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Cytotoxicity of Antifolate Inhibitors of Thymidylate and Purine Synthesis to WiDr Colonic Carcinoma Cells¹

Departments of Biochemistry and Molecular Biology [R. G. M.], Molecular Pharmacology and Toxicology [N. L. L., R. G. M.], and the Norris Comprehensive Cancer Center [S. G. S., R. G. M.], University of Southern California, Los Angeles, California 90033

² To whom requests for reprints should be addressed, at Massey Cancer Center, Medical College of Virginia, Box 230, Richmond, VA 23298.

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 µM drug, or a 24-h exposure to very low concentrations (0.04 µM). On the other hand, the cytotoxicity of DDATHF was substantially lower, with 2 logs of cell kill requiring >>100 µM with 4 h of exposure or 40 µM 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.

¹ This work was supported in part by Grants CA 27605 and CA 36054 from the NIH, Department of Health and Human Services.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3/18/93. Accepted 9/21/93.

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