Abstract
The ability of the protein kinase C down-regulator bryostatin 1 to potentiate 1-β-d-arabinofuranosylcytosine (ara-C)-induced apoptosis was examined in human leukemia cells (U937) over-expressing the antiapoptotic protein Bcl-xL. Coadministration of bryostatin 1 with ara-C resulted in enhanced cytosolic release of cytochrome c and Smac/DIABLO, procaspase-3 and -9 activation, loss of mitochondrial membrane potential (Δψm), poly(ADP-ribosyl)phosphorylase degradation, apoptosis, and loss of clonogenic survival in U937/Bcl-xL cells, although effects were not as marked as in empty-vector control cells. Whereas the broad caspase inhibitor ZVAD-fluoromethyl ketone blocked ara-C/bryostatin 1-mediated caspase activation, loss of Δψm,and apoptosis in U937 cells, it failed to diminish cytochrome c release. In contrast, ectopic expression of Bcl-xL blocked cytochromec redistribution as well as all other events involved in ara-C/bryostatin 1-mediated apoptosis. The ability of ectopic expression of cytokine response modifier A to attenuate, albeit partially, bryostatin 1-mediated potentiation of ara-C-related apoptosis suggested a contributory role for activation of the extrinsic pathway in this phenomenon. Finally, the F0F1ATPase inhibitor oligomycin effectively blocked cytochromec release as well as loss of Δψm and apoptosis in U937/Bcl-xL cells. Together, these findings support the concept that bryostatin 1 potentiates ara-C lethality in human leukemia cells ectopically expressing Bcl-xL by diminishing the capacity of this antiapoptotic protein to antagonize cytochrome c release. In addition, they raise the possibility that activation of caspase cascades operating independently of Bcl-xL-associated mitochondrial actions may also contribute to enhanced lethality.
Footnotes
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This work was supported by awards CA63753 and CA83705 from the National Institutes of Health and Award LSA 6630-01 from the Leukemia and Lymphoma Society of America. Portions of this work have been presented in preliminary form at the meeting of the American Association for Cancer Research, New Orleans, LA, March 31–April 4, 2001.
- Abbreviations:
- Δψm
- mitochondrial membrane potential
- apaf-1
- apoptosis-activating factor-1
- AIF
- apoptosis-inducing factor
- PKC
- protein kinase C
- ara-C
- 1-β-d-arabinofuranosylcytosine
- PBS
- phosphate-buffered saline
- DiOC6
- 3,3-dihexyloxacarbocyanine iodide
- pNA
- p-nitroanilide
- AMC
- 7-amino-4-methylcoumarin
- PAGE
- polyacrylamide gel electrophoresis
- PARP
- poly(ADP-ribosyl)phosphorylase
- fmk
- fluoromethyl ketone
- TNF
- tumor necrosis factor
- UCN-01
- 7-hydroxystaurosporine
- CrmA
- cytokine response modifier A
- Received October 9, 2001.
- Accepted January 7, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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