Satsuki Tsuji

Isolation of temperature-sensitive CHO-K1 cell mutants exhibiting chromosomal instability and reduced DNA synthesis at nonpermissive temperature

Twenty-five temperature-sensitive (ts) mutants were isolated from Chinese hamster CHO-K1 cells after mutagenization withN-methyl-N′-nitro-N-nitrosoguanidine. Of 13 complementation groups identified, nine exhibited chromosomal instability at a nonpermissive temperature. They were classified into three major classes according to inducibility of sister chromatid exchange (SCE) and/or chromosomal aberration (CA): class 1 resulted in predominant SCEs, class 2 manifested both SCEs and CAs, and class 3 exhibited higher induction of CAs. Flow cytometric analysis of the mutants exhibiting chromosomal instability indicated that many of the mutants were arrested in the S or S to G2 phases of the cell cycle at the nonpermissive temperature, accompanied by a decrease in the rate of DNA synthesis. These results imply that ts defects are related to some points in DNA replication and might be responsible for the induction of SCEs and/or CAs at the nonpermissive temperature.

Introduction of a foreign gene into medakafish using the particle gun method

We developed a procedure to introduce a foreign gene into fertilized eggs of medakafish (Oryzias latipes) using the particle gun method, which is one of the easiest and most reliable techniques for gene transfer. A plasmid construct with the green fluorescence protein (GFP) gene driven by the madakafish beta-actin gene promoter was successfully introduced into eggs, and the expression of GFP was observed in 20% of the primary transfectant (chimera) fish. In addition, germ line transmission of GFP was observed in 13% of the GFP-positive primary transfectant fish. The new application described here should enable us to investigate gene expression using the fish model on a routine basis without high technical sophistication. J. Exp. Zool. 287:285-293, 2000.

Perceived risk of nuclear power and other risks during the last 25 years in Japan.

Abstract The present study described the results of three “fixed-point” surveys on perceived risk related to a list of social and individual risk events during 25 years in Japan. Female clerical staff and researchers were asked to rank 30 items related to various types of technologies and human activities according to their subjective judgments on the order of perceived magnitude of risk in 1983, 1992, and 2007. A similar survey was undertaken for Japanese citizens using web-based questionnaires in 2007. In general, the risk perceptions of the Japanese people, irrespective of gender, age, and occupation, have been uniform during the last 25 years. The female clerical staffs have consistently judged nuclear power as most risky during the last 25 years, whereas researchers’ judgment fluctuated with events such as the Chernobyl accident. The ranking of the risk of motor vehicles fell during the 25-y period, whereas those of health risks with food preservatives, x-rays, and antibiotics rose transiently in the 1992 survey. During the 15 years from 1992 to 2007, people tended to learn how to accommodate themselves to these technologies with low risks in exchange for high benefits, except in the case of nuclear power. Nuclear power was regarded as a high-risk item by the Japanese even before the Fukushima nuclear power plant accident in March 2011. This partly explains that the crisis inevitably provokes further high risk perception in Japan, although the overall health threat to the human population in Japan is estimated to be relatively limited so far.

Heritable unstable DNA sequences and hypermethylation associated with fragile X syndrome in Japanese families

Fragile X syndrome, associated with the fragile site at Xq27.3 (FRAXA), is the most common form of familial mental retardation. The fragile X mutation has recently been characterized as a heritable unstable DNA sequence, p(CCG)n/p(CGG)n, in the FRAXA locus. In the present study, a correlation between fragile X‐genotypes in the FRAXA locus and hypermethylation of an adjacent CpG island was examined in four Japanese families with fragile X syndrome. We show here that the heritable unstable DNA sequences in the fragile X chromosome usually increase in size when transmitted by female carriers, and that the degree of methylation in the CpG island correlated with the increased sizes of the unstable DNA sequences. When a hypermethylated full mutation was transmitted by a male to his daughters, both the size of the unstable DNA sequence and the degree of the methylation reduced to the premutation range. Our observations suggest that female meiosis has a greater potential for amplifying unstable DNA sequences and that amplified DNA sequences can be transmitted through germ cells, while male germ cells seem not to be able to tolerate highly amplified unstable DNA sequences.

Effect of SCID mutation on the occurrence of mouse Pc-1 (Ms6-hm) germline mutations.

