Activation of multidomain and BH3-only pro-apoptotic Bcl-2 family members in p53-defective cells.
In the p53-deficient human B lymphoma Namalwa cell line that quickly undergoes apoptosis after DNA topoisomerase I inhibitor (camptothecin, CPT) treatment, we observed rapid and slight induction of the pro-apoptotic BH3-only Bik, Bim-EL, Bim-L and Bim-S proteins. In contrast, the expression levels of Bad and multidomain Bax-alpha and Bak remained mostly unchanged after CPT treatment. However, multiple pro-apoptotic proteins, including Bax-alpha, Bak, Bik, Bim-EL and Bim-L, translocated rapidly to the mitochondria after CPT treatment. Gel filtration chromatography experiments demonstrated that somes of the pro-apoptotic proteins assemble themselves into high molecular weight protein complexes. The protein composition of these oligomers was further analyzed by co-immunoprecipitation experiments performed on highly purified mitochondrial fractions, which revealed the formation of Bax/Bak, Bax/VDAC1, Bak/VDAC1, Bim/VDAC1 and Bim/Bcl-2 complexes after DNA damage induction. Thus, it appeared that induction, mitochondrial translocation and assembly in multimeric protein complexes of several pro-apoptotic members of the Bcl-2 family correlated with the rapid activation of apoptosis in a p53-independent pathway after CPT-mediated DNA strand breaks.
Paquet
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C
Centre de recherche, Centre hospitalier de l'Université de Montréal, Hôpital Notre Dame and Institut du Cancer de Montréal, Montreal (Que.) Canada.
Schmitt
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E
Beauchemin
M
M
Bertrand
R
R
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; D002166:Camptothecin / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016158:Genes, p53; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008854:Microscopy, Electron; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins; D011233:Precipitin Tests; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D020298:U937 Cells; D051028:bcl-2-Associated X Protein
D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; C000606775:BCL2L11 protein, human; C096820:BIK protein, human; D000072224:Bcl-2-Like Protein 11; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D002166:Camptothecin