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Induction of HL-60 Leukemia Cell Differentiation by the Novel Antifolate 5,10-Dideazatetrahydrofolic Acid¹

Departments of Pharmacology and Pediatrics and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06510

The novel tetrahydrofolate, 5,10-dideazatetrahydrofolic acid (DDATHF), was designed as an inhibitor of folate metabolism at a site other than dihydrofolate reductase. DDATHF has been shown to inhibit glycinamide ribonucleotide transformylase, a folate-requiring enzyme that catalyzes the first of two one-carbon transfer reactions in the de novo purine nucleotide biosynthetic pathway. Incubation of HL-60 promyelocytic leukemia cells with 5 x 10^-8 to 10^-5 M DDATHF resulted in a marked inhibition of growth after 48 h, with a complete cessation of cellular replication by day 4. Cell cycle analyses of DDATHF-treated HL-60 cells demonstrated an initial block in early S phase by day 3 followed by an accumulation of cells in the G₁ and G₂ + M phases of the cell cycle. Inhibition of growth was accompanied by a concentration-dependent increase in the percentage of mature myeloid cells that expressed nitroblue tetrazolium positivity, and a small increase in nonspecific esterase activity. Induction of differentiation and inhibition of growth by DDATHF were completely prevented by hypoxanthine and 5(4)-amino-4(5)-imidazole carboxamide, suggesting that depletion of intracellular purine nucleotide pools has an important role in the biological effects of this inhibitor. This possibility was confirmed by the finding that DDATHF caused a pronounced reduction in intracellular GTP and ATP levels within 2 h, with maximum decreases being observed by 24 h, a time interval which preceded the inhibition of cellular proliferation by this agent. Pyrimidine nucleoside triphosphate levels were markedly increased under these conditions. The findings indicate the importance of purine nucleotides to both the inhibition of growth and the induction of differentiation of HL-60 leukemia cells by DDATHF.

¹ This research was supported in part by USPHS Grants CA-02817 and CA-42300 from the National Cancer Institute.

² To whom requests for reprints should be adressed, at Department of Pharmacology, Yale University School of Medicine, P. O. Box 3333, New Haven, CT 06510-8066.

Received 11/ 8/88. Revised 5/11/89. Accepted 6/ 7/89.