Increase in topoisomerase‐II‐mediated dna breaks and cytotoxicity of VP16 in human U937 lymphoma cells pretreated with low doses of methotrexate

Abstract

Pre‐treatment with low, non‐toxic concentrations (0.04μM) of methotrexate (MTX) for 16 hr increased etoposide (VP16)‐induced growth inhibition and cytotoxicity in the U937 human histiocytic lymphoma cell line. VP16 cytotoxicity was significantly potentiated when the drug was given for 2 hr immediately after MTX pre‐treatment or between 2 and 4 hr or 4 and 6 hr after recovery from MTX pre‐treatment. By 24 hr after recovery from MTX, no potentiation was evident. The increased cytotoxicity of VP16 was associated with an increase in drug‐induced DNA breaks as assessed by the alka‐line elution method after proteinase K digestion. The amount of DNA single‐strand breaks (DNA SSB) increased when the drug was given 0, 2, and 4 hr after MTX pre‐treatment. DNA SSBs induced by the drug between 6 and 24 hr after MTX pre‐treatment were similar to those seen in cells without pre‐treatment. The amount of DNA double‐strand breaks (DNA DSB) caused by VP16 increased significantly when the drug was given 4 hr after recovery from MTX pre‐treatment. VP16‐induced DNA DSBs were still higher 6 hr after MTX pre‐treatment, but by 24 hr they were similar to those observed in MTX‐untreated cells. Flow cytometric analysis showed that MTX pre‐treatment was causing an accumulation of U937 cells at the G₁‐S boundary of the cell cycle. When MTX was removed, a wave of synchronization followed. Using Western blot electrophoresis and polyclonal antibodies to antitopoisomerase II, we found that MTX pre‐treatment raised the cellular topoisomerase II content. Our findings suggest that the potentiation of VP16 cytotoxicity on U937 cells by low, non‐toxic MTX pre‐treatment is due to a larger fraction of S‐phase cells containing a higher concentration of topoisomerase II, which is the putative target of VP16 action.