[CANCER RESEARCH 59, 2190 –2194, May 1, 1999] Poly(ADP-ribosyl)ation of p53 during Apoptosis in Human Osteosarcoma Cells 1 Cynthia M. Simbulan-Rosenthal, Dean S. Rosenthal, RuiBai Luo, and Mark E. Smulson 2 Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, DC 20007 ABSTRACT Spontaneous apoptosis in human osteosarcoma cells was observed to be associated with a marked increase in the intracellular abundance of p53. Immunoprecipitation and immunoblot analysis revealed that, together with a variety of other nuclear proteins, p53 undergoes extensive poly- (ADP-ribosyl)ation early during the apoptotic program in these cells. Subsequent degradation of poly(ADP-ribose) (PAR), attached to p53 pre- sumably by PAR glycohydrolase, the only reported enzyme to degrade PAR, was apparent concomitant with the onset of proteolytic processing and activation of caspase-3, caspase-3-mediated cleavage of poly(ADP- ribose) polymerase (PARP), and internucleosomal DNA fragmentation during the later stages of cell death. The decrease in PAR covalently bound to p53 also coincided with the marked induction of expression of the p53-responsive genes bax and Fas. These results suggest that poly- (ADP-ribosyl)ation may play a role in the regulation of p53 function and implies a regulatory role for PARP and/or PAR early in apoptosis. INTRODUCTION p53, a tumor suppressor nuclear phosphoprotein, reduces the occur- rence of mutations by mediating cell cycle arrest in G 1 or G 2 -M or inducing apoptosis in cells that have accumulated substantial DNA dam- age, thus, preventing progression of cells through S phase before DNA repair is complete (1–3). One of the earliest nuclear events that follows DNA strand breakage during DNA repair in response to agents such as -irradiation, carcinogens, or alkylating agents is the poly(ADP-ribo- syl)ation of various proteins that are localized near DNA strand breaks. PARP 3 catalyzes the poly(ADP-ribosyl)ation of nuclear proteins only when bound to single- or double-stranded DNA ends (4 – 6) and cycles on and off the DNA ends during DNA repair in vitro (7–10). In addition to undergoing automodification, PARP catalyzes the poly(ADP-ribosyl) ation of such nuclear proteins as histones, topoisomerases I and II (11, 12), SV40 large T antigen (13), DNA polymerase , proliferating cell nuclear antigen, and 15 protein components of the DNA synthesome (12). The modification of nucleosomal proteins also alters the nucleoso- mal structure of the DNA containing strand breaks and promotes access of various replicative and repair enzymes to these sites (14, 15). Additionally, depletion of PARP by antisense RNA expression has indicated that poly(ADP-ribosyl)ation plays an auxiliary role in the repair of DNA strand breaks (16, 17), in preferential gene repair (18), in the survival of cells after exposure to various alkylating agents, in gene amplification (19), in differentiation-linked DNA replication (12, 20, 40), and recently, in an early stage of apoptosis (21). Given that PARP is only catalytically active when bound to DNA strand breaks, when PARP undergoes caspase-3-mediated cleavage into M r 89,000 and M r 24,000 fragments during drug-induced (22) or spontaneous (23, 24) apoptosis, separation of its DNA binding domain from its catalytic site essentially inactivates the enzyme. PARP has also been implicated in the induction of p53 expression during apoptosis (25) . The specific proteolytic cleavage of PARP by caspase-3 is a key apoptotic event because PARP cleavage and inactivation as well as subsequent apoptotic events are blocked by a peptide inhibitor of this protease (23, 26). We have shown recently that a transient poly(ADP-ribosyl)ation of nuclear proteins in intact human osteosarcoma cells occurs early in apoptosis, prior to commitment to cell death, and is subsequently followed by cleavage and inactivation of PARP (24). No PAR is synthesized at the later stages of apoptosis, despite the presence of a large number of DNA strand breaks at this