Selective Exclusion by the Polyamine Transporter as a Mechanism for Differential Radioprotection of Amifostine Derivatives

Amifostine metabolites WR-1065 and the disulfide WR-33278are thiol-containing polyamine analogues with potent radio- and chemoprotective properties. Some studies suggest that amifostine exerts differential cytoprotection in normal versus neoplastic tissues, but this finding remains controversial. To assess the role of the polyamine transport system in radioprotection by amifostine derivatives, human DU-145 prostate cancer cells were transfected with a cDNA that encodes antizyme (OAZ), a polyamine-inducible protein that suppresses polyamine transport under control of a minimal heat shock promoter. Selected clones expressing OAZ displayed heat shock-dependent suppression of polyamine uptake. When added to culture medium under nonreducing conditions, both WR-1065 and WR-33278 were detected as the disulfide form. Each derivative protected both parental and OAZ-transfected DU-145 cells from X-ray-induced cell killing at 37°C. When cultures were heat shocked at 42°C, both derivatives protected parental, but not OAZ-transfected cells from radiation-induced cell killing. Treatment of DU-145 cells with difluoromethylornithine (DFMO) suppressed intracellular putrescine and spermidine content, but increased the uptake of WR-33278-derived aminothiols. The concentration-dependent radioprotection of DU-145 cells by WR-33278 was enhanced by DFMO. Addition of exogenous putrescine reduced WR-33278-mediated radioprotection in both DFMO-treated and untreated DU-145 cells. These data demonstrate that negative regulation of the polyamine transporter, mediated by polyamines or antizyme, suppresses the uptake and radioprotective activity of amifostine derivatives. Selective exclusion of amifostine derivatives by the polyamine transporter could account for differential radio- or chemoprotection in normal versus neoplastic tissues in specific situations.