitochondrial function to perpetuate genomic instability.     

Non-targeted and delayed effects of exposure to ionizing radiation: II. Radiation-induced genomic instability and bystander effects in vivo,clastogenic factors and transgenerational effects

The goal of this review is to summarize the evidence for non-targeted and delayed effects of exposure to ionizing radiation in vivo. Currently, human health risks associated with radiation exposures are based primarily on the assumption that the detrimental effects of radiation occur in irradiated cells. Over the years a number of non-targeted effects of radiation exposure in vivo have been described that challenge this concept. These include radiation-induced genomic instability, bystander effects, clastogenic factors produced in plasma from irradiated individuals that can cause chromosomal damage when cultured with nonirradiated cells, and transgenerational effects of parental irradiation that can manifest in the progeny. These effects pose new challenges to evaluating the risk(s) associated with radiation exposure and understanding radiation-induced carcinogenesis.     

Untargeted effects of ionizing radiation: implications for radiation pathology

The dogma that genetic alterations are restricted to directly irradiated cells has been challenged by observations in which effects of ionizing radiation, characteristically associated with the consequences of energy deposition in the cell nucleus, arise in non-irradiated cells. These, so called, untargeted effects are demonstrated in cells that have received damaging signals produced by irradiated cells (radiation-induced bystander effects) or that are the descendants of irradiated cells (radiation-induced genomic instability). Radiation-induced genomic instability is characterized by a number of delayed adverse responses including chromosomal abnormalities, gene mutations and cell death. Similar effects, as well as responses that may be regarded as protective, have been attributed to bystander mechanisms. Whilst the majority of studies to date have used in vitro systems, some adverse non-targeted effects have been demonstrated in vivo. However, at least for haemopoietic tissues, radiation-induced genomic instability in vivo may not necessarily be a reflection of genomically unstable cells. Rather the damage may reflect responses to ongoing production of damaging signals; i.e. bystander responses, but not in the sense used to describe the rapidly induced effects resulting from direct interaction of irradiated and non-irradiated cells. The findings are consistent with a delayed and long-lived tissue reaction to radiation injury characteristic of an inflammatory response with the potential for persisting bystander-mediated damage. An important implication of the findings is that contrary to conventional radiobiological dogma and interpretation of epidemiologically-based risk estimates, ionizing radiation may contribute to malignancy and particularly childhood leukaemia by promoting initiated cells rather than being the initiating agent. Untargeted mechanisms may also contribute to other pathological consequences.     

Radiation-induced genomic instability: a role for secreted soluble factors in communicating the radiation response to non-irradiated cells

Radiation induced genomic instability can be described as the increased rate of genomic alterations occurring in the progeny of an irradiated cell. Its manifestations are the dynamic ongoing production of chromosomal rearrangements, mutations, gene amplifications, transformation, microsatellite instability, and/or cell killing. In this prospectus, we present the hypothesis that cellular exposure to ionizing radiation can result in the secretion of soluble factors by irradiated cells and/or their progeny, and that these factors can elicit responses in other cells thereby initiating and perpetuating ongoing genomic instability.     

Karyotypic instability and centrosome aberrations in the progeny of finite life-span human mammary epithelial cells exposed to sparsely or densely ionizing radiation

The human breast is sensitive to radiation carcinogenesis, and genomic instability occurs early in breast cancer development. This study tests the hypothesis that ionizing radiation elicits genomic instability in finite life-span human mammary epithelial cells (HMEC) and asks whether densely ionizing radiation is a more potent inducer of instability. HMEC in a non-proliferative state were exposed to X rays or 1 GeV/nucleon iron ions followed by delayed plating. Karyotypic instability and centrosome aberrations were monitored in expanded clonal isolates. Severe karyotypic instability was common in the progeny of cells that survived X-ray or iron-ion exposure. There was a lower dose threshold for severe karyotypic instability after iron-ion exposure. More than 90% of X-irradiated colonies and >60% of iron-ion-irradiated colonies showed supernumerary centrosomes at levels above the 95% upper confidence limit of the mean for unirradiated clones. A dose response was observed for centrosome aberrations for each radiation type. There was a statistically significant association between the incidence of karyotypic instability and supernumerary centrosomes for iron-ion-exposed colonies and a weaker association for X-irradiated colonies. Thus genomic instability occurs frequently in finite life-span HMEC exposed to sparsely or densely ionizing radiation and may contribute to radiation-induced breast cancer.     

