To identify critical events associated with heat-induced cell killing, we examined foci formation of ␥H2AX (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 ␥H2AX 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 ␥H2AX 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 ␥H2AX foci is correlated with the temperature dependence of cell killing. During periods when cells were exposed to heat, the cell cycledependent pattern of cell killing was the same as the cell cycle pattern of ␥H2AX foci formation. We also found that thermotolerance was due to a depression in the number of ␥H2AX 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.
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.
Induction of WAF1 expression was investigated after heat treatment (44°C, 30 min) in two human glioblastoma cell lines with the wild-type or a mutant p53 gene. WAF1 accumulation was induced by heat treatment in A-172 cells carrying the wild-type p53 gene but not in T98G cells carrying the mutant p53 gene. We examined whether this phenomenon was due to the induction of WAF1 expression. Northern blot analysis showed that heat treatment not only activated WAF1 but also upregulated p53 expression only in A-172 cells carrying the wild-type p53 gene. Gel mobility shift assay indicated an increase in p53 DNA binding activity after heat treatment. These findings suggest that the WAF1 expression is heat-inducible in human glioblastoma cells and that this induction may be due to signal transduction mediated by p53 in response to heat stress.
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 Ringer's 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.
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 eects of a PARP inhibitor, 3-aminobenzamide (3AB), on the p53 pathway were investigated in human glioblastoma cells with dierent p53 status. Consistent with previous studies, irradiation with g-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 g-ray-induced WAF1 gene expression. Moreover, 3AB but not its analog inhibited irradiationinduced activation of sequence-speci®c DNA binding of wtp53 as detected using 32 P-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 ®ndings suggested that poly(ADP-ribosyl)ation is required for rapid accumulation of p53, activation of p53 sequence-speci®c DNA binding and its transcriptional activity after DNA damage.
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