Trial of novel thermo-gene therapy for malignant brain tumors-Aiming for enhancement of hyperthermia using p53 introduction-

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We evaluated combination effect of p53 introduction and hyperthermia on malignant glioma cells. Cell lines used are U251-MG, U373-MG (mutant p53), and D54, U87-MG (wild-type p53). 1)First, optimal MOl of Ad/p53 vector was determined. 2)Heat shock was applied at 43℃ for 2h using liquid incubator. 3)In in vitro experiment, additive effect of both treatments was observed in all the cell lines used. 4)In cell cycle analysis, no change of distribution was observed by heat shock alone in either U373-MG or D54 cells. However, there was an increase in both sub-G1 and G1 by p53 introduction alone. The effect of combination treatment was similar to that of p53 introduction alone. 5)In DNA fragmentation assay, apoptosis was induced by p53 introduction alone but not by heat shock alone. Higher percentage of apoptosis was induced by the combination treatment, which was likely to be caused by heat shock. This enhancing effect by heat shock was observed regardless of p53 status of tumor cells. 6)Apoptosis induction was caspase-dependent in U373-MG and U251-MG cells, but was caspase-independent in D54 cells. 7)p21 protein was over-expressed only after p53 introduction. 8)As in vivo experiment, we established U373-MG subcutaneous tumor model using nude mice. 9)Treatments were given to the following four groups. A)control-vector(CTR) and 37℃ heat shock(HS), b)CTR and 43℃ HS, c)p53-vector(p53V) and 37℃ HS, d)p53V and 43℃ HS. Results showed that no significant inhibitory effect on tumor growth was observed in groups (b),(c),and (d)compared with control-group (a).For future experiments, to achieve more efficient hyperthermia, A)repeating of heat shock, and B)heating with needle electrode, should be considered. Regarding gene transfer, C)gene expression under the control of tetracycline, and D) use of liposome with higher transfer efficiency, should be also be considered.