Mutation induction in human cells after low dose X ray exposure.

Mutations induced after low dose ionising radiation exposure have been intensively analysed not only for radiation risk estimation but also for basic understanding of cellular responses. Human lymphoblastoid TK6-20C cells were irradiated with 100 mGy of X rays and mutation at the heterozygous thymidine kinase (TK) locus was selected by trifluorothymidine (TFT) resistance. Although the mutation frequency at the TK locus increased from 5.6 x 10(-6) to 7.4 x 10(-6), this increase was not statistically significant. However, molecular analysis of TK mutants exhibiting loss of heterozygocity (LOH) demonstrated a clear effect of such low dose IR exposure. Exposure to 100 mGy X ray increased the fraction of hemizygous-LOH from 10% to 42%. In previous experiments, a similar tendency in the increase of hemizygous-LOH was also observed in TK6 cells after exposure to a 2 Gy dose of X rays. This type of LOH can be considered as a result of end-joining repair of DNA double strand breaks.

Cellular responses to low dose heavy-ion exposure in human cells.

The human lymphoblastoid cell line TK6 was used to study the cellular responses after low-dose (100, 200, 500 mGy) or high-dose (3 Gy) of X rays, C (22 keV.micron-1) and Fe (1000 keV.micron-1) ion exposures. p53 protein induction in individual cells was determined by indirect immunofluorescence staining. Cell-cycle progression after heavy-ion exposure was determined by using a laser scanning cytometer. A characteristic pattern of cell-cycle progression was observed with 3 Gy exposure of Fe ions but not with 100 mGy. Similarly such a pattern with 100 mGy C ion exposure did not match that with 3 Gy. The proportion of p53-induced cells is proportional to the probability of cell being hit by a primary heavy ion. The observed low-dose effect can be reflected in the probability of a hit, although detailed nature about their energy deposition must be considered for more precise estimation of such an effect. New detection methodology must be developed for identification of heavy-ion specific cellular responses.

Cellular responses by exposure to heavy-ions.

To better understand cellular responses in human lymphoblastoid cell TK6 after exposure to C-ion (22 keV/micrometer) and Fe-ion (1000 keV/micrometer), both protein induction and cell-cycle progression have been extensively analyzed by the recently developed techniques. While proceeding this line of analyses, we realized the importance of studying low-dose effect, in relation to the genetic alterations. Adaptive response by 5~20 cGy of such C- or Fe-ion irradiation to both lethal and mutagenic effects of the challenging X-ray exposure (1~3 Gy) was difficult to be seen in this TK6 cells, but surprisingly, a relatively high level of p53 and its related proteins induction was observed after low-dose irradiations of heavy-ions. Here, we focus to introduce the above results of genetic and biochemical studies to elucidate the adaptive response.

Cell cycle-dependent proteolysis and phosphorylation of human Mcm10.

Mcm10 (Dna43) is an essential protein for chromosomal DNA replication in Saccharomyces cerevisiae. Recently, we identified a human Mcm10 homolog that interacts with the mammalian Orc2 and Mcm2-7 complex. We additionally demonstrated that human Mcm10 binds nuclease-resistant nuclear structures during S phase and dissociates from them in G(2) phase. In this study, we have further characterized the subcellular localization, modification, and expression levels of human Mcm10 protein throughout the cell cycle. Human Mcm10 protein decreased in late M phase, remained low during G(1) phase, started to accumulate, and bound chromatin at the onset of S phase. Proteasome inhibitors stabilized Mcm10 levels, suggesting that proteolysis is involved in the down-regulation of the protein in late M/G(1) phase. Dissociation of Mcm10 from chromatin in G(2)/M phase was concomitant with alterations in the electrophoretic mobility of the protein. Treatment with lambda phosphatase revealed that mobility shifts were due to hyperphosphorylation. These results indicate that human Mcm10 is regulated by proteolysis and phosphorylation in a cell cycle-dependent manner. It is further suggested that mammalian Mcm10 is involved in S phase progression, and not the formation of a prereplicative complex, as previously proposed from data on the S. cerevisiae protein.

Judgement on "hit or non-hit" of CHO cells exposed to accelerated heavy-ions (Fe- or Ar-ions) using division delay and CR-39 plastics as an indicator.

