Pre-treatment with low, non-toxic concentrations (0.04 microM) 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 alkaline 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 pretreatment. 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.