Cell-cidal effect of the combination therapy of p53 gene under the control of radiation- and hypoxia-sensitized promoter and heavy particle irradiation in glioma

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AIM : Malignant gliomas, especially the glioma cells under hypoxic microenvironments, are refractory to conventional radiotherapy and chemotherapy. The aims of this study were to examine the efficacy of combination of radiation/hypoxia dual-sensitive promoter-regulated p53 gene and high-LET radiation for killing glioma cells with mutant p53 under normoxic and hypoxic conditions and to compare the effectiveness as a 'trigger' to activate therapeutic gene expression between high-LET and low-LET radiation. METHODS : The chimeric promoter Egr-HRE was generated by insertion of CArG elements derived from Egr-1 gene and hypoxia response elements(HREs) derived from erythropoietin gene. p53 expression vector was constructed by cloning of the Egr-HRE promoter upstream of p53 gene. After transfecting the plasmid into p53-mutant U373 human glioma cell line, the cells were exposed to hypoxia (0.1% O_2, 24 h) and high-LET (1 Gy) or low-LET radiation (1 Gy). The expression level of p53 and the cell killing effect was determined by Western blotting and colony formation assay, respectively. RESULTS : The results showed that the exposure of the cells to high-LET carbon ions and hypoxia greatly enhanced the expression of p53 and accordingly was most effective in killing the glioma cells. When compared with low-LET radiation, high-LET carbon ions were more effective in enhancing p53 expression and killing the cells. CONCLUSION : The results suggest that the combination of radiation/hypoxia dual promoter-regulated p53 gene and high-LET carbon ions is a promising strategy for effective gene therapy of malignant gliomas and that high-LET radiation is more effective to activate therapeutic gene expression than low-LET radiation.