Induction of DNA DSB and its rejoining in clamped and non-clamped tumours after exposure to carbon ion beams in comparison to X rays.

We studied double-strand breaks (DSB) induction and rejoining in clamped and non-clamped transplanted tumours in mice leg after exposure to 80 keV µm(-1) carbon ions and X rays. The yields of DSB in the tumours were analysed by a static-field gel electrophoresis. The OER of DSB after X rays was 1.68±0.31, and this value was not changed after 1 h rejoining time (1.40±0.26). These damages in oxygenated conditions were rejoined 60-70% within 1 h in situ. No difference was found between the exposure to X rays and carbon ions for the induction and rejoining of DSB. Thus, the values of OER and rejoined fraction after exposure to carbon ions were similar to those after X rays, and the calculated relative biological effectivenesses of carbon ion were around 1 under both oxygen conditions. The yields of DSB in vivo depend on exposure doses, oxygen conditions and rejoining time, but not on the types of radiation quality.

Cellular signal transduction events as a function of linear energy transfer (LET).

In order to obtain a deeper insight into the molecular mechanism controlling the cellular response to high-linear energy transfer (LET) radiation, the number and size of pATM (S1981) and gamma-H2AX foci were compared in cultures of diploid human fibroblasts after exposure to charged particles of varying species, energy and LET at the NIRS-HIMAC-facility (Chiba, Japan). Particle LET ranged from 2.2 to 300 keV/mum, and a low fluence of 7.3 x 10(4) cm(-2) was chosen. Therefore, about 1 out of 7 nuclei was traversed by a particle. Doses and LET were verified with thermoluminescence detectors (LiF:Mg, Ti) evaluated according to the high temperature ratio method. Two hours after irradiation, fibroblasts were fixed and the subcellular distribution of pATM (S1981) and gamma-H2AX was visualised by immunofluorescence or histochemical staining using phosphorylation-specific antibodies. It was found that the number of pATM (S1981) foci per nucleus was higher after exposure to higher-LET particles. Irradiation with the two highest LET beams (Fe-ions, 197 and 300 keV/mum) gave a significant increase in the number of pATM foci, whereas ions with an LET lower than 30 keV/mum yielded similar numbers of pATM foci compared with unirradiated control samples. These data show that the early cellular response to high-LET radiation is modulated by the energy deposition of the particle. Therefore, the correlation between the microdosimetric aspect of energy deposition and biologic consequences at low radiation doses deserves further study.

Single extreme low dose/low dose rate irradiation causes alteration in lifespan and genome instability in primary human cells.

To investigate the long-term biological effect of extreme low dose ionising radiation, we irradiated normal human fibroblasts (HFLIII) with carbon ions (290 MeV u(-1), 70 keV microm(-1)) and gamma-rays at 1 mGy (total dose) once at a low dose rate (1 mGy 6-8 h(-1)), and observed the cell growth kinetics up to 5 months by continuous culturing. The growth of carbon-irradiated cells started to slow down considerably sooner than that of non-irradiated cells before reaching senescence. In contrast, cells irradiated with gamma-rays under similar conditions did not show significant deviation from the non-irradiated cells. A DNA double strand break (DSB) marker, gamma-H2AX foci, and a DSB repair marker, phosphorylated DNA-PKcs foci, increased in number when non-irradiated cells reached several passages before senescence. A single low dose/low dose rate carbon ion exposure further raised the numbers of these markers. Furthermore, the numbers of foci for these two markers were significantly reduced after the cells became fully senescent. Our results indicate that high linear energy transfer (LET) radiation (carbon ions) causes different effects than low LET radiation (gamma-rays) even at very low doses and that a single low dose of heavy ion irradiation can affect the stability of the genome many generations after irradiation.

Elevated breast cancer risk in irradiated BALB/c mice associates with unique functional polymorphism of the Prkdc (DNA-dependent protein kinase catalytic subunit) gene.

