5-fluorouridine-5--triphosphate and Colonic-Neoplasms

To explore the determinants of cytotoxicity during prolonged exposure to pharmacologically relevant concentrations of 5-fluorouracil (FUra), we studied the effects of FUra at concentrations ranging from 0.1 to 1 microM in HCT 116 and HT 29 colon cancer cells grown in the presence of physiologic levels of leucovorin. A 5- and 7-day exposure to 1 microM FUra reduced cell growth to 46% and 20% of control in HT 29 cells and to 74% and 38% of control in HCT 116 cells. Concurrent exposure to thymidine (10 or 20 microM) or uridine (1 mM) provided partial protection against FUra toxicity in HT 29 cells, but did not protect HCT 116 cells. After a 24-h exposure to 1 microM [3H]FUra, free 5-fluoro-2'-deoxyuridine-5' -monophosphate (FdUMP) and FUDP. + FUTP levels were 0.7 and 144 pmol/10(6) cells in HT 29 cells, respectively, and 3.9 and 178 pmol/10(6) cells in HCT 116 cells. FdUMP and FUDP + FUTP pools increased by 5.7- and 2.0-fold in HT 29 cells and by 1.7- and 3.3-fold in HCT 116 cells over the next 48 h, but did not accumulate thereafter. After a 24-h exposure to 1 microM [3H]FUra, FUra-RNA levels were 158 and 280 fmol/microgram in HT 29 and HCT 116 cells, respectively; FUra-RNA levels increased over time, and reached 700 and 1156 fmol/microgram at day 5. Concurrent exposure to 1 mM uridine for 72 h did not diminish [3H]FUra-RNA incorporation. Upon removal of [3H]FUra following a 24-h exposure, FUra-RNA levels remained relatively stable with 57-78% retained at 120 h. A low level of [3H]FUra-DNA incorporation was detected in HT 29 cells. Thymidylate synthase (TS) catalytic activity in control cells was 2-fold higher in HCT 116 cells compared to HT 29 cells (47 vs. 23 pmol/min/mg). Total TS content increased 1.5- to 3-fold over control in both cell lines during FUra exposure, and ternary complex formation was evident for up to 96 h-dTTP pools were not depleted in FUra-treated cells, suggesting that residual TS catalytic activity was sufficient to maintain dTTP pools relative to demand. Surprisingly, the partial inhibition of TS was accompanied by a striking accumulation of immunoreactive "dUMP" pools in both lines; dUTP pools also increased 2-to 3-fold. In summary, the gradual and stable accumulation of FUra in RNA noted in both lines may account for the thymidine-insensitive component of FUra toxicity. Because dTTP pools were not appreciably diminished, the interference with nascent DNA chain elongation and induction of single-strand breaks in newly synthesized D

Topics: Cell Division; Cell Survival; Colonic Neoplasms; Deoxyribonucleotides; Deoxyuracil Nucleotides; DNA Damage; DNA, Neoplasm; Dose-Response Relationship, Drug; Fluorodeoxyuridylate; Fluorouracil; Gene Expression Regulation, Enzymologic; Humans; Leucovorin; Thymidylate Synthase; Tumor Cells, Cultured; Uridine Triphosphate

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.

Topics: Adenosine Triphosphate; Aged; Aged, 80 and over; Colon; Colonic Neoplasms; Fluorodeoxyuridylate; Fluorouracil; Humans; Intestinal Mucosa; Middle Aged; Orotate Phosphoribosyltransferase; Pentosyltransferases; Phosphoribosyl Pyrophosphate; Pyrimidine Phosphorylases; Rectal Neoplasms; Ribosemonophosphates; Uracil Nucleotides; Uridine Triphosphate

The nucleoside transport inhibitor dipyridamole can increase the cytotoxicity of 5-fluorouracil in a human colon cancer cell line (HCT 116) without affecting the total amount of fluorouracil incorporated into the acid soluble and insoluble fractions (J. L. Grem and P. H. Fischer, Cancer Res., 45: 2967-2972, 1985). We now report that dipyridamole altered the pattern of fluorouracil metabolism and provided a selective increase in intracellular fluorodeoxyuridine monophosphate (FdUMP) levels. At 2 and 4 h after exposure to fluorouracil and dipyridamole, FdUMP levels were approximately 5-fold higher in the presence of dipyridamole. The ratio of FdUMP to fluorouridine triphosphate at 4 h was substantially increased in the presence of dipyridamole (0.4 +/- 0.05) compared to fluorouracil alone (0.08 +/- 0.03). In cells preloaded with fluorodeoxyuridine (FdUrd), dipyridamole potently inhibited the efflux of FdUrd, leading to an increased retention of intracellular FdUMP. One h following removal of [6-3H]FdUrd, the FdUMP levels were increased 8-fold in the presence of dipyridamole, and the half-life of intracellular FdUMP was increased from 24 to 78 min. We have previously shown that the addition of sufficient thymidine (25 microM) can prevent the augmentation of fluorouracil toxicity produced by dipyridamole. In these studies, the addition of 25 microM thymidine reduced the FdUMP levels to less than half of those measured in the presence of fluorouracil plus dipyridamole for the first 8 h of exposure, and reduced the FdUMP levels to 6% of the FdUMP levels seen with fluorouracil and dipyridamole after 24 h of exposure. Thymidine prevented the enhanced intracellular retention of FdUMP produced by dipyridamole in cells preloaded with FdUrd. In addition, thymidine inhibited the accumulation of FdUMP in cells exposed to FdUrd. In cancer cells which significantly catabolize FdUMP, the ability of dipyridamole to block the efflux of FdUrd may provide an effective means of selectively increasing FdUMP levels and enhancing the toxicity of fluorouracil. Furthermore, dipyridamole blocked the efflux of deoxyuridine and prolonged the intracellular half-life of deoxyuridine monophosphate. In cells prelabeled with [2'-3H]dUrd, transfer of tritium to FdUrd and FdUMP occurred in cells exposed to fluorouracil and dipyridamole. These data suggest that blockade of nucleoside efflux can enhance the availability of deoxyribose-1-phosphate donors for the synthesis of FdUrd. Thus, dipyri

Topics: Cells, Cultured; Colonic Neoplasms; Deoxyuracil Nucleotides; Deoxyuridine; Dipyridamole; DNA; Floxuridine; Fluorodeoxyuridylate; Fluorouracil; Humans; Ribonucleotides; Time Factors; Uridine Triphosphate