Takaji Ikushima

Radioadaptive response: efficient repair of radiation-induced DNA damage in adapted cells.

To verify the hypothesis that the induction of a novel, efficient repair mechanism for chromosomal DNA breaks may be involved in the radioadaptive response, the repair kinetics of DNA damage has been studied in cultured Chinese hamster V79 cells with single-cell gel electrophoresis. The cells were adapted by priming exposure with 5 cGy of gamma-rays and 4-h incubation at 37 degrees C. There were no indication of any difference in the initial yields of DNA double-strand breaks induced by challenging doses from non-adapted cells and from adapted cells. The rejoining of DNA double-strand breaks was monitored over 120 min after the adapted cells were challenged with 5 or 1.5 Gy, doses at the same level to those used in the cytogenetical adaptive response. The rate of DNA damage repair in adapted cells was higher than that in non-adapted cells, and the residual damage was less in adapted cells than in non-adapted cells. These results indicate that the radioadaptive response may result from the induction of a novel, efficient DNA repair mechanism which leads to less residual damage, but not from the induction of protective functions that reduce the initial DNA damage.

Radio-adaptive response: characterization of a cytogenetic repair induced by low-level ionizing radiation in cultured Chinese hamster cells

Pretreatment with low doses of beta-rays from incorporated tritiated thymidine ([3H]dThd) or of Co-60 gamma-rays (1 or 5 cGy) rendered actively growing Chinese hamster V79 cells more resistant to the induction of micronuclei or sister-chromatid exchanges (SCEs) by a subsequent high dose of gamma-rays (1 Gy). This adaptive response to ionizing radiation (radio-adaptive response) can be induced by an optimal range of low doses of 3H beta-rays, but not by much lower or higher adapting doses. Full expression of the adaptive response induced by the exposure to low doses of 60Co gamma-rays occurred 4 h after the adapting dose. The cells pre-exposed to low doses of gamma-rays showed cross-resistance to challenge doses of gamma-rays themselves and also of mitomycin C (MMC) and near ultraviolet light (UV-B, 313 nm), but not to those of ethyl methanesulfonate (EMS) or cis-platinum (II) diammine dichloride (cisplatin) for SCE induction. These results suggest that the radio-adaptive response mechanistically couples to the repair network which copes with chromatin lesions induced by MMC and UV-B.

Effects of an inhibitor of topoisomerase II, ICRF-193 on the formation of ultraviolet-induced chromosomal aberrations

Treatments of Chinese hamster V79 cells during one cell cycle with a new type of topoisomerase II inhibitor, ICRF-193, which does not accumulate cleavable topoisomerase-DNA complexes induced both chromosome- and chromatid-type aberrations with high frequencies. Furthermore, ICRF-193 synergistically enhanced the yield of UVB-induced chromatid-type aberrations, chromatid exchanges in particular. Treated with ICRF-193 for the last 3 h before harvest, cells showed frequent incidence of chromatid-type aberrations and synergistic enhancement of UVB-induced chromatid-type aberrations, chromatid exchanges in particular. These results suggest that spontaneous and UVB-induced lesions might be ultimately transformed into chromatid-type aberrations by topoisomerase II-dependent checkpoint process(es) in the G2 phase of the cell cycle.

Radiobiological studies in plants—XI: Further studies on somatic mutations induced by X-rays at the al locus of diploid oats*

Abstract The effects of X-ray dose rate, fractionated exposure, and DNP treatment on somatic mutation frequency were studied using a mutant strain of diploid oats heterozygous for the albino gene. Various X-ray treatments were conducted on both the dry and 24-hr pre-soaked seeds. Higher dose rates were more effective in inducing mutations than lower dose rates. Fractionated exposure induced less mutations than non-fractionated exposure. Similar fractionated exposure but accompanied with a DNP treatment had about the same efficiency as the non-fractionated exposure. Consequently, it was concluded that many of the induced somatic mutations were induced by chromosomal deficiencies. The number of white stripes per leaf decreased with the progress of leaf order. This phenoemenon appears to be due to the difference in numbers of the primordial or initial cells at the time of irradiation. A similar relationship was observed between the average ratio of white stripe width to leaf width and leaf order. It seems possible to consider that a considerable elimination of mutant cells occurred in the course of leaf development. Distinct white stripes were observed in leaf 3 and upper leaves but never in leaf 1 and 2 after irradiations of dry seeds. However, such distinct stripes were also observed in leaf 2 after irradiations of 24-hr pre-soaked seeds. This phenomenon is discussed giving two possible explanations.

