INCREASED SENSITIVITY TO THE PRODRUG 5'-DEOXY-5-FLUOROURIDINE AND MODULATION OF 5-FLUORO-2'-DEOXYURIDINE SENSITIVITY IN MCF-7 CELLS TRANSFECTED WITH THYMIDINE PHOSPHORYLASE

Platelet-derived endothelial cell growth factor (PD-ECGF) is identical to human thymidine phosphorylase (dThdPase). The human MCF-7 breast c ancer cell line was transfected with the dThdPase cDNA and expressed a 45 kDa protein that was detected with anti-dThdPase antibody. Cell ly sates possessed elevated dThdPase activity and cells had up to 165-fol d increased sensitivity to the prodrug 5'-deoxy-5-fluorouridine (5'-DF UR) in vitro. Sensitivity to 5-fluorouracil (5-FU) and 5-fluoro-2'-deo xyuridine (5-FUdR) was unchanged. Recombinant dThdPase was shown to ca talyse directly the phosphorolytic cleavage of 5'-DFUR to 5-FU. Exogen ous thymidine (dThd) reversed the toxicity of 5-FUdR on the parental l ine (1 mu M dThd increased the IC50 value 1000-fold), but the dThd res cue was substantially modulated in the dThdPase-expressing clone 4 (1 mu M dThd raised the IC50 value 3-fold). We observed a substantial 'by stander' killing effect when small proportions of dThdPase-expressing cells were mixed with parental MCF-7 cells. dThdPase activity was on a verage 27-fold higher in breast tumours than in normal breast. The lev els in wild-type MCF-7 are similar to the low end of the tumour expres sion. Thus, in some tumours resistance to 5'-DFUR therapy could be due to low dThdPase activity, and transfection to raise the dThdPase. lev els within the broad tumour range or above it should markedly enhance sensitivity to the prodrug. These results confirm that dThdPase is a m ajor pathway in the metabolic activation of 5'-DFUR, and the bystander effect suggests that this may be a suitable enzyme for gene therapy-d irected enzyme/prodrug activation therapy.