Increase in topoisomerase-II-mediated DNA breaks and cytotoxicity of VP16 in human U937 lymphoma cells pretreated with low doses of methotrexate.
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.
Lorico
A
A
Division of Experimental Oncology I, Centro di Riferimento Oncologico, Aviano, Italy.
Boiocchi
M
M
Rappa
G
G
Sen
S
S
Erba
E
E
D'Incalci
M
M
D015153:Blotting, Western; D002460:Cell Line / Q000187:drug effects; D004247:DNA / Q000032:analysis / Q000187:drug effects; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000032:analysis / Q000187:drug effects*; D004277:DNA, Single-Stranded / Q000032:analysis / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D004357:Drug Synergism; D004591:Electrophoresis, Polyacrylamide Gel; D005047:Etoposide / Q000627:therapeutic use / Q000633:toxicity*; D005434:Flow Cytometry; D006801:Humans; D016403:Lymphoma, Large B-Cell, Diffuse / Q000188:drug therapy* / Q000235:genetics; D008727:Methotrexate / Q000008:administration & dosage*; D013997:Time Factors; D059005:Topoisomerase II Inhibitors; D014407:Tumor Cells, Cultured / Q000187:drug effects
D004273:DNA, Neoplasm; D004277:DNA, Single-Stranded; D059005:Topoisomerase II Inhibitors; D005047:Etoposide; D004247:DNA; D008727:Methotrexate