Bcl-2 and Mitochondrial Oxygen Radicals

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)-␣. 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 C 6 -ceramide or TNF-␣ 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 C 6 -ceramide and TNF-␣. In particular, mitochondria changed in appearance, clustering in the perinuclear region and Bcl-2 expression prevented these changes from occurring.

Effect of Bcl-2 Overexpression on Mitochondrial Structure and Function

“…This ⌬⌿ effect could explain many changes observed in Bcl-2-overexpressing mitochondria, including increased reactive oxygen species generation (14 -16), enhanced Ca 2ϩ uptake capacity, and larger quantities of reduced pyridine nucleotides (10,14,23). To understand the mechanism through which Bcl-2 apparently enhances ⌬⌿, we performed measurements of mitochondrial uptake of the ⌬⌿-sensitive probe safranin O and calibrated the data by using K ϩ gradients and applying the Nernst equation (see "Experimental Procedures" and Ref.…”

“…Cytochrome c normally interacts closely with the inner membrane and must be displaced to the intermembrane space to be released into the cytosol (28). Finally, larger matrix volumes may explain why Bcl-2ϩ cells present higher quantities of matrix-soluble components such as NADPH, NADH, and glutathione (10,14,23). The antioxidant effects of NADPH and GSH are related to the increased resistance Bcl-2ϩ cells present to oxidative damage (10,14,16,23).…”

Reactive species mechanisms of cellular hypoxia-reoxygenation injury

“…Ca 2ϩ leaves mitochondria during reoxygenation through pore formation, reversal of uniport influx carrier, the Ca 2ϩ /H ϩ antiport system, or channel-mediated Ca 2ϩ pathways. Increased intracellular Ca 2ϩ also appears to be related to oxidation of adenine nucleotides or by thiol oxidation, because overexpression of the antiapoptotic protein Blc-2, which has antioxidant properties, in mitochondrial membranes inhibits Ca 2ϩ efflux due to oxidants (31 (44). Removal of Ca 2ϩ from the medium or Ca 2ϩ chelation prevented killing of rat hepatocytes by rotenone and anoxia, but not by cyanide.…”

“…Bcl-2 overexpression allows cells to adapt to ROS overproduction (31). Apoptosis in an endothelial reperfusion model also required activation of NF-B, because NF-B decoy oligodeoxynucleotides decreased the number of TUNEL (TdT-mediated dUTP nick end labeling)-positive cells.…”

Lipids, cardiolipin and apoptosis: a greasy licence to kill

“…3 This was connected to the previously reported anti-oxidant activity associated with Bcl-2 expression, 3 which is probably linked to the catabolism of fatty acids. 18,19 Lipid peroxidation represents a grey area in which the protective action of Bcl-2 resembles that of some enzymes, especially phospholipid hydroperoxide glutathione peroxidase, that directly counteract damaging oxygen radicals. 11,16 Again, a lipid connection emerges for the Bcl-2 proteins.…”