Hatsumi Nagasawa

Unexpected sensitivity to the induction of mutations by very low doses of alpha-particle radiation: evidence for a bystander effect.

We examined the induction of HPRT mutations in CHO cells exposed to low fluences of (238)Pu alpha particles from a specially constructed irradiator. The dose-response relationship was linear over the dose range of 5 cGy-1.2 Gy. However, unexpected sensitivity, leading to a significantly higher frequency of mutations than would be predicted by a back extrapolation from the data for higher doses, was observed in the dose range below 5 cGy, where the mean number of alpha-particle traversals per nucleus was significantly less than one (0.05-0.3). The frequency of mutations induced by a single alpha particle traversing the nucleus of a cell was increased nearly fivefold at the lowest fluence studied. The data are consistent with the conclusion that the enhanced efficiency of each nuclear traversal at low particle fluences is the result of mutations arising in nonirradiated, bystander cells.

Ku70: A Candidate Tumor Suppressor Gene for Murine T Cell Lymphoma

We present evidence that inactivation of the Ku70 gene leads to a propensity for malignant transformation both in vitro and in vivo. In vitro, Ku70-/- mouse fibroblasts displayed an increased rate of sister chromatid exchange and a high frequency of spontaneous neoplastic transformation. In vivo, Ku70-/- mice, known to be defective in B but not T lymphocyte maturation, developed thymic and disseminated T cell lymphomas at a mean age of 6 months with CD4+CD8+ tumor cells. These findings directly demonstrate that Ku70 deficiency facilitates neoplastic growth and suggest a novel role of the Ku70 locus in tumor suppression.

Radiation-induced genomic instability: delayed mutagenic and cytogenetic effects of X rays and alpha particles.

The frequency of mutations at the Hprt locus was measured in clonal populations of Chinese hamster ovary cells derived from single cells surviving exposure to 0-12 Gy of X rays or 2 Gy of alpha particles. Approximately 8-9% of 446 clonal populations examined 23 population doublings after irradiation showed high frequencies of late-arising mutations as indicated by mutant fractions 10(2)-10(4)-fold above background. The frequency with which such clones occurred was similar for alpha-particle irradiation and X irradiation, with no apparent dose dependence for X irradiation over the range of 4-12 Gy. The molecular structure of Hprt mutations was determined by analysis by multiplex polymerase chain reaction of all nine exons. Of mutations induced directly after exposure to X rays, 75% involved partial or total gene deletions. Only 19-23% of late-arising (delayed) mutations were associated with deletions, the preponderance of these being partial deletions involving one or two exons. This spectrum was very similar to that for spontaneously arising mutations. To determine whether delayed mutations were non-clonal, the spectrum of exons deleted was examined among 29 mutants with partial deletions derived from a single clonal population. The results indicated that at least 15 of these mutants arose independently. To examine the relationship between the occurrence of delayed mutations and chromosomal instability, 60 Hprt mutant subclones isolated from a clonal population showing a high frequency of delayed mutations were serially cultivated in vitro. Of these, 14 showed a slow-growth phenotype with a high frequency of polyploid cells (10-38%) and a markedly enhanced frequency of non-clonal chromosomal rearrangements including both chromosome-type and chromatid-type aberrations. These clones also showed a 3- to 30-fold increase in the frequency of ouabain-resistant mutations; no ouabain-resistant mutants were induced directly by X irradiation. These results suggest that among clones showing a high frequency of delayed mutations there may be a subpopulation of cells that are particularly unstable; selection for the slow-growth phenotype has the effect of selecting for this chromosomally unstable subpopulation.

Heat-induced Lethality and Chromosomal Damage in Synchronized Chinese Hamster Cells Treated with 5-bromodeoxyuridine

SummarySynchronous Chinese hamster cells exposed to heat treatments of 45·5°c were very sensitive during S phase when chromosomal damage could account for cell killing, i.e. as for x-irradiated cells, the 37 per cent survival value for heated cells was observed when there was about one aberration per cell. However, the aberration frequency was very low and could not account for cell killing when the cells were heated, either during mitosis when they were most sensitive in terms of lethality, or during G1 when they were most resistant. Furthermore, about 50 per cent of the deletions in these cells heated in mitosis or G, occurred at the secondary constriction of the X chromosome. Incorporation of BUdR into DNA prior to synchronizing the cells had only an additive interaction with heat administered during G1 or S. This interaction was characterized by a reduction in the shoulder of the dose-response curves observed for both chromosomal aberrations and cell killing.These findings contrast greatly with those ...

