Protein microarray analysis of apoptosis-related protein expression following heat shock in human tongue squamous cell carcinomas containing different p53 phenotypes.

PURPOSE: Hyperthermia is useful in the treatment of human head and neck cancers, because it is relatively easy to regulate temperatures when compared to tumors located in deep organs. In this study, attention was focused on p53 as a possible predictive indicator for the efficacy of hyperthermic cancer therapy. METHODS: Two kinds of cell lines were used. These were derived from a human squamous cell carcinoma (SAS) and had identical genetic backgrounds except for their p53 gene status. It was previously reported that the heat sensitivity and frequency of apoptosis in wild-type p53 cells (SAS/neo) were clearly elevated when compared with mutated p53 cells (SAS/mp53). In order to study the expression of apoptosis related proteins after heat treatment, protein microarray analysis was used. RESULTS: The expression of apoptosis inhibitory proteins such as Bcl-2, Bcl-xL, NF-kappaB, COX2, STAT3, IL-6, and IKKalpha/1 was seen to increase after heat treatment in SAS/mp53 cells, but not in SAS/neo cells. CONCLUSION: The result of these observations indicates that apoptosis inhibitory proteins (such as Bcl-2, Bcl-xL, IL-6, etc.) were highly induced in SAS/mp53 cells after heat treatment when compared to control SAS/neo cells.

LY294002, an inhibitor of PI-3K, enhances heat sensitivity independently of p53 status in human lung cancer cells.

The aim of this study was to ascertain whether LY294002, an inhibitor of PI-3K, enhances heat sensitivity in human cancer cells regardless of their p53 status. Colony formation assays showed that LY294002 enhanced heat sensitivity in two human lung cancer cell lines; H1299/wild-type p53 (wtp53) and H1299/mutated p53 (mp53) cells. These cell lines have identical genetic backgrounds except for their p53 status. LY294002 suppressed the heat-induced accumulation of heat shock protein 27 (hsp27) and heat shock protein 72 (hsp72) in these cell lines. Heat-induced apoptosis was observed more frequently in H1299/wtp53 cells than in H1299/mp53 cells, and was enhanced by LY294002 in both cell lines. In addition, both the heat-induced phosphorylation of Akt and the accumulation of survivin were suppressed by LY294002. These results suggest that LY294002 inhibits anti-apoptosis signaling through hsp27 and hsp72 as well as cell survival signaling through Akt and survivin. LY294002 appears to be an attractive candidate for a p53-independent heat sensitizer in hyperthermic cancer therapy.

Glycerol enhances radiosensitivity in a human oral squamous cell carcinoma cell line (Ca9-22) bearing a mutant p53 gene via Bax-mediated induction of apoptosis.

Radiotherapy for oral squamous cell carcinomas is limited in its efficacy and in its ability to improve the survival rate in patients at an advanced stage. A protocol is described here which may elevate the therapeutic efficacy of radiation for these cells. The addition of glycerol to the culture medium prior to irradiation of an oral squamous cell carcinoma cell line (Ca9-22) bearing a mutant p53 (mp53) gene was found to increase the radiosensitivity of these cells. A colony formation assay was used to evaluate the effect of glycerol on the radiation sensitivity of Ca9-22 cells. Apoptosis was analyzed using Hoechst 33342 staining, Western blotting, and a DNA ladder formation assay. Glycerol, when present in the culture medium, enhanced the radiation sensitivity and extent of apoptosis following X-irradiation in the Ca9-22 cells, although neither X-rays or glycerol alone increased the extent of apoptosis. Bax protein was accumulated after treatment with X-rays plus glycerol, but not after exposure to X-rays or glycerol alone. A gel mobility-shift assay showed that glycerol restored the DNA-binding activity of mp53 for a p53-consensus sequence to levels similar to that of wild-type p53. These findings suggest that pre-treatment with glycerol may enhance the effectiveness of radiotherapy against oral squamous cell carcinomas bearing an mp53 gene mutation.

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 gammaH2AX (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 gammaH2AX 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 degrees C to 45.5 degrees C, we observed a linear increase with time in the number of gammaH2AX foci. An inflection point at 42.5 degrees 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 gammaH2AX 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 gammaH2AX foci formation. We also found that thermotolerance was due to a depression in the number of gammaH2AX 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.

High-LET radiation enhanced apoptosis but not necrosis regardless of p53 status.

