Endogenous bax translocation in SH-SY5Y human neuroblastoma cells and cerebellar granule neurons undergoing apoptosis.
Changes at the mitochondria are an early, required step in apoptosis in various cell types. We used western blot analysis to demonstrate that the proapoptotic protein Bax translocated from the cytosolic to the mitochondrial fraction in SH-SY5Y human neuroblastoma cells undergoing staurosporine- or EGTA-mediated apoptosis. Levels of mitochondrial Bax increased 15 min after staurosporine treatment. In EGTA-treated cells, increased levels of mitochondrial Bax were seen at 4 h, consistent with a slower onset of apoptosis in EGTA versus staurosporine treatments. We also demonstrate the concomitant translocation of cytochrome c from the mitochondrial to the cytosolic fractions. We correlated these translocations with changes in caspase-3-like activity. An increase in caspase-3-like activity was evident 2 h after staurosporine treatment. Inhibition of the mitochondrial permeability transition had no effect on Bax translocation or caspase-3-like activity in staurosporine-treated SH-SY5Y cells. In primary cultures of cerebellar granule neurons undergoing low K(+)-mediated apoptosis, Bax translocation to the mitochondrial fraction was evident at 3 h. Cytochrome c release into the cytosol was not significant until 8 h after treatment. These data support a model of apoptosis in which Bax acts directly at the mitochondria to allow the release of cytochrome c.
McGinnis
K M
KM
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109, USA.
Gnegy
M E
ME
Wang
K K
KK
D017209:Apoptosis / Q000502:physiology*; D001692:Biological Transport / Q000502:physiology; D020169:Caspases / Q000378:metabolism; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D003574:Cytochrome c Group / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D010539:Permeability; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors; D014018:Tissue Distribution / Q000502:physiology; D014407:Tumor Cells, Cultured / Q000187:drug effects; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D003574:Cytochrome c Group; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D020169:Caspases; D019311:Staurosporine