HPRT Mutants Induced in Bystander Cells by Very Low Fluences of Alpha Particles Result Primarily from Point Mutations

Abstract Huo, L., Nagasawa, H. and Little, J. B. HPRT Mutants Induced in Bystander Cells by Very Low Fluences of Alpha Particles Result Primarily from Point Mutations. Radiat. Res. 156, 521–525 (2001). We have shown previously that damage signals may be transmitted from irradiated cells to nonirradiated cells in monolayer cultures, leading to changes in gene expression and an enhanced frequency of mutations in these “bystander” cells. The present study was designed to test the hypothesis that mutations occurring in bystander cells result from a different mechanism than those occurring in irradiated cells, and thus show differences in molecular structure. Structural changes in the HPRT gene of Chinese hamster ovary (CHO) cells were determined by multiplex PCR analysis. A total of 790 mutant clones derived from monolayer cultures exposed to mean doses of 0, 0.5 or 10 cGy of α-particle radiation (0, 3% or 44%, respectively, of nuclei traversed by one or more α particles) were examined. Whereas mutations induced by 10 cGy included a high frequency of deletions, nearly all mutations occurring in bystander cells in cultures irradiated with 0.5 cGy involved point mutations, confirming our hypothesis that they are induced by a different mechanism.

Effect of confluent holding on potentially lethal damage repair, cell cycle progression, and chromosomal aberrations in human normal and ataxia-telangiectasia fibroblasts

The effects of confluent holding recovery on survival, chromosomal aberrations, and progression through the life cycle after subculture of human diploid fibroblasts X-irradiated during density inhibition of growth have been examined. The responses of three normal strains were determined and compared with those of four ataxia-telangiectasia (AT), an AT heterozygote, and two hereditary retinoblastoma strains. The capacity for potentially lethal damage repair (PLDR) was slightly reduced in retinoblastoma cells and almost absent in AT cells, but normal in an AT heterozygote. The decline in chromosomal aberrations seen in normal cells during confluent holding was absent in AT cells, consistent with the lack of PLDR. Following subculture, all irradiated AT fibroblasts progressed through the cell cycle to the first mitosis with no delay. AT heterozygotic and retinoblastoma cells showed both an enhanced delay in the initiation of DNA synthesis and a large fraction of cells irreversibly blocked in G1 as compared with normal cells. Both the delayed entry into S and the G1 block were reduced by confluent holding. These results indicate that AT homozygotic and heterozygotic cells respond quite differently to X irradiation.

Comparison of kinetics of X-ray-induced cell killing in normal, ataxia telangiectasia and hereditary retinoblastoma fibroblasts.

Survival, cumulative labeling indices and chromosomal aberrations were studied in normal, ataxia telangiectasia (AT) and hereditary retinoblastoma fibroblasts after X-irradiation during density-inhibition of growth and immediate release by subculture to low density. The D0 of the survival curves were: normal strains, 150-160 rad; Retinoblastoma strains AG 1880, 95 rad; AG 1978, 40-50 rad (sensitive fraction); AT5BI, 45 rad. Mainly chromosome-type Aberrations were induced in normal and retinoblastoma cells. The frequency of X-ray-induced chromosomal aberrations was much higher in AT5BI cells, and 33-45% were of the chromatid type. Normal and retinoblastoma cells showed a measureable X-ray induced G1 delay before entering S. In addition, a fraction of the cells showed an apparently irreversible G1 block; these cells did not initiate DNA synthesis up to 120 h post-irradiation and subculture. The G1 block was much more marked in retinoblastoma cells; after 400 rad about 70% of retinoblastoma cells did not enter S as compared with only 20% of normal cells. Neither a G1 delay nor a G1 block was observed in AT cells irradiated with up to 400 rad despite their hypersensitivity to cell killing by X-rays and evidence of severe chromosome damage. These results suggest different mechanisms for the X-ray hypersensitivity of AT and retinoblastoma cells.

