Overexpression of bcl-2 Results in Reduction of Cytochrome c Content and Inhibition of Complex I Activity C. S. Schwarz,* B. O. Evert,* J. Seyfried,† M. Schaupp,† W. S. Kunz,‡ S. Vielhaber,‡ T. Klockgether,* and U. Wu ¨ llner* ,1 *Department of Neurology and ‡Department of Epileptology, University of Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany; and †Department of Neurology, University of Tu ¨ bingen, Hoppe-Seyler-Str. 3, D-72076 Tu ¨ bingen, Germany Received December 29, 2000 Bcl-2 has been shown to exert its antiapoptotic ac- tivity predominantly at the level of mitochondria by preventing cytochrome c release. Whether Bcl-2 is in- volved in the regulation of mitochondrial function prior to an apoptotic stimulus remains elusive. Using functional and spectrophotometric measurements in an inducible PC12-Tet-on-bcl-2 cell line we demon- strate that induction of Bcl-2 overexpression rapidly reduced cytochrome b and c levels as well as complex I activity. To confirm that these changes were specific for Bcl-2 we generated a bcl-2 antisense construct un- der the control of the tetracycline responsive promo- tor. Transient transfection with this antisense plasmid prevented both the decrease of cytochrome b and c levels and the loss of complex I activity. The decrease of cytochrome b levels was paralleled by a decrease of cytochrome b mRNA levels while Northern blot analy- sis of cytochrome c mRNA expression did not reveal any overt changes in Bcl-2 cells. We propose that the antiapoptotic properties of Bcl-2 are related to the reduction of mitochondrial complex I activity and low- ered mitochondrial cytochrome b and c levels. © 2001 Academic Press Key Words: Bcl-2; mitochondria; cytochrome b; cyto- chrome c; complex I. Members of the Bcl-2 family of proteins function either to promote or to repress apoptosis. Antiapoptotic members such as Bcl-2 and Bcl-X L inhibit programmed cell death whereas proapoptotic proteins such as Bax and Bad can accelerate the death program (1). Bcl-2 has been mainly localized to the inner mitochondrial membrane (2, 3). Bcl-2 is targeted to the outer mito- chondrial membrane via a COOH-terminal signal an- chor sequence; deletion of its transmembrane domain renders Bcl-2 cytosolic and impairs its ability to pre- vent apoptotic cell death (4). Specific apoptotic stimuli alter the mitochondrial membranes to release cytochrome c into the cytosol where it activates caspase-9 resulting in cleavage of cellular substrates and finally cell death. The efflux of cytochrome c has been shown to involve the mitochon- drial porin channel, also called voltage-dependent an- ion channel (VDAC), which can be modulated by direct binding of Bcl-X L and Bax (5). A direct role of Bcl-2 however has not yet been demonstrated. Microinjec- tion of cytochrome c into the cytosol of various cell types results in apoptosis that cannot be inhibited by Bcl-2 (6). Bcl-2 thus appears to act predominantly up- stream of cytochrome c release in its prevention of apoptosis (7, 8). However, the exact biochemical mech- anism involved in Bcl-2 function is still subject of in- tense investigation. Previous studies of Bcl-2 mainly focussed on the effects of Bcl-2 overexpression with respect to an apop- totic stimulus. Yet information on the immediate cel- lular changes evoked by Bcl-2 overexpression prior to an apoptotic stimulus are scarce and whether Bcl-2 is involved in the regulation of mitochondrial function under physiological conditions remains elusive. To investigate immediate changes of mitochondrial function induced by Bcl-2 overexpression we generated an inducible PC12 bcl-2 overexpressing cell line using the Tet-on expression system (9). We demonstrate that induction of Bcl-2 within 24 h significantly decreased cytochrome c and b protein levels. The activity of com- plex I of the respiratory chain subsequently decreased to less than 50% of control after 72 h. Transient trans- fection with bcl-2 antisense prevented both the de- crease of cytochrome levels and of complex I activity. Our data suggest that Bcl-2 overexpression modulates mitochondrial independent of an apototic stimulus. 1 To whom correspondence should be addressed. Fax: 49 228 2875024. E-mail: wuellner@uni-bonn.de. Biochemical and Biophysical Research Communications 280, 1021–1027 (2001) doi:10.1006/bbrc.2001.4242, available online at http://www.idealibrary.com on 1021 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.