The course of etoposide-induced apoptosis from damage to DNA and p53 activation to mitochondrial release of cytochrome c.
Treatment of L929 fibroblasts by the topoisomerase II inhibitor etoposide killed 50% of the cells within 72 h. The cell killing was preceded by the release of cytochrome c from the mitochondria. Simultaneous treatment of the cells with wortmannin, cycloheximide, furosemide, cyclosporin A, or decylubiquinone prevented the release of cytochrome c and significantly reduced the loss of viability. Etoposide caused the phosphorylation of p53 within 6 h, an effect prevented by wortmannin, an inhibitor of DNA-dependent protein kinase (DNA-PK). The activation of p53 by etoposide resulted in the up-regulation of the pro-apoptotic protein Bax, a result that was prevented by the protein synthesis inhibitor cycloheximide. The increase in the content of Bax was followed by the translocation of this protein from the cytosol to the mitochondria, an event that was inhibited by furosemide, a chloride channel inhibitor. Stably transfected L929 fibroblasts that overexpress Akt were resistant to etoposide and did not translocate Bax to the mitochondria or release cytochrome c. Bax levels in these transfected cells were comparable with the wild-type cells. The release of cytochrome c upon translocation of Bax has been attributed to induction of the mitochondrial permeability transition (MPT). Cyclosporin A and decylubiquinone, inhibitors of MPT, prevented the release of cytochrome c without affecting Bax translocation. These data define a sequence of biochemical events that mediates the apoptosis induced by etoposide. This cascade proceeds by coupling DNA damage to p53 phosphorylation through the action of DNA-PK. The activation of p53 increases Bax synthesis. The translocation of Bax to the mitochondria induces the MPT, the event that releases cytochrome c and culminates in the death of the cells.
Karpinich
Natalie O
NO
Department of Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Tafani
Marco
M
Rothman
Ronald J
RJ
Russo
Matteo A
MA
Farber
John L
JL
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival; D002478:Cells, Cultured; D018118:Chloride Channels / Q000037:antagonists & inhibitors; D003513:Cycloheximide / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004249:DNA Damage; D004232:Diuretics / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects; D005665:Furosemide / Q000494:pharmacology; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology*; D010766:Phosphorylation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000494:pharmacology; D015854:Up-Regulation; D000077191:Wortmannin; D051028:bcl-2-Associated X Protein
D000730:Androstadienes; C494827:Bax protein, mouse; D018118:Chloride Channels; D003574:Cytochrome c Group; D004232:Diuretics; D004791:Enzyme Inhibitors; D019384:Nucleic Acid Synthesis Inhibitors; D011500:Protein Synthesis Inhibitors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016271:Proto-Oncogene Proteins c-myc; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D014451:Ubiquinone; C060262:2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone; D005047:Etoposide; D005665:Furosemide; D003513:Cycloheximide; D000077191:Wortmannin