Apoptotic and necrotic cell death are well characterized and are influenced by intracellular ATP levels. Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated by DNA strand breaks, physiologically participates in DNA repair. Overactivation of PARP after cellular insults can lead to cell death caused by depletion of the enzyme's substrate ␤-nicotinamide adenine dinucleotide and of ATP. In this study, we have differentially elicited apoptosis or necrosis in mouse fibroblasts. Fibroblasts from PARP-deficient (PARP ؊͞؊ ) mice are protected from necrotic cell death and ATP depletion but not from apoptotic death. These findings, together with cell death patterns in PARP ؊͞؊ animals receiving other types of insults, indicate that PARP activation is an active trigger of necrosis, whereas other mechanisms mediate apoptosis. P oly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) is a nuclear enzyme activated by DNA strand breaks, which transfers 50-200 branched chains of ADP-ribose to a variety of nuclear proteins including PARP itself (1, 2). With moderate amounts of DNA damage, PARP is thought to participate in the DNA repair process (3). However, with excessive activation of PARP, its substrate ␤-nicotinamide adenine dinucleotide (NAD ϩ ) is depleted, and, in efforts to resynthesize NAD ϩ , ATP is also depleted such that cells may die from energy loss (4-6). A role for PARP overactivation in cell death is indicated by the protection against cell death observed after treatment with PARP inhibitors (7,8) and the pronounced protection against neuronal ischemia (9, 10), myocardial ischemia (11), and diabetic pancreatic damage (12)(13)(14) Cells can die by energy-dependent apoptosis or by necrosis (18,19). Because overactivation of PARP depletes ATP, we wondered whether this mode of cell death might be selectively associated with necrosis rather than apoptosis. Accordingly, we have compared cellular models of necrosis and apoptosis. To minimize contaminating variables, we employed a single cell type in which different stimuli can elicit necrosis or apoptosis differentially. We used fibroblasts in which DNA-damaging agents such as N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG) and hydrogen peroxide (H 2 O 2 ) elicit necrosis at high concentration, whereas activation of Fas leads to apoptosis (16,20,21). We show that PARP Ϫ͞Ϫ fibroblasts are protected from necrotic but not apoptotic death and that necrosis but not apoptosis is associated with ATP depletion. Materials and MethodsCell Culture. Mouse embryonic fibroblasts (MEFs) derived from both wild-type and PARP knockout mice were kindly provided by Z. Q. Wang (Institute of Molecular Pathology, Vienna). MEFs were cultured at 37°C (5% CO 2 ) in DMEM supplemented with 10% (vol͞vol) FBS, 2 mM L-glutamine, 100 units͞ml penicillin, and 100 g͞ml streptomycin. MEFs were seeded in 6-well plates at Ϸ70% conf luence (exponential phase of growth). Cell numbers were similar for PARP Ϫ͞Ϫ and PARP ϩ͞ϩ MEFs within a given experiment. MNNG was prepared freshly in DMSO such that the final co...

The polyamines are small organic cations that are absolutely required for eukaryotic cell growth. Although their growth requirements are well established, the molecular functions of the polyamines are ill-defined. Oxidative damage to DNA by reactive oxygen species is a continual problem that cells must guard against to survive. The polyamine spermine, which is normally found in millimolar concentrations in the nucleus, is shown here to function directly as a free radical scavenger, and adducts formed as a result of this function are identified. These data suggest that spermine is a major natural intracellular compound capable of protecting DNA from free radical attack.The cellular polycationic polyamines are ubiquitous in nature and are absolutely required for eukaryotic cell growth (1, 2). However, very few specific molecular functions of polyamines have been described (3, 4). Some of the attributes ascribed to polyamines, particularly spermine, include the regulation of gene expression (5), the stabilization of chromatin (6-8), the prevention of endonuclease-mediated DNA fragmentation (9), and the inhibition of DNA damage (8, 10-13). Lovaas has recently reviewed many of these properties (14). Although protection of DNA damage by polyamines has been demonstrated, the mechanisms by which spermine functions in this protection are not known. Most of the proposals suggest that polyamine action is a result of charge neutralization and conformational changes (8,15). Because spermine is thought to be intimately associated with chromatin (7), we sought to investigate if spermine had a direct role in protecting DNA from free radical attack. Free radical and other reactive oxygen species (ROS) generation through normal cellular metabolism and by exogenous insult is a constant problem for which cells have developed multiple protective mechanisms to survive(16). Here we demonstrate that spermine, which is normally found in millimolar concentrations in the nucleus (17), can function directly as a free radical scavenger. MATERIALS AND METHODSAssays for DNA Strand Breaks. DNA strand breakage was measured by the conversion to open circular and linear forms of supercoiled ⌽X-174 RF1 double-stranded DNA (New England Biolabs). To assess DNA cleavage, 0.2 g of DNA was incubated in the presence of 30 M H 2 O 2 and 10 M CuCl 2 in PBS (pH 7.4) in a total volume of 30 l as described (18,19). Polyamines or antioxidants were coincubated as indicated. Following incubation the samples were separated by electrophoresis in a 1% agarose gel containing 40 mM Tris-acetate and 1 mM EDTA in a horizontal slab gel apparatus using Tris/acetate gel buffer. The gel was stained with ethidium bromide (2 g/ml) for 10 min, followed by destaining in water for 10 min, and was then photographed by UV translumination. The gels were photographed using an Eagle Eye digital camera (Stratagene). A single strand break in supercoiled double-stranded DNA results in the formation of open circular DNA, and double strand breaks result in the formation of linea...

