Metabolic effects and mode of cytotoxicity of 5-deazaacyclotetrahydrofolate (5-DACTHF, BW543U76), a glycineamide ribonucleotide transformlyase inhibitor, were studied in MOLT-4 cells, a human T-cell leukemia line. 5-DACTHF inhibits purine synthesis with 50% inhibitory concentration values of 0.5 µm and 0.08 µm following 6- or 24-h exposure to drug, respectively. At 6 h, adenine nucleotide synthesis is preferentially inhibited over guanine nucleotide synthesis. A similar effect was observed with another glycineamide ribonucleotide transformylase inhibitor, 5,10-dideazatetrahydrofolate. GTP was depleted to 40% of control and ATP to 10% of control by 5 µm 5-DACTHF. After a transitory increase, UTP and CTP were depleted to 30% of control. Deoxynucleotides were also depleted by the drug; dCTP was depleted to the greatest extent, followed by dATP, dTTP, and dGTP, respectively. MOLT-4 cell growth was inhibited by 5-DACTHF with a 50% inhibitory concentration of 0.066 µm. Complete reversal was effected by hypoxanthine, and there was no reversal by thymidine. The drug was cytotoxic to MOLT-4 cells in the range 0.25 to 5.0 µm, but a minimum of 48 h was required for trypan blue-staining dead cells to appear. The rate and extent of kill with the thymidylate synthase inhibitor 2-methyl-10-propargyl-5,8-dideazafolate was greater than with 5-DACTHF, which indicates that kill by inhibition of thymidylate synthase is more effective than that by inhibition of purine synthesis. Electron microscopy of MOLT-4 cells exposed to 5-DACTHF showed electron-dense mitochondria and nuclear changes reminiscent of apoptosis. These morphological changes were accompanied by the appearance of DNA strand breaks at ∼180-base pair intervals (internucleosomal breaks). Concomitant proteolysis of nuclear proteins poly(ADP-ribose) polymerase and lamin B was observed.

1

The studies performed in the laboratory of S. H. K. were funded in part by grants from the NIH (CA 50325) and American Cancer Society.

This content is only available via PDF.