time. By depleting 3T3-L1 and Jurkat T cells of PARP by antisense RNA expression, or with the use of immortalized fibroblasts derived from PARP knockout (PARP-/-) mice, we demonstrated that prevention of this early activation of PARP blocks various biochemical and morphological changes associated with apoptosis (21), thus correlating the early poly(ADP-ribosyl)ation with later events in the cell death cascade. p53 is induced by a variety of apoptotic stimuli and is required for apoptosis in many cell systems (27); overexpression of p53 is sufficient to induce apoptosis in various cell types (28). Interestingly, p53 can use transcription activation of target genes and/or direct protein-protein in- teraction to initiate p53-dependent apoptosis. It was shown recently that p53 is poly(ADP-ribosyl)ated in vitro by purified PARP, and that binding of p53 to a specific p53 consensus sequence prevents its covalent mod- ification (29). We now show for the first time that modification of p53 by poly(ADP-ribosyl)ation also occurs in vivo, and that it represents one of the early acceptors of poly(ADP-ribosyl)ation during apoptosis in human osteosarcoma cells. Given that the in vivo half-life of PAR chains on an acceptor has been estimated to be about 1–2 min, we have additionally explored how this posttranslational modification of p53 is altered at the onset of caspase-3-mediated cleavage and inactivation of PARP during the later stages of the death program. MATERIALS AND METHODS Cell Culture and Induction of Apoptosis. Human osteosarcoma cells (23, 24) were cultured in DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 g/ml). Cell cultures were maintained as exponentially growing cells in a humidified 5% CO 2 incubator. Spontaneous apoptosis was induced by allowing the cells to grow for 10 days without any medium changes, as described previously (23, 24). Immunoprecipitation and Immunoblot Analysis. For immunoblot anal- ysis, SDS-PAGE and transfer of proteins (30 g/lane) to nitrocellulose mem- branes were performed according to standard procedures. The membranes were stained with Ponceau S (0.5%) to confirm equal loading and transfer. Membranes were then incubated with polyclonal antibodies to CPP32 (1:5000 dilution; a gift from Dr. D. Nicholson, Merck), to PARP (1:5000 dilution; BioMol), to Fas (1:200 dilution; Santa Cruz Biotechnology), or to Bax (1:100 dilution; Calbiochem) and to mAbs to human p53 (Ab-5; 1:10 dilution; Calbiochem) or to PAR (1:250 dilution; Ref. 30). The anti-p53 antibody recognizes wild-type but not mutant p53. The membranes were subsequently probed with appropriate peroxidase-labeled antibodies (1:3000 dilution), and immune complexes were detected by enhanced chemiluminescence (Pierce). Immunoprecipitation was performed with another monoclonal antibody to p53 (Ab-1; Calbiochem), according to procedures described previously (31). Briefly, equal amounts of cell extracts (10 g) were precleared overnight at 4°C with 200 l of EBC buffer [50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 0.5% NP40, and 0.1 TIU/ml aprotinin] and 10 l of protein A-Sepharose beads Received 11/23/98; accepted 3/2/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by Grants CA25344 and CA13195 from the National Cancer Institute, by the United States Air Force Office of Scientific Research Grant AFOSR- 89-0053, and by the United States Army Medical Research and Development Command Contract DAMD17-90-C-0053 (to M. E. S.) and DAMD 17-96-C-6065 (to D. S. R). 2 To whom requests for reprints should be addressed, at Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Basic Science Building, Room 351, 3900 Reservoir Road NW, Washington, DC 20007. Phone: (202) 687-1718; Fax: (202) 687-7186; E-mail: smulson@bc.georgetown.edu. 3 The abbreviations used are: PARP, poly(ADP-ribose) polymerase; PAR, poly(ADP- ribose); mAb, monoclonal antibody. 2190 Research. on November 29, 2015. © 1999 American Association for Cancer cancerres.aacrjournals.org Downloaded from