The cell killing effect of ionizing radiation depends on the degree of linear energy transfer (LET). The relative biological effectiveness (RBE) reaches a maximum at LET of around 100-200 keV/micron and decreases at higher levels. The ion clusters produced by high-LET radiation are not uniformly distributed. The incidence of non-hit cell events is higher in high LET irradiation than in the cases of low-LET irradiation. This fact could explain the decrease in the cell killing effect at higher levels of LET irradiation. Since the cell killing effect may be related to the nuclear traversal of heavy-ions, it is necessary to establish methods to distinguish the hit cells from the non-hit cells, especially in case with high LET irradiation. Using time-lapse photography, we first examined the hit events by observing the division delay in the cells caused by high-LET irradiation. In addition, we explored the use of CR-39 plastics to detect the exact position of heavy-ion traversal on the surface of a flask where cells were growing. When Chinese hamster ovary (CHO-K1) cells were exposed to 4 Gy of accelerated Fe-ions (2000 keV/micron) or Ar (1640 keV/micron)-ions, the surviving fraction decreased to about 30% in both cases of irradiation. Eighty percent of the irradiated cells, suffered a division delay in contrast to the remaining 20% of the cells which showed a normal division time (12-13 hrs). The later 20% of the cells is considered to be a population of cells which were not actually traversed by heavy-ions. The difference between the higher values of the surviving fraction (approximately 30%) and the non-hit cell population (20%) indicates that some hit cells can grow even after being hit by heavy-ions. The fraction of recovered cells determined by the time-lapse photography method was 10%, and this value closely correlated with the difference between the surviving fraction and the non-hit cells. We used the Poisson distribution of the hit-events by heavy-ions among the cell population in order to calculate the fraction of cells receiving at least a single-hit in the cell nucleus (130 micron 2 in average size). From this calculation we determined that 80% of the cells had a single hit to their nuclei by a heavy-ion which induced such early cellular responses as division delay. Our finding in the experiments using CR-39 plastics as a detector for hit-sites further supported the idea that the hit lethality of a cell is related to heavy-ion traversal through its nucleus. This study indicates the possible usefulness of both the division delay and CR-39 plastic methods for evaluating the biological effects of heavy-ions, especially when these two methods are combined.

Effects of ionic valency of interacting metal elements in ion uptake by carrot (Daucas carota cv. U.S. harumakigosun).

Interaction of elements in the course of element uptake by carrot (Daucas carota cv. U.S. harumakigosun) exerted by the addition of elements, such as Rb, Zn, and Al, was investigated. For the purpose of precise evaluation of uptake behavior, the simultaneous determination of absorption of Na, Be, Sr, Mn, Co, Zn, Ce, Pm, and Gd was conducted by the multitracer technique. For root uptakes, Al exhibited its influence on the uptake of essential elements and on the uptake of toxic or unbeneficial ones, presumably as a result of the large electric valency that caused cell membrane disintegrity. On the other hand, Zn as a divalent cation only affected the uptake of essential and beneficial elements. Rubidium, which is a monovalent cation, did not exhibit any effect on the uptake of other ions. Concerning shoot uptakes, inhibition by Zn and Al, but not by Rb, was observed for the uptake of Sr, Mn, Co, and Zn. From the present investigation, it is suggested that there exists an interaction between added ions and the elements taken into plants and that the degree of interaction increases in the increasing order of ionic valency: M+ (Rb), M2+ (Zn), and M3+ (Al).

Mutation induction by heavy-ion irradiation of gpt delta transgenic mice.

Using the new transgenic mice produced by mating gpt delta with p53 knockout, mutation induction by heavy-ion irradiation and the effect of p53 background on such induction were studied. After the whole body irradiation with 10 Gy of 135 MeV/u carbon-ion beam, the genomic DNA was isolated from the different organs and the lambda DNA was rescued as a phage. Mutations in the transgene on the lambda DNA were determined by the spi(-) selection (deletion assay). The spi(-) mutation was induced by the above irradiation, but enhancement of the mutant frequency by the knockout of p53 gene was found not in the phages recovered from liver but in those from kidney. We are now making an effort to determine the nature of spi(-) mutation to confirm such p53 effect.