Poly(ADP-ribose) polymerase-1 (PARP-1, EC 2.4.2.30), a nuclear enzyme activated by DNA strand breaks, physiologically participates in DNA repair. Excessive activation of PARP-1 by cellular insults depletes its substrate ␤-nicotinamide adenine dinucleotide and ATP, leading to cell death. PARP-1-deficient (PARP-1 ؊͞؊ ) mice are protected from several forms of inflammation. In the present study, we demonstrate in PARP-1 ؊͞؊ glial cells a loss of several stress-activated transcription factors as well as decreased expression of genes for cytokines and cellular adhesion molecules. We also show that augmented expression of some of these genes is independent of PARP-1 catalytic activity. These findings indicate that PARP-1 plays a pivotal role in the initial inflammatory response by modulating transcription of inflammation-linked genes.P oly(ADP-ribose) polymerase-1 (PARP-1, EC 2.4.2.30) is a nuclear enzyme activated by DNA strand breaks, which adds branched chains of up to 200 ADP-ribose units to a variety of nuclear proteins, especially PARP-1 itself. PARP-1 participates in the DNA repair process after its activation by DNA damage. Several isozymes of PARP have been identified. However, the initially described form of the enzyme, PARP-1, is the most abundant, because PARP activity is reduced markedly in most tissues of PARP-1-deficient (PARP-1 Ϫ͞Ϫ ) mice (1). PARP-1 plays a major role in tissue damage (2) and necrotic cell death (3), because PARP-1 Ϫ͞Ϫ mice are protected from cerebral ischemia (4, 5), streptozotocin-induced diabetes (6-8), myocardial ischemia (9-11), and several forms of inflammation (9,(12)(13)(14). In many instances cellular death seems related to PARP-1 overactivation and ATP depletion. Tissue insults lead to DNA damage, which can arise from the formation of nitricoxide derivatives such as peroxynitrite (2,15). This massive DNA damage leads to pronounced overactivation of PARP-1. Because PARP-1 is an extremely abundant enzyme, its overactivation results in depletion of its substrate ␤-nicotinamide adenine dinucleotide (NAD ϩ ). In efforts to resynthesize NAD ϩ , ATP is depleted, and the cell dies from energy loss.An alternative way in which PARP-1 may influence the stress͞inflammation response involves regulation of transcription factors and associated gene transcription. PARP-1 has been reported either to activate or repress transcription activity (16). PARP-1 influences on transcription activity may involve direct protein-protein interaction with PARP-1 or the catalytic activity of the PARP-1 enzyme, which can poly(ADP-ribosyl)ate transcription factors. Transcription factors such as AP-2 (17), B-MYB (18), Oct-1 (19), YY-1 (20), and TEF-1 (21) have been shown to bind directly to PARP-1. On the other hand, transcription factors such as p53 (22), fos (23), and RNA polymerases I (24) and II (25) are poly(ADP-ribosyl)ated. NF-B transcription activation after stress͞inflammatory stimuli is reduced in PARP-1 Ϫ͞Ϫ cells (13,26). PARP-1 participates in the activation of NF-B independently of energy depleti...