Chromosome analysis in childhood cancer survivors and their offspring--no evidence for radiotherapy-induced persistent genomic instability

Suggestions that the induction of genomic instability could play a role in radiation-induced carcinogenesis and heritable disease prompted the investigation of chromosome instability in relation to radiotherapy for childhood cancer. Chromosome analysis of peripheral blood lymphocytes at their first in vitro division was undertaken on 25 adult survivors of childhood cancer treated with radiation, 26 partners who acted as the non-irradiated control group and 43 offspring. A statistically significant increase in the frequency of dicentrics in the cancer survivor group compared with the partner control group was attributed to the residual effect of past radiation therapy. However, chromatid aberrations plus chromosome gaps, the aberrations most associated with persistent instability, were not increased. Therefore, there was no evidence that irradiation of the bone marrow had resulted in instability being transmitted to descendant cells. Frequencies of all aberration categories were significantly lower in the offspring group, compared to the partner group, apart from dicentrics for which the decrease did not reach statistical significance. The lower frequencies in the offspring provide no indication of transmissible instability being passed through the germline to the somatic cells of the offspring. Thus, in this study, genomic instability was not associated with radiotherapy in those who had received such treatment, nor was it found to be a transgenerational radiation effect.     

Strong association between cancer and genomic instability

After a first wave of radiation-induced chromosomal aberrations, a second wave appears 20–30 cell generations after radiation exposure and persists thereafter. This late effect is usually termed “genomic instability”. A better term is “increased genomic instability”. This effect has been observed in many cell systems in vitro and in vivo for quite a number of biological endpoints. The radiation-induced increase in genomic instability is apparently a general phenomenon. In the development of cancer, several mutations are involved. With increasing genomic instability, the probability for further mutations is enhanced. Several studies show that genomic instability is increased not only in the cancer cells but also in “normal” cells of cancer patients e.g. peripheral lymphocytes. This has for example been shown in uranium miners with bronchial carcinomas, but also in untreated head and neck cancer patients. The association between cancer and genomic instability is also found in individuals with a genetic predisposition for increased radiosensitivity. Several such syndromes have been found. In all cases, an increased genomic instability, cancer proneness and increased radiosensitivity coincide. In these syndromes, deficiencies in certain DNA-repair pathways occur as well as deregulations of the cell cycle. Especially, mutations are seen in genes encoding proteins, which are involved in the G₁/S-phase checkpoint. Genomic instability apparently promotes cancer development. In this context, it is interesting that hypoxia, increased genomic instability and cancer are also associated. All these processes are energy dependent. Some strong evidence exists that the structure and length of telomeres is connected to the development of genomic instability.     

WR-1065, the active metabolite of amifostine, mitigates radiation-induced delayed genomic instability

Compounds that can protect cells from the effects of radiation are important for clinical use, in the event of an accidental or terrorist-generated radiation event, and for astronauts traveling in space. One of the major concerns regarding the use of radio-protective agents is that they may protect cells initially, but predispose surviving cells to increased genomic instability later. In this study we used WR-1065, the active metabolite of amifostine, to determine how protection from direct effects of high- and low-LET radiation exposure influences genomic stability. When added 30 min before irradiation and in high concentrations, WR-1065 protected cells from immediate radiation-induced effects as well as from delayed genomic instability. Lower, nontoxic concentrations of WR-1065 did not protect cells from death; however, it was effective in significantly decreasing delayed genomic instability in the progeny of irradiated cells. The observed increase in manganese superoxide dismutase protein levels and activity may provide an explanation for this effect. These results confirm that WR-1065 is protective against both low- and high-LET radiation-induced genomic instability in surviving cells.     

WR-1065, the active metabolite of amifostine,mitigates radiation-induced delayed genomic instability

Compounds that can protect cells from the effects of radiation are important for clinical use, in the event of an accidental or terrorist-generated radiation event, and for astronauts traveling in space. One of the major concerns regarding the use of radio-protective agents is that they may protect cells initially, but predispose surviving cells to increased genomic instability later. In this study we used WR-1065, the active metabolite of amifostine, to determine how protection from direct effects of high- and low-LET radiation exposure influences genomic stability. When added 30 min before irradiation and in high concentrations, WR-1065 protected cells from immediate radiation-induced effects as well as from delayed genomic instability. Lower, nontoxic concentrations of WR-1065 did not protect cells from death; however, it was effective in significantly decreasing delayed genomic instability in the progeny of irradiated cells. The observed increase in manganese superoxide dismutase protein levels and activity may provide an explanation for this effect. These results confirm that WR-1065 is protective against both low- and high-LET radiation-induced genomic instability in surviving cells.     