The human homolog of Saccharomyces cerevisiae Mcm10 interacts with replication factors and dissociates from nuclease-resistant nuclear structures in G(2) phase.

Mcm10 (Dna43), first identified in Saccharomyces cerevisiae, is an essential protein which functions in the initiation of DNA synthesis. Mcm10 is a nuclear protein that is localized to replication origins and mediates the interaction of the Mcm2-7 complex with replication origins. We identified and cloned a human cDNA whose product was structurally homologous to the yeast Mcm10 protein. Human Mcm10 (HsMcm10) is a 98-kDa protein of 874 amino acids which shows 23 and 21% overall similarity to Schizosaccharomyces pombe Cdc23 and S. cerevisiae Mcm10, respectively. The messenger RNA level of HsMcm10 increased at the G(1)/S-boundary when quiescent human NB1-RGB cells were induced to proliferate as is the case of many replication factors. HsMcm10 associated with nuclease-resistant nuclear structures throughout S phase and dissociated from it in G(2) phase. HsMcm10 associated with human Orc2 protein when overexpressed in COS-1 cells. HsMcm10 also interacted with Orc2, Mcm2 and Mcm6 proteins in the yeast two-hybrid system. These results suggest that HsMcm10 may function in DNA replication through the interaction with Orc and Mcm2-7 complexes.

Inactivation of aerobic and hypoxic cells from three different cell lines by accelerated (3)He-, (12)C- and (20)Ne-ion beams.

The LET-RBE spectra for cell killing for cultured mammalian cells exposed to accelerated heavy ions were investigated to design a spread-out Bragg peak beam for cancer therapy at HIMAC, National Institute of Radiological Sciences, Chiba, prior to clinical trials. Cells that originated from a human salivary gland tumor (HSG cells) as well as V79 and T1 cells were exposed to (3)He-, (12)C- and (20)Ne-ion beams with an LET ranging from approximately 20-600 keV/micrometer under both aerobic and hypoxic conditions. Cell survival curves were fitted by equations from the linear-quadratic model and the target model to obtain survival parameters. RBE, OER, alpha and D(0) were analyzed as a function of LET. The RBE increased with LET, reaching a maximum at around 200 keV/micrometer, then decreased with a further increase in LET. Clear splits of the LET-RBE or -OER spectra were found among ion species and/or cell lines. At a given LET, the RBE value for (3)He ions was higher than that for the other ions. The position of the maximum RBE shifts to higher LET values for heavier ions. The OER value was 3 for X rays but started to decrease at an LET of around 50 keV/micrometer, passed below 2 at around 100 keV/micrometer, and then reached a minimum above 300 keV/micrometer, but the values remained greater than 1. The OER was significantly lower for (3)He ions than the others.

Transcription of the catalytic 180-kDa subunit gene of mouse DNA polymerase alpha is controlled by E2F, an Ets-related transcription factor, and Sp1.

We have isolated a genomic DNA fragment spanning the 5'-end of the gene encoding the catalytic subunit of mouse DNA polymerase alpha. The nucleotide sequence of the upstream region was G/C-rich and lacked a TATA box. Transient expression assays in cycling NIH 3T3 cells demonstrated that the GC box of 20 bp (at nucleotides -112/-93 with respect to the transcription initiation site) and the palindromic sequence of 14 bp (at nucleotides -71/-58) were essential for basal promoter activity. Electrophoretic mobility shift assays showed that Sp1 binds to the GC box. We also purified a protein capable of binding to the palindrome and identified it as GA-binding protein (GABP), an Ets- and Notch-related transcription factor. Transient expression assays in synchronized NIH 3T3 cells revealed that three variant E2F sites near the transcription initiation site (at nucleotides -23/-16, -1/+7 and +17/+29) had no basal promoter activity by themselves, but were essential for growth-dependent stimulation of the gene expression. These data indicate that E2F, GABP and Sp1 regulate the gene expression of this principal replication enzyme.

Mutation induction by heavy ion irradiation of gpt delta transgenic mice.

