Six cell lines differing in histological origin were studied regarding the growth inhibitory effect of fluoropyrimidines in relation to their metabolism. The human colon carcinoma cell line WiDr was most sensitive to 5-fluorouracil (FUra) (50% growth inhibitory concentration, 0.7 microM) and to its analogue 5'deoxy-5-fluorouridine (5'dFUR) (50% growth inhibitory concentration, 18 microM). The murine B16 melanoma cell line was moderately sensitive to FUra but least sensitive to 5'dFUR. The 50% growth inhibitory concentration values in the human melanoma cell lines IGR3 and M5, the transformed human intestine cell line intestine 407 and the rat hepatoma cell line H35 varied for FUra between 1.7 and 5.0 microM, and for 5'dFUR between 54 and 160 microM. Several enzymes from pyrimidine metabolism responsible for FUra metabolism were measured with FUra as a substrate. The activity of uridine phosphorylase, which catalyzes the conversion of 5'dFUR to FUra, was lowest in B16 cells correlating with the low sensitivity to 5'dFUR. When adenosine 5'-triphosphate was included in the reaction mixture for uridine phosphorylase, FUra was rapidly channeled into FUra nucleotides via its nucleoside. The rate of channeling appeared to correlate with the nucleoside phosphorylase activity in the various cell lines. In several cell lines activities of nucleotide-degrading enzymes were rather high and interfered with the measurement of orotate phosphoribosyl transferase (OPRT) with FUra as substrate. Addition of the phosphatase inhibitor glycerol-2-phosphate partly prevented breakdown of the newly formed 5-fluorouridine 5'-monophosphate and enabled measurement of OPRT. The WiDr cell line had a relatively high OPRT activity which could explain its sensitivity to FUra. The activity of thymidylate synthase was measured at a suboptimal concentration of 1 microM and at the optimal concentration of 10 microM deoxyuridine 5'-phosphate. With all cell lines the ratio between the activities at 10 and 1 microM was between 2.3 and 3.6. The activity of thymidylate synthase was lowest in WiDr and IGR3 cells and 3-4 times higher in M5 and Intestine 407 cells. The inhibition of 0.01 microM 5-fluorodeoxyuridine 5'-monophosphate was 80-90% at 1 microM deoxyuridine 5'-phosphate and 50-70% at 10 microM deoxyuridine 5'-phosphate with all cell lines. At 0.1 microM 5-fluorodeoxyuridine 5'-monophosphate enzyme activity was inhibited by 95-100%. The incorporation of FUra into RNA was relatively low in IGR3 cells and 3-5 times higher in all other cell lines.(ABSTRACT TRUNCATED AT 400 WORDS)
The metabolism of 5-fluorouracil (5-FU) was studied in biopsy specimens of primary colorectal cancer and healthy colonic mucosa obtained from previously untreated patients immediately after surgical removal. The conversion of 5-FU to anabolites was measured under saturating substrate (5-FU) and cosubstrate concentrations. For all enzymes, the activity was about threefold higher in tumor tissue compared with healthy mucosa of the same patient. The activity of pyrimidine nucleoside phosphorylase with deoxyribose-1-phosphate (dRib-1-P) was about tenfold higher (about 130 and 1200 nmol/hr/mg protein in tumors) than with ribose-1-phosphate (Rib-1-P), both in tumor and mucosa. Synthesis of the active nucleotides (5-fluoro-uridine-5′-monophosphate [FUMP] and 5-fluoro-2′deoxyuridine-5′-monophosphate [FdUMP]) was studied by adding physiologic concentrations of adenosine triphosphate (ATP) to the reaction mixture; the rate of FdUMP synthesis was 50% of that of FUMP (about 4 and 7 nmol/hr/mg protein in tumors). Direct synthesis of FUMP from 5-FU in the presence of 5-phosphoribosyl-1-pyrophosphate (PRPP) was about 2 nmol/hr/mg protein. With the natural substrate for this reaction, orotic acid, the activity was about 14-fold higher. To obtain insight into the recruitment of precursors for these cosubstrates, the authors also tested the enzyme activity of pyrimidine nucleoside phosphorylase with inosine and ribose-5-phosphate (Rib-5-P, as precursors for Rib-1-P) and deoxyinosine (as a precursor for dRib-1-P); enzyme activities were approximately 7%, 7%, and 3%, respectively, of that with the normal substrates, both in tumors and mucosa. However, when ATP and Rib-5-P were combined, the synthesis of FUMP was about 70% of that with PRPP, but only in tumors. In normal tissues no activity was detectable. These data suggest a preference of colon tumor over colon mucosa for the conversion of 5-FU to active nucleotides by a direct pathway; a selective antitumor effect of 5-FU may be related to this difference.
