Kazue Yuki

Evidence for the Involvement of Double-Strand Breaks in Heat-Induced Cell Killing

To identify critical events associated with heat-induced cell killing, we examined foci formation of γH2AX (histone H2AX phosphorylated at serine 139) in heat-treated cells. This assay is known to be quite sensitive and a specific indicator for the presence of double-strand breaks. We found that the number of γH2AX foci increased rapidly and reached a maximum 30 minutes after heat treatment, as well as after X-ray irradiation. When cells were heated at 41.5°C to 45.5°C, we observed a linear increase with time in the number of γH2AX foci. An inflection point at 42.5°C and the thermal activation energies above and below the inflection point were almost the same for cell killing and foci formation according to Arrhenius plot analysis. From these results, it is suggested that the number of γH2AX foci is correlated with the temperature dependence of cell killing. During periods when cells were exposed to heat, the cell cycle-dependent pattern of cell killing was the same as the cell cycle pattern of γH2AX foci formation. We also found that thermotolerance was due to a depression in the number of γH2AX foci formed after heating when the cells were pre-treated by heat. These findings suggest that cell killing might be associated with double-strand break formation via protein denaturation.

Glycerol as a chemical chaperone enhances radiation-induced apoptosis in anaplastic thyroid carcinoma cells

IntroductionAnaplastic thyroid carcinoma, which is one of the most aggressive, malignant tumors in humans, results in an extremely poor prognosis despite chemotherapy and radiotherapy. The present study was designed to evaluate therapeutic effects of radiation by glycerol on p53-mutant anaplastic thyroid carcinoma cells (8305c cells). To examine the effectiveness of glycerol in radiation induced lethality for anaplastic thyroid carcinoma 8305c cells, we performed colony formation assay and apoptosis analysis.ResultsApoptosis was analyzed with Hoechst 33342 staining and DNA ladder formation assay. 8305c cells became radiosensitive when glycerol was added to culture medium before X-ray irradiation. Apoptosis was induced by X-rays in the presence of glycerol. However, there was little apoptosis induced by X-ray irradiation or glycerol alone. The binding activity of whole cell extracts to bax promoter region was induced by X-rays in the presence of glycerol but not by X-rays alone.ConclusionThese findings suggest that glycerol is effective against radiotherapy of p53-mutant thyroid carcinomas.

Sensitization by glycerol for CDDP-therapy against human cultured cancer cells and tumors bearing mutated p53 gene

To clarify effective chemotherapeutic regimens against cancer, we examined the effects of glycerol on apoptosis induced by CDDP treatment using cultured human cancer cells (in vitro) and transplanted tumor in mice (in vivo). Human tongue cell carcinoma (SAS) cells transfected with mutated p53 gene (SAS/mp53) showed CDDP-resistance compared with the cells with neo control gene (SAS/neo). When those cultured cells were pre-treated with glycerol, CDDP-induced apoptosis was enhanced by glycerol in SAS/mp53 cells but not in SAS/neo cells.In tumor-transplanted mice, the glycerol treatment to tumors enhanced growth delay induced by CDDP in mp53 tumors transplanted with SAS/mp53 cells, but not in wtp53 tumors transplanted with SAS/neo cells. When transplanted tumors were treated with CDDP alone, the cells positive for active caspase-3, 85kDa PARP and apoptosis were observed by immunohistochemical staining in wtp53 tumors but not in mp53 tumors. When the tumors were treated with CDDP combined with glycerol, positive cells were observed not only in wtp53 tumors but also in mp53 tumors. These results showed that the CDDP-induced growth inhibition of the tumors is p53-dependent and that the enhanced growth delay by glycerol may be due to the increased apoptosis. Glycerol might be available for cancer chemotherapy in patients with m{p53} tumors.

C-terminal peptides of p53 molecules enhance radiation-induced apoptosis in human mutant p53 cancer cells

We propose here a novel p53-targeting radio-cancer therapy using p53 C-terminal peptides for patients having mutated p53. Hoechst 33342 staining showed that X-ray irradiation alone efficiently induced apoptotic bodies in wild-type p53 (wtp53) human head and neck cancer cells transfected with a neo control vector (SAS/neo cells), but hardly induced apoptotic bodies in mutation-type p53 (mp53) cells transfected with a vector carrying the mp53 gene (SAS/mp53). In contrast, transfection of p53 C-terminal peptides (amino acid residues 361-382 or 353-374) via liposomes caused a remarkable increase of apoptotic bodies in X-ray-irradiated SAS/mp53 cells, but did not enhance apoptotic bodies in X-ray-irradiated SAS/neo cells. In immunocytochemical analysis, positively stained cells for active type caspase-3 were observed at high frequency after X-ray irradiation in the SAS/mp53 cells pre-treated with p53 C-terminal peptides. In SAS/neo cells, positively stained cells for active type caspase-3 were observed with X-ray irradiation alone. Furthermore, protein extracts from X-ray-irradiated SAS/mp53 cells showed higher DNA-binding activity of p53 to p53 consensus sequence when supplemented in vitro with p53 C-terminal peptides than extracts from non-irradiated SAS/mp53 cells. These results suggest that radiation treatment in the presence of p53 C-terminal peptides is more effective for inducing p53-mediated apoptosis than radiation treatment alone or p53 C-terminal peptide treatment alone, especially in mp53 cancer cells. This novel tool for enhancement of apoptosis induction in mp53 cells might be useful for p53-targeted radio-cancer therapy.