Using the new transgenic mice produced by mating gpt delta with p53 knockout, mutation induction by heavy-ion irradiation and the effect of p53 background on such induction were studied. After the whole body irradiation with 10 Gy of 135 MeV/u carbon-ion beam, the genomic DNA was isolated from the different organs and the lambda DNA was rescued as a lambda phage. Mutations in the transgene on the lambda DNA were determined by the spi(-) selection (deletion assay). The spi(-) mutation was induced by the above irradiation, but enhancement of the mutant frequency by the knockout of p53 gene was found not in the phages recovered from liver but in those from kidney. We are now making an effort to determine the nature of spi(-) mutation to confirm such p53 effect.

Mutation spectrum of MSH3-deficient HHUA/chr.2 cells reflects in vivo activity of the MSH3 gene product in mismatch repair.

The endometrial tumor cell line HHUA carries mutations in two mismatch repair (MMR) genes MSH3 and MSH6. We have established an MSH3-deficient HHUA/chr.2 cell line by introducing human chromosome 2, which carries wild-type MSH6 and MSH2 genes, to HHUA cells. Introduction of chromosome 2 to HHUA cells partially restored G:G MMR activity to the cell extract and reduced the frequency of mutation at the hypoxanthine-guanine phosphoribosyltransferase (hprt*) locus to about 3% that of the parental HHUA cells, which is five-fold the frequency in MMR-proficient cells, indicating that the residual mutator activity in HHUA/chr.2 is due to an MSH3-deficiency in these cells. The spectrum of mutations occurring at the HPRT locus of HHUA/chr.2 was determined with 71 spontaneous 6TG(r) clones. Base substitutions and +/-1 bp frameshifts were the major mutational events constituting, respectively, 54% and 42% of the total mutations, and more than 70% of them occurred at A:T sites. A possible explanation for the apparent bias of mutations to A:T sites in HHUA/chr.2 is haploinsufficiency of the MSH6 gene on the transferred chromosome 2. Comparison of the mutation spectra of HHUA/chr.2 with that of the MSH6-deficient HCT-15 cell line [S. Ohzeki, A. Tachibana, K. Tatsumi, T. Kato, Carcinogenesis 18 (1997) 1127-1133.] suggests that in vivo the MutSalpha (MSH2:MSH6) efficiently repairs both mismatch and unpaired extrahelical bases, whereas MutSbeta (MSH2:MSH3) efficiently repairs extrahelical bases and repairs mismatch bases to a limited extent.

Complex hprt deletion events are recovered after exposure of human lymphoblastoid cells to high-LET carbon and neon ion beams.

Hypoxanthine phosphoribosyltransferase gene (hprt) mutations were induced in human TK-6 lymphoblastoid cells by irradiation at a linear energy transfer (LET) of 250 or 310 keV/micron for carbon and neon ions, respectively. At such a high level of LET, ions will lose most of their total energy and stop shortly after passing through the cell. The hprt mutations were analyzed by multiplex PCR, long-PCR and DNA sequencing of both genomic and cDNA. Over half of the C ion-induced hprt mutations (10 of 19) were point mutations, in contrast to 15% of the mutations induced by Ne ions (three of 20). The remaining 47 and 85% of the C and Ne ion-induced mutants, respectively, are deletion events. The latter events include three complex losses of multiple non-contiguous exon regions in both ion irradiation collections. We note that mutations involving the exon 6 region are frequent in the Ne ion collection: all three of the complex events retained the exon 6 region with flanking deletion of sequence and three other mutants involved deletion of this region. It may be concluded that these high-LET C and Ne ion irradiations produce different mutational spectra.

Extension of in vitro life-span of gamma-irradiated human embryo cells accompanied by chromosome instability.

We studied the effect of repeated irradiation with a low dose rate (about 0.0012 cGy per min) of 60Co gamma-rays on the in vitro life-span of human embryo (HE) cells. HE cells were cultured in an incubator that was set in a 60Co gamma-ray-irradiation room, and the irradiation was repeated throughout the life-span of the HE cells (for about 150 to 160 days) on every day base. During this period, the cells accumulated 106 to 123 cGy. The life-span of the irradiated cells prolonged 1.14 to 1.35 times when compared to that of non-irradiated cells. The incidence of cells with chromosome bridge and micronuclei significantly increased in the irradiated cells. Although the number of chromosomes gradually changed with repetition of culture in both non-irradiated and irradiated cells, the frequencies of aneuploid cells in irradiated cells were about two times higher than that in non-irradiated cells. These results indicate that repeated irradiation with a low dose of gamma-rays produces chromosome instability, and it may be a cause of numerical chromosome abnormalities and life-span extension of irradiated cells.

