SENSITIVITY OF HUMAN, MURINE, AND RAT-CELLS TO 5-FLUOROURACIL AND 5'-DEOXY-5-FLUOROURIDINE IN RELATION TO DRUG-METABOLIZING-ENZYMES

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  • 1 January 1986
    • journal article
    • research article
    • Vol. 46  (1) , 20-28
Abstract
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 .mu.M) and to its analogue 5''deoxy-5-fluorouridine (5''dFUR) (50% growth inhibitory concentration, 18 .mu.M). 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 .mu.M, and for 5''dFUR between 54 and 160 .mu.M. 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 .mu.M and at the optimal concentration of 10 .mu.M deoxyuridine 5''-phosphate. With all cell lines the ratio between the activities at 10 and 1 .mu.M 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 .mu.M 5-fluorodeoxyuridine 5''-monophosphate was 80-90% at 1 .mu.M deoxyuridine 5''-phosphate and 50-70% at 10 .mu.M deoxyuridine 5''-phosphate with all cell lines. At 0.1 .mu.M 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. Incorporation of FUra into DNA showed the same pattern. In the three melanoma cell lines N-(phosphonacetyl)-L-asparatate decreased the concentration of normal pyrimidine nucleotides, which facilitated the detection of 5-fluorouridine 5''-triphosphate. However, the amount of 5-fluorouridine 5''-triphosphate in the presence of N-(phosphonacetyl)-L-aspartate was not higher than in its absence. The amount of 5-fluorouridine 5''-triphosphate was comparable in the three cell lines although they showed a completely different enzyme pattern. We conclude that the inhibition of thymidylate synthase by 5-fluorodeoxyuridine 5''-monophosphate and incorporation of FUra into RNA contribute to FUra toxicity to a different extent in the various cell lines tested. These factors do not solely determine the sensitivity to FUra or 5''dFUR. A very low uridine phosphorylase activity is limiting for conversion of 5''dFUR to FUra but a high uridine phosphorylase activity does not correlate with a high sensitivity to either 5''dFUR or FUra. OPRT appears to play an appreciable role in the sensitivity of several cell lines to both FUra and 5''dFUR.