Female BALB/c mice are unusually radiosensitive and more susceptible than C57BL/6 and other tested inbred mice to ionizing radiation (IR)-induced mammary tumors. This breast cancer susceptibility is correlated with elevated susceptibility for mammary cell transformation and genomic instability following irradiation. In this study, we report the identification of two BALB/c strain-specific polymorphisms in the coding region of Prkdc, the gene encoding the DNA-dependent protein kinase catalytic subunit, which is known to be involved in DNA double-stranded break repair and post-IR signal transduction. First, we identified an A --> G transition at base 11530 resulting in a Met --> Val conversion at codon 3844 (M3844V) in the phosphatidylinositol 3-kinase domain upstream of the scid mutation (Y4046X). Second, we identified a C --> T transition at base 6418 resulting in an Arg --> Cys conversion at codon 2140 (R2140C) downstream of the putative leucine zipper domain. This unique PrkdcBALB variant gene is shown to be associated with decreased DNA-dependent protein kinase catalytic subunit activity and with increased susceptibility to IR-induced genomic instability in primary mammary epithelial cells. The data provide the first evidence that naturally arising allelic variation in a mouse DNA damage response gene may associate with IR response and breast cancer risk.

Inhibition of repair of radiation-induced DNA double-strand breaks by nickel and arsenite.

The effect of arsenite or nickel on the repair of DNA double-strand breaks (DSBs) was studied in gamma-irradiated Chinese hamster ovary cells using pulsed-field gel electrophoresis. After treatment with nickel chloride or arsenite for 2 h, cells were irradiated with gamma rays at a dose of 40 Gy, and the numbers of DNA DSBs were measured immediately after irradiation as well as at 30 min postirradiation. Both arsenite and nickel(II) inhibited repair of DNA DSBs in a concentration-dependent manner; 0.08 mM arsenite significantly inhibited the rejoining of DSBs, while 76 mM nickel was necessary to observe a clear inhibition. The mean lethal concentrations for the arsenite and nickel(II) treatments were approximately 0.12 and 13 mM, respectively. This indicates that the inhibition of repair by arsenite occurred at a concentration at which appreciable cell survival occurred, but that nickel(II) inhibited repair only at cytotoxic concentrations at which the cells lost their proliferative ability. These novel observations provide insight into the mechanisms underlying the effects of combined exposure to arsenite and ionizing radiation in our environment.

A deficiency in DNA repair and DNA-PKcs expression in the radiosensitive BALB/c mouse.

We have studied the efficiency of DNA double strand break (DSB) rejoining in primary cells from mouse strains that show large differences in in vivo radiosensitivity and tumor susceptibility. Cells from radiosensitive, cancer-prone BALB/c mice showed inefficient end joining of gamma ray-induced DSBs as compared with cells from all of the other commonly used strains and F1 hybrids of C57BL/6 and BALB/c mice. The BALB/c repair phenotype was accompanied by a significantly reduced expression level of DNA-PKcs protein as well as a lowered DNA-PK activity level as compared with the other strains. In conjunction with published reports, these data suggest that natural genetic variation in nonhomologous end joining processes may have a significant impact on the in vivo radiation response of mice.

Biological effects of naturally occurring and man-made fibres: in vitro cytotoxicity and mutagenesis in mammalian cells.

Cytotoxicity and mutagenicity of tremolite, erionite and the man-made ceramic (RCF-1) fibre were studied using the human-hamster hybrid A(L) cells. Results from these fibres were compared with those of UICC Rhodesian chrysotile fibres. The A(L) cell mutation assay, based on the S1 gene marker located on human chromosome 11, the only human chromosome contained in the hybrid cell, has been shown to be more sensitive than conventional assays in detecting deletion mutations. Tremolite, erionite and RCF-1 fibres were significantly less cytotoxic to A(L) cells than chrysotile. Mutagenesis studies at the HPRT locus revealed no significant mutant yield with any of these fibres. In contrast, both erionite and tremolite induced dose-dependent S1- mutations in fibre-exposed cells, with the former inducing a significantly higher mutant yield than the latter fibre type. On the other hand, RCF-1 fibres were largely non-mutagenic. At equitoxic doses (cell survival at approximately 0.7), erionite was found to be the most potent mutagen among the three fibres tested and at a level comparable to that of chrysotile fibres. These results indicate that RCF-1 fibres are non-genotoxic under the conditions used in the studies and suggest that the high mesothelioma incidence previously observed in hamster may either be a result of selective sensitivity of hamster pleura to fibre-induced chronic irritation or as a result of prolonged fibre treatment. Furthermore, the relatively high mutagenic potential for erionite is consistent with its documented carcinogenicity.

