lometrexol and Colonic-Neoplasms

The class of folate antimetabolites typified by (6R)-dideazatetrahydrofolate (lometrexol, DDATHF) are specific inhibitors of de novo purine synthesis because of potent inhibition of glycinamide ribonucleotide formyltransferase (GART) but do not induce detectable levels of DNA strand breaks. As such, they are a test case of the concept that ribonucleotide depletion can be sensed by p53, resulting in a G(1) cell cycle block. The GART inhibitors have been proposed previously to be cytotoxic in tumor cells lacking p53 function but only cytostatic in p53 wild-type tumor cells. We have investigated this concept. Cell cycle progression into and through S phase was slowed by DDATHF, but both p53 +/+ and -/- human colon carcinoma cells entered and completed one S phase in the presence of drug. This inability of p53 to initiate a G(1) arrest after DDATHF treatment was mirrored by an independence of the cytotoxicity of DDATHF on p53 function. We conclude that carcinoma cells are killed equally well by DDATHF and related compounds whether or not the p53 pathway is intact and that the utility of GART inhibitors would not be limited to p53-negative tumors.

Topics: Alleles; Animals; Cell Division; Colonic Neoplasms; Folic Acid Antagonists; G1 Phase; Glutamates; HeLa Cells; Humans; Hydroxymethyl and Formyl Transferases; Leukemia L1210; Mice; Mitosis; Neoplasms; Phosphoribosylglycinamide Formyltransferase; Purines; Pyrimidines; S Phase; Tetrahydrofolates; Tumor Cells, Cultured; Tumor Suppressor Protein p53

(6R)5,10-Dideazatetrahydrofolic acid (DDATHF, Lometrexol), a potent antitumor drug in vivo and in vitro, is an inhibitor of the two folate-dependent enzymes in the de novo purine biosynthesis pathway: glycinamide ribonucleotide (GAR) and amino imidazole carboxamide (AICAR) transformylases. A single dose of DDATHF (50 mg/kg, i.p.) in C57/BL6 mice caused a prolonged depletion of purine nucleotides (ATP and GTP) in colon 38 tumor and only a temporary effect in liver. GAR transformylase activity was higher in colon 38 tumor than in liver, but a kinetic analysis on the purified enzyme showed no differences in Ki values for DDATHF or Km values for the folate substrate. As a consequence of de novo purine synthesis inhibition, there was a 2- to 3-fold elevation of 5-phosphoribosyl-1-pyrophosphate pools in colon 38 tumor between 4 and 12 hr after DDATHF administration. When DDATHF (50 mg/kg) was administered 4 or 8 hr prior to 5-fluorouracil (5-FU; 85 mg/kg, i.p., weekly), these biochemical effects significantly increased the antitumor activity of 5-FU, with a modest increase in toxicity. Lower doses of DDATHF (25 and 37.5 mg/kg) when combined with 5-FU also resulted in an improved antitumor activity without additional toxicity. The two different schedules of administration for DDATHF, 4 and 8 hr prior to 5-FU, showed no differences in antitumor activity or toxicity.

Topics: Acyltransferases; Animals; Antineoplastic Agents; Colonic Neoplasms; Drug Synergism; Female; Fluorouracil; Hydroxymethyl and Formyl Transferases; Mice; Mice, Inbred C57BL; Phosphoribosyl Pyrophosphate; Phosphoribosylglycinamide Formyltransferase; Purine Nucleotides; Tetrahydrofolates

We have studied the cytotoxicity of 5,10-dideazatetrahydrofolate (DDATHF) and of D-1694 to human WiDr colonic carcinoma cells as a model system for the effects of pure inhibitors of either the de novo purine synthesis pathway or thymidylate synthesis. The growth of this cell line was inhibited by very low concentrations of either agent and the lethality of DDATHF and D-1694 was completely prevented by continuous exposure to either hypoxanthine or thymidine, respectively, indicating that these compounds were very potent metabolic inhibitors, each specific for one of these pathways. D-1694 was highly cytotoxic (> 3 logs of kill) after a 4-h exposure to 1 microM drug, or a 24-h exposure to very low concentrations (0.04 microM). On the other hand, the cytotoxicity of DDATHF was substantially lower, with 2 logs of cell kill requiring >> 100 microM with 4 h of exposure or approximately 40 microM for 72 h of exposure. Maximal cell kill induced by D-1694 was 5-6 logs, consistent with elimination of all viable cells except preexisting mutants. A maximum of 2-3 logs of cell kill was observed with DDATHF. Exposure of WiDr cells to either D-1694 or DDATHF caused striking cellular changes, but the morphologies of cells treated with the two drugs were remarkably different. D-1694-treated cells detached from the dish within 1-2 days after a megaloblastosis, whereas DDATHF-treated cells remained adherent to the dishes for at least 10 days after treatment. The addition of thymidine to D-1694-treated cultures or hypoxanthine to DDATHF-treated cells after up to 20 h of drug exposure completely prevented cytotoxicity of either drug. With longer exposures, cytotoxicity of both drugs progressively increased in spite of such rescue. Our results indicate that substantial (99-99.9%) tumor cell kill can be induced by a pure inhibitor of purine synthesis, but that the rate of commitment to cell death and the extent of cell kill is greater with a pure inhibitor of thymidylate synthesis.

Topics: Acyltransferases; Antineoplastic Agents; Cell Division; Cell Survival; Colonic Neoplasms; Folic Acid Antagonists; Humans; Hydroxymethyl and Formyl Transferases; Hypoxanthine; Hypoxanthines; Methotrexate; Phosphoribosylglycinamide Formyltransferase; Quinazolines; Tetrahydrofolates; Thiophenes; Thymidine; Thymidylate Synthase; Tumor Cells, Cultured