TY - JOUR
T1 - Accumulation of DNA strand breaks in cells exposed to methotrexate or N10-propargyl-5,8-dideazafolic acid
AU - Lorico, A.
AU - Toffoli, G.
AU - Boiocchi, M.
AU - Erba, E.
AU - Broggini, M.
AU - Rappa, G.
AU - D'Incalci, M.
PY - 1988
Y1 - 1988
N2 - N10-Propargyl-5,8-dideazafolic acid (CB 3717), a new antifolate which directly 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 becacuse of inhibition of DNA synthesis and blockage at the G1-S boundary. In NIH/3T3R treated for 16 h with 2 μ 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.
AB - N10-Propargyl-5,8-dideazafolic acid (CB 3717), a new antifolate which directly 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 becacuse of inhibition of DNA synthesis and blockage at the G1-S boundary. In NIH/3T3R treated for 16 h with 2 μ 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.
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M3 - Article
C2 - 3349474
AN - SCOPUS:0023880857
VL - 48
SP - 2036
EP - 2041
JO - Journal of Cancer Research
JF - Journal of Cancer Research
SN - 0008-5472
IS - 8
ER -