ENHANCEMENT OF 5-FLUORO-2'-DEOXYURIDINE ANTINEOPLASTIC ACTIVITY BY 5-BENZYLOXYBENZYLOXYBENZYLACYCLOURIDINE IN A HUMAN COLON-CARCINOMA CELL-LINE

The pyrimidine acyclonucleoside benzyloxybenzyloxybenzylacyclouridine (BBBAU) showed growth inhibitory activity against the human colon canc er HCT-8 cell line with a 50% inhibitory concentration of 55-mu-M. Unl ike its parent compounds, BBBAU was an extremely weak inhibitor of uri dine phosphorylase. This acyclonucleoside analogue is an inhibitor of thymidylate synthase (TS) as determined by inhibition of [6-H-3]-2'-de oxyuridine incorporation into DNA, inhibition of H-3 release from [5-H -3]-2'-deoxyuridine, and decrease in both the free and total TS 5'-flu oro-2'-deoxyuridine 5'-monophosphate binding sites. Kinetic analysis r evealed that BBBAUMP, the monophosphate analogue of BBBAU, is a compet itive inhibitor of purified human recombinant TS with a K(i) of 8.0-mu -M. Nucleoside transport and uptake studies revealed that BBBAU (30-mu -M) inhibited the initial rate of transport and the total uptake of th ymidine (25-mu-M). In contrast, while BBBAU (30-mu-M) inhibited the in itial rate of transport of 5-fluoro-2'-deoxyuridine (FdUrd, 25-mu-M), its intracellular accumulation was increased. BBBAU (10 and 50-mu-M, r espectively) potentiated FdUrd growth inhibition of HCT-8 cells and si gnificantly enhanced the cytotoxic effects of FdUrd (0.3 and 1-mu-M, r espectively) against HCT-8 cells using a clonogenic assay system. This combination resulted in additive inhibitory effects on TS activity re sulting in greater depletion of dTTP pools. Moreover, the incorporatio n of radiolabeled FdUrd into the DNA fraction of HCT-8 cells was enhan ced. The potential importance of this novel combination for human colo n cancer chemotherapy is discussed.