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J Biol Chem, Vol. 274, Issue 42, 29831-29837, October 15, 1999
From the Department of Biochemistry and Molecular Biology, Monash
University, Clayton 3168 Victoria, Australia
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 C6-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 C6-ceramide and TNF-. In
particular, mitochondria changed in appearance, clustering in the
perinuclear region and Bcl-2 expression prevented these changes from occurring.
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