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 (0.04 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.

• Authors: A Lorico, M Boiocchi, G Rappa, S Sen, E Erba, M D'Incalci
• Journal: International journal of cancer (Int J Cancer) Vol. 45 Issue 1 Pg. 156-62 (Jan 15 1990) ISSN: 0020-7136 [Print] United States
• PMID: 2153635 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• DNA, Neoplasm
• DNA, Single-Stranded
• Topoisomerase II Inhibitors
• Etoposide
• DNA
• Methotrexate

Topics

• Blotting, Western
• Cell Line (drug effects)
• DNA (analysis, drug effects)
• DNA Damage
• DNA, Neoplasm (analysis, drug effects)
• DNA, Single-Stranded (analysis, drug effects)
• Dose-Response Relationship, Drug
• Drug Evaluation, Preclinical
• Drug Synergism
• Electrophoresis, Polyacrylamide Gel
• Etoposide (therapeutic use, toxicity)
• Flow Cytometry
• Humans
• Lymphoma, Large B-Cell, Diffuse (drug therapy, genetics)
• Methotrexate (administration & dosage)
• Time Factors
• Topoisomerase II Inhibitors
• Tumor Cells, Cultured (drug effects)