Genomic instability and enhanced radiosensitivity in Hsp70.1- and Hsp70.3-deficient mice

Heat shock proteins (HSPs) are highly conserved among all organisms from prokaryotes to eukaryotes. In mice, the HSP genes Hsp70.1 and Hsp70.3 are induced by both endogenous and exogenous stressors, such as heat and toxicants. In order to determine whether such proteins specifically influence genomic instability, mice deficient for Hsp70.1 and Hsp70.3 (Hsp70.1/3(-/-) mice) were generated by gene targeting. Mouse embryonic fibroblasts (MEFs) prepared from Hsp70.1/3(-/-) mice did not synthesize Hsp70.1 or Hsp70.3 after heat-induced stress. While the Hsp70.1/3(-/-) mutant mice were fertile, their cells displayed genomic instability that was enhanced by heat treatment. Cells from Hsp70.1/3(-/-) mice also display a higher frequency of chromosome end-to-end associations than do control Hsp70.1/3(+/+) cells. To determine whether observed genomic instability was related to defective chromosome repair, Hsp70.1/3(-/-) and Hsp70.1/3(+/+) fibroblasts were treated with ionizing radiation (IR) alone or heat and IR. Exposure to IR led to more residual chromosome aberrations, radioresistant DNA synthesis (a hallmark of genomic instability), increased cell killing, and enhanced IR-induced oncogenic transformation in Hsp70.1/3(-/-) cells. Heat treatment prior to IR exposure enhanced cell killing, S-phase-specific chromosome damage, and the frequency of transformants in Hsp70.1/3(-/-) cells in comparison to Hsp70.1/3(+/+) cells. Both in vivo and in vitro studies demonstrate for the first time that Hsp70.1 and Hsp70.3 have an essential role in maintaining genomic stability under stress conditions.     

Lack of specificity of chromosome breaks resulting from radiation-induced genomic instability in Chinese hamster cells

In V79 Chinese hamster cells, radiation-induced genomic instability results in a persistently increased frequency of micronuclei, dicentric chromosomes and apoptosis and in decreased colony-forming ability. These manifestations of radiation-induced genomic instability may be attributed to an increased rate of chromosome breakage events many generations after irradiation. This chromosomal instability does not seem to be a property which has been inflicted on individual chromosomes at the time of irradiation. Rather, it appears to be secondary to an increased level of non-specific clastogenic factors in the progeny of most if not all irradiated cells. This conclusion is drawn from the observations presented here, that all the chromosomes in surviving V79 cells are involved in the formation of dicentric chromosome aberrations 1 or 2 weeks after irradiation with about equal probability if corrections are made for chromosome length. Received: 5 March 1998 / Accepted in revised form: 1 July 1998     

Persistent genomic instability in the yeast Saccharomyces cerevisiae induced by ionizing radiation and DNA-damaging agents

A "hypermutable" genome is a common characteristic of cancer cells, and it may contribute to the progressive accumulation of mutations required for the development of cancer. It has been reported that mammalian cells surviving exposure to gamma radiation display several highly persistent genomic instability phenotypes which may reflect a hypermutability similar to that seen in cancer. These phenotypes include an increased mutation frequency and a decreased plating efficiency, and they continue to be observed many generations after the radiation exposure. The underlying causes of this genomic instability have not been fully determined. We show here that exposure to gamma radiation and other DNA-damaging treatments induces a similar genomic instability in the yeast Saccharomyces cerevisiae. A dose-dependent increase in intrachromosomal recombination was observed in cultures derived from cells surviving gamma irradiation as many as 50 generations after the exposure. Increased forward mutation frequencies and low colony-forming efficiencies were also observed. Persistently elevated recombination frequencies in haploid cells were dominant after these cells were mated to nonirradiated partners, and the elevated recombination phenotype was also observed after treatment with the DNA-damaging agents ultraviolet light, hydrogen peroxide, and ethyl methanesulfonate. Radiation-induced genomic instability in yeast may represent a convenient model for the hypermutability observed in cancer cells.     