Specificity of mutations in the PMS2-deficient human tumor cell line HEC-1-A.

The spectrum of mutations was determined at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in the human uterine tumor cell line HEC-1-A which is defective in the mismatch repair gene hPMS2. The mutation frequency at the hprt locus in HEC-1-A was about two orders higher than that in wild type repair-proficient cells. The fifty-eight mutations detected were exclusively point mutations, with frameshifts of one base deletion/addition predominating (66%) the remaining were base substitutions. All the frameshift mutations occurred at sites of monotonous repeating sequences, including six consecutive guanine bases site which was the hot spot for the addition of one G that contributed 60% of the total mutations. Although the observed specificity of mutations in HEC-1-A apparently resembled that of the hMLH1-deficient cell line HCT116 [Ohzeki, S., Tachibana, A., Tatsumi, T., Kato, T., 1997. Spectra of spontaneous mutations at the hprt locus in colorectal carcinoma cell lines defective in mismatch repair. Carcinogenesis, 18, 1127-1133.], the pronounced increase of +/-1 bp frameshifts and the reduced incidence of C-->T transitions at the CpG site suggest that the hPMS2 gene product may have an additional function in the mismatch repair process independent of it's role in the hMutLalpha heterodimer.

Repair of DNA double-strand breaks and cell killing by charged particles.

It has been suggested that it is not simple double-strand breaks (dsb) but the non-reparable breaks which correlate well with the high biological effectiveness of high LET radiations for cell killing (Kelland et al., 1988; Radford, 1986). We have compared the effects of charged particles on cell death in 3 pairs of cell lines which are normal or defective in the repair of DNA dsbs. For the cell lines SL3-147, M10, and SX10 which are deficient in DNA dsb repair, RBE values were close to unity for cell killing induced by charged particles with linear energy transfer (LET) up to 200 keV/micrometer and were even smaller than unity for the LET region greater than 300 keV/micrometer. The inactivation cross section (ICS) increased with LET for all 3 pairs. The ICS of dsb repair deficient mutants was always larger than that of their parents for all the LET ranges, but with increasing LET the difference in ICS between the mutant and its parent became smaller. Since a small difference in ICS remained at LET of about 300 keV/micrometer, dsb repair may still take place at this high LET, even if its role is apparently small. These results suggest that the DNA repair system does not play a major role in protection against the attack of high LET radiations and that a main muse of cell death is non-reparable dsb which are produced at a higher yield compared with low LET radiations. No correlation was observed between DNA content or nuclear area and ICS.

Dependence of induction of interphase death of Chinese hamster ovary cells exposed to accelerated heavy ions on linear energy transfer.

Induction of interphase death was examined in Chinese hamster ovary cells exposed to accelerated heavy ions (carbon, neon, argon and iron) of various linear energy transfers (LETs) (10-2000 keV/microm). The fraction of cells that underwent interphase death was determined by observing individual cells with time-lapse photography (direct method) as well as by counting cells undergoing interphase death made visible by the addition of caffeine (indirect method). After exposure to X rays, interphase death increased linearly with dose above a threshold of about 10 Gy, whereas it increased at a higher rate without a threshold after exposure to high-LET heavy ions. The relative biological effectiveness (RBE) compared to X rays, as determined at the 50% level of induction, increased with LET, reached a maximum at an LET of approximately 230 keV/microm and then decreased with further increase in LET. The range of LET values corresponding to the maximum RBE appears to be narrower for interphase death than for reproductive death (120-230 keV/microm), as assayed using loss of colony-forming ability as a criterion. The inactivation cross section for interphase cell death reached a plateau of 5-10 microm2. This means that the probability for the induction of interphase death by traversal of a single heavy-ion track through the nucleus (size: about 130 microm2) is about 0.04-0.08.