Relative contribution of DNA repair, cell cycle checkpoints, and cell death to survival after DNA damage in Drosophila larvae

BACKGROUND: Components of the DNA damage checkpoint are essential for surviving exposure to DNA damaging agents. Checkpoint activation leads to cell cycle arrest, DNA repair, and apoptosis in eukaryotes. Cell cycle regulation and DNA repair appear essential for unicellular systems to survive DNA damage. The relative importance of these responses and apoptosis for surviving DNA damage in multicellular organisms remains unclear. RESULTS: After exposure to ionizing radiation, wild-type Drosophila larvae regulate the cell cycle and repair DNA; grp (DmChk1) mutants cannot regulate the cell cycle but repair DNA; okra (DmRAD54) mutants regulate the cell cycle but are deficient in repair of double strand breaks (DSB); mei-41 (DmATR) mutants cannot regulate the cell cycle and are deficient in DSB repair. All undergo radiation-induced apoptosis. p53 mutants regulate the cell cycle but fail to undergo apoptosis. Of these, mutants deficient in DNA repair, mei-41 and okra, show progressive degeneration of imaginal discs and die as pupae, while other genotypes survive to adulthood after irradiation. Survival is accompanied by compensatory growth of imaginal discs via increased nutritional uptake and cell proliferation, presumably to replace dead cells. CONCLUSIONS: DNA repair is essential for surviving radiation as expected; surprisingly, cell cycle regulation and p53-dependent cell death are not. We propose that processes resembling regeneration of discs act to maintain tissues and ultimately determine survival after irradiation, thus distinguishing requirements between muticellular and unicellular eukaryotes.     

Exploring the link between ceramide and ionizing radiation

The aim of radiotherapy is to eradicate cancer cells with ionizing radiation; tumor cell death following irradiation can be induced by several signaling pathways, most of which are triggered as a consequence of DNA damage, the primary and major relevant cell response to radiation. Several lines of evidence demonstrated that ceramide, a crucial sensor and/or effector of different signalling pathways promoting cell cycle arrest, death and differentiation, is directly involved in the molecular mechanisms underlying cellular response to irradiation. Most of the studies strongly support a direct relationship between ceramide accumulation and radiation-induced cell death, mainly apoptosis; for this reason, defining the contribution of the multiple metabolic pathways leading to ceramide formation and the causes of its dysregulated metabolism represent the main goal in order to elucidate the ceramide-mediated signaling in radiotherapy. In this review, we summarize the current knowledge concerning the different routes leading to ceramide accumulation in radiation-induced cell response with particular regard to the role of the enzymes involved in both ceramide neogenesis and catabolism. Emphasis is placed on sphingolipid breakdown as mechanism of ceramide generation activated following cell irradiation; the functional relevance of this pathway, and the role of glycosphingolipid glycohydrolases as direct targets of ionizing radiation are also discussed. These new findings add a further attractive point of investigation to better define the complex interplay between sphingolipid metabolism and radiation therapy.     

DNA hypomethylation and unusual chromosome instability in cell lines from ICF syndrome patients

The ICF syndrome (immunodeficiency, centromeric region instability, facial anomalies) is a unique DNA methylation deficiency disease diagnosed by an extraordinary collection of chromosomal anomalies specifically in the vicinity of the centromeres of chromosomes 1 and 16 (Chr1 and Chr16) in mitogen-stimulated lymphocytes. These aberrations include decondensation of centromere-adjacent (qh) heterochromatin, multiradial chromosomes with up to 12 arms, and whole-arm deletions. We demonstrate that lymphoblastoid cell lines from two ICF patients exhibit these Chr1 and Chr16 anomalies in 61% of the cells and continuously generate 1qh or 16qh breaks. No other consistent chromosomal abnormality was seen except for various telomeric associations, which had not been previously noted in ICF cells. Surprisingly, multiradials composed of arms of both Chr1 and Chr16 were favored over homologous associations and cells containing multiradials with 3 or >4 arms almost always displayed losses or gains of Chr1 or Chr16 arms from the metaphase. Our results suggest that decondensation of 1qh and 16qh often leads to unresolved Holliday junctions, chromosome breakage, arm missegregation, and the formation of multiradials that may yield more stable chromosomal abnormalities, such as translocations. These cell lines maintained the abnormal hypomethylation in 1qh and 16qh seen in ICF tissues. The ICF-specific hypomethylation occurs in only a small percentage of the genome, e.g., ICF brain DNA had 7% less 5-methylcytosine than normal brain DNA. The ICF lymphoblastoid cell lines, therefore, retain not only the ICF-specific pattern of chromosome rearrangements, but also of targeted DNA hypomethylation. This hypomethylation of heterochromatic DNA sequences is seen in many cancers and may predispose to chromosome rearrangements in cancer as well as in ICF.     

