Bcl-2 and mdr-1 gene expression during doxorubicin-induced apoptosis in murine leukemic P388 and P388/R84 cells.
The induction of apoptosis by doxorubicin (DOX) alone or in the presence of efflux blockers, verapamil (VPL) or trifluoperazine (TFP), and of bcl-2 and mdr-1 gene expression was analyzed in murine leukemic P388 and doxorubicin resistant P388/R84 cells. Incubation with DOX (0.1-1 microM) for 24 hours induced apoptosis in sensitive cells but not in the resistant P388/R84 cells. Flow cytometric analysis of apoptosis by terminal dideoxynucleotidyl (TdT) assay showed that 1 microM DOX induced apoptosis in 70% of P388 cells and in less than 5% of P388/R84 cells. When P388/R84 resistant cells were co-incubated with DOX and efflux blockers (10 microM VPL or 15 microM TFP), enhanced cellular DOX accumulation was accompanied by apoptosis. Quantitative analysis of DNA fragmentation by 14C-thymidine incorporation and gel electrophoresis of fragmented DNA confirmed the results from TdT assay and showed enhancement of DOX-induced DNA fragmentation in the presence of efflux blockers (VPL or TFP). DOX + VPL and DOX + TFP combination treatments induced apoptosis in upto 55% and 83% of P388/R84 cells, respectively. mdr-1 mRNA and P-gp expression were not altered during DOX-induced apoptosis. The results suggest that in murine leukemic cells, down-regulation of bcl-2 mRNA expression occurs during DOX-induced apoptosis and it depends on the cellular drug retention determined by mdr-1/P-gp drug efflux.
Ramachandran
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Department of Radiation Oncology, Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, FL 33136, USA.
You
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Krishan
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D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053938:DNA Fragmentation / Q000187:drug effects; D004317:Doxorubicin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D019450:Genes, MDR; D019254:Genes, bcl-2; D007941:Leukemia P388 / Q000188:drug therapy; D051379:Mice; D014268:Trifluoperazine / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
D014268:Trifluoperazine; D004317:Doxorubicin; D014700:Verapamil