On Western blot analysis, phosphorylation of serine 15 seems to be re quired for this process. New cancer therapies for patients with mp53 containing tumors are recently being developed. Especially, reports concerning to transfection of p53 gene into tumor and molecules which activate latent p53, change the conformation most of mp53 or restore the function of mp53 are on the increase. We have recently reported that glycerol has an ability to restore normal function to mp53, leading to WAF1 induction. There after, new compounds which rescue mp53 conformation and function have been reported by other laboratory. Among the reported molecules, glycerol is easy to be rec ognized as the most useful molecule for cancer therapy, because it is widely used as a convenient reagent in clini cal course already.

Furthermore, as reported in this paper, glycerol has an ability to enhance bax expression in mp53 cells as a chemical chaperone through phosphorylation of p53 at serine 15 by PI3 K family and conformational change of mp53. Thus, it is expected that the cancer ther apy combined hyperthermia and glycerol is efficient for patients with mp53 containing tumors, in which p53 de pendent bax expression is less frequently induced. As we have reported elsewhere, glycerol is also effec tive in inducing a conformational change of p53 and re storing normal function to mp53, leading to enhanced radiosensitivity. The enhanced radiosensitivity is closely related to the induction of p53 dependent apop tosis. Further, we have recently obtained results showing that glycerol functions as a chemical chaperone in p53 de pendent CDDP sensitivity.

Our re sults obtained from previous and the present studies show that glycerol is available for cancer therapy such as hyper thermia, radiotherapy or chemotherapy which induces the activation of p53 even in mp53 tumors. Materials and Methods Cells Human glioblastoma A 172 cells were cultured at 37?C in Dulbeccos Modified Eagle medium containing 10% fetal bovine serum, penicillin, streptomycin and kan amycin. Plasmids A 172 cells were transfected with the plasmids pC53 SCX3, pC53 248 or pCMV Neo Bam. Be fore transfection, these plasmids were digested with Hin dIII and linearized. A 172 cells were electroporated three times at 600 V with linearized DNA. The transfectants were selected by G418 and incubated at 37?C through all experiments.

Glycerol treatment Cells were treated with glycerol 48 h before heating and then were incubated at 37?C for 6 or 10 h in the presence of glycerol until sampling. In the case Brefeldin_A of cell survival assay, the medi um with glycerol was changed with glycerol free one after 10 h incubation and thereafter cells were incubated for ten to fourteen days at 37?C in glycerol free medium. In in vit ro treatment, whole cell extracts from intact cells were treated with glycerol for 30 min at 37?C.