Effects of X-irradiation on cell-cycle progression, induction of chromosomal aberrations and cell killing in ataxia telangiectasia (AT) fibroblasts

Survival, cumulative labeling indices, chromosomal aberrations and cell-cycle distribution by flow microfluorometry (FMF) were studied in fibroblasts from normal and three ataxia telangiectasia (AT) families after X-irradiation during density-inhibition of growth and immediate release by subculture to low density. Homozygotic AT (proband) fibroblasts were very hypersensitive to cell killing by X-irradiation (D0 = 40-45 rad). Fibroblasts from AT heterozygotes (parents) were minimally hypersensitive, with D0s (100-110 rad) slightly lower than those for normal fibroblasts (D0 = 120-140 rad). There were three different response groups for a G1 phase block induced by 400 rad of X-rays: (1) minimal or no G1 block was observed in AT homozygote cell strains; (2) 10-20% of the cells were blocked in G1 in normal cell strains; and (3) 50% or more of the cells were blocked in AT heterozygote strains. FMF profiles and cumulative labeling indices showed that homozygotic AT cells irradiated in plateau phase moved into the S-phase following subculture with no additional delay over non-irradiated controls. Homozygotic AT cells showed not only a 4-5 times higher frequency of X-ray-induced chromosomal aberrations than normal strains, but approximately 30% of these were of the chromatid-type. There were no differences in the frequency or type of X-ray-induced chromosomal aberrations between normal and heterozygotic AT cells.

Involvement of the Nonhomologous End Joining DNA Repair Pathway in the Bystander Effect for Chromosomal Aberrations

Abstract Little, J. B., Nagasawa, H., Li, G. C. and Chen, D. J. Involvement of the Nonhomologous End Joining DNA Repair Pathway in the Bystander Effect for Chromosomal Aberrations. Radiat. Res. 159, 262–267 (2003). Cells of mouse knockout cell lines for Ku80 (now known as Xrcc5), Ku70 (now known as G22p1), DNA-PKcs (now known as Prkdc) and PARP (now known as Adprt) were synchronized in G1 phase and exposed to very low fluences of α particles. The frequency of gross chromosomal aberrations was scored at the first postirradiation metaphase. At the two lowest doses examined, aberrations were induced in 4–9% of wild-type cells and 36–55% of Xrcc5−/− cells, whereas only 2–3% of the nuclei were traversed by an α particle and thus received any radiation exposure. G22p1−/− cells responded similarly to Xrcc5−/− cells, whereas Prkdc−/− and Adprt−/− cells showed an intermediate effect. The frequency of aberrations per nuclear traversal increased approximately 30-fold for Xrcc5−/− and G22p1−/− cells at the lowest mean dose examined (0.17 cGy), compared with 10-fold in Prkdc−/− cells and 3-fold in wild-type cells. Based on these and other findings, we hypothesize that the marked sensitization of repair-deficient bystander cells to the induction of chromosomal aberrations is a consequence of unrejoined DNA double-strand breaks occurring as a result of clustered damage arising from opposed oxidative lesions and single-strand breaks.

Relationship of DNA repair to chromosome aberrations, sister-chromatid exchanges and survival during liquid-holding recovery in X-irradiated mammalian cells

The repair of X-ray-induced DNA single strand breaks and DNA--protein cross-links was investigated in stationary phase, contact-inhibited mouse cells by the alkaline-elution technique. Approx. 90% of X-ray-induced single strand breaks were rejoined during the first hour of repair, whereas most of the remaining breaks were rejoined more slowly during the next 5 h. At early repair times, the number of residual non-rejoined single strand breaks was approx. proportional to the X-ray dose. DNA--protein cross-links were removed at a slower rate (T1/2 approx. 10--12 h). Cells were held in stationary growth for various periods of time after irradiation before subculture at low density to score for colony survival (potentially lethal damage repair), chromosome aberrations in the first mitosis, and sister-chromatid exchanges in the second mitosis. Both cell killing and the frequency of chromosome aberrations decreased during the first several hours of recovery, reaching a minimum level by 6 h; this decrease correlated temporally with the repair of the slowly rejoining DNA-strand breaks. Relatively few sister-chromatid exchanges were observed when the cells were subcultured immediately after X-ray. The exchange frequency rose to maximum levels after a 4-h recovery interval, and returned to control levels after 12 h of recovery. The possible relationship of DNA repair to these changes in survival, chromosome aberrations, and sister-chromatid exchanges during liquid-holding recovery is discussed.