Bimodal induction of sister-chromatid exchanges by luminol, an inhibitor of poly(ADP-ribose) synthetase, during the S-phase of the cell cycle.

The cell cycle dependence of sister chromatid exchanges (SCEs) induced by luminol, a new potent inhibitor of poly(ADP-ribose) synthetase, was studied in Chinese hamster V79 cells. Continuous treatment with luminol during two whole cell cycles in the presence of 5-bromo-2′-deoxyuridine (BrdUrd), or in the first or second cycle induced SCEs very efficiently in a linear dosedependent manner. However, no enhancement of SCE levels was observed after luminol treatment in a cycle preceding BrdUrd treatment, in contrast to results found with other strong SCE inducers such ascis-diammine-dichloroplatinum (II) (CDDP) and mitomycin C (MMC). Luminol was about ten times as effective in inducing SCEs as 3-aminobenzamide (3AB)′, an inhibitor of the NAD+ site of poly(ADP-ribose) synthetase. The induction of SCEs by luminol was restricted to the Sphase of the cell cycle with peaks at an early and a late stage, corresponding to the biphasic replication of DNA. The mechanism of SCE appears to be the same at the early and late stages of S-phase for luminol-induced SCE formation.

A developmental study of diploid oats by means of radiation-induced somatic mutations*

Abstract The developmental behaviour of radiation-induced mutant cells in X 1 plants was studied using a strain of diploid oats heterozygous for the albino gene, al . Dry and 24-hr pre-soaked seeds were irradiated with X-rays, γ-rays and thermal neutrons. Distinct white stripes were observed in leaf 3 and upper leaves when dry seeds were irradiated, but from leaf 2 when pre-soaked seeds were treated. The different response in the pre-soaked seeds might be caused by the radiation-induced loss of reproductive integrity of most of the sensitized leaf 2 primordial cells followed by reorganization of the primordia in the embryos. The number of white stripes per leaf decreased with the progression of leaf order. From the relative widths of the stripes, the number of primordial or initial cells for each leaf in the mature embryos was estimated to be about 30 or less for leaf 3 and at least three to five for leaf 4 to 6. Since the stripes in leaf 3 from the treated dry seeds were induced independently from those in the upper leaves, leaf 3 seems to have been derived from an already differentiated primordial cell group, although such a group could not be detected histologically. The white stripes were more frequently observed near the centre of the leaf blade rather than in the marginal part with wider stripes tending to appear in the marginal region, indicating a differential development in the lateral direction. The tillers on the same orthostichy tended to behave similarly with regard to the appearance of the white stripes or sectors. The present study demonstrates that mutations having an appropriate phenotype induced in somatic tissues provide a useful means for detailed study of organogenesis in higher plants.

TWO KINDS OF CHROMOSOME REJOININGS IN X-RAYED TWO-ROWED BARLEY

Abstract In order to determine whether or not some rejoining of broken ends of chromo-somes occurs within a matter of seconds after irradiation, X-ray irradiation was performed on the seeds of two-rowed barley presoaked for 24 hr, changing the exposure and the dose rate or fractionating the exposure. All the exposures or dose rates used were measured simultaneously with or immediately before the treatments, and all the exposure times and the intervals in fractionation experiments were accurately measured. In each lot, the frequency of induced dicentric bridges was scored at anaphase of the first mitotic division in 2000 to 6700 cells fixed 48 hr after the start of presoaking. All the experiments were carried out at 20 ± 1 °C. The frequency of dicentric bridges increased with exposure, giving a curve close to square-of-exposure (Figs. 2 and 4), and effects of dose rate and of fractionation were clearly observed (Fig. 1 and Table 3). It was also made clear that there are two kinds of X-ray induced chromosome breaks, one of which can rejoin within an extremely short period such as 20 to 30 sec after irradiation, and another can rejoin after a longer time such as 15 to 30 min (Fig. 3). The latter breaks were induced about 20 per cent more frequently than the former breaks. From the experiment using DNP, it was presumed that the energy required for rejoining is supplied from oxidative phosphorylation.

