Development of novel heat-induced cytokine gene therapy against malignant gliomas

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1) First, we constructed LacZ gene expression vector under the control of heat shock protein 70 (hsp70) promoter (hsp70/LacZ). Then three human glioma cell lines (U251-MG, T98G, NP2) and one mouse glioma cell line (203) were transfected with hsp70/LacZ vector using lipofection method. After selection with G418, the stable transfectants were obtained. Using these transfectants, the expression of beta-galactosidase after heating was measured. The condition of heating was 41℃ for 2h, 42℃ for 1h, or 43℃ for 1h. The results showed that the peak of the beta-galactosidase expression was between 3h and 24h after heating, even though the degree of its expression varied among the cell lines. (In contrast to other cell lines, the expression was positive in U251-MG cells even with 37℃ heating as a negative control.)2) As a next step, we constructed CD40 ligand (CD40L) gene expression vector under the control of hsp70 promoter (hsp70/CD40L). Then we transfected the vector into mouse 203 glioma cell … More or human NP2 glioma cell by lipofection method. After the selection with G418, we obtained stable transfectants, hsp70/CD40L-203 or hsp70/CD40L-NP2. Using each transfectants, we measured the expression of CD40L protein in the supernatant of the culture medium after heating using ELISA. The results revealed that the peak expression of CD40L protein was between 3h and 48h after heating.3) Furthermore, we constructed IFN-gamma gene expression vector under the control of hsp70 (hsp70/IFN-gamma). The stable transfectant with this vector (hsp70/IFN-gamma-203) was established after the selection with G418. Using this transfectant, we measured the expression of IFN-gamma protein in the supernatant of the culture medium after heating at 42℃ for 1h using ELISA. The result showed that the expression peaked at 24h after the heating. There was no expression of IFN-gamma observed with the treatment of 37℃ heating as a negative control.4) Taken together, in most of the glioma cell lines except for U251-MG cells, the peak expression of the inserted gene by heating was observed between 3h and 48h after heating, even though the expression pattern varied depending on each cell line. Therefore, this gene expression system using hsp70 promoter can be a good candidate for heat-inducible gene treatments. Less