Apoptotic protease activating factor 1 (Apaf-1)-independent cell death suppression by Bcl-2.
Reportedly, antiapoptotic Bcl-2 family proteins suppress apoptosis by binding to and inhibiting members of the CED-4 family of caspase activators. To explore this question, we used embryonic stem (ES) cells in which one (-/+) or both (-/-) copies of the gene encoding apoptotic protease activating factor 1 (Apaf-1), a CED-4 homologue, were disrupted by homologous recombination. Stable clones of heterozygous (-/+) and homozygous (-/-) Apaf-1 knockout ES cells that overexpressed Bcl-2 were generated. Withdrawal of serum growth factors or stimulation of heterozygous ES cells with staurosporine (STS), ultraviolet (UV)B irradiation, etoposide (VP16), or cisplatin induced apoptosis followed by cell death (determined by failure to exclude propidium iodide dye). These cell death stimuli also induced activation of several types of caspases and loss of mitochondrial membrane potential (DeltaPsi) in heterozygous (+/-) Apaf-1 knockout ES cells. In addition, overexpression of Bcl-2 protected against these events in Apaf-1-expressing ES cells. In contrast, STS, UVB, and VP16 induced little or no caspase activation and apoptosis in homozygous (-/-) Apaf-1 knockout ES cells. Nevertheless, Apaf-1-deficient ES cells subjected to these cell death stimuli or deprived of growth factors did eventually die through a nonapoptotic mechanism associated with loss of DeltaPsi. Moreover, Bcl-2 overprotection preserved DeltaPsi, reduced the percentage of Apaf-1(-/)- ES cells undergoing cell death, and increased clonigenic survival. The extent of Bcl-2-mediated cytoprotection was not significantly different for heterozygous (-/+) versus homozygous (-/-) Apaf-1 knockout cells. Furthermore, although Bcl-2 could be readily coimmunoprecipitated with Bax, associations with Apaf-1 were undetectable under conditions where Apaf-1 interactions with procaspase-9 were observed. We conclude that Bcl-2 has cytoprotective functions independent of Apaf-1, preserving mitochondrial function through a caspase-independent mechanism.
Haraguchi
M
M
Burnham Institute Program on Apoptosis and Cell Death Regulation, La Jolla, California 92037, USA.
Torii
S
S
Matsuzawa
S i
Si
Xie
Z
Z
Kitada
S
S
Krajewski
S
S
Yoshida
H
H
Mak
T W
TW
Reed
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects; D053477:Apoptotic Protease-Activating Factor 1; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000235:genetics / Q000502:physiology / Q000528:radiation effects; D002999:Clone Cells; D004789:Enzyme Activation; D015870:Gene Expression; D019254:Genes, bcl-2; D051379:Mice; D018345:Mice, Knockout; D008854:Microscopy, Electron; D009842:Oligopeptides / Q000737:chemistry; D011506:Proteins / Q000235:genetics* / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000502:physiology*; D013234:Stem Cells / Q000166:cytology / Q000502:physiology; D013379:Substrate Specificity; D014466:Ultraviolet Rays; D051028:bcl-2-Associated X Protein
D000970:Antineoplastic Agents; C506593:Apaf1 protein, mouse; D053477:Apoptotic Protease-Activating Factor 1; C494827:Bax protein, mouse; D009842:Oligopeptides; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D020169:Caspases