Combination of radiation and suicide gene therapy with radio-sensitized promoter/Endotherial precursor cell in glioma

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In the first year we verified that the gene expression located at the downstream of radio-sensitized promoter would actually amplify by radiation. We constructed the EGFP&HSV-tk vectors containing E_4/CMV chimeric radio-sensitized promoter (E_4: four-repeated structure of CArG element located at Egr-1 gene). E_4/EGFP was transferred into U251-MG/U373-MG human glioma cell line respectively and the expression of EGFP was evaluated by optical observation under fluorescent microscopy and Western blotting after 3Gy irradiation. As a result, we verified that the expression of EGFP was amplified about 1.3〜1.4 times after 6〜54 hours compared to pre-irradiation stage. Then E4/HSV-tk and HSV-tk were transferred into U251-MG/U373-MG human glioma cell line respectively, and the degree of cell proliferation was evaluated by MTT assay after irradiation (1,3,5Gy) following GCV administration. The results were 1)the radio-sensitized promoter also could be activated by physical/chemical stimuli. 2)the radiation dose-dependent apoptosis was observed only in the E_4/HSV-tk-administrative group but not statistically significant.In the second year, we replaced the promoter with E_<9ns-2>, stronger than E_4 in radio-sensitization and did the same trial. Contrary to the expectation, however, the expression of EGFP was not significantly amplified in the E_<9ns-2>/EGFP -administrative groups. In the MTT assay, we also could not verified the significant inhibition of the cell proliferation among E_<9ns-2>/HSV-tk-administrative glioma cell lines after irradiation. We are going to promote our previously scheduled plan by using E_4/CMV as the radio-sensitized promoter, transferring E_4/HSV-tk into C6 and endothelial precursor cell line.