Increase in topoisomerase-II-mediated dna breaks and cytotoxicity of VP16 in human U937 lymphoma cells pretreated with low doses of methotrexate
✍ Scribed by Aurelio Lorico; Mauro Boiocchi; Germana Rappa; Soumitra Sen; Eugenio Erba; Maurizio D'Incalci
- Publisher
- John Wiley and Sons
- Year
- 1990
- Tongue
- French
- Weight
- 619 KB
- Volume
- 45
- Category
- Article
- ISSN
- 0020-7136
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✦ Synopsis
Pre-treatment with low, non-toxic concentrations ( 0 . 0 4 ~~) of methotrexate (MTX) for I6 hr increased etoposide (VP 16)induced growth inhibition and cytotoxicity in the U937 human histiocytic lymphoma cell line. VP I6 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 in- crease 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 VPl6 increased significantly when the drug was given 4 hr after recovery from MTX pre-treatment. VPI64nduced DNA DSBs were still higher 6 hr after MTX pre-treatment, but by 24 hr they were similar t o those observed in MTX-untreated cells. Flow cytometric analysis showed that MTX pre-treatment was causing an accumulation of U937 cells at the G,-S boundary of the cell cycle. When MTX was removed, a wave of synchronization followed. Us- ing 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 VPl6 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 VPl6 action.
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