The antitumor activity of cytostatic 5'-deoxy-5-fluorouridine (5'-dFUrd) depends on its being converted to 5-fluorouracil (5-FUra) by the enzyme thymidine phosphorylase (dThdPase, EC 2.42.4). We prepared mouse anti-human dThdPase monoclonal antibodies to serve as tools for clinical studies with this drug. Partially purified dThdPase obtained form HCT116 human colon cancer cells grown in athymic mice was used as and antigen for the immunization of mice. Six hybridomas were cloned which produced anti-human dThdPase antibodies, as detected by Western blot analysis with human dThdPase. With these antibodies, we developed an ELISA method sensitive enough to measure dThdPase levels, even in tumor tissue samples weighing as little as 10 mg. In addition, one monoclonal antibody was suitable for immunologically staining the enzyme in tumor tissues. Thus, these anti-human dThdPase monoclonal antibodies could be used to measure levels of the enzyme in tumor cells, which is essential for the activation of 5'-dFUrd.
The present study shows that various cytokines such as tumor necrosis factor (TNF alpha), interleukin-1 alpha (IL-1 alpha), and interferon-gamma (IFN gamma) make tumor cells much more susceptible to the cytostatic 5'-deoxy-5-fluorouridine (5'-dFUrd) than to 5-fluorouracil (5-FUra) and other cytostatics. These three cytokines increased the susceptibility of human cancer cell lines (COLO201, MKN45 and WiDr) but did not affect that of normal fibroblast WI38 cells. The cytokine mixture induced a 50-fold increase in the susceptibility of COLO201 to 5'-dFUrd, whereas a 12-fold increase and a less than 5-fold enhancement in the susceptibility to 5-FUra and other cytostatics, respectively, were observed. The increased susceptibility would be a result of the induction of thymidine phosphorylase (TdR Pase), which is the essential enzyme for the conversion of 5'-dFUrd to 5-FUra. The cytokine mixture increased TdR Pase activity by up to 47 times and greatly induced its mRNA expression in the cancer cell lines. These results suggest that the therapeutic benefit of 5'-dFUrd would be improved by its use in combination with the cytokines.
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