G2/M-phase arrest and death by apoptosis of HL60 cells irradiated with exponentially decreasing low-dose-rate gamma radiation.

Cells of the TP53-deficient human leukemia cell line HL60 continue to progress throughout the cell cycle and arrest in the G2/M phase during protracted exposure to exponentially decreasing low-dose-rate radiation. We have hypothesized that G2/M-phase arrest contributes to the extent of radiation-induced cell death by apoptosis as well as to overall cell killing. To test this hypothesis, we used caffeine and nocodazole to alter the duration of G2/M-phase arrest of HL60 cells exposed to exponentially decreasing low-dose-rate irradiation and measured the activity of G2/M-phase checkpoint proteins, redistribution of cells in the phases of the cell cycle, cell death by apoptosis, and overall survival after irradiation. The results from these experiments demonstrate that concomitant exposure of HL60 cells to caffeine (2 mM) during irradiation inhibited radiation-induced tyrosine 15 phosphorylation of the G2/M-phase transition checkpoint protein CDC2/p34 kinase and reduced G2/M-phase arrest by 40-46% compared to cells irradiated without caffeine. Radiation-induced apoptosis also decreased by 36-50% in cells treated with caffeine and radiation compared to cells treated with radiation alone. Radiation survival was significantly increased by exposure to caffeine. In contrast, prolongation of G2/M-phase arrest by pre-incubation with nocodazole enhanced radiation-induced apoptosis and overall radiation-induced cell killing. To further study the role of cell death by apoptosis in the response to exponentially decreasing low-dose-rate irradiation, HL60 cells were transfected with the BCL2 proto-oncogene. The extent of G2/M-phase arrest was similar for parental, neomycin-transfected control and BCL2-transfected cells during and after exponentially decreasing low-dose-rate irradiation. However, there were significant differences (P < 0.01) in the extent of radiation-induced apoptosis of parental and neomycin- and BCL2-transfected cells after irradiation, with significantly less radiation-induced apoptosis and higher overall survival in BCL2-transfected cells than similarly irradiated control cells. These data demonstrate that radiation-induced G2/M-phase arrest and subsequent induction of apoptosis play an important role in the response of HL60 cells to low-dose-rate irradiation and suggest that it may be possible to increase radiation-induced apoptosis by altering the extent of G2/M-phase arrest. These findings are clinically relevant and suggest a novel therapeutic strategy for increasing the efficacy of brachytherapy and radioimmunotherapy.     

Protein tyrosine kinase inhibitors modulate radiosensitivity and radiation-induced apoptosis in K562 cells.

