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Accumulation of DNA Strand Breaks in Cells Exposed to Methotrexate or N¹⁰-Propargyl-5,8-dideazafolic Acid¹

Division of Experimental Oncology 1, Centro di Riferim. Oncologico, Aviano, PN [A. L., G. T., M. Bo., G. R.], and Istituto di Ricerche Farmacologiche "Mario Negri," Milan [E. E., M. Br., M. D.], Italy

N¹⁰-Propargyl-5,8-dideazafolic acid (CB 3717), a new antifolate which directly inhibits thymidylate synthase and which is now under early clinical investigation, was compared with methotrexate (MTX) for its antiproliferative activity and mode of action on M14 human melanoma cell line and NIH/3T3 murine fibroblasts transfected with human c-Ha-ras oncogene (NIH/3T3R). CB 3717 was as active as MTX on both cell lines in inhibiting colony formation, but 20–100 times less potent. After 24 h of exposure both drugs caused an accumulation of cells in the G₁ phase of the cell cycle, probably because of inhibition of DNA synthesis and blockage at the G₁-S boundary. In NIH/3T3R treated for 16 h with 2 µM MTX or 200 µM CB 3717, we found DNA single-strand breaks amounting to approximately 130 and 140 rad equivalents, respectively, and a considerable number of DNA double-strand breaks, far more than expected if they had been the result of the proximity of single-strand breaks on the two complementary DNA strands. No DNA-protein cross-links were detected. When cells were incubated in drug-free medium for 8 h, there was a further accumulation of single-strand breaks, possibly due to the effects of the drug retained intracellularly as polyglutamyl derivative. Simultaneous treatment with 1.77 µM cycloheximide prevented DNA damage produced by both drugs. Thymidine (10 µM), renewed in the culture medium every 24 h, also prevented DNA damage and cytotoxicity. Since after 16 h treatment with MTX or CB 3717 cells were completely viable, as assessed by [³H]thymidine release, trypan blue exclusion test, and ⁵¹Cr release, DNA damage appears to be an early event preceding cell death and may be a feature of the killing ability of the drugs. The involvement of a protein in the formation of DNA breaks is suggested by the fact that when protein synthesis was inhibited with cycloheximide DNA damage was no longer seen.

¹ The study was partially supported by the Italian Association for Cancer Research and by the Italian National Research Council, Rome, Italy, Progetto Oncologia Np. 86.00653.44.

² To whom requests for reprints should be addressed, at Division of Experimental Oncology 1, Centro di Riferim. Oncologico, 33081 Aviano, PN, Italy.

Received 12/10/86. Revised 6/30/87. Revised 11/16/87. Accepted 12/22/87.

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