Fumio Yatagai

Inactivation of Aerobic and Hypoxic Cells from Three Different Cell Lines by Accelerated 3He-, 12C- and 20Ne-Ion Beams

Abstract Furusawa, Y., Fukutsu, K., Aoki, M., Itsukaichi, H., Eguchi-Kasai, K., Ohara, H., Yatagai, F., Kanai, T. and Ando, K. Inactivation of Aerobic and Hypoxic Cells from Three Different Cell Lines by Accelerated 3He-, 12C- and 20Ne-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 3He-, 12C- and 20Ne-ion beams with an LET ranging from approximately 20–600 keV/μm 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, α and D0 were analyzed as a function of LET. The RBE increased with LET, reaching a maximum at around 200 keV/μm, 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 3He 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/μm, passed below 2 at around 100 keV/μm, and then reached a minimum above 300 keV/μm, but the values remained greater than 1. The OER was significantly lower for 3He ions than the others.

Transcriptional Infidelity Promotes Heritable Phenotypic Change in a Bistable Gene Network

Bistable epigenetic switches are fundamental for cell fate determination in unicellular and multicellular organisms. Regulatory proteins associated with bistable switches are often present in low numbers and subject to molecular noise. It is becoming clear that noise in gene expression can influence cell fate. Although the origins and consequences of noise have been studied, the stochastic and transient nature of RNA errors during transcription has not been considered in the origin or modeling of noise nor has the capacity for such transient errors in information transfer to generate heritable phenotypic change been discussed. We used a classic bistable memory module to monitor and capture transient RNA errors: the lac operon of Escherichia coli comprises an autocatalytic positive feedback loop producing a heritable all-or-none epigenetic switch that is sensitive to molecular noise. Using single-cell analysis, we show that the frequency of epigenetic switching from one expression state to the other is increased when the fidelity of RNA transcription is decreased due to error-prone RNA polymerases or to the absence of auxiliary RNA fidelity factors GreA and GreB (functional analogues of eukaryotic TFIIS). Therefore, transcription infidelity contributes to molecular noise and can effect heritable phenotypic change in genetically identical cells in the same environment. Whereas DNA errors allow genetic space to be explored, RNA errors may allow epigenetic or expression space to be sampled. Thus, RNA infidelity should also be considered in the heritable origin of altered or aberrant cell behaviour.

Contributions of Direct and Indirect Actions in Cell Killing by High-LET Radiations

Abstract Hirayama, R., Ito, A., Tomita, M., Tsukada, T., Yatagai, F., Noguchi, M., Matsumoto, Y., Kase, Y., Ando, K., Okayasu, R., and Furusawa, F. Contributions of Direct and Indirect Actions in Cell Killing by High-LET Radiations. Radiat. Res. 171, 212–218 (2009). The biological effects of radiation originate principally in damages to DNA. DNA damages by X rays as well as heavy ions are induced by a combination of direct and indirect actions. The contribution of indirect action in cell killing can be estimated from the maximum degree of protection by dimethylsulfoxide (DMSO), which suppresses indirect action without affecting direct action. Exponentially growing Chinese hamster V79 cells were exposed to high-LET radiations of 20 to 2106 keV/μm in the presence or absence of DMSO and their survival was determined using a colony formation assay. The contribution of indirect action to cell killing decreased with increasing LET. However, the contribution did not reach zero even at very high LETs and was estimated to be 32% at an LET of 2106 keV/μm. Therefore, even though the radiochemically estimated G value of OH radicals was nearly zero at an LET of 1000 keV/μm, indirect action by OH radicals contributed to a substantial fraction of the biological effects of high-LET radiations. The RBE determined at a survival level of 10% increased with LET, reaching a maximum value of 2.88 at 200 keV/μm, and decreased thereafter. When the RBE was estimated separately for direct action (RBED) and indirect action (RBEI); both exhibited an LET dependence similar to that of the RBE, peaking at 200 keV/μm. However, the peak value was much higher for RBED (5.99) than RBEI (1.89). Thus direct action contributes more to the high RBE of high-LET radiations than indirect action does.

Let dependence of cell death, mutation induction and chromatin damage in human cells irradiated with accelerated carbon ions

We investigated the LET dependence of cell death, mutation induction and chromatin break induction in human embryo (HE) cells irradiated by accelerated carbon-ion beams. The results showed that cell death, mutation induction and induction of non-rejoining chromatin breaks detected by the premature chromosome condensation (PCC) technique had the same LET dependence. Carbon ions of 110 to 124keV/micrometer were the most effective at all endpoints. However, the number of initially induced chromatin breaks was independent of LET. About 10 to 15 chromatin breaks per Gy per cell were induced in the LET range of 22 to 230 keV/micrometer. The deletion pattern of exons in the HPRT locus, analyzed by the polymerase chain reaction (PCR), was LET-specific. Almost all of the mutants induced by 124 keV/micrometer beams showed deletion of the entire gene, while all mutants induced by 230keV/micrometer carbon-ion beams showed no deletion. These results suggest that the difference in the density distribution of carbon-ion track and secondary electron with various LET is responsible for the LET dependency of biological effects.

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 G2 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 G1 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/G1 phase. Dissociation of Mcm10 from chromatin in G2/M phase was concomitant with alterations in the electrophoretic mobility of the protein. Treatment with λ 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.