Asbestos and DNA double strand breaks.

A radiosensitive DNA repair-deficient xrs-5 cell line was used to study asbestos cytotoxicity and DNA double strand breaks (DSBs). Although xrs-5 cells did not show any increase in sensitivity to chrysotile fibers in short-term (4-h) treatment when compared with wild-type CHO cells, longer-term exposure (24 h) gave significantly lower cell survival accompanied by a cell growth delay as well as a higher DNA DSB induction in this mutant cell line. These results suggest an important role played by DNA DSBs at the initial stage of asbestos injury.

Overview and initial results of the very long baseline interferometry space observatory programme

High angular resolution images of extragalactic radio sources are being made with the Highly Advanced Laboratory for Communications and Astronomy (HALCA) satellite and ground-based radio telescopes as part of the Very Long Baseline Interferometry (VLBI) Space Observatory Programme (VSOP). VSOP observations at 1.6 and 5 gigahertz of the milli-arc-second-scale structure of radio quasars enable the quasar core size and the corresponding brightness temperature to be determined, and they enable the motions of jet components that are close to the core to be studied. Here, VSOP images of the gamma-ray source 1156+295, the quasar 1548+056, the ultraluminous quasar 0014+813, and the superluminal quasar 0212+735 are presented and discussed.

Wortmannin inhibits repair of DNA double-strand breaks in irradiated normal human cells.

Wortmannin, a specific inhibitor of PI-3 kinase, was recently found to be an effective radiosensitizer in cells of various human and murine cell lines. Another study indicated that wortmannin inhibited repair of DNA double-strand breaks (DSBs) in irradiated Chinese hamster ovary cells using the neutral elution assay. To further clarify the mechanism behind radiosensitization by wortmannin, we have studied DSB repair in gamma-irradiated normal human fibroblasts using pulsed-field gel electrophoresis. The rejoining of DSBs in irradiated cells was significantly inhibited when 20 microM or more of wortmannin was added to the cells. The colony formation assay in cultures treated with wortmannin showed that the radiosensitization occurred in a manner that was dependent on the drug concentration. However, significant sensitization was observed only with a concentration of wortmannin of 20 microM or higher, reflecting the results of DSB rejoining studies. No marked reduction in plating efficiencies was observed for cells treated with wortmannin alone. The studies of the levels of expression of DNA-dependent protein kinase (DNA-PK) indicated that, while there were no significant changes in expression of Ku protein, the expression of the DNA-PK catalytic subunit (DNA-PKcs) was reduced markedly in cultures treated with wortmannin using an antibody against the C-terminus region of DNA-PKcs. In addition, no reduction in the levels of expression of DNA-PKcs was observed in cells treated with wortmannin using an antibody which recognizes a mid-region of this large protein. These results together with those of related studies suggest that wortmannin radiosensitizes normal human cells by inhibiting DSB repair and that this inhibition is a consequence of an inactivation of kinase activity and/or a structural change caused by binding of wortmannin to the C-terminus region of DNA-PKcs.

Microsatellite instability in human mammary epithelial cells transformed by heavy ions.

We analyzed DNA and proteins obtained from normal and transformed human mammary epithelial cells for studying the neoplastic transformation by high-LET irradiation in vitro. We also examined microsatellite instability in human mammary cells transformed to various stages of carcinogenesis, such as normal, growth variant and tumorigenic, using microsatellite marker D5S177 on the chromosome 5 and CYl7 on the Chromosome 10. Microsatellite instabilities were detected in the tumorigenic stage. These results suggest that microsatellite instability may play a role in the progression of tumorigenecity. The cause of the genomic instability has been suggested as abnormalities of DNA-repair systems which may be due to one of the three reasons: 1) alterations of cell cycle regulating genes. 2) mutations in any of the DNA mismatch repair genes, 3) mutation in any of the DNA strand breaks repair genes. No abnormality of these genes and encoded proteins, however was found in the present studies. These studies thus suggest that the microsatellite instability is induced by an alternative mechanism.

The phosphatidylinositol 3-kinase inhibitor wortmannin sensitizes quiescent but not proliferating MG-63 human osteosarcoma cells to radiation.

