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The action of fluoropyrimidine (FP) drugs at thymidylate synthase (TS) is associated with enhanced chemotherapeutic response. Calcium leucovorin (CF) increases the cytotoxicity of the FP drugs, 5-fluorouracil and 5-fluorodeoxyuridine, in human laryngeal carcinoma HEp-2 cells by directing the action of these drugs at TS. Thus, the effect of CF on the cytotoxicity and site of action of the FP, 5-fluorouridine (FUrd), was investigated in HEp-2 cells. The cytotoxicity of FUrd was unaffected by CF. Moreover, CF was unable to alter the growth-limiting target of FUrd to TS. HEp-2 cells convert FUrd to FdUMP, the FP metabolite that is the direct inhibitor of TS; thus, the inability of CF to modulate FUrd action is not due to lack of inhibitor formation. In addition, greater than 90 percent of TS activity is inhibited at concentrations of FUrd that inhibit HEp-2 cell growth by 50 percent. Thus, while TS is significantly inhibited by FUrd, it is not the growth-limiting target of this drug. It is likely that the RNA-directed effects of FUrd are so extensive that CF, which maximizes TS-directed action, is ineffective at reducing the cytotoxicity further. An approach to overcoming the RNA-directed effects of FUrd is suggested.

N6-(delta2-Isopentenyl)adenosine (IPAR) inhibited severely the incorporation of uridine and cytidine into S-180 cells in culture. When IPAR and the nucleosides were simultaneously present in the medium the inhibition was competitive (Ki 3.4 muM) and indicated inhibition of transport. However, the inhibition occurred even in the absence of extracellular IPAR if the cells had been preincubated with IPAR. Since 5'-IPAMP was the product which accumulated in large quantities in S-180 cells when incubated with IPAR, the effects of this AMP analog of the intracellular metabolism of uridine had to be considered. No direct correlation between the amount of intracellular IPAMP and the degree of inhibition of uridine utilization was observed and the relative distribution of uridine nucleotides in the acid soluble pool of the cells was unaltered in cells treated with IPAR. Also, IPAMP was not an inhibitor of uridine kinase in a cell free system nor was the activity of this enzyme affected by treatment of cells with IPAR. In addition, a profound inhibition of uridine utilization was also observed in a resistant subline of S-180 cells, which is unable to form IPAMP. These data suggest that IPAMP was not the inhibitory agent. Furthermore, the observation that the inhibition in both sensitive and resistant cells was caused even by a 15-second exposure to 100 muM IPAR, followed by rinsing, suggests that IPAR itself is the effective agent. It is concluded that IPAR exerts its inhibitory effect on uridine and cytidine utilization by becoming lodged in the cell membrane and thereby preventing the passage of these nucleosides into the cells. It is also shown that the inhibition of uridine and cytidine utilization by IPAR and by other potent nucleoside uptake inhibitors is unrelated to inhibition of growth or RNA-synthesis when the cells do not depend on an extracellular source of a nucleoside for growth.

The purpose of this study was to compare the pools of free FdUMP derived from 5-fluorouracil (FUra) and of dUMP synthesized de novo in Hep-2 and S-180 cells, their relationship to inhibition of thymidylate synthase (dTMP synthase; EC 2.1.1.45), and the effect of excess folinic acid (CF) on these parameters. These cells differ 50-fold in their sensitivity to FUra and, in the absence of thymidine, dTMP synthase is the growth-limiting site of action of FUra in S-180 cells, but in Hep-2 cells this site becomes growth-limiting only in the presence of excess folates. In both cells after a 3-hr incubation with varied concentrations of FUra, FdUMP comprised only 0.1-0.2% of the total acid-soluble pools derived from FUra. The changes in dUMP and FdUMP pools paralleled each other, dUMP being 1000-2000 times higher than FdUMP. The pools of dUMP increased only when dTMP synthase was significantly inhibited. This occurred in S-180 cells above 3 microM FUra and in Hep-2 cells above 30 microM, where the residual dTMP synthase was similar in both cells. Under these conditions, the dUMP and FdUMP pools in Hep-2 cells were 2 and 4 times higher, respectively, than in S-180 cells. After FUra removal, both pools continued to increase, dUMP and FdUMP pools in Hep-2 cells rising 6-fold and 10-fold higher, respectively, than in S-180 cells. The dTMP synthase inhibition and the high nucleotide pools in Hep-2 were short-lived, whereas in S-180 cells the inhibition and the pools were maintained longer. Excess CF retarded the recovery of dTMP synthase after FUra removal only in Hep-2 cells and led to a further increase in dUMP and FdUMP pools in these cells, while having no effect in S-180 cells. These data indicate that a high capacity of cells to accumulate free FdUMP does not alone guarantee that dTMP synthase inhibition will be growth-limiting. The relationship shown here between excess CF, dTMP synthase recovery, and the nucleotide pools suggests that some cell types, such as Hep-2, in spite of high levels of FdUMP, require in addition an excess of folates to retard dTMP synthase recovery and make it growth-limiting.