PURPOSE: We analyzed the death pattern of human lung cancer cells harboring different p53 statuses after irradiation with different levels of linear energy transfer (LET). METHODS AND MATERIALS: We used three kinds of human lung cancer cell lines with identical genotypes, except for the p53 gene. These cells were exposed to X-rays or accelerated carbon-ion beams. The cellular sensitivities were determined by a colony-forming assay. The detection and quantification of cell death (apoptosis and necrosis) were evaluated and compared by acridine orange/ethidium bromide double staining for fluorescence microscopy. RESULTS: We found that (1) there was no significant difference in cellular sensitivity to LET radiation >70 KeV/microm, although wild-type p53 cell sensitivity to X-rays was higher than that of mutated p53 or p53-null cells; (2) low-LET radiation effectively induced apoptosis in wild-type p53 cells as compared with mutated p53 and p53-null cells; and (3) high-LET radiation induced p53-independent apoptosis. CONCLUSIONS: Our findings suggest that high-LET radiotherapy is expected to be a valid application for patients carrying mutated p53 cancer cells. We proposed that the elucidation of the p53-independent apoptosis-related genes might provide new insights into radiotherapy for cancer.

Glycerol enhances CDDP-induced growth inhibition of thyroid anaplastic carcinoma tumor carrying mutated p53 gene.

To increase the chemo-sensitivity of anaplastic thyroid carcinoma, we examined the effects of glycerol on the tumor growth after CDDP treatment. The cultured cells of an anaplastic thyroid carcinoma cell line (8305c) carrying a mutated p53 gene (mp53) were transplanted into the thighs of nude mice. Tumor growth was evaluated until 24 days after intraperitoneal injection of CDDP and/or pre-injection of glycerol to the tumor. We treated the mice with half the tumor volume of glycerol (1.2 M) and/or CDDP at 6 mg/kg (BW) either of which hardly inhibited tumor growth by itself. When we treated the mice with the combination of glycerol and CDDP at these concentrations, however, a clear delay of the tumor growth was observed. We also immunohistochemically analyzed the effects of glycerol on the induction of caspase-3 activity and apoptosis. Cells positive for cleavage to active caspase-3 and 85 kDa PARP, and apoptosis were hardly observed in the tumors when they were treated with glycerol or CDDP alone. In contrast, when they were treated with CDDP combined with glycerol, such positive cells were significantly increased. It has been shown that glycerol synergistically enhanced the effects of CDDP as a tumor suppressive agent through the induction of caspase-3-mediated apoptosis in 8305c tumors. Therefore, glycerol might be useful for chemotherapy in patients with mp53 cancer cells.

Apoptosis-related gene expression after hyperthermia in human tongue squamous cell carcinoma cells harboring wild-type or mutated-type p53.

Hyperthermia is useful for the treatment of human head and neck cancer, as it is relatively easy to perform thermoregulation when compared with deep organs. In this study, we focused attention on the p53 as a predictive indicator of hyperthermic cancer therapy. We used two kinds of cell lines of a human squamous cell carcinoma (SAS) with identical backgrounds of function except for the p53 protein. We assayed the heat sensitivity, frequency of apoptosis, and apoptosis-related gene expression after heat treatment using DNA array. The SAS/neo (wild-type p53; wtp53) cells were sensitive to heat, and the induction of Caspase-3 activation and apoptosis in the wtp53 cells was clearly high compared with the SAS/mp53 (mutated p53; mp53) cells. The gene expression of apoptosis suppressive-genes such as IL-12 p35 decreased in the wtp53 cells, and IL-12 R beta1 increased in the mp53 cells, though apoptosis-promotive genes of Caspase-9, CD30 and CD40 were induced p53-independently by hyperthermia. It is suggested that heat-induced apoptosis was suppressed by IL-12-related genes in the mp53 cells. These findings strongly imply that p53 status is a useful candidate for a predictive indicator of the effectiveness in hyperthermic therapy.

Analysis of apoptosis-related gene expression after X-ray irradiation in human tongue squamous cell carcinoma cells harboring wild-type or mutated p53 gene.

Mutations in the p53 tumor suppressor gene have recently been reported to have an impact on clinical trials of several human tumors, including head and neck cancers. To confirm the p53-dependence of X-ray induced apoptosis, we used two cell lines derived from a human squamous cell carcinoma (SAS) with identical genetic backgrounds, except for the p53 gene, which are SAS/mp53 cells with mp53 and SAS/neo cells with wtp53. We previously reported that the radiosensitivity, Caspase-3 activity and apoptosis frequency in SAS/neo cells were clearly high as compared with SAS/mp53 cells. In order to elucidate the expression of apoptosis-related genes after irradiation, we used cDNA array analysis. The expressions of apoptosis-inductive genes, such as DFF40, Caspase-3, Caspase-8, Caspase-9, Caspase-10 and CRADD, were increased by X-ray irradiation in SAS cells with wtp53, but not in SAS cells expressing mp53. These results suggest that the X-ray sensitivity of wtp53 cells may come from the expression of these apoptosis-related genes.