Ken Ohnishi

Induction of radioresistance by a nitric oxide-mediated bystander effect.

Abstract Matsumoto, H., Hayashi, S., Hatashita, M., Ohnishi, K., Shioura, H., Ohtsubo, T., Kitai, R., Ohnishi, T. and Kano, E. Induction of Radioresistance by a Nitric Oxide-Mediated Bystander Effect. To elucidate whether nitric oxide secreted from irradiated cells affects cellular radiosensitivity, we examined the accumulation of inducible nitric oxide synthase, TP53 and HSP72, the concentration of nitrite in the medium of cells after X irradiation, and cellular radiosensitivity using two human glioblastoma cell lines, A-172, which has a wild-type TP53 gene, and a transfectant of A-172 cells, A-172/mp53, bearing a mutated TP53 gene. Accumulation of inducible nitric oxide synthase was caused by X irradiation of the mutant TP53 cells but not of the wild-type TP53 cells. Accumulation of TP53 and HSP72 in the wild-type TP53 cells was observed by cocultivation with irradiated mutant TP53 cells, and the accumulation was abolished by the addition of an inhibitor for inducible nitric oxide synthase, aminoguanidine, to the medium. Likewise, accumulation of these proteins was observed in the wild-type TP53 cells after exposure to conditioned medium from irradiated mutant TP53 cells, and the accumulation was abolished by the addition of a specific nitric oxide scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, to the medium. The radiosensitivity of wild-type TP53 cells was reduced when the cells were cultured in conditioned medium from irradiated mutant TP53 cells compared to conventional fresh growth medium. Collectively, these findings indicate the potential importance of an intercellular signal transduction pathway initiated by nitric oxide in the cellular response to ionizing radiation.

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.

Hypertonic saline resuscitation reduces apoptosis and tissue damage of the small intestine in a mouse model of hemorrhagic shock.

&NA; The effect of hypertonic saline resuscitation on intestinal damage and the incidence of apoptosis after hemorrhagic shock were investigated. After anesthesia, male BALB/c mice weighing 24‐34 g were hemorrhaged to the mean arterial pressure of 40 ± 5 mmHg for 90 min. Animals were randomly assigned to four groups: 1) resuscitation with 4 mL/kg of 7.5% NaCl (hypertonic saline; HS) + shed blood (SB); 2) resuscitation with two times the volume of shed blood of lactated Ringers solution (2LR) + SB; 3) sham (catheter only); or 4) control (no treatment). Intestinal damage was graded based on the extent of the vacuolation at the basal area of the intestinal villi. Apoptosis of the small intestines was examined with the terminal deoxynucleotidyl transferase‐mediated deoxyuridine 5‐triphosphate nick‐end labeling method and with DNA laddering. Caspase‐3 activation, heat shock protein (HSP) 70, and HSP40 were assessed by western blotting. Apoptosis of the small intestine and intestinal damage were significantly lower (P < 0.01) in the HS+SB group compared with the 2LR+SB group 2 h and 6 h after hemorrhagic shock and resuscitation, respectively. This corresponded with more DNA fragmentation in the small intestine of the 2LR+SB group compared with the HS+SB group 2 h after hemorrhage and resuscitation. In addition, we observed less caspase‐3 activation in the small intestine of the HS+SB group compared with the 2LR+SB group at 2 h after resuscitation. The content of HSP40 and HSP70 in the HS+SB group was similar to that in controls, but slightly decreased in the 2LR+SB group. HS resuscitation reduced intestinal damage and apoptosis after hemorrhagic shock, suggesting that HS resuscitation may improve the outcome after hemorrhagic shock by reducing apoptosis and damage to the small intestine.

Poly(ADP-ribosyl)ation is required for p53-dependent signal transduction induced by radiation.

p53 and poly(ADP-ribose) polymerase (PARP) are both DNA damage recognition proteins and can be functionally activated by DNA strand breaks. To understand the functional interaction between these two proteins, the effects of a PARP inhibitor, 3-aminobenzamide (3AB), on the p53 pathway were investigated in human glioblastoma cells with different p53 status. Consistent with previous studies, irradiation with γ-rays induced both p53 and WAF1 accumulation in A-172 cells (wtp53) but not in T98G cells (mp53). However, the presence of 3AB but not its analog suppressed radiation-induced accumulation of wtp53 and the expression of WAF1 and MDM2. Similar results were also obtained from U87MG, another human glioblastoma cell line with wtp53 status. Northern blotting analysis showed that 3AB inhibited the γ-ray-induced WAF1 gene expression. Moreover, 3AB but not its analog inhibited irradiation-induced activation of sequence-specific DNA binding of wtp53 as detected using 32P-labeled or biotin-labeled p53 consensus sequence (p53CON). However, immunoblotting with an anti-poly(ADP-ribose) antibody showed that p53 proteins of the p53CON-bound fraction did not contain poly(ADP-ribose) (PAR). These findings suggested that poly(ADP-ribosyl)ation is required for rapid accumulation of p53, activation of p53 sequence-specific DNA binding and its transcriptional activity after DNA damage.