Twenty-one cell lines (six human lines, and nine mouse lines) were compared with respect to inhibition of growth by N-6-(delta-2-isopentenyl)adenosine (IPAR). Six of these, mouse Sarcoma 180, Ehrilich ascites carcinoma, mammary adenocarconoma (TA3), leukemia L1210, mouse kidney, and canine kidney cells, differed by up to 16-fold with respect to their sensitivity and were chosen for further study. One factor contributing to the resistance was a slower formation of intracellular 5-monophosphate of IPAR (IPAMP) due to reduced adenosine kinase activity. Because of this slower formation of IPAMP in the resistant cells, a higher extracellular IPAR was required for the maintenance of equal intracellular IPAMP levels. Regardless of the degree of resistance, the rate of decay of intracellular IPAMP was similar and very rapid, with a half-life of 37 plus or minus 5 min. In the sensitive cells IPAMP was cleaved back to IPAR, while in the resistant cells IPAR was cleaved further to the free base, N-6-(delta-2-isopentenyl)adenine (IPA), which accumulated in the medium. The rate of formation of IPA constitutes an irreversible inactivation of IPAR, because IPA is not converted back to IPAMP and is not growth inhibitory. In one of the resistant cells (mouse kidney) the inactivation was so rapid that in 1 hr 25% of the extracellular (30 muM) IPAR was converted to IPA.

Analogues of pyridoxal and of pyridoxal phosphate in which the 4-CHO group is replaced with CH = CH2 were synthesized and were found to be potent inhibitors of pyridoxal kinase and pyridoxine phosphate oxidase of rat liver. They also inhibited the growth of mouse Sarcoma 180 and mammary adenocarcinoma TA3 in cell culture. Saturation of the vinyl double bond, replacement of the 5-CH2OH with methyl, methylation of the phenolic hydroxyl, or conversion to the N-oxide resulted in diminution or loss of all these activities. Similarly, the introduction of a beta-methyl group into the vinyl analogues of pyridoxal reduced all these inhibitory activities. The 4-vinyl anatogue of pyridoxal was shown to be a substrate of pyridoxal kinase and the product a potent inhibitor of pyridoxine oxidase, competing with pyridoxal phosphate. The affinity of this phosphorylated pyridoxal analogue to some apoenzymes varied greatly, indicating striking differences among the cofactor binding sites of these enzymes. The growth inhibitory effects of these analogues on cells in culture correlated well with their effects on pyridoxal kinase and pyridoxine phosphate oxidase in cell-free systems.

The growth-inhibitory effect of 6-methylmercaptopurine riboside (MMPR) against leukemia L1210 cells in culture was dramatically potentiated by the addition of guanine nucleosides to the medium. In the presence of either deoxyguanosine or guanosine, the concentration of MMPR that caused 50% inhibition of growth was 35 times lower than in the absence of these nucleosides. Similar potentiation was also observed against Sarcoma 180 cells in culture by guanosine. The metabolic basis of this synergism was approached in a study of the incorporation of [14C]glycine into 5'-phosphoribosyl-N-formylglycinamide in Sarcoma 180 cells. The results show that the site of inhibition resulting in synergism is an early step in purine biosynthesis, probably phosphoribosyl pyrophosphate amidotransferase (EC 2.4.2.14). In the L1210 cell system, the addition of hypoxanthine to the medium prevented the potentiation of MMPR by guanine nucleosides supporting the conclusion that the site of the synergistic interaction involves purine biosynthesis de novo. While hypoxanthine partially reversed the growth-inhibitory effects of MMPR, an even higher degree of protection was observed in the presence of both uridine and hypoxanthine, suggesting that MMPR may have additional sites of action concerned with pyrimidine metabolism.