Induction of a large deletion in mitochondrial genome of mouse cells by X-ray irradiation

Abstract Large deletions and point mutations of mitochondrial DNA (mtDNA) is causally associated with mitochondrial diseases, and accumulates with age in human tissues. In order to find out the role of oxidative stress in the generation of large mtDNA deletions, using normal Balb and severe combined immunodeficiency (SCID) mouse cells, we assessed whether X-ray irradiation induces large mtDNA deletions. Cultured cells were irradiated with X-rays and assayed for a large mtDNA deletion using a PCR technique with a specific pair of primers. X-ray doses as low as 1 Gy were effective for the induction of the large mtDNA deletion (5823 bp long), which corresponds to the human 4977 bp common deletion. The breakpoints were flanked by the 5-bp long tandem repeats, 5′-TACCC-3′. A dose-dependent induction of the large mtDNA deletion was observed, the yield being higher in normal cells than in SCID cells. The fraction of large mtDNA deletion increased in the normal cells but decreased in the SCID cells within 7 days after X-ray irradiation. As the SCID cells carry a mutation in the gene encoding DNA–PKcs, the key enzyme in DNA double-strand break repair, it can be concluded that repair of DNA strand breaks may be involved in the formation of X-ray induced large mtDNA deletions.

Post-treatment effects of DNA topoisomerase inhibitors on UVB- and X-ray-induced chromosomal aberration formations

Post-treatments with nogalamycin, an inhibitor of DNA topoisomerase I, for last 3h of the culture (during the G2 phase) drastically enhanced the yield of ultraviolet light B (UVB)-induced exchange-type chromatid aberrations, while showing little effect on the formation of breakage-type aberrations in Chinese hamster V79 cells. These results are very similar to those obtained with ICRF-193, an inhibitor of topoisomerase II, with respect to the effect on UVB-induced chromatid aberrations. Thus, both types of topoisomerases may suppress the formation of exchange-type chromatid aberrations in the G2 phase which is suggested to be the principal stage of the cell cycle for chromatid aberration formation.In human lymphocytes irradiated with X-rays before phytohaemagglutinin (PHA) stimulation, post-treatments with nogalamycin through the whole cell cycle enhanced only the yield of dicentrics, while showing little effect on the yields of any other chromosome-type aberrations. Nogalamycin added 6h after PHA stimulation to irradiated cells also showed almost the same effects, whereas, addition of nogalamycin 24h after PHA stimulation showed no effect on X-ray-induced chromosome-type aberrations. These results suggest that X-ray-induced DNA damage lead to chromosome-type aberrations before the start of S phase and topoisomerase I may suppress the formation of dicentrics, exchange-type chromosome aberrations. Post-treatments with ICRF-193 showed no effect on the formation of X-ray-induced chromosome-type aberrations, suggesting nonparticipation of topoisomerase II in this process.

Involvement of G2-dependent DNA double-strand break repair in the formation of ultraviolet light B-induced chromosomal aberrations

Wortmannin, an inhibitor of DNA double-strand break (DSB) repair added 19 h before harvest enhanced the incidence of ultraviolet light B (UVB)-induced chromatid aberrations in Chinese hamster V79 cells. Posttreatment with wortmannin for last 3 h of culture also enhanced the yield of breakage-type chromatid aberrations and suppressed the yield of exchange-type chromatid aberrations almost completely. Thus, the inhibition of DSB repair in the G2 phase stimulated the breakage-type aberration formation, while suppressing the exchange-type aberration formation. We propose the model of UVB-induced chromatid-type aberration formation which might be fully related to G2-dependent DSB repair pathway.