Drug concentration-dependent DNA lesions are induced by the lipid-soluble antifolate, piritrexim (BW301U)

The lipid-soluble antagonist, 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidi ne (piritrexim; BW301U), induced misincorporation of dUMP in human B (SB)- and T (MOLT-4)-lymphoblastoid cells, and in human promyelocytic leukemia cells (HL-60). Analysis by alkaline sucrose gradients and alkaline elution indicated that ³H-DNA that had been labeled for 15 min distributed into progressively smaller DNA fragment sizes in a drug concentration-dependent manner from 0 μM to 50 μM piritrexim. The phenomenon was observed regardless of the labeled nucleotide precursor employed for detection of newly synthesized DNA ([³H]deoxyuridine, [³H]deoxyadenosine, or [³H]deoxycytidine). In contrast, formaldehyde denaturation and sedimentation of DNA in neutral denaturing sucrose gradients released only 3-4% of the newly synthesized DNA as 3S-6S fragments (80-200 nucleotides), whereas the remaining population of newly synthesized DNA pelleted to the bottom of the tube. Failure to detect DNA fragmentation under neutral conditions to the extent observed under alkaline conditions indicated the presence of apurinic and apyrimidinic sites in DNA-lesions which would be expected in DNA undergoing excision-repair of misincorporated dUMP. Cytotoxicity resulting from dUMP misincorporation was consistent with the enhanced toxicity of piritrexim which was observed when HL-60 cells or MOLT-4 cells were exposed concurrently to exogenous deoxyuridine. Deoxyuridine-enhanced toxicity was demonstrated to be concentration dependent for both cell lines when piritrexim concentrations were marginally toxic. The cytotoxic effect of dUMP misincorporation was further substantiated by the observation that MOLT-4 cells treated with 0.5 μM piritrexim alone eventually developed resistance to the drug, whereas treatment with both piritrexim and 10 μM deoxyuridine prevented the selection of piritrexim-resistant cells.