Mouse Pc-1 (Ms6-hm) is a hypervariable minisatellite locus that is unstable during intergenerational transmission. This hyper-instability of Pc-1 is useful for detecting germline mutation using a small number of experimental animals, although its molecular mechanism has not yet been elucidated. We examined the effect of severe combined immune deficiency (SCID) mutation on the spontaneous germline mutation at the Pc-1 locus using the CB17 mouse strain. Our results showed that the frequency of spontaneous germline mutation at Pc-1 in the offspring of wild-type parents was 9.7%. In F1 between SCID male and wild-type female, however, the frequency of germline mutation was drastically increased to 42.3%. When SCID female mice were mated with wild-type male, the frequency of germline mutation in F1 was slightly increased to 13.6%. These results suggest that DNA protein kinase catalytic subunit (DNA-PKcs), deficiency of which causes SCID mutation, plays an important role in the stable transmission of a genome containing hypervariable tandem repeats to progeny in male germ cells.

Induction of distamycin A-inducible rare fragile sites and increased sister chromatid exchanges at the fragile site

SummaryExpression of distamycin A-inducible rare fragile sites by AT-specific DNA-ligands was examined in lymphoblastoid cell lines derived from heterozygous carriers for the fra(8)(q24), fra(16)(pl2), and fra(16)(q22) sites. The sensitivity of fragile site expression to the inducers was different at these fragile sites. The expression of fra(8)(q24) was induced markedly by Hoechst 33258, but not by distamycin A or berenil. An increased expression of fra(16)(p12) was found following treatment with Hoechst 33258 or berenil, but not with distamycin A. At fra(16)(q22), distamycin A markedly induced the fragile site, but Hoechst 33258 and berenil did not. Since their response to the different inducers was similar to that found in cultured lymphocytes, lymphoblastoid cell lines appear to retain their inherent properties. Although BrdUrd alone did nto induce any fragile sites, concomitant treatment with BrdUrd plus the inducer was synergistically effective in inducing all the fragile sites. An increased frequency of sister chromatid exchanges was observed at fra(16)(p12) following simultaneous treatment with BrdUrd and berenil, mainly when the site was expressed as an isochromatid gap. Thus, the induced fra (16)(pl2) site is a hot spot for the formation of sister chromatid exchanges, as found in other reported fragile sites.

Chromosomal instability in mutagen-sensitive mutants isolated from mouse lymphoma L5178Y cells II. Abnormal induction of sister-chromatid exchanges and chromosomal aberrations by mutagens in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS 1)

To determine the mutual relationships between cell survival and induction of sister-chromatid exchanges (SCEs) as well as chromosomal aberrations (CAs), mutagen-induced SCEs and CAs were analyzed in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS 1) isolated from mouse lymphoma L5178Y cells. The levels of CA induction in both mutants strictly corresponded to the sensitivity to lethal effects of mutagens, except that caffeine-induced CAs in M10 are considerably lower than those in L5178Y. The results clearly indicate that except for caffeine-induced CAs in M10, mutagen-induced lethal lesions are responsible for CA induction. In contrast, SCE induction in mutants was complicated. In M10, hypersensitive to killing by gamma-rays, methyl methanesulfonate (MMS), and 4-nitroquinoline 1-oxide (4NQO), but not sensitive to UV or caffeine, the frequency of SCEs induced by gamma-rays was barely higher than that in L5178Y, and the frequencies of MMS- and UV-induced SCEs were similar to those in L5178Y, but 4NQO- and caffeine-induced SCEs were markedly lower than those in L5178Y. MS 1, which is hypersensitive to MMS and caffeine, but not sensitive to UV or 4NQO, responded to caffeine with an enhanced frequency of SCEs and had a normal frequency of MMS-induced SCEs, but a reduced frequency of UV- and 4NQO-induced SCEs. Thus, susceptibility to SCE induction by mutagens is not necessarily correlated with sensitivity of mutants to cell killing and/or CA induction by mutagens. Furthermore, the spontaneous levels of SCEs are lower in M10 and higher in MS 1 than that in L5178Y (Tsuji et al., 1987). Based on these results, we speculate that M10 may be partially defective in the processes for the formation of SCEs caused by mutagens. On the other hand, MS 1 may modify SCE formation-related lesions induced by UV and 4NQO to some repair intermediates that do not cause SCE formation. In addition, MMS-induced lethal lesions in MS 1 may not be responsible for SCE induction whereas caffeine-induced lethal lesions are closely correlated with SCE induction. Thus, the lesions or mechanisms involved in SCE production are in part different from those responsible for cell lethality or CA production.