We studied the modulating effect of protein tyrosine kinase inhibitors on the response of cells of the human chronic myelogenous leukemia cell line K562 to radiation. The radiosensitivity of the cells was increased by treatment with herbimycin A and decreased by treatment with genistein. This modulating effect of protein tyrosine kinase inhibitors on radiation sensitivity was associated with the alteration of the mode of radiation-induced cell death. After X irradiation, the cells arrested in the G(2) phase of the cell cycle, but these TP53(-/-) cells were unable to sustain cell cycle arrest. This G(2)-phase checkpoint deficit caused cell death. The morphological pattern of cell death was characterized by swelling of the cytoplasmic compartments, cytosolic vacuolation, disruption of the plasma membrane, less evident nuclear condensation, and faint DNA fragmentation, all of which were consistent with oncosis or cytoplasmic apoptosis. The nonreceptor protein tyrosine kinase inhibitor herbimycin A accelerated the induction of typical apoptosis by X irradiation, which was demonstrated by morphological assessments using nuclear staining and electron microscopy as well as oligonucleosomal fragmentation and caspase 3 activity. Herbimycin A is known to be a selective antagonist of the BCR/ABL kinase of Philadelphia chromosome-positive K562 cells; this kinase blocks the induction of apoptosis after X irradiation. Our results showed that the inhibition of protein tyrosine kinase by herbimycin A enhanced radiation-induced apoptosis in K562 cells. This effect was associated with the activation of caspase 3 and rapid abrogation of the G(2)-phase checkpoint with progression out of G(2) into G(1) phase. In contrast, the receptor-type protein tyrosine kinase inhibitor genistein protected K562 cells from all types of radiation-induced cell death through the inhibition of caspase 3 activity and prolonged maintenance of G(2)-phase arrest. Further investigations using this model may give valuable information about the mechanisms of radiation-induced apoptosis and about the radiosensitivity and radioresistance of chronic myelogenous leukemia cells having the Philadelphia chromosome.     

Radioadaptation in Indian muntjac fibroblast cells induced by low intensity laser irradiation.

Earlier reports have indicated that an adaptive, protective response to ionizing radiation is inducible by pre-treatment with low intensity laser irradiation (LILI). We have investigated the potential of LILI to induce an adaptive response against the damaging effects of ionizing radiation in Indian muntjac fibroblasts. LILI at 660, but not 820 nm, at 11.5 and 23.0 J/cm2, induced an apparent adaptive response in the form of a reduction in the frequency of radiation-induced chromosome aberrations, but not in cell survival. There was also a trend towards a reduction in the level of single-stranded and double-stranded DNA breaks induced by ionizing radiation when cells were preconditioned with LILI. However, this did not contribute to the reduced chromosome aberration frequency. Further analysis revealed that the reduced aberration frequency was caused by a laser-induced extension of G2 delay. The adaptive response was therefore the result of cell cycle modulation by LILI, at a wavelength where there is no known DNA damaging effect to induce the checkpoint mechanisms that are normally responsible for altering cell cycle progression.     

Differences in repair profiles of interstitial telomeric sites between normal and DNA double-strand break repair deficient Chinese hamster cells

Interstitial Telomeric Repeat Sequence (ITRS) blocks are recognized as hot spots for spontaneous and ionizing radiation-induced chromosome breakage and recombination. Background and ionizing radiation-induced DNA breaks in large blocks of ITRS from Chinese hamster cell lines were analyzed using the DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) procedure. Our results indicate an extremely alkali-sensitivity of ITRS. Furthermore, it appears that ITRS blocks exhibit a particular chromatin structure, being enriched in short unpaired DNA segments. These segments could be liable to severe topological stress in highly compacted areas of the genome resulting in their spontaneous fragility and thus explaining their alkali-sensitivity. The induction and repair kinetics of DNA single-strand breaks (ssb) and DNA double-strand breaks (dsb) induced by ionizing radiation were assessed by DBD-FISH on neutral comets using Chinese hamster cells deficient in either DNA-PKcs or Rad51C. Our results indicate that the initial rejoining rate of dsb within ITRS is slower than that in the whole genome, in wild-type cells, demonstrating an intragenomic heterogeneity in dsb repair. Interestingly, in the absence of DNA-PKcs activity, the rejoining rate of dsb within ITRS is not modified, unlike in the whole genome. This was also found in the case of Rad51C mutant cells. Our results suggest the possibility that different DNA sequences or chromatin organizations may be targeted by specific dsb repair pathways. Furthermore, it appears that additional unknown dsb repair pathways may be operational in mammalian cells.     

