Importance of Poly(ADP-ribose) Polymerase and Its Cleavage in Apoptosis
LESSON FROM AN UNCLEAVABLE MUTANT*
- F. Javier Oliver‡,
- Guadalupe de la Rubia,
- Véronique Rolli,
- M. Carmen Ruiz-Ruiz§,
- Gilbert de Murcia and
- Josiane Ménissier-de Murcia
- From the UPR 9003 du Centre National de la Recherche Scientifique “Cancérogénèse et Mutagénèse Moléculaire et Structurale,” Laboratoire Conventionné du Commissariat à l’Energie Atomique, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brant, F-67400 Illkirch-Graffenstaden, France and §Instituto de Parasitologia y Biomedicina, Consejo Superior de Investigaciones Cientificas, 18001-Granada, Spain
Abstract
We have studied the apoptotic response of poly(ADP-ribose) polymerase (PARP)−/− cells to different inducers and the consequences of the expression of an uncleavable mutant of PARP on the apoptotic process. The absence of PARP drastically increases the sensitivity of primary bone marrow PARP−/− cells to apoptosis induced by an alkylating agent but not by a topoisomerase I inhibitor CPT-11 or by interleukin-3 removal. cDNA of wild type or of an uncleavable PARP mutant (D214A-PARP) has been introduced into PARP−/− fibroblasts, which were exposed to anti-CD95 or an alkylating agent to induce apoptosis. The expression of D214A-PARP results in a significant delay of cell death upon CD95 stimulation. Morphological analysis shows a retarded cell shrinkage and nuclear condensation. Upon treatment with an alkylating agent, expression of wild-type PARP cDNA into PARP-deficient mouse embryonic fibroblasts results in the restoration of the cell viability, and the D214A-PARP mutant had no further effect on cell recovery. In conclusion, PARP−/− cells are extremely sensitive to apoptosis induced by triggers (like alkylating agents), which activates the base excision repair pathway of DNA, and the cleavage of PARP during apoptosis facilitates cellular disassembly and ensures the completion and irreversibility of the process.
Footnotes
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↵* This work was supported by L’Association pour la Recherche Contre le Cancer, by Electricité de France, by CNRS (grant ACC-SV Radiations ionisantes), and by the Commissariat à l’Energie Atomique.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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↵‡ Recipient of a postdoctoral fellowship from L’Association pour la Recherche Contre le Cancer and the Biomedicine and Health Program of the European Community. To whom correspondence should be addressed. Tel.: 33 0388 65 53 69; Fax: 33 0388 65 53 43; E-mail:Javier.Oliver{at}esbs1.u-strasbg.fr.
- Abbreviations:
- PARP
-
poly(ADP-ribose) polymerase
- MEF
-
mouse embryonic fibroblast
- PBS
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phosphate-buffered saline
- MMS
-
N-methylmethanesulfonate
- MNU
-
methylnitrosourea
- wt
-
wild-type
- X-gal
-
5-bromo-4-chloro-3-indolyl β-d-galactopyranoside
- BM
-
bone marrow
- BER
-
base excision repair
- GFP
-
green fluorescence protein.
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- Received May 7, 1998.
- Revision received September 29, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