Possible role of exosomes containing RNA in mediating nontargeted effect of ionizing radiation

Communication between irradiated and un-irradiated (bystander) cells can cause damage in cells that are not directly targeted by ionizing radiation, a process known as the bystander effect. Bystander effects can also lead to chromosomal/genomic instability within the progeny of bystander cells, similar to the progeny of directly irradiated cells. The factors that mediate this cellular communication can be transferred between cells via gap junctions or released into the extracellular media following irradiation, but their nature has not been fully characterized. In this study we tested the hypothesis that the bystander effect mediator contains an RNA molecule that may be carried by exosomes. MCF7 cells were irradiated with 2 Gy of X rays and the extracellular media was harvested. RNase treatment abrogated the ability of the media to induce early and late chromosomal damage in bystander cells. Furthermore, treatment of bystander cells with exosomes isolated from this media increased the levels of genomic damage. These results suggest that the bystander effect, and genomic instability, are at least in part mediated by exosomes and implicate a role for RNA.     

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.     

The death-inducing effect and genomic instability

Exposure to ionizing radiation can induce a heritable change in the unirradiated progeny of irradiated cells. This non-targeted effect of ionizing radiation manifests as genomic instability, and although there is some debate as to the role of genomic instability in the carcinogenic process, it is thought by some to be an early step in radiation carcinogenesis. Although the mechanism of induction of genomic instability is not clearly understood, evidence suggests that secreted factors from irradiated cells may be involved. We have previously identified another non-targeted effect of ionizing radiation, the death-inducing effect. Exposure of unirradiated GM10115 cells to medium from chromosomally unstable clones was generally found to be cytotoxic. However, occasionally cells will survive in medium from unstable clones and can be clonally expanded. The absolute yield of survivors is independent of the initial number of cells plated when cell densities reached 5,000 or more cells/dish. After cytogenetic analysis of the surviving colonies, we found chromosomal instability in three of 40 clones analyzed, while some clones exhibited increased micronucleus frequency and HPRT mutation frequency. These data suggest that our chromosomally unstable GM10115 cells secrete factors that are cytotoxic to the majority of stable, parental cells but are also capable of inducing a heritable change in some of the survivors that can manifest as delayed genomic instability. These results suggest a mechanism whereby instability can be perpetuated through the influences of potentially cytotoxic factors produced by genomically unstable clones.     

Transgenerational transmission of radiation induced genomic instability

Stability of genome is one of the evolutionary important trait of cells. Various mutations (gene, chromosomal, genomic) as well as artificial manipulations with genomes (inbreeding, DNA transfection, introduction of Br-DU in DNA) cause the genetic instability. Ionizing radiation is known as the factor which induced instability of genome in late mitotic descendants of cells after in vitro and in vivo exposure. Radiation induced genetic instability can be transmitted through germline cells. On the cell level both types of radiation induced genomic instability are manifested in elevated frequency of mutations, chromosome aberrations, micronuclei, increased radiosensitivity, disappearance of adaptive response, changes in gene expression. In studies of 1970-1980 years clear evidences on the different morphological and functional injuries in tissues of irradiated organisms as well as in tissues of the progeny of exposed parents were obtained. On the organism level the instability of mitotic and of meiotic progeny of irradiated cells is resulted in increased risk of cancer and of other somatic diseases. It seems to be useful to review the earlier radiobiology literature where delayed and transgenerational effects of ionizing radiation on tissues and on organisms level were clearly shown in animals. For the estimation of pathogenic role of radiation induced genomic instability in humans, particularly in children of exposed parents the parallel study of the same human cohorts using clinical parameters and various characteristic of genomic instability seems to be very important.     

Cyclin E2 induces genomic instability by mechanisms distinct from cyclin E1

Cyclins E1 drives the initiation of DNA replication, and deregulation of its periodic expression leads to mitotic delay associated with genomic instability. Since it is not known whether the closely related protein cyclin E2 shares these properties, we overexpressed cyclin E2 in breast cancer cells. This did not affect the duration of mitosis, nor did it cause an increase in p107 association with CDK2. In contrast, cyclin E1 overexpression led to inhibition of the APC complex, prolonged metaphase and increased p107 association with CDK2. Despite these different effects on the cell cycle, elevated levels of either cyclin E1 or E2 led to hallmarks of genomic instability, i.e., an increased proportion of abnormal mitoses, micronuclei and chromosomal aberrations. Cyclin E2 induction of genomic instability by a mechanism distinct from cyclin E1 indicates that these two proteins have unique functions in a cancer setting.     