DNMTs are required for delayed genome instability caused by radiation

The ability of ionizing radiation to initiate genomic instability has been harnessed in the clinic where the localized delivery of controlled doses of radiation is used to induce cell death in tumor cells. Though very effective as a therapy, tumor relapse can occur in vivo and its appearance has been attributed to the radio-resistance of cells with stem cell-like features. The molecular mechanisms underlying these phenomena are unclear but there is evidence suggesting an inverse correlation between radiation-induced genomic instability and global hypomethylation. To further investigate the relationship between DNA hypomethylation, radiosensitivity and genomic stability in stem-like cells we have studied mouse embryonic stem cells containing differing levels of DNA methylation due to the presence or absence of DNA methyltransferases. Unexpectedly, we found that global levels of methylation do not determine radiosensitivity. In particular, radiation-induced delayed genomic instability was observed at the Hprt gene locus only in wild-type cells. Furthermore, absence of Dnmt1 resulted in a 10-fold increase in de novo Hprt mutation rate, which was unaltered by radiation. Our data indicate that functional DNMTs are required for radiation-induced genomic instability, and that individual DNMTs play distinct roles in genome stability. We propose that DNMTS may contribute to the acquirement of radio-resistance in stem-like cells.     

DNMTs are required for delayed genome instability caused by radiation

The ability of ionizing radiation to initiate genomic instability has been harnessed in the clinic where the localized delivery of controlled doses of radiation is used to induce cell death in tumor cells. Though very effective as a therapy, tumor relapse can occur in vivo and its appearance has been attributed to the radio-resistance of cells with stem cell-like features. The molecular mechanisms underlying these phenomena are unclear but there is evidence suggesting an inverse correlation between radiation-induced genomic instability and global hypomethylation. To further investigate the relationship between DNA hypomethylation, radiosensitivity and genomic stability in stem-like cells we have studied mouse embryonic stem cells containing differing levels of DNA methylation due to the presence or absence of DNA methyltransferases. Unexpectedly, we found that global levels of methylation do not determine radiosensitivity. In particular, radiation-induced delayed genomic instability was observed at the Hprt gene locus only in wild-type cells. Furthermore, absence of Dnmt1 resulted in a 10-fold increase in de novo Hprt mutation rate, which was unaltered by radiation. Our data indicate that functional DNMTs are required for radiation-induced genomic instability, and that individual DNMTs play distinct roles in genome stability. We propose that DNMTS may contribute to the acquirement of radio-resistance in stem-like cells.     

Interstitial telomeric repeats are not preferentially involved in radiation-induced chromosome aberrations in human cells

Telomeric repeat sequences, located at the end of eukaryotic chromosomes, have been detected at intrachromosomal locations in many species. Large blocks of telomeric sequences are located near the centromeres in hamster cells, and have been reported to break spontaneously or after exposure to ionizing radiation, leading to chromosome aberrations. In human cells, interstitial telomeric sequences (ITS) can be composed of short tracts of telomeric repeats (less than twenty), or of longer stretches of exact and degenerated hexanucleotides, mainly localized at subtelomeres. In this paper, we analyzed the radiation sensitivity of a naturally occurring short ITS localized in 2q31 and we found that this region is not a hot spot of radiation-induced chromosome breaks. We then selected a human cell line in which approximately 800 bp of telomeric DNA had been introduced by transfection into an internal euchromatic chromosomal region in chromosome 4q. In parallel, a cell line containing the plasmid without telomeric sequences was also analyzed. Both regions containing the transfected plasmids showed a higher frequency of radiation-induced breaks than expected, indicating that the instability of the regions containing the transfected sequences is not due to the presence of telomeric sequences. Taken together, our data show that ITS themselves do not enhance the formation of radiation-induced chromosome rearrangements in these human cell lines.     