Recent genetic and biochemical studies indicate that DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break (dsb) repair and V(D)J recombination. Since the catalytic subunit of DNA-PK (DNA-PKcs) has high sequence homology with phosphatidylinositol 3-kinase (PI 3-kinase), we examined the effect of wortmannin, a specific inhibitor of PI 3-kinase, on the survival of human tumor cells after X-irradiation. The present study demonstrates that wortmannin at 20 microM is an effective radiosensitizer of quiescent (Q), but not proliferating (P) cells. In addition, the rejoining of DNA dsb is significantly inhibited in Q, but not in P cells. Finally, we found that Q cell extracts have approximately five-fold less DNA-PK activity than those of P cells. After a 2 h exposure to wortmannin, the DNA-PK activity of Q cell extracts was considerably lower than that of P cells. This can explain why wortmannin sensitizes Q, but not P cells to radiation.

In vitro rejoining of double-strand breaks in cellular DNA by factors present in extracts of HeLa cells.

We described previously a cell-free assay, that could be employed to study the rejoining of radiation-induced DNA double-strand breaks (dsb) in agarose embedded nuclei by activities present in an extract prepared from exponentially growing HeLa cells. Here, we extend the study and present an in vitro assay for rejoining of radiation-induced DNA dsb that employs 'naked' DNA prepared from agarose-embedded cells as a substrate and extract of HeLa cells as an enzyme source. There is no detectable residual protein on substrate DNA after extensive lysis with ionic detergents and treatment with proteases, as determined by SDS-PAGE and silver staining. We demonstrate that rejoining of dsb is absolutely dependent on cell extract and that, under optimal reaction conditions, it proceeds to an extent and with kinetics similar to those observed in intact cells. Dsb rejoining in this assay requires Mg 2+ and is inhibited by high concentrations of either K+ or Na+. This assay complements the nuclei assay for DNA dsb repair previously developed, and may be preferable to the latter in the purification of factors involved in DNA dsb repair, as it employs as substrate DNA deprived of proteins.

Defective repair of DNA double-strand breaks and chromosome damage in fibroblasts from a radiosensitive leukemia patient.

A radiation-sensitive fibroblast culture (180BR) established from an acute lymphoblastic leukemia patient who died following radiotherapy is defective in the repair of radiation-induced DNA double-strand breaks. The cells also show a reduced capacity to repair interphase chromosome damage visualized by means of premature chromosome condensation and metaphase chromosome aberrations measured by fluorescence in situ hybridization on chromosome 4. This case represents the first example in humans where hypersensitivity to ionizing radiation can be ascribed directly to a defect in DNA and chromosome repair, and the defect may underlie the cancerous phenotype observed.

Induction of DNA double-strand breaks by restriction enzymes in X-ray-sensitive mutant Chinese hamster ovary cells measured by pulsed-field gel electrophoresis.

This investigation was designed to determine whether the cytotoxic effects of different restriction endonucleases are related to the number and type of DNA double-strand breaks (DSBs) they produce. Chinese hamster ovary (CHO) K1 and xrs-5 cells, a radiosensitive mutant of CHO K1, were exposed to restriction endonucleases HaeIII, HinfI, PvuII and BamHI by electroporation. These enzymes represent both blunt and sticky end cutters with differing recognition sequence lengths. The number of DSBs was measured by pulsed-field gel electrophoresis (PFGE). Two forms of PFGE were employed: asymmetric field-inversion gel electrophoresis (AFIGE) for measuring the kinetics of DNA breaks by enzyme digestion and clamped homogeneous gel electrophoresis (CHEF) for examining the size distributions of damaged DNA. The amount of DNA damage induced by exposure to all four restriction enzymes was significantly greater in xrs-5 compared to CHO K1 cells, consistent with the reported DSB repair deficiency in these cells. Since restriction endonucleases produce DSBs alone as opposed to the various types of DNA damage induced by X rays, these results confirm that the repair defect in this mutant involves the rejoining of DSBs. Although the cutting frequency was directly related to the length of the recognition sequence for four restriction enzymes, there was no simple correlation between the cytotoxic effect and the amount of DNA damage produced by each enzyme in either cell line. This finding suggests that the type or nature of the cutting sequence itself may play a role in restriction enzyme-induced cell killing.

