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Enhancement of 5-Fluoro-2'-deoxyuridine Antineoplastic Activity by 5-Benzyloxybenzyloxybenzylacyclouridine in a Human Colon Carcinoma Cell Line¹

Division of Biology and Medicine, Brown University and Roger Williams General Hospital, Providence, Rhode Island 02908

The pyrimidine acyclonucleoside benzyloxybenzyloxybenzylacyclouridine (BBBAU) showed growth inhibitory activity against the human colon cancer HCT-8 cell line with a 50% inhibitory concentration of 55 µM. Unlike its parent compounds, BBBAU was an extremely weak inhibitor of uridine phosphorylase. This acyclonucleoside analogue is an inhibitor of thymidylate synthase (TS) as determined by inhibition of [6-³H]-2'-deoxyuridine incorporation into DNA, inhibition of ³H release from [5-³H]-2'-deoxyuridine, and decrease in both the free and total TS 5'-fluoro-2'-deoxyuridine 5'-monophosphate binding sites. Kinetic analysis revealed that BBBAUMP, the monophosphate analogue of BBBAU, is a competitive inhibitor of purified human recombinant TS with a K₁ of 8.0 µM. Nucleoside transport and uptake studies revealed that BBBAU (30 µM) inhibited the initial rate of transport and the total uptake of thymidine (25 µM). In contrast, while BBAU (30 µM) inhibited the initial rate of transport of 5-fluoro-2'-deoxyuridine (FdUrd, 25 µM), its intracellular accumulation was increased. BBBAU (10 and 50 µM, respectively) potentiated FdUrd growth inhibition of HCT-8 cells and significantly enhanced the cytotoxic effects of FdUrd (0.3 and 1 µM, respectively) against HCT-8 cells using a clonogenic assay system. This combination resulted in additive inhibitory effects on TS activity resulting in greater depletion of dTTP pools. Moreover, the incorporation of radiolabeled FdUrd into the DNA fraction of HCT-8 cells was enhanced. The potential importance of this novel combination for human colon cancer chemotherapy is discussed.

¹ This investigation was supported by USPHS Grants CA 39427, CA 13943, and CA 20892 from the National Cancer Institute.

² Present address: Medicine Branch, Building 10, Room 12N226, National Cancer Institute, Bethesda, MD 20892.

³ To whom requests for reprints should be addressed, at the Division of Biology and Medicine, Brown University, Box G, Providence, RI 02912.

Received 3/ 5/90. Accepted 1/24/92.