Apoptosis, or programmed cell death, plays important roles in development, homeostasis, and immunological competence.It is characterized by marked morphological changes such as membrane blebbing, chromatin condensation, nuclear breakdown, and the appearance of membrane-associated apoptotic bodies, as well as by internucleosomal DNA fragmentation. The enzyme poly(ADP-ribose) polymerase (PARP) 1 catalyzes the poly(ADP-ribosyl)ation of various nuclear proteins with NAD as substrate, and, because it is activated by binding to DNA ends or strand breaks, PARP has been suggested to contribute to cell death by depleting the cell of NAD and ATP (1). PARP undergoes proteolytic cleavage into 89-and 24-kDa fragments that contain the active site and the DNA-binding domain of the enzyme, respectively, during drug-induced apoptosis in a variety of cells (2). More recently, PARP has been implicated in the induction of both p53 expression and apoptosis (3), with the specific proteolysis of the enzyme thought to be a key apoptotic event (4 -6).Caspase-3, a member of the caspase family of 10 or more aspartate-specific cysteine proteases that play a central role in the execution of the apoptotic program (7), is primarily responsible for the cleavage of PARP during cell death (4, 5). Other caspases, such as caspase-7, also cleave PARP in vivo, but at lower efficiencies. Composed of two subunits of 17 and 12 kDa that are derived from a common proenzyme (CPP32), caspase-3 is related to interleukin-1␤-converting enzyme and CED-3, which is required for apoptosis in Caenorhabditis elegans (8).In human osteosarcoma cells that undergo confluence-associated apoptosis over a 10-day period, caspase-3-like activity, measured with a specific [35 S]PARP-cleavage assay in vitro, peaks at 6 -7 days after initiation of apoptosis, concomitant with the onset of internucleosomal DNA fragmentation (4).We recently examined the time course of PARP activation and cleavage during apoptosis in intact osteosarcoma cells by immunofluorescence microscopy with antibodies to PARP, to the 24-kDa cleavage product of PARP, and to poly(ADP-ribose) (PAR) (9). We observed a transient burst of synthesis of PAR from NAD that increased early and was maximal 3 days after initiation of apoptosis, prior to the appearance of internucleosomal DNA cleavage (at day 7) and before the cells became irreversibly committed to apoptosis. During this early period, expression of full-length PARP was detected by both immunofluorescence and immunoblot analysis. The amounts of both PAR and PARP decreased thereafter, and at 6 days, the 24-kDa cleavage product of PARP was detected both immunocytochemically and by immunoblot analysis. PAR was not observed during days 8 to 10, despite the presence of abundant DNA strand breaks, potential activators of PARP, during this time.

Ceramide, a sphingolipid generated by the hydrolysis of membrane-associated sphingomyelin, appears to play a role as a gauge of apoptosis. A further metabolite of ceramide, sphingosine 1-phosphate (SPP), prevents ceramide-mediated apoptosis, and it has been suggested that the balance between intracellular ceramide and SPP levels may determine the cell fate (

Burn wound progression refers to the phenomenon of continued tissue necrosis in the zone of stasis after abatement of the initial thermal insult. A multitude of chemical and mechanical factors contribute to the local pathophysiologic process of burn wound progression. Prolonged inflammation results in an accumulation of cytotoxic cytokines and free radicals, along with neutrophil plugging of dermal venules. Increased vascular permeability and augmentations of interstitial hydrostatic pressure lead to edema with vascular congestion. Hypercoagulability with thrombosis further impairs blood flow, while oxidative stress damages endothelial cells and compromises vascular patency. A number of studies have investigated the utility of various agents in modulating these mechanisms of burn wound progression. However, as many of studies have used animal models of burn injury, often with administration of therapy preburn, obscuring the clinical applicability of the results to burn patients is of questionable benefit. An understanding of the complex, interrelated mediators of burn wound progression and their ultimate point of convergence in effecting tissue necrosis—cell apoptosis or oncosis—will allow for the future development of therapeutic interventions.

3T3-L1 preadipocytes have been shown to exhibit a transient increase in poly(ADP-ribose) polymerase (PARP) protein and activity, as well as an association of PARP with DNA polymerase alpha, within 12-24 h of exposure to inducers of differentiation, whereas 3T3-L1 cells expressing PARP antisense RNA showed no increase in PARP and are unable to complete the round of DNA replication required for differentiation into adipocytes. The role of PARP in differentiation-linked DNA replication has now been further clarified at both the cellular and enzymological levels. Flow cytometric analysis revealed that control 3T3-L1 cells progressed through one round of DNA replication prior to the onset of terminal differentiation, whereas cells expressing PARP antisense RNA were blocked at the G0/G1 phase of the cell cycle. Confocal microscope image analysis of control S phase cells demonstrated that PARP was localized within distinct intranuclear granular foci associated with DNA replication centers. On the basis of these results, purified replicative complexes from other cell types that had been characterized for their ability to catalyze viral DNA replication in vitro were analyzed for the presence of PARP. PARP exclusively copurified through a series of centrifugation and chromatography steps with core proteins of an 18-21S multiprotein replication complex (MRC) from human HeLa cells, as well as with the corresponding mouse MRC from FM3A cells. The MRC were shown to contain DNA polymerases alpha and delta, DNA primase, DNA helicase, DNA ligase, and topoisomerases I and II, as well as accessory proteins such as PCNA, RF-C, and RP-A. Finally, immunoblot analysis of MRCs from both cell types with monoclonal antibodies to poly (ADP-ribose) revealed the presence of approximately 15 poly(ADP-ribosyl)ated proteins, some of which were further confirmed to be DNA polymerase alpha, DNA topoisomerase I, and PCNA by immunoprecipitation experiments. These results suggest that PARP may play a regulatory role within the replicative apparatus as a molecular nick sensor controlling the progression of the replication fork or modulates component replicative enzymes or factors in the complex by directly associating with them or by catalyzing their poly(ADP-ribosyl)ation.