Transient Poly(ADP-ribosyl)ation of Nuclear Proteins and Role of Poly(ADP-ribose) Polymerase in the Early Stages of Apoptosis*
- Cynthia M. Simbulan-Rosenthal,
- Dean S. Rosenthal,
- Sudha Iyer,
- A. Hamid Boulares and
- Mark E. Smulson‡
- From the Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, D. C. 20007
Abstract
A transient burst of poly(ADP-ribosyl)ation of nuclear proteins occurs early, prior to commitment to death, in human osteosarcoma cells undergoing apoptosis, followed by caspase-3-mediated cleavage of poly(ADP-ribose) polymerase (PARP). The generality of this early burst of poly(ADP-ribosyl)ation has now been investigated with human HL-60 cells, mouse 3T3-L1, and immortalized fibroblasts derived from wild-type mice. The effects of eliminating this early transient modification of nuclear proteins by depletion of PARP protein either by antisense RNA expression or by gene disruption on various morphological and biochemical markers of apoptosis were then examined. Marked caspase-3-like PARP cleavage activity, proteolytic processing of CPP32 to its active form, internucleosomal DNA fragmentation, and nuclear morphological changes associated with apoptosis were induced in control 3T3-L1 cells treated for 24 h with anti-Fas and cycloheximide but not in PARP-depleted 3T3-L1 antisense cells exposed to these inducers. Similar results were obtained with control and PARP-depleted human Jurkat T cells. Whereas immortalized PARP +/+ fibroblasts showed the early burst of poly(ADP-ribosyl)ation and a rapid apoptotic response when exposed to anti-Fas and cycloheximide, PARP −/− fibroblasts exhibited neither the early poly (ADP-ribosyl)ation nor any of the biochemical or morphological changes characteristic of apoptosis when similarly treated. Stable transfection of PARP −/− fibroblasts with wild-type PARP rendered the cells sensitive to Fas-mediated apoptosis. These results suggest that PARP and poly(ADP-ribosyl)ation may trigger key steps in the apoptotic program. Subsequent degradation of PARP by caspase-3-like proteases may prevent depletion of NAD and ATP or release certain nuclear proteins from poly(ADP-ribosyl)ation-induced inhibition, both of which might be required for late stages of apoptosis.
Footnotes
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↵* This work was supported in part by National Cancer Institute Grants CA25344 and CA13195, the United States Air Force Office of Scientific Research Grant AFOSR-89-0053, and the United States Army Medical Research and Development Command Contracts DAMD17-90-C-0053 (to M. E. S.) and DAMD 17-96-C-6065 (to D. S. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Basic Science Bldg., Rm. 351, 3900 Reservoir Rd., NW, Washington, DC 20007. Tel.: 202-687-1718; Fax: 202-687-7186; E-mail:smulson{at}bc.georgetown.edu.
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↵1 The abbreviations used are: PARP, poly(ADP-ribose) polymerase; PAR, poly(ADP-ribose); MNU,N-methyl-N-nitrosourea; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Pipes, 1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline; TNF-α, tumor necrosis factor-α.
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- Received January 21, 1998.
- Revision received March 9, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