Transfection with mutant p53 gene inhibits heat-induced apoptosis in a head and neck cell line of human squamous cell carcinoma

PURPOSE To confirm that human cancer cells show p53-dependent heat sensitivity through an apoptosis-related mechanism, we examined the heat sensitivity and Bax-mediated apoptosis after heating in a human squamous cell carcinoma cell line, SAS, with identical genetic backgrounds except for the p53 status. MATERIALS AND METHODS We performed colony formation assay, Western blotting and analyses of apoptosis, using the SAS cells transfected with pC53-248 vector with mutant p53 gene (SAS/Trp248 cells) or the cells transfected with pCMV-Neo-Bam vector (SAS/neo cells) as a control. RESULTS SAS/Trp248 cells showed heat resistance due to the dominant negative nature of mp53, compared with SAS/neo cells. The incidence of DNA ladders and apoptotic bodies increased markedly after heating in SAS/neo cells, but increased very little in SAS/Trp248 cells. CONCLUSION These results suggest that heat resistance brought by mp53-transfection is p53-dependent and closely correlates with the induction of apoptosis in human squamous cell carcinomas.

p53-dependent induction of WAF1 by cold shock in human glioblastoma cells

Induction of WAF1 expression was investigated in human glioblastoma cell lines differing in p53 gene statuses after cold shock treatment. Accumulation of both wild-type (wt) and mutant p53 (mp53) was induced by cold shock at 4°C for 60 min, however, WAF1 accumulation was induced by cold shock in A-172 cells carrying the wtp53 but not in T98G cells carrying the mp53. Inactivation of wtp53 by a dominant negative p53 mutant (p53Trp248) abolished cold shock-induced WAF1 expression in A-172 transfectant cells. Furthermore, no WAF1 expression was induced by cold shock in p53-deficient human osteosarcoma Saos-2 cells. Northern blot analysis showed that the WAF1 but not p53 gene was activated by cold shock only in A-172 cells. These findings suggest that WAF1 expression is cold shock-inducible in human glioblastoma cells, and that this induction may be due to signal transduction mediated by p53 in response to non-genotoxic stress, cold shock.

Radiation response of apoptosis in C57BL/6N mouse spleen after whole-body irradiation.

Purpose : Primary conditioning low dose irradiation suppresses the molecular responses against secondary challenge high dose irradiation; this phenomenon has been termed the radioadaptive response. The mechanism of the radioadaptive response is not yet clear. This study was undertaken to elucidate the radiation response of apoptosis in mouse spleen after whole-body irradiation. Materials and methods : The induction of apoptosis was analysed in the spleens of C57BL/6N mice after chronic irradiation with γ-rays at 1.5 Gy (0.001 Gy/min for 25 h) followed by challenge irradiation with X-rays at 3.0Gy (1 Gy/min). Results : Accumulation of p53 and Bax, and the induction of apoptosis were observed dose-dependently in mouse spleen 12 h after acute irradiation at a high dose-rate. However, it was found that there was significant suppression of the accumulation of p53 and Bax, and induction of apoptosis 12 h after challenge irradiation at 3.0Gy at a high dose-rate following chronic preirradiation at 1.5Gy at a low dose-rate. In addition, the combination of pre-irradiation at 1.5Gy at a high dose-rate and challenge irradiation at 3.0Gy at a high dose-rate could not suppress the accumulation of p53 and Bax or the induction of apoptosis. Conclusions : Chronic pre-irradiation at a low dose-rate suppressed Bax-mediated apoptosis. These findings suggest that the radioadaptive response in mouse spleen may be due to a suppression of p53-mediated apoptosis.

Effects of a heat shock protein inhibitor KNK437 on heat sensitivity and heat tolerance in human squamous cell carcinoma cell lines differing in p53 status