DNA damage caused by ionizing radiation in embryonic diploid fibroblasts WI-38 induces both apoptosis and senescence

Cellular response to ionizing radiation-induced damage depends on the cell type and the ability to repair DNA damage. Some types of cells undergo apoptosis, whereas others induce a permanent cell cycle arrest and do not proliferate. Our study demonstrates two types of response of embryonic diploid fibroblasts WI-38 to ionizing radiation. In the WI-38 cells p53 is activated, protein p21 increases, but the cells are arrested in G2 phase of cell cycle. Some of the cells die by apoptosis, but in remaining viable cells p16 increases, senescence associated DNA-damage foci occur, and senescence-associated beta-galactosidase activity increases, which indicate stress-induced premature senescence.     

Folic acid deficiency increases chromosomal instability, chromosome 21 aneuploidy and sensitivity to radiation-induced micronuclei

Folic acid deficiency can lead to uracil incorporation into DNA, hypomethylation of DNA, inefficient DNA repair and increase chromosome malsegregation and breakage. Because ionising radiation increases demand for efficient DNA repair and also causes chromosome breaks we hypothesised that folic acid deficiency may increase sensitivity to radiation-induced chromosome breakage. We tested this hypothesis by using the cytokinesis-block micronucleus assay in 10 day WIL2-NS cell cultures at four different folic acid concentrations (0.2, 2, 20, and 200 nM) that span the "normal" physiological range in humans. The study showed a significant dose-dependent increase in frequency of binucleated cells with micronuclei and/or nucleoplasmic bridges with decreasing folic acid concentration (P<0.0001, P=0.028, respectively). These biomarkers of chromosomal instability were also increased in cells irradiated (1.5 Gy gamma-rays) on day 9 relative to un-irradiated controls (P<0.05). Folic acid deficiency and gamma-irradiation were shown to have a significant interactive effect on frequency of cells containing micronuclei (two-way ANOVA, interaction P=0.0039) such that the frequency of radiation-induced micronucleated cells (i.e. after subtracting base-line frequency of un-irradiated controls) increased with decreasing folic acid concentration (P-trend<0.0001). Aneuploidy of chromosome 21, apoptosis and necrosis were increased by folic acid deficiency but not by ionising radiation. The results of this study show that folate status has an important impact on chromosomal stability and is an important modifying factor of cellular sensitivity to radiation-induced genome damage.     

Stable loss of global DNA methylation in the radiation-target tissue--a possible mechanism contributing to radiation carcinogenesis?

Radiation-induced lymphomagenesis and leukemogenesis are complex processes involving both genetic and epigenetic changes. Although genetic alterations during radiation-induced lymphoma- and leukemogenesis are fairly well studied, the role of epigenetic changes has been largely overlooked. Rodent models are valuable tools for identifying molecular mechanisms of lymphoma and leukemogenesis. A widely used mouse model of radiation-induced thymic lymphoma is characterized by a lengthy "pre-lymphoma" period. Delineating molecular changes occurring during the pre-lymphoma period is crucial for understanding the mechanisms of radiation-induced leukemia/lymphoma development. In the present study, we investigated the role of radiation-induced DNA methylation changes in the radiation carcinogenesis target organ--thymus, and non-target organ--muscle. This study is the first report on the radiation-induced epigenetic changes in radiation-target murine thymus during the pre-lymphoma period. We have demonstrated that acute and fractionated whole-body irradiation significantly altered DNA methylation pattern in murine thymus leading to a massive loss of global DNA methylation. We have also observed that irradiation led to increased levels of DNA strand breaks 6 h following the initial exposure. The majority of radiation-induced DNA strand breaks were repaired 1 month after exposure. DNA methylation changes, though, were persistent and significant radiation-induced DNA hypomethylation was observed in thymus 1 month after exposure. In sharp contrast to thymus, no significant persistent changes were noted in the non-target muscle tissue. The presence of stable DNA hypomethylation in the radiation-target tissue, even though DNA damage resulting from initial genotoxic radiation insult was repaired, suggests of the importance of epigenetic mechanisms in the development of radiation-related pathologies. The possible role of radiation-induced DNA hypomethylation in radiation-induced genome instability and aberrant gene expression in molecular etiology of thymic lymphomas is discussed.     