Mitotic metaphase cells from different cell lines cause different levels of expression of the alpha-form of interphase chromosome breaks in irradiated CHO cells.

We previously showed that HeLa mitotic metaphase cells, when used as inducers of premature chromosome condensation (PCC), uncover two times more chromosome breaks in irradiated interphase cells than CHO mitotic metaphase cells, and have at the same time a 2.5-fold higher mitosis promoting factor (MPF) activity. In a different set of experiments, we provided evidence that ionizing radiation induces two forms of interphase chromosome breaks, the alpha- and the beta-form, that can be discriminated from each other based on their kinetics of rejoining, their sensitivities to postirradiation treatments, and the genetic requirements of their repair. Here we demonstrate that HeLa mitotic metaphase cells increase the radiation yield of interphase chromosome breaks by specifically uncovering interphase chromosome damage of the alpha-form. Using xrs-5 cells that constitutively express chromosome breaks of the beta-form, we also show that the choice of metaphase cells does not affect the expression of the beta-form of interphase chromosome breaks. These observations add yet another qualitative and quantitative difference between alpha- and beta-forms of interphase chromosome breaks, and suggest that separation of radiation damage into two components will be helpful in the description of radiation action in living cells. The finding that different types of mitotic inducer cells give widely different yields of interphase chromosome breaks indicates that a quantitative comparison of the results obtained by different investigators should be made with caution and should consider the type of mitotic cells used.

Evidence that the product of the xrs gene is predominantly involved in the repair of a subset of radiation-induced interphase chromosome breaks rejoining with fast kinetics.

We classified interphase chromosome breaks into alpha and beta forms to study the requirement for the xrs gene product in the repair of each of these forms of damage. The alpha form of damage comprises radiation-induced interphase chromosome breaks whose rejoining is slow and sensitive to treatment with beta-arabinofuranosyladenine (beta-araA), whereas the beta form of damage comprises interphase chromosome breaks whose rejoining is fast and sensitive to treatment in hypertonic medium. Interphase chromosome breaks of the alpha form are visualized in plateau-phase cells by premature chromosome condensation (PCC) carried out in the absence of any treatment during the condensation period. More interphase chromosome breaks of the alpha form can be uncovered by treatment with beta-araA during the period of PCC. Interphase chromosome breaks of the beta form are not visualized in experiments using standard PCC protocols but can be uncovered by treatment in hypertonic growth medium during the period allowed for PCC. In the present report, we show that the yield of interphase chromosome breaks of the alpha form is similar in CHO and xrs-5 cells and demonstrate that xrs-5 cells rejoin this type of interphase chromosome breaks with an efficiency similar to that observed in repair-proficient CHO cells. Furthermore, we provide evidence supporting the notion that xrs-5 cells are deficient in the rejoining of the beta form of interphase chromosome breaks. These results strongly suggest that the product of the xrs gene is required predominantly in the repair of the beta form of interphase chromosome damage and emphasize the need for discrimination between different forms of interphase chromosome breaks in irradiated cells.

Mitosis-promoting factor activity of inducer mitotic cells may affect radiation yield of interphase chromosome breaks in the premature chromosome condensation assay.

We measured mitosis-promoting factor (MPF) activity in two cell lines, CHO and HeLa, extensively used at mitosis as inducers in the assay of premature chromosome condensation to study the yield and the repair kinetics of radiation damage in interphase chromosomes of diverse cell lines. We found a 2.5-fold higher MPF activity in HeLa as compared to CHO mitotic cells per mg of crude extract protein. HeLa mitotic cells, when used as inducers of premature chromosome condensation, uncovered two times more interphase chromosome breaks in irradiated, nonstimulated human lymphocytes as compared to CHO mitotic cells. A 2-fold increase in the yield of interphase chromosome breaks with HeLa mitotics was also observed in G1 cells from plateau-phase CHO cultures. Thus, MPF activity may be a contributing factor of the process that transforms radiation-induced DNA damage to chromosome breaks, and subsequently to other types of lethal chromosome aberrations. We speculate that the level and the control in the cell cycle of MPF activity may influence the radiosensitivity of cells to killing. The results strongly suggest that a direct comparison between the yields of interphase chromosome breaks measured in different laboratories may not be possible unless similar inducer cells with similar MPF activity are used.

