Enhanced cytotoxicity with methotrexate in conjunction with hypoxanthine in L1210 cells in culture

Summary

By inhibiting dihydrofolate reductase, methotrexate (MTX) depletes cellular stores of reduced folates, resulting in the inhibition of DNA and RNA synthesis. Inhibition of RNA synthesis arrests cells in the G₁ phase of the cell cycle, preventing these cells from entering S phase and rendering them insensitive to MTX. Because MTX cytotoxicity can be enhanced by concurrent administration of hypoxanthine (HX), we examined the hypothesis that this modulation can allow normal rates of RNA synthesis and cell cycle progression from G₁ to S phase. For L1210 cells exposed to MTX for 12 h or 24 h, the addition of HX enhanced the cytotoxicity of MTX; however, no enhancement was observed with a 6-h exposure. Inhibition of RNA synthesis by MTX was prevented by concurrent administration of HX. The effect of HX on cell cycle progression was first examined using flow cytometry, which indicated that MTX treatment alone or with concurrent HX caused a buildup of cells with aG₁ content of DNA. Because this technique may fail to distinguish between cells in late G₁ phase, the G₁/S border, or early S, the method of premature chromosome condensation was used to determine cell cycle position based on chromatin morphology. A shift to a higher degree of chromatin decondensation was observed when HX was coadministered with MTX during a 12-h exposure, suggesting progression from G₁ towards S. This correlated with the enhancement of MTX cytotoxicity by HX after 12 h exposure. The results of these studies suggest that HX potentiates MTX cytotoxicity by maintaining RNA synthesis, allowing cells that mightordinarily be arrested in G₁ to progress into the cytotoxic S phase.