Bcl-2 and mitochondrial oxygen radicals. New approaches with reactive oxygen species-sensitive probes.
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)-alpha. 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-alpha 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-alpha. In particular, mitochondria changed in appearance, clustering in the perinuclear region and Bcl-2 expression prevented these changes from occurring.
Esposti
M D
MD
Department of Biochemistry, Monash University, Clayton 3168 Victoria, Australia. maurolit@hotmail.com
Hatzinisiriou
I
I
McLennan
H
H
Ralph
S
S
D002518:Ceramides / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D018384:Oxidative Stress; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D017382:Reactive Oxygen Species; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D002518:Ceramides; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D009249:NADP; D006861:Hydrogen Peroxide