The Nijmegen breakage syndrome protein is essential for Mre11 phosphorylation upon DNA damage.

The Nijmegen breakage syndrome (NBS), a chromosomal instability disorder, is characterized in part by cellular hypersensitivity to ionizing radiation. Repair of DNA double-strand breaks by radiation is dependent on a multifunctional complex containing Rad50, Mre11, and the NBS1 gene product, p95 (NBS protein, nibrin). The role of p95 in these repair processes is unknown. Here it is demonstrated that Mre11 is hyperphosphorylated in a cell cycle-independent manner in response to treatment of cells with genotoxic agents including gamma irradiation. This response is abrogated in two independently established NBS cell lines that have undetectable levels of the p95 protein. NBS cells are also deficient for radiation-induced nuclear foci containing Mre11, while those with Rad51 are unaffected. An analysis of the kinetic relationship between Mre11 phosphorylation and the appearance of its radiation-induced foci indicates that the former precedes the latter. Together, these data suggest that specific phosphorylation of Mre11 is induced by DNA damage, and p95 is essential in this process, perhaps by recruiting specific kinases.     

Vorinostat induces reactive oxygen species and DNA damage in acute myeloid leukemia cells

Histone deacetylase inhibitors (HDACi) are promising anti-cancer agents, however, their mechanisms of action remain unclear. In acute myeloid leukemia (AML) cells, HDACi have been reported to arrest growth and induce apoptosis. In this study, we elucidate details of the DNA damage induced by the HDACi vorinostat in AML cells. At clinically relevant concentrations, vorinostat induces double-strand breaks and oxidative DNA damage in AML cell lines. Additionally, AML patient blasts treated with vorinostat display increased DNA damage, followed by an increase in caspase-3/7 activity and a reduction in cell viability. Vorinostat-induced DNA damage is followed by a G2-M arrest and eventually apoptosis. We found that pre-treatment with the antioxidant N-acetyl cysteine (NAC) reduces vorinostat-induced DNA double strand breaks, G2-M arrest and apoptosis. These data implicate DNA damage as an important mechanism in vorinostat-induced growth arrest and apoptosis in both AML cell lines and patient-derived blasts. This supports the continued study and development of vorinostat in AMLs that may be sensitive to DNA-damaging agents and as a combination therapy with ionizing radiation and/or other DNA damaging agents.     

Damaging and protective cell signalling in the untargeted effects of ionizing radiation

The major adverse consequences of radiation exposures are attributed to DNA damage in irradiated cells that has not been correctly restored by metabolic repair processes. However, the dogma that genetic alterations are restricted to directly irradiated cells has been challenged by observations in which effects of ionizing radiation arise in non-irradiated cells. These, so called, untargeted effects are demonstrated in cells that are the descendants of irradiated cells either directly or via media transfer (radiation-induced genomic instability) or in cells that have communicated with irradiated cells (radiation-induced bystander effects). Radiation-induced genomic instability is characterized by a number of delayed responses including chromosomal abnormalities, gene mutations and cell death. Bystander effects include increases or decreases in damage-inducible and stress-related proteins, increases or decreases in reactive oxygen and nitrogen species, cell death or cell proliferation, cell differentiation, radioadaptation, induction of mutations and chromosome aberrations and chromosomal instability. The phenotypic expression of untargeted effects and the potential consequences of these effects in tissues reflect a balance between the type of bystander signals produced and the responses of cell populations to such signals, both of which may be significantly influenced by cell type and genotype. Thus, in addition to targeted effects of damage induced directly in cells by irradiation, a variety of untargeted effects may also make important short-term and long-term contributions to determining overall outcome after radiation exposures.     

How does radiation kill cells?

Recent advances in the understanding of intracellular signaling after genotoxic injury have led to a better understanding of the pathways that influence radiation-induced cell death. Particular progress has been made in defining molecular controls of apoptosis and radiation-induced cell cycle arrest, as well as the possible role of telomerase activity in stabilizing DNA breaks.     

