Apoptotic Protease Activating Factor 1 (Apaf-1)-Independent Cell Death Suppression by Bcl-2

doi:10.1084/jem.191.10.1709

Published online 15 May 2000.

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© The Rockefeller University Press, 0022-1007/2000/5/1709/ $5.00
The Journal of Experimental Medicine, Volume 191, Number 10, May 15, 2000 1709-1720

Original Article

Apoptotic Protease Activating Factor 1 (Apaf-1)–Independent Cell Death Suppression by Bcl-2

Misako Haraguchi^a, Seiji Torii^a, Shu-ichi Matsuzawa^a, Zhihua Xie^a, Shinichi Kitada^a, Stanislaw Krajewski^a, Hiroki Yoshida^b, Tak W. Mak^b, and John C. Reed^a

^a Burnham Institute Program on Apoptosis and Cell Death Regulation, La Jolla, California 92037
^b Department of Cellular and Molecular Biology, Ontario Cancer Institute, Toronto, Ontario, Canada M5G 2C1
The Burnham Institute, Program on Apoptosis and Cell Death Regulation, 10901 North Torrey Pines Rd., La Jolla, CA 92037.858-646-3194858-646-3140

jreed{at}burnham-inst.org

Reportedly, antiapoptotic Bcl-2 family proteins suppress apoptosisby binding to and inhibiting members of the CED-4 family ofcaspase activators. To explore this question, we used embryonicstem (ES) cells in which one (–/+) or both (–/–)copies of the gene encoding apoptotic protease activating factor1 (Apaf-1), a CED-4 homologue, were disrupted by homologousrecombination. Stable clones of heterozygous (–/+) andhomozygous (–/–) Apaf-1 knockout ES cells that overexpressedBcl-2 were generated. Withdrawal of serum growth factors orstimulation of heterozygous ES cells with staurosporine (STS),ultraviolet (UV)B irradiation, etoposide (VP16), or cisplatininduced apoptosis followed by cell death (determined by failureto exclude propidium iodide dye). These cell death stimuli alsoinduced activation of several types of caspases and loss ofmitochondrial membrane potential ( ${Delta}$ ${Psi}$ ) in heterozygous (+/–)Apaf-1 knockout ES cells. In addition, overexpression of Bcl-2protected against these events in Apaf-1–expressing EScells. In contrast, STS, UVB, and VP16 induced little or nocaspase activation and apoptosis in homozygous (–/–)Apaf-1 knockout ES cells. Nevertheless, Apaf-1–deficientES cells subjected to these cell death stimuli or deprived ofgrowth factors did eventually die through a nonapoptotic mechanismassociated with loss of ${Delta}$ ${Psi}$ . Moreover, Bcl-2 overprotection preserved ${Delta}$ ${Psi}$ , reduced the percentage of Apaf-1^–/– ES cells undergoingcell death, and increased clonigenic survival. The extent ofBcl-2–mediated cytoprotection was not significantly differentfor heterozygous (–/+) versus homozygous (–/–)Apaf-1 knockout cells. Furthermore, although Bcl-2 could bereadily coimmunoprecipitated with Bax, associations with Apaf-1were undetectable under conditions where Apaf-1 interactionswith procaspase-9 were observed. We conclude that Bcl-2 hascytoprotective functions independent of Apaf-1, preserving mitochondrialfunction through a caspase-independent mechanism.

Key Words: Bcl-2 • apoptotic protease activating factor 1 • caspase • apoptosis • mitochondria

Abbreviations used in this paper: Apaf-1, apoptotic proteaseactivating factor 1; CP, cisplatin; ${Delta}$ ${Psi}$ , mitochondrial transmembranepotential; ES, embryonic stem; PI, propidium iodide; STS, staurosporine.

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