Interferon-alpha-induced apoptosis in U266 cells is associated with activation of the proapoptotic Bcl-2 family members Bak and Bax.
We have recently reported that the cytokine interferon-alpha (IFNalpha), commonly used in the treatment of cancer, induced a caspase-dependent apoptosis in tumor cell lines. The signaling mechanisms involved have not been defined. Here, we show that both proapoptotic Bcl-2 family members Bak and Bax were activated by IFNalpha, strictly in correlation with the induction of apoptosis. Using double stainings, we demonstrated that Bak was activated prior to cytochrome c (cyt c) release and caspase-3 activation, whereas activated Bax was only found in cells with released cyt c, mitochondrial depolarization, as well as activated caspase-3. Furthermore, IFNalpha-induced activation of Bak, and to a large extent also of Bax, was dependent on caspase activity. With the use of a panel of specific caspase inhibitors we found, however, that none of caspases-1 to -10 were responsible for this activation. Neither was the Ca(2+)-dependent protease calpain nor the stress-activated p38 SAPK pathway significantly involved. Overexpression of Bcl-2 blocked apoptosis induced by IFNalpha totally abolished Bak activation, as well as decreased the amount of activated Bax. We conclude that IFNalpha induces Bak and Bax activation via distinct mechanisms involving an unknown protease, and that their activation is regulated by Bcl-2.
Panaretakis
Theocharis
T
Department of Oncology and Pathology, Cancer Centre Karolinska R8:03, Karolinska Hospital and Institute, Stockholm S-171 76, Sweden.
Pokrovskaja
Katja
K
Shoshan
Maria C
MC
Grandér
Dan
D
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D051023:BH3 Interacting Domain Death Agonist Protein; D002051:Burkitt Lymphoma / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003574:Cytochrome c Group / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009101:Multiple Myeloma / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D048051:p38 Mitogen-Activated Protein Kinases
C494772:BAK1 protein, human; C494826:BAX protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002352:Carrier Proteins; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D016898:Interferon-alpha; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D002154:Calpain; D020169:Caspases