Complementation of a DNA repair deficiency in six human tumor cell lines by chromosome 11

Summary Human tumor cells, after x-irradiation during the G₂ phase of the cell cycle, show an abnormally high frequency of persistent chromatid breaks and gaps resulting from deficient DNA repair. Addition of a single human chromosome 11 from normal fibroblasts by micro-cell fusion to cell lines from six different tumors resulted in efficient repair of the radiation-induced damage to the level in normal cells. For one of the cell lines, addition of the long arm of chromosome 11 was sufficient to restore repair efficiency. In four of the six tumor lines, restoration of efficient DNA repair by chromosome 11 was associated with tumor suppression in nude mice. These results suggest that chromosome 11 carries a DNA repair gene or genes that complement the repair deficiency of tumor cells and that this gene for at least one tumor is localized to the long arm.     

Glucose-6-phosphate dehydrogenase and the oxidative pentose phosphate cycle protect cells against apoptosis induced by low doses of ionizing radiation

The initial and rate-limiting enzyme of the oxidative pentose phosphate shunt, glucose-6-phosphate dehydrogenase (G6PD), is inhibited by NADPH and stimulated by NADP(+). Hence, under normal growth conditions, where NADPH levels exceed NADP(+) levels by as much as 100-fold, the activity of the pentose phosphate cycle is extremely low. However, during oxidant stress, pentose phosphate cycle activity can increase by as much as 200-fold over basal levels, to maintain the cytosolic reducing environment. G6PD-deficient (G6PD(-)) cell lines are sensitive to toxicity induced by chemical oxidants and ionizing radiation. Compared to wild-type CHO cells, enhanced sensitivity to ionizing radiation was observed for G6PD(-) cells exposed to single-dose or fractionated radiation. Fitting the single-dose radiation response data to the linear-quadratic model of radiation-induced cytotoxicity, we found that the G6PD(-) cells exhibited a significant enhancement in the alpha component of radiation-induced cell killing, while the values obtained for the beta component were similar in both the G6PD(-) and wild-type CHO cell lines. Here we report that the enhanced alpha component of radiation-induced cell killing is associated with a significant increase in the incidence of ionizing radiation-induced apoptosis in the G6PD(-) cells. These data suggest that G6PD and the oxidative pentose phosphate shunt protect cells from ionizing radiation-induced cell killing by limiting the incidence of radiation-induced apoptosis. The sensitivity to radiation-induced apoptosis was lost when the cDNA for wild-type G6PD was transfected into the G6PD(-) cell lines. Depleting GSH with l-BSO enhanced apoptosis of K1 cells while having no effect in the G6PD(-) cell line     

Characteristics of genomic instability in clones of TK6 human lymphoblasts surviving exposure to 56Fe ions

Genomic instability in the human lymphoblast cell line TK6 was studied in clones surviving 36 generations after exposure to accelerated 56Fe ions. Clones were assayed for 20 characteristics, including chromosome aberrations, plating efficiency, apoptosis, cell cycle distribution, response to a second irradiation, and mutant frequency at two loci. The primary effect of the 56Fe-ion exposure on the surviving clones was a significant increase in the frequency of unstable chromosome aberrations compared to the very low spontaneous frequency, along with an increase in the phenotypic complexity of the unstable clones. The radiation-induced increase in the frequency of unstable chromosome aberrations was much greater than that observed previously in clones of the related cell line, WTK1, which in comparison to the TK6 cell line expresses an increased radiation resistance, a mutant TP53 protein, and an increased frequency of spontaneous unstable chromosome aberrations. The characteristics of the unstable clones of the two cell lines also differed. Most of the TK6 clones surviving exposure to 56Fe ions showed unstable cytogenetic abnormalities, while the phenotype of the WTK1 clones was more diverse. The results underscore the importance of genotype in the characteristics of instability after radiation exposure.