Purpose: The effects of a heat shock protein (hsp) inhibitor KNK437 (N‐formyl‐3,4‐methylenedioxy‐benzylidene‐γ‐butyrolactam) were examined on the heat sensitivity and heat tolerance of human cancer cells with special reference to p53 status. Materials and methods: Human squamous cell carcinoma (SAS) and glioblastoma cell lines (A‐172) transfected with mutant p53 (mp53) or control neo genes were used. KNK437 was added in culture medium at a final concentration of 50, 100 or 300 µM 1 h before heating (42°C). Surviving fractions of cells were measured by use of a clonogenic assay. Effects of KNK437 on the accumulation of heat shock proteins and DNA binding activity of heat shock factor 1 were examined with Western blot analysis and gel mobility‐shift assay, respectively. Heat‐induced apoptotic bodies were detected by Hoechst 33342 staining. Results: The mp53‐transfected SAS (SAS/mp53) and A‐172 (A‐172/mp53) cells were more resistant to heat than the neomycin (neo)‐transfected SAS (SAS/neo) and A‐172 (A‐172/neo) cells. The constitutive amount of hsp27 was larger in SAS/mp53 than in SAS/neo cells. Clear differences in the constitutive amounts of hsp40, hsp72 and hsp90 were not observed between SAS/mp53 and SAS/neo cells. KNK437 enhanced the heat sensitivity in SAS/mp53 and A‐172/mp53 cells more effectively than in neo control cells. Heat tolerance was suppressed by KNK437 in SAS/mp53 and SAS/neo cells and also in A‐172/mp53 and A‐172/neo cells. Along with suppression of heat tolerance, KNK437 suppressed heat‐induced accumulation of both hsp27 and hsp72. Heat‐induced apoptotic bodies were enhanced by KNK437 in SAS/mp53 and SAS/neo cells. Conclusion: The results suggest a possible mechanism for the heat sensitivity of SAS cells. Heat sensitivity depends on p53 status regulating the amount of hsp27. Heat tolerance is suppressed by KNK437 through the suppression of heat‐induced accumulations of hsp27 and hsp72 and the induction of p53‐independent apoptosis.

Low-dose-rate radiation attenuates the response of the tumor suppressor TP53.

Acute low-dose irradiation (0.1-1 Gy, 1.33 Gy/min) of cells of a human glioblastoma cell line, A-172, induced a dose-dependent monophasic accumulation of TP53 (formerly known as p53) and CDKN1A (formerly known as WAF1). In contrast, chronic gamma irradiation (0.001 Gy/min) produced a clear biphasic response of accumulation TP53 with the first peak at 1.5 h (0.09 Gy) and the second peak at 10 h (0.54 Gy). Significantly, when the cells were preirradiated with a chronic dose of gamma irradiation for 24 h (1.44 Gy) or 50 h (3 Gy), they no longer responded to an acute challenging dose to produce a dose-dependent response of the TP53 pathway. These findings suggest that chronic irradiation at low dose rate alters the TP53-dependent signal transduction pathway. Wearing away of the TP53 pathway by chronic exposure to radiation may have important implications for radiation protection.

siRNA targeting NBS1 or XIAP increases radiation sensitivity of human cancer cells independent of TP53 status.

Abstract Ohnishi, K., Scuric, Z., Schiestl, R. H., Okamoto, N., Takahashi, A. and Ohnishi, T. siRNA Targeting NBS1 or XIAP Increases Radiation Sensitivity of Human Cancer Cells Independent of TP53 Status. Radiat. Res. 166, 454–462 (2006). NBS1 is essential for the repair of radiation-induced DNA double-strand breaks (DSBs) in yeast and higher vertebrate cells. In this study, we examined whether suppressed NBS1 expression by small interference RNA (siRNA) could enhance radiation sensitivity in cancer cells with different TP53 status. We used human non-small cell lung cancer cells differing in TP53 gene status (H1299/wtp53 cells bearing wild-type TP53 or H1299/mp53 cells bearing mutant TP53). A DNA cassette expressing siRNA targeted for the NBS1 gene was transfected into those cell lines, and radiation sensitivity was examined with a colony-forming assay. Cellular levels of NBS1 and other proteins were analyzed using Western blotting. We found that the radiation sensitivity of H1299/wtp53 and H1299/mp53 cells was enhanced by transfection of the DNA cassette. In the NBS1-siRNA-transfected cells, we observed decreased constitutive expression of NBS1 protein and decreased radiation-induced accumulation of phosphorylated NBS1 protein. In addition, radiation-induced expression of the transcription factor NF-κB (NFKB) and XIAP (X-chromosome-linked inhibitor of apoptosis protein) was suppressed by NBS1-siRNA. Enhanced X-ray sensitivity after NBS1-siRNA transfection was achieved in TP53 wild-type cells and sensitivity was even more pronounced in TP53 mutant cells. The transfection of siRNA targeted for XIAP also enhanced X-ray sensitivity even more for TP53 mutant cells compared to TP53 wild-type cells. Our data suggest that the sensitization to radiation results from NBS1-siRNA-mediated suppression of DNA repair and/ or X-ray-induced cell survival signaling pathways through NFKB and XIAP. siRNA targeting appears to be a novel radiation-sensitizing agent, particularly in human TP53 mutant cancer cells.