Wild-type p53-function is not required for hyperthermia-enhanced cytotoxicity of cisplatin

The objective was to test the hypothesis that wild-type p53-function is required for the enhancement of the cytotoxicity of cis-diammine-dichloroplatinum(II) (cDDP) cytotoxicity by hyperthermia (HT). Human colorectal carcinoma cells (RKO) with wild-type p53-function and transfectants with HPV16-E6 or with a dominant negative mutant p53 were used. Cells were treated with HT (60min at 41°C, 43°C, 45°C: HT41, HT43, HT45), with various doses of cDDP alone or with a combined treatment, simultaneously applied. Survival was determined by clonogenic assays. Levels and localization of p53 were analysed with immunocytochemistry and Western blotting. The extent of HT41-enhanced cytotoxicity of cDDP was similar in all cell lines studied. Immunocytochemistry of wild-type p53 cells showed that p53 is transferred to the nucleus within 5h after HT43, whilst after HT41 no significant effects were observed. Cell fractionation experiments of wild-type p53 cells showed that, immediately after HT43/ HT45, nuclear p53-levels increased as compared to controls, but could not be extracted from the matrix. The extractability was restored 3-5h after treatment. No significant differences in p53-levels were observed after HT41. These results indicate that, although HT43/HT45 might shortly inactivate p53-function, probably by protein aggregation to the nuclear matrix, the HT-enhanced cDDP-cytotoxicity does not depend on p53-function.