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 aut ofluorescence 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 Dau di. We evaluated for the first time the effect of Bcl-2 expression on the i ntracellular distribution and production of hydrogen peroxide, under basal conditions and after treatment with apoptosis inducing agents, ceramide ana logs and tumor necrosis factor (TNF)-alpha. Increased availability of mitoc hondrial NAD(P)H was detected in Bcl-2-expressing cells and was correlated with an increased constitutive mitochondrial production of hydrogen peroxid e. Although production of hydrogen peroxide was increased by either C-6-cer amide or TNF-alpha in Bcl-2 negative Daudi cells commensurate with the earl y phases of apoptosis, this increase did not occur in Bcl-2-expressing cell s. Thus, Bcl-2 appears to allow cells to adapt to an increased state of oxi dative stress, fortifying the cellular anti-oxidant defenses and counteract ing the radical overproduction imposed by different cell death stimuli. Fur thermore, we report altered cytological features of mitochondria during the early phases of apoptosis induced by C-6-ceramide and TNF-alpha. In partic ular, mitochondria changed in appearance, clustering in the perinuclear reg ion and Bcl-2 expression prevented these changes from occurring.