A single low dose of X-rays induces high frequencies of genetic instability (aneuploidy) and heritable damage (apoptosis), dependent on cell type and p53 status

We harvested and analyzed cells from four different non-transformed cell lines surviving a single X-ray exposure. Evidence of radiation-induced karyotype instability was observed in 100% of C3H 10T1/2 fibroblast clones and 11.3% of V79 fibroblast clones. Heritable damage: predisposition to apoptosis, but not karyotype instability, was induced in TK6 (p53(wt/wt)) and WTK1 (p53(mut/mut)) human B-lymphoblastoid cell clones. The studies indicate: (1) genetic instability and/or heritable damage are induced in cells exposed to radiation at a high frequency, and induction of genetic instability is not limited to morphologically transformed cells [Radiat. Res. 138 (1994) S105; Radiat. Environ. Biophys. 36 (1998) 255]; (2) sensitivity to genetic instability and heritable damage depend on cell type; (3) checkpoint stringency and p53 status significantly influence the frequency of radiation-induced genetic instability and heritable damage; (4) in some cell lines, damage induced by low doses of radiation (below 2 Gy) leads to heritable cytotoxic and genotoxic effects in 100% of cells exposed. The data suggest that mammalian cells misinterpret damage induced by ionizing radiation as if it were a physiological cell signal. This contrasts strongly with the response of mammalian cells to damage induced by other types of DNA-toxic agents where damage-specific repair mechanisms are activated.     

Genomic instability in human lymphocytes irradiated with individual charged particles: involvement of tumor necrosis factor alpha in irradiated cells but not bystander cells

Exposure to ionizing radiation can increase the risk of cancer, which is often characterized by genomic instability. In environmental exposures to high-LET radiation (e.g. 222Ra), it is unlikely that many cells will be traversed or that any cell will be traversed by more than one alpha particle, resulting in an in vivo bystander situation, potentially involving inflammation. Here primary human lymphocytes were irradiated with precise numbers of 3He2+ ions delivered to defined cell population fractions, to as low as a single cell being traversed, resembling in vivo conditions. Also, we assessed the contribution to genomic instability of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFA). Genomic instability was significantly elevated in irradiated groups (> or = two-fold over controls) and was comparable whether cells were traversed by one or two 3He2+ ions. Interestingly, substantial heterogeneity in genomic instability between experiments was observed when only one cell was traversed. Genomic instability was significantly reduced (60%) in cultures in which all cells were irradiated in the presence of TNFA antibody, but not when fractions were irradiated under the same conditions, suggesting that TNFA may have a role in the initiation of genomic instability in irradiated cells but not bystander cells. These results have implications for low-dose exposure risks and cancer.     

Bystander-mediated genomic instability after high LET radiation in murine primary haemopoietic stem cells

Communication between irradiated and unirradiated (bystander) cells can result in responses in unirradiated cells that are similar to responses in their irradiated counterparts. The purpose of the current experiment was to test the hypothesis that bystander responses will be similarly induced in primary murine stem cells under different cell culture conditions. The experimental systems used here, co-culture and media transfer, are similar in that they both restrict communication between irradiated and bystander cells to media borne factors, but are distinct in that with the media transfer technique, cells can only communicate after irradiation, and with co-culture, cells can communication before, during and after irradiation. In this set of parallel experiments, cell type, biological endpoint, and radiation quality and dose, were kept constant. In both experimental systems, clonogenic survival was significantly decreased in all groups, whether irradiated or bystander, suggesting a substantial contribution of bystander effects (BE) to cell killing. Genomic instability (GI) was induced under all radiation and bystander conditions in both experiments, including a situation where unirradiated cells were incubated with media that had been conditioned for 24h with irradiated cells. The appearance of delayed aberrations (genomic instability) 10-13 population doublings after irradiation was similar to the level of initial chromosomal damage, suggesting that the bystander factor is able to induce chromosomal alterations soon after irradiation. Whether these early alterations are related to those observed at later timepoints remains unknown. These results suggest that genomic instability may be significantly induced in a bystander cell population whether or not cells communicate during irradiation.     