Technical note: comparison of yields and repair kinetics of interphase chromosome breaks visualized by Sendai-virus or PEG-mediated cell fusion in irradiated CHO cells.

We examined the initial yields and the kinetics of rejoining of interphase chromosome breaks in irradiated plateau-phase Chinese hamster ovary cells by means of premature chromosome condensation (PCC) using either Sendai virus or polyethylene glycol (PEG) as fusogens. We found a yield of 2.2 chromosome breaks/cell/Gy independently of the method used to induce PCC. Rejoining of interphase chromosome breaks also proceeded with identical kinetics in cells fused using either Sendai virus or PEG. In an additional set of experiments, we compared the kinetics of rejoining of interphase chromosome breaks in cells synchronized in G1 phase by elutriation to that measured in plateau-phase cells, using either Sendai virus or PEG as fusogens. Here again, the rejoining kinetics were not affected by the fusogen used, and were similar in synchronized G1 and plateau-phase cells. These observations suggest that both methods of fusion give equivalent results in terms of yields and rejoining kinetics of interphase chromosome breaks. They also suggest that differences in the fusogens or the metabolic state of the cells cannot explain differences in the yields, and probably also the kinetics of the rejoining of interphase chromosome breaks that have been reported elsewhere. Cell line characteristics and other as of yet unidentified technical parameters may underlie these differences.

Ionizing radiation induces two forms of interphase chromosome breaks in Chinese hamster ovary cells that rejoin with different kinetics and show different sensitivity to treatment in hypertonic medium or beta-araA.

We have shown previously that incubation of irradiated plateau-phase CHO cells in hypertonic growth medium during the period normally allowed for chromosome condensation, in the premature chromosome condensation (PCC) assay, uncovers a form of interphase chromosome breaks that rejoin with fast kinetics (t1/2 = 1.5 min). Here, we report that incubation with beta-arabinofuranosyladenine (beta-araA), an inhibitor of DNA, chromosome, and cellular repair processes, during the same period uncovers a different form of interphase chromosome breaks that rejoin with slower kinetics (t1/2 = longer than 15-20 min). The yield of interphase chromosome breaks increased from 2.0 breaks/cell/Gy in untreated control cells to 3.6 breaks/cell/Gy in cells exposed to 1 mM beta-araA, and was the same as that observed in cells treated in hypertonic medium (500 mM NaCl). Simultaneous exposure to beta-araA and hypertonic medium increased the yield of interphase chromosome breaks further to 5.3 breaks/cell/Gy. This increase was consistent with an additive effect of each treatment on the overall yield of breaks, and suggested that hypertonic medium and beta-araA affect distinct and independent subsets of radiation-induced interphase chromosome breaks. We tested further the notion of independence by measuring rejoining of interphase chromosome breaks sensitive to hypertonic treatment in the presence of 1 mM beta-araA, and vice versa, rejoining of interphase chromosome breaks sensitive to beta-araA during and after treatment in hypertonic medium (500 mM NaCl, 20 min); under both sets of conditions each treatment caused maximal expression of prematurely condensed chromosome breaks responding sensitively to it when given immediately after irradiation. There was no change in the rejoining kinetics of interphase chromosome breaks sensitive to hypertonic treatment in the presence of beta-araA, and no change in the rejoining kinetics of interphase chromosome breaks sensitive to beta-arA in cells treated in hypertonic medium. These results are consistent with the hypothesis that exposure to ionizing radiation leads to the induction of two forms of prematurely condensed chromosome breaks that can be distinguished from each other on the basis of their repair kinetics and their differential sensitivity to treatment with beta-araA or hypertonic medium. In direct analogy to a classification proposed previously for potentially lethal damage (PLD) based on a similar set of experiments, we introduce the terms alpha form and beta form of interphase chromosome breaks for the slow, beta-araA-sensitive, and the fast, hypertonic treatment-sensitive form, respectively. We also propose that there is a correlation between alpha and beta form of interphase chromosome breaks and alpha and beta form of PLD, and present evidence suggesting that fast and slowly repairing DNA double-strand breaks underlie fast and slowly repairing interphase chromosome breaks.(ABSTRACT TRUNCATED AT 400 WORDS)