Activation of Bak, Bax, and BH3-only proteins in the apoptotic response to doxorubicin.
The anthracyclin doxorubicin (DXR) is a major antitumor agent known to cause cellular damage via a number of mechanisms including free radical formation and inhibition of topoisomerase II. It is not clear, however, how the subsequent lesions may lead to the apoptotic death of the cell. We have here examined the effects of DXR on activation of pro-apoptotic members of the Bcl-2 family, all of which are connected to the mitochondrial events of apoptosis. In two human cell lines (lymphoma and myeloma), clinically relevant concentrations of DXR were found to induce apoptosis, first observed after 24 h of treatment. Apoptosis correlated with modulation of Bak and Bax to their active conformations. bax- as well as bak-deficient mouse embryo fibroblasts were resistant to DXR compared with wild-type mouse embryo fibroblasts further supporting a role for these proteins as main DXR-induced apoptosis regulators. Furthermore, using immunocytochemistry as well as chemical blocking of putative apical pathways we could demonstrate that Bak is activated prior to Bax. In the human cell lines, DXR was furthermore found to induce high protein levels of Bik, another BH3-only protein. DXR-induced apoptosis was completely blocked in Bcl-2-overexpressing U266 cells. Interestingly, in Bcl-2-transfected cells Bak activation was also blocked, while Bax was still partially active in agreement with differential regulation of these two proteins. Furthermore, co-incubation of the phosphatidylinositol 3-kinase (PI3K)-inhibitor LY294002 potentiated the apoptotic response to DXR. This enhanced apoptosis was preceded by enhanced Bak and Bax activation, and both responses as well as apoptosis were blocked in transfectants overexpressing Bcl-2. In summary, several pieces of evidence suggest that DXR induces apoptosis through a sequential and differential activation of Bak and Bax.
Department of Oncology and Pathology, Cancer Centrum Karolinska (CCK), Karolinska Hospital and Institute, S-171 76 Stockholm, Sweden.
Theocharis
T
Panaretakis
Katja
K
Pokrovskaja
Maria C
MC
Shoshan
Dan
D
Grandér
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D015153:Blotting, Western; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009025:Morpholines / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein
D000970:Antineoplastic Agents; C494773:Bak1 protein, mouse; C494827:Bax protein, mouse; D002867:Chromones; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D004317:Doxorubicin