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Biochemical Determinants of Responsiveness to 5-Fluorouracil and Its Derivatives in Xenografts of Human Colorectal Adenocarcinomas in Mice¹

Division of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101

The level of thymidylate synthetase (EC 2.1.1.45) and its activity have been measured in a series of human colorectal adenocarcinomas growing as xenografts in immune-deprived mice. Enzyme activity varied between 8.4 and 124 pmol per mg protein per hr; within each tumor line, this activity correlated with the capacity to bind [6-³H]5-fluoro-2'-deoxyuridine 5'-monophosphate ([6-³H]FdUMP), which varied between 0.16 and 1.68 pmol [6-³H]FdUMP bound per g tissue. Highest and lowest activities were measured in tumor lines that were insensitive to 5-fluorouracil, 5-fluorouridine, and 5-fluoro-2'-deoxyuridine. The ratio of the maximum free FdUMP concentration to thymidine 5'-monophosphate synthetase-binding activity did not differentiate fluorinated pyrimidine-responsive lines from those innately insensitive. Maximum potential binding of [6-³H]FdUMP in vitro was measured without addition of dl-L-5,10-methylenetetrahydrofolate (CH₂FH₄) in cytosol from two tumor lines, both of which demonstrated some sensitivity to fluorinated pyrimidine therapy. The other four insensitive tumor lines required CH₂FH₄ to be added in order to attain maximum [6-³H]FdUMP binding. Similar data were obtained using nitrocellulose membrane filtration to isolate both covalent and noncovalent complexes. Direct measurement of thymidine 5'-monophosphate synthetase activity after incubation of tumor cytosols with FdUMP, with or without added CH₂FH₄, showed that, in nonresponsive tumors, maximum enzyme inhibition was achieved only in the presence of exogenous cofactor. It is suggested that the availability of cofactor may prove important in the formation of the ternary complex CH₂FH₄:thymidine 5'-monophosphate synthetase:FdUMP when high concentrations of FdUMP are present for only short periods of time.

¹ This work was supported by Grants CA21677 from the National Cancer Institute, by Grants ACS IN99F and CH-172 from the American Cancer Society, and by the American Lebanese Syrian Associated Charities (ALSAC).

² To whom requests for reprints should be addressed:

Received 5/22/80. Accepted 10/ 1/80.

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