Bcl-2 and Mitochondrial Oxygen Radicals
NEW APPROACHES WITH REACTIVE OXYGEN SPECIES-SENSITIVE PROBES*
- From the Department of Biochemistry and Molecular Biology, Monash University, Clayton 3168 Victoria, Australia
Abstract
Investigations into the capacity of the Bcl-2 protein to prevent apoptosis have targeted mitochondria as key sites of the preventative action accorded by Bcl-2 to cells. Using novel approaches with fluorescence probes and autofluorescence detection of endogenous NAD(P)H, we have examined the effects of expressing Bcl-2 in the Bcl-2 negative Burkitt’s lymphoma cell line Daudi. We evaluated for the first time the effect of Bcl-2 expression on the intracellular distribution and production of hydrogen peroxide, under basal conditions and after treatment with apoptosis inducing agents, ceramide analogs and tumor necrosis factor (TNF)-α. Increased availability of mitochondrial NAD(P)H was detected in Bcl-2-expressing cells and was correlated with an increased constitutive mitochondrial production of hydrogen peroxide. Although production of hydrogen peroxide was increased by either C6-ceramide or TNF-α in Bcl-2 negative Daudi cells commensurate with the early phases of apoptosis, this increase did not occur in Bcl-2-expressing cells. Thus, Bcl-2 appears to allow cells to adapt to an increased state of oxidative stress, fortifying the cellular anti-oxidant defenses and counteracting the radical overproduction imposed by different cell death stimuli. Furthermore, we report altered cytological features of mitochondria during the early phases of apoptosis induced by C6-ceramide and TNF-α. In particular, mitochondria changed in appearance, clustering in the perinuclear region and Bcl-2 expression prevented these changes from occurring.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton 3168 Victoria, Australia. Tel.: 61-3-99051431; Fax: 61-3-99054699; E-mail: mauro1it@hotmail.com.
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↵2 H. McLennan and M. Degli Esposti, unpublished results.
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↵3 M. Degli Esposti, N. Waterhouse, and D. Green, unpublished results.
- Abbreviations:
- ROS
-
reactive oxygen species
- CM-H2XRos
-
reduced chloromethyl-tetramethyl rosamine (reduced MitoTracker Red®)
- FCCP
-
carbonyl cyanidep-trifluoromethoxyphenylhydrazone
- DCFDA
-
dichlorodihydrofluorescein diacetate
- TNF
-
tumor necrosis factor
- PBS
-
phosphate-buffered saline
- DTMR
-
dihydrotetramethyl-rosamine
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- Received November 16, 1998.
- Revision received May 10, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
