Metabolic effects and mode of cytotoxicity of 5-deazaacyclotetrahydrofolate (5-DACTHF, BW543U76), a
glycineamide ribonucleotide transformylase 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 microM and 0.08 microM 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 microM
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 microM. 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 microM, 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 approximately 180-base pair intervals (internucleosomal breaks). Concomitant proteolysis of
nuclear proteins poly(ADP-ribose) polymerase and
lamin B was observed.