LET dependence of cell death and chromatin-break induction in normal human cells irradiated by neon-ion beams

We investigated the LET dependence of cell death and chromatin-break induction in normal human embryo cells irradiated by accelerated neon-ion beams. Neon-ion beams were generated by the Riken Ring Cyclotron (RRC) at the Institute of Physical and Chemical Research, Japan. Chromatin breaks were measured by counting the number of chromatin fragments detected by the premature chromosome condensation (PCC) technique. The results indicated that cell death and the induction of remaining chromatin breaks showed a qualitatively similar LET dependence. The LET RBE curves for both cell death and the induction of remaining chromatin breaks had a broad peak in the LET range of 120-300 keV/microm and steeply downward trend up to 340 keV/microm. These results suggest that there is a good correlation between cell death and the induction of remaining chromatin breaks by neon-ion beams with different LET values.

Delayed Cell Cycle Progression in Human Lymphoblastoid Cells after Exposure to High-LET Radiation Correlates with Extremely Localized DNA Damage

Abstract Goto, S., Watanabe, M. and Yatagai, F. Delayed Cell Cycle Progression in Human Lymphoblastoid Cells after Exposure to High-LET Radiation Correlates with Extremely Localized DNA Damage. Radiat. Res. 158, 678–686 (2002). To compare the genotoxic effects of high-LET ionizing radiation to those of low-LET radiation, we investigated the responses of human lymphoblastoid cells to DNA damage TK6 after treatment with either low-LET X rays or high-LET iron ions (1000 keV/μm). A highly localized distribution of γH2AX/RAD51 foci was observed in the nuclei of cells irradiated with iron ions, in sharp contrast to cells exposed to X rays, where the distribution of foci was much more uniform. This implied the occurrence of a relatively high frequency of closely spaced double-strand breaks, i.e. clustered DNA damage, after iron-ion exposure. Despite the well-established notion that clustered DNA damage is refractory to repair compared to isolated DNA lesions, there were no significant differences in the levels of clonogenic survival and apoptosis between cells treated with iron ions or X rays. Strikingly, however, cells accumulated in G2/M phase to a much lesser extent after iron-ion exposure than after X-ray exposure. This differential accumulation could be attributed to a much slower evacuation of the S-phase compartment in the case of cells irradiated with iron ions. Taken together, our results indicate that, relative to the situation for low-LET X rays, exposure to high-LET iron ions results in a substantially greater inhibition of S-phase progression as a result of a higher frequency of DNA replication-blocking clustered DNA damage.

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.

The role of DNA repair on cell killing by charged particles

It can be noted that it is not simple double strand breaks (dsb) but the non-reparable breaks that are associated with high biological effectiveness in the cell killing effect for high LET radiation. Here, we have examined the effectiveness of fast neutrons and low (initial energy = 12 MeV/u) or high (135 MeV/u) energy charged particles on cell death in 19 mammalian cell lines including radiosensitive mutants. Some of the radiosensitive lines were deficient in DNA dsb repair such as LX830, M10, V3, and L5178Y-S cells and showed lower values of relative biological effectiveness (RBE) for fast neutrons if compared with their parent cell lines. The other lines of human ataxia-telangiectasia fibroblasts, irs 1, irs 2, irs 3 and irs1SF cells, which were also radiosensitive but known as proficient in dsb repair, showed moderated RBEs. Dsb repair deficient mutants showed low RBE values for heavy ions. These experimental findings suggest that the DNA repair system does not play a major role against the attack of high linear energy transfer (LET) radiations. Therefore, we hypothesize that a main cause of cell death induced by high LET radiations is due to non-reparable dsb, which are produced at a higher rate compared to low LET radiations.

Radiosensitization by Hyperthermia in the Chicken B-Lymphocyte Cell Line DT40 and its Derivatives Lacking Nonhomologous End Joining and/or Homologous Recombination Pathways of DNA Double-Strand Break Repair

Abstract Yin, H. L., Suzuki, Y., Matsumoto, Y., Tomita, M., Furusawa, Y., Enomoto, A., Morita, A., Aoki, M., Yatagai, F., Suzuki, T., Hosoi, Y., Ohtomo, K. and Suzuki, N. Radiosensitization by Hyperthermia in the Chicken B-Lymphocyte Cell Line DT40 and its Derivatives Lacking Nonhomologous End Joining and/or Homologous Recombination Pathways of DNA Double-Strand Break Repair. Radiat. Res. 162, 433–441 (2004). Hyperthermia has a radiosensitizing effect, which is one of the most important biological bases for its use in cancer therapy with radiation. Although the mechanism of this effect has not been clarified in molecular terms, possible involvement of either one or both of two major DNA double-strand break (DSB) repair pathways, i.e. nonhomologous end joining (NHEJ) and homologous recombination (HR), has been speculated. To test this possibility, we examined cells of the chicken B-lymphocyte cell line DT40 and its derivatives lacking NHEJ and/or HR: KU70−/−, DNA-PKcs−/−/−, RAD54−/− and KU70−/−/RAD54−/−. Radiosensitization by hyperthermia could be seen in all of the mutants, including KU70−/−/RAD54−/−, which lacked both NHEJ and HR. Therefore, radiosensitization by hyperthermia cannot be explained simply by its inhibitory effects, if any, on NHEJ and/or HR alone. However, in NHEJ-defective KU70−/− and DNA-PKcs−/−/−, consisting of two subpopulations with distinct radiosensitivity, the radiosensitive subpopulation, which is considered to be cells in G1 and early S, was not sensitized. Substantial sensitization was seen only in the radioresistant subpopulation, which is considered to be cells in late S and G2, capable of repairing DSBs through HR. This observation did not exclude possible involvement of NHEJ in G1 and early S phase and also suggested inhibitory effects of hyperthermia on HR. Thus partial contribution of NHEJ and HR in radiosensitization by hyperthermia, especially that depending on the cell cycle stage, remains to be considered.