Rapid and reliable diagnosis of murine myeloid leukemia (ML) by FISH of peripheral blood smear using probe of PU. 1, a candidate ML tumor suppressor

BackgroundMurine myeloid leukemia (ML) provides a good animal model to study the mechanisms of radiation-induced leukemia in humans. This disease has been cytogenetically characterized by a partial deletion of chromosome 2 with G-banding. For the rapid diagnosis of ML, this study reports a FISH method using spleen cells and peripheral blood smears from ML mice exposed to gamma rays and neutrons with PU.1, a candidate ML tumor suppressor, as a probe.ResultsAmong mice that were tentatively diagnosed with ML by clinical findings and blood smear examination, 85% carried spleen cells showing the loss of PU.1 although the frequency of these abnormal cells varied among individuals. Mice with very low frequencies of cells showing the loss of one copy of PU.1 (one-PU.1 frequency) were later diagnosed pathologically not with ML but with blastic or eosinophilic leukemia. Some neutron-irradiated mice had cells showing translocated PU.1, although no pathological features differentiated these ML mice from ML mice expressing the simple loss of PU.1.The one-PU.1 frequency can be detected from spleen metaphase cells, spleen interphase cells, and blood smears. There was a good correlation between the one-PU.1 frequency in spleen metaphase cells and that in spleen interphase cells (r = 0.96) and between one-PU.1 frequency in spleen interphase cells and that in blood cells (r = 0.83).ConclusionThe FISH method was capable of detecting aberration of copy number of the PU.1 gene on murine chromosome 2, and using a peripheral blood smear is more practical and less invasive than conventional pathological diagnosis or the cytogenetic examination of spleen cells.

Chromosomal instability in mutagen-sensitive mutants isolated from mouse lymphoma L5178Y cells I. Five different genes participate in the formation of baseline sister-chromatid exchanges and spontaneous chromosomal aberrations

In a search for cell mutants that show an increase or a decrease in the frequency of baseline sister-chromatid exchanges (SCEs) or spontaneous chromosomal aberrations (CAs), large numbers of mutagen-sensitive clones previously isolated from mouse lymphoma L5178Y cells were analyzed. In addition to two SCE mutants (ES 4 and AC 12) previously reported, three other mutants were identified as an SCE mutant. An ethyl methanesulfonate-sensitive mutant ES 2 and an alkylating agent-sensitive mutant MS 1 exhibited, respectively, 1.4-fold and 1.8-fold higher baseline SCE frequencies than did the parental L5178Y. In contrast, M10, which is sensitive to X-ray and 4-nitroquinoline 1-oxide, showed a reduced frequency of baseline SCEs (0.65-fold). These 5 mutants including ES 4 and AC 12 had 3--9-fold increases in spontaneous CA frequencies. Measurement of baseline SCE formation in inter-mutant hybrids revealed that M10 mutation is dominant, MS 1 and ES 4 mutations are semidominant, and ES 2 and AC 12 mutations are recessive. Because SCE frequencies in hybrids formed between pairs of 4 mutants (ES 2, MS 1, ES 4 and AC 12) were significantly lower than those in the tetraploid mutant cells, these 4 mutants probably belong to different complementation groups. Since M10 behaved dominantly with respect to SCE phenotype, it was not possible to determine by complementation test whether it belongs to a different group from the other mutants. However, the finding that M10 is complemented by other mutants for EMS sensitivity indicates that the M10 mutation is different from the other mutations. From these results, it is concluded that at least 4 different genes participate in the formation of high levels of baseline SCEs. The defects in ES 2, MS 1, ES 4, and AC 12 produce common lesions responsible for the formation of both SCEs and CAs. In contrast, the defect in M10 is associated with a high increase in spontaneous CA frequency, but conversely associated with a decrease in baseline SCE frequency. This suggests that M10 is defective in the process involved in the formation of baseline SCEs.

Investigation of new cytogenetic biomarkers specific to high-LET radiation using in vivo and in vitro exposed human lymphocytes

Purpose: To find detectable cytogenetic biomarkers that can offer information about the radiation quality of in vivo exposure retrospectively. Materials and methods: Chromosome-type aberrations of peripheral lymphocytes of uterine cancer patients that received internal γ- and external X-ray therapy or carbon beam therapy and of victims severely exposed to neutrons and γ-rays in a criticality accident that occurred in Tokai-mura, Japan were analysed. Data obtained from in vitro irradiation experiments using 60Co γ-rays and 10 MeV neutrons were compared with the in vivo exposure data. Results: The ratio of acentric rings to dicentric chromosomes (termed RaD ratio) and that of excess fragments to dicentrics (termed EfD ratio) showed significant (p < 0.05) differences between the two groups of cancer patients, and these ratios for accidental victims were in between the values of the two groups of cancer patients. The in vitro studies using doses equivalent to 1 – 3 Gy of γ-rays have confirmed that the EfD ratios were increased with the high LET (linear energy transfer) and RaD ratios decreased. Conclusion: The present data show that the RaD and EfD ratios can be used as cytogenetic biomarkers of exposure to high-LET radiation at least within a few years of exposure.