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Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1235-1246
Cytochrome c Induces Caspase-Dependent Apoptosis in Intact
Hematopoietic Cells and Overrides Apoptosis Suppression Mediated by
bcl-2, Growth Factor Signaling, MAP-Kinase-Kinase, and
Malignant Change
John M. Garland and
Claudius Rudin
From the Institute for Clinical Science, Exeter University, Noy Scott
House, Wonford, Exeter, UK; the Department of Haematology, Royal Devon
& Exeter NHS Trust, Wonford, Exeter, UK.
It has been shown that cytochrome c is released from mitochondria
during apoptosis, activates pro-caspase CPP32 (caspase III), and
induces DNA fragmentation in mixtures of cytosolic extracts and
isolated nuclei. To establish whether cytochrome c can primarily induce
apoptosis in intact cells, we used direct electroporation of cytochrome
c into murine interleukin-3 (IL-3)-dependent cells. Electroporation of
micromolar external concentrations of cytochrome c rapidly
induced apoptosis (2 to 4 hours) that was concentration-dependent, did
not affect mitochondrial transmembrane potential, and was independent
of cell growth. Only certain isoforms of cytochrome c were apoptogenic;
yeast cytochrome c and other redox proteins were inactive. Cytochrome
c-induced apoptosis was dependent on heme attachment to the apo-enzyme
and was completely abolished by caspase inhibitors. Nonapoptogenic
isoforms of cytochrome c did not compete for apoptogenic cytochrome c.
Although apoptosis induced by IL-3 withdrawal was inhibited by bcl-2
overexpression and expression of an activated MAP-kinase-kinase
(MAP-KK), cytochrome c induced apoptosis in the presence of IL-3
signaling, bcl-2 over-expression, expression of activated MAP-KK, and
the combined antiapoptotic action of all three. Cytochrome c also
induced apoptosis in the leukemic cell line WEHI 3b. However, human
HL60 and CEM cells were resistant to cytochrome c-induced apoptosis.
HL60 cells did not electroporate, but CEM cells were efficiently
electroporated. Our studies with IL-3-dependent cells confirm that the
apoptogenic attributes of cytochrome c are identical in intact cells to
those in cell extracts. We conclude that cytochrome c can be a prime initiator of apoptosis in intact growing cells and acts downstream of
bcl-2 and mitochondria, but that other cells are resistant to its
apoptogenic activity. The system described offers a novel, simple
approach for investigating regulation of apoptosis by cytochrome c and
provides a model linking growth factor signaling to metabolism, survival, and apoptosis control.
© 1998 by The American Society of Hematology.
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