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.     

Differences in degradation lead to asynchronous expression of cyclin E1 and cyclin E2 in cancer cells

Cyclin E1 is expressed at the G₁/S phase transition of the cell cycle to drive the initiation of DNA replication and is degraded during S/G₂M. Deregulation of its periodic degradation is observed in cancer and is associated with increased proliferation and genomic instability. We identify that in cancer cells, unlike normal cells, the closely related protein cyclin E2 is expressed predominantly in S phase, concurrent with DNA replication. This occurs at least in part because the ubiquitin ligase component that is responsible for cyclin E1 downregulation in S phase, Fbw7, fails to effectively target cyclin E2 for proteosomal degradation. The distinct cell cycle expression of the two E-type cyclins in cancer cells has implications for their roles in genomic instability and proliferation and may explain their associations with different signatures of disease.     

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.     

Opposite Roles for p38MAPK-Driven Responses and Reactive Oxygen Species in the Persistence and Resolution of Radiation-Induced Genomic Instability

We report the functional and temporal relationship between cellular phenotypes such as oxidative stress, p38MAPK-dependent responses and genomic instability persisting in the progeny of cells exposed to sparsely ionizing low-Linear Energy Transfer (LET) radiation such as X-rays or high-charge and high-energy (HZE) particle high-LET radiation such as ⁵⁶Fe ions. We found that exposure to low and high-LET radiation increased reactive oxygen species (ROS) levels as a threshold-like response induced independently of radiation quality and dose. This response was sustained for two weeks, which is the period of time when genomic instability is evidenced by increased micronucleus formation frequency and DNA damage associated foci. Indicators for another persisting response sharing phenotypes with stress-induced senescence, including beta galactosidase induction, increased nuclear size, p38MAPK activation and IL-8 production, were induced in the absence of cell proliferation arrest during the first, but not the second week following exposure to high-LET radiation. This response was driven by a p38MAPK-dependent mechanism and was affected by radiation quality and dose. This stress response and elevation of ROS affected genomic instability by distinct pathways. Through interference with p38MAPK activity, we show that radiation-induced stress phenotypes promote genomic instability. In contrast, exposure to physiologically relevant doses of hydrogen peroxide or increasing endogenous ROS levels with a catalase inhibitor reduced the level of genomic instability. Our results implicate persistently elevated ROS following exposure to radiation as a factor contributing to genome stabilization.     

High-resolution mapping of mitotic DNA synthesis regions and common fragile sites in the human genome through direct sequencing

DNA replication stress, a feature of human cancers, often leads to instability at specific genomic loci, such as the common fragile sites (CFSs). Cells experiencing DNA replication stress may also exhibit mitotic DNA synthesis (MiDAS). To understand the physiological function of MiDAS and its relationship to CFSs, we mapped, at high resolution, the genomic sites of MiDAS in cells treated with the DNA polymerase inhibitor aphidicolin. Sites of MiDAS were evident as well-defined peaks that were largely conserved between cell lines and encompassed all known CFSs. The MiDAS peaks mapped within large, transcribed, origin-poor genomic regions. In cells that had been treated with aphidicolin, these regions remained unreplicated even in late S phase; MiDAS then served to complete their replication after the cells entered mitosis. Interestingly, leading and lagging strand synthesis were uncoupled in MiDAS, consistent with MiDAS being a form of break-induced replication, a repair mechanism for collapsed DNA replication forks. Our results provide a better understanding of the mechanisms leading to genomic instability at CFSs and in cancer cells.Subject terms: Cancer, DNA damage and repair     

The Werner syndrome protein contributes to induction of p53 by DNA damage.

Mutations in the p53 tumor-suppressor gene promote increased genomic instability and cancer. Mutations in the WRN gene, encoding a DNA helicase, underlie the segmental progeroid Werner syndrome (WS). WS is also associated with increased genomic instability and elevated cancer risk. The p53 and WRN proteins can engage in direct protein-protein interactions. We report that excess WRN elicits increased cellular p53 levels and potentiates p53-mediated apoptosis. Importantly, cells derived from WS patients exhibit an attenuated and delayed induction of p53 by UV or by the topoisomerase I inhibitor camptothecin. These results suggest that WRN may participate in the activation of p53 in response to certain types of DNA damage. Furthermore, the failure to induce p53 effectively may contribute to enhanced genomic instability and elevated cancer risk in WS patients.