Hydrogen peroxide mediates the radiation-induced mutator phenotype in mammalian cells

Chronic oxidative stress has been associated with genomic instability following exposure to ionizing radiation. However, results showing direct causal linkages between specific ROS (reactive oxygen species) and the ionizing radiation-induced mutator phenotype are lacking. The present study demonstrates that ionizing radiation-induced genomically unstable cells (characterized by chromosomal instability and an increase in mutation and gene amplification frequencies) show a 3-fold increase in steady-state levels of hydrogen peroxide, but not superoxide. Furthermore, stable clones isolated from parallel studies showed significant increases in catalase and GPx (glutathione peroxidase) activity. Treatment of unstable cells with PEG-CAT (polyethylene glycol-conjugated catalase) reduced the mutation frequency and mutation rate in a dose-dependent fashion. In addition, inhibiting catalase activity in the stable clones using AT (3-aminotriazole) increased mutation frequency and rate. These results clearly demonstrate the causal relationship between chronic oxidative stress mediated by hydrogen peroxide and the mutator phenotype that persists for many generations following exposure of mammalian cells to ionizing radiation.     

Detection of chromosomal instability in alpha-irradiated and bystander human fibroblasts

There is increasing evidence biological responses to ionizing radiation are not confined to those cells that are directly hit, but may be seen in the progeny at subsequent generations (genomic instability) and in non-irradiated neighbors of irradiated cells (bystander effects). These so called non-targeted phenomena would have significant contributions to radiation-induced carcinogenesis, especially at low doses where only a limited number of cells in a population are directed hit. Here we present data using a co-culturing protocol examining chromosomal instability in alpha-irradiated and bystander human fibroblasts BJ1-htert. At the first cell division following exposure to 0.1 and 1Gy alpha-particles, irradiated populations demonstrated a dose dependent increase in chromosome-type aberrations. At this time bystander BJ1-htert populations demonstrated elevated chromatid-type aberrations when compared to controls. Irradiated and bystander populations were also analyzed for chromosomal aberrations as a function of time post-irradiation. When considered over 25 doublings, all irradiated and bystander populations had significantly higher frequencies of chromatid aberrations when compared to controls (2-3-fold over controls) and were not dependent on dose. The results presented here support the link between the radiation-induced phenomena of genomic instability and the bystander effect.     

Increases in oxidative stress in the progeny of X-irradiated cells

A number of phenotypes persist in the progeny of irradiated cells for many generations including delayed reproductive death, cell transformation, genomic instability, and mutations. It appears likely that persistent phenotypes are inherited by an epigenetic mechanism, although very little is known about the nature of such a mechanism or how it is established. One hypothesis is that radiation causes a heritable increase in oxy-radical activity. In the present study, intracellular levels of reactive oxygen species (ROS) in human lymphoblast clones derived from individually X-irradiated cells were monitored for about 55 generations after exposure. A number of clones derived from irradiated cells had an increase in dichlorofluorescein (DCF) fluorescence at various times. Cells with abrogated TP53 expression had a decreased oxidant response. Flow cytometry analysis of clones with increased fluorescence did not detect increases in the sub-G(1) fraction or decreased cell viability compared to nonirradiated clones, indicating that increased levels of apoptosis and cell death were not present. The oxidative stress response protein heme oxygenase 1 (HO1) was induced in some cultures derived from X-irradiated cells but not in cultures derived from unirradiated cells. The expression of the dual specificity mitogen-activated protein (MAP) kinase phosphatase (MPK1/CL100), which is inducible by oxidative stress and has a role in modulating ERK signaling pathways, was also increased in the progeny of some irradiated cells. Finally, there was an increase in the phosphorylated tyrosine content of a prominent protein band of about 45 kDa. These results support the hypothesis that increased oxy-radical activity is a persistent effect in X-irradiated mammalian cells and further suggest that this may lead to changes in the expression of proteins involved in signal transduction.