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Gimeracil, an inhibitor of dihydropyrimidine dehydrogenase (DPYD), partially inhibits homologous recombination (HR) repair and has a radiosensitizing effect as well as enhanced sensitivity to Camptothecin (CPT). DPYD is the target protein for radiosensitization by Gimeracil. We investigated the mechanisms of sensitization of radiation and CPT by DPYD inhibition using DLD-1 cells treated with siRNA for DPYD. We investigated the focus formation of various kinds of proteins involved in HR and examined the phosphorylation of RPA by irradiation using Western blot analysis. DPYD depletion by siRNA significantly restrained the formation of radiation-induced foci of Rad51 and RPA, whereas it increased the number of foci of NBS1. The numbers of colocalization of NBS1 and RPA foci in DPYD-depleted cells after radiation were significantly smaller than in the control cells. These results suggest that DPYD depletion is attributable to decreased single-stranded DNA generated by the Mre11/Rad50/NBS1 complex-dependent resection of DNA double-strand break ends. The phosphorylation of RPA by irradiation was partially suppressed in DPYD-depleted cells, suggesting that DPYD depletion may partially inhibit DNA repair with HR by suppressing phosphorylation of RPA. DPYD depletion showed a radiosensitizing effect as well as enhanced sensitivity to CPT. The radiosensitizing effect of DPYD depletion plus CPT was the additive effect of DPYD depletion and CPT. DPYD depletion did not have a cell-killing effect, suggesting that DPYD depletion may not be so toxic. Considering these results, the combination of CPT and drugs that inhibit DPYD may prove useful for radiotherapy as a method of radiosensitization.

To study mechanisms which could be involved in the reverse dose rate effect observed during mutation induction after exposure to high LET radiation, synchronized mouse L5178Y cells were exposed to carbon 290 MeV/n beams with different LET values at the G2/M, G1, G1/S or S phases in the cell cycle. The frequency of Hprt-deficient (6-thioguanine-resistant) mutant induction was subsequently determined. The results showed that after exposure to high LET value radiation (50.8 and 76.5 keV/μm), maximum mutation frequencies were seen at the G2/M phase, but after exposure to lower LET radiation (13.3 keV/μm), the highest mutation frequencies were observed at the G1 phase. The higher LET beam always produced higher mutation frequencies in the G2/M phase than in the G1 phase, regardless of radiation dose. These results suggest that cells in the G2/M phase is hyper-sensitive for mutation induction from high LET radiation, but not to mutation induction from low LET radiation. Molecular analysis of mutation spectra showed that large deletions (which could include almost entire exons) of the mouse Hprt gene were most efficiently induced in G2/M cells irradiated with high LET radiation. These entire exon deletions were not as frequent in cells exposed to lower LET radiation. This suggests that inappropriate recombination repair might have occurred in response to condensed damage in condensed chromatin in the G2/M phase. In addition, by using a hyper-sensitive mutation detection system (GM06318-10 cells), a reverse dose-rate effect was clearly observed after exposure to carbon beams with higher LET values (66 keV/μm), but not after exposure to beams with lower LET values (13.3 keV/μm). Thus, G2/M sensitivity towards mutation induction, and the dependence on radiation LET values could both be major factors involved in the reverse dose rate effect produced by high LET radiation.

DNA double strand break (DSB) is one of the most critical types of damage which is induced by ionizing radiation. In this review, we summarize current progress in investigations on the function of DSB repair-related proteins. We focused on recent findings in the analysis of the function of proteins such as 53BP1, histone H2AX, Mus81-Eme1, Fanc complex, and UBC13, which are found to be related to homologous recombination repair or to non-homologous end joining. In addition to the function of these proteins in DSB repair, the biological function of nuclear foci formation following DSB induction is discussed.

The BRCT domain is an evolutionarily conserved domain found in a large number of proteins involved in DNA repair, recombination and damage checkpoints. To elucidate the function of the PTIP protein which carries 6 BRCT domains, we constructed constitutive (chPTIP-/-/-) and conditional PTIP-null mutants (tet-off hPTIP, chPTIP-/-/-) in chicken DT40 cells. In contrast to mouse PTIP-null mutant cells. DT40 PTIP-null mutants are viable, but appear to have a defective proliferative capacity, since a high proportion of dead cells appear during culture growth and colony formation ability in methyl cellulose containing medium was less than 1% of that of wild type cells. Conditional PTIP mutants express human PTIP (hPTIP) in the absence of doxycycline (Dox) but expression levels decrease to almost undetectable levels within 48 hours after the addition of Dox. Starting from the 7th day after the addition of Dox, the cell culture s begin to produce deal cells and show chromatid-type gaps. Conditional PTIP mutants become sensitive to the lethal effects of ionizing radiation (IR) at 1 to 2 days after the addition of Dox [D10(+Dox)/D10(-Dox)=0.8], but not to the effects of UV and MMS, and the cells display low levels of resistance to MMC. The hPTIP polypeptide consisting of the two N-terminal BRCT domains was able to extensively complement cell proliferation defects and high radiation sensitivity of constitutive PTIP mutants. Since PTIP mutants showed a higher sensitivity to IR, we examined whether they were defective in double strand break repair or damage induced checkpoints. Western blot analysis revealed that the phosphorylation kinetics of H2AX after IR irradiation was similar in PTIP mutants and in wild type cells. Rad51 foci formation and disappearance after IR irradiation was normal, and homologous recombination operated normally in PTIP mutants. These results indicate that the PTIP protein is not involved in double strand break repair. The G2 block after IR irradiation also operated normally in PTIP mutants. However IR-induced phosphorylation of S345 in Chk1 was higher in PTIP mutants than in wild type cells. This enhanced phosphorylation of Chk1 S345 was not observed after UV irradiation in PTIP mutants. These results suggested that repair of IR induced DNA damage or IR induced signaling was defective in PTIP mutants., 13th International Congress of Radiation Research