Transient Poly(ADP-ribosyl)ation of Nuclear Proteins and Role of Poly(ADP-ribose) Polymerase in the Early Stages of Apoptosis

Abstract

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.

Poly(ADP-ribose) polymerase upregulates E2F-1 promoter activity and DNA pol α expression during early S phase

“…To determine whether depletion of PARP by gene disruption similarly blocks reentry of cells into S phase, we studied immortalized ®broblasts that were derived from wild-type (control) and PARP knockout (PARP7/7) mice (Wang et al, 1995). These PARP7/7 cells were previously con®rmed to be devoid of detectable PARP and poly(ADP-ribose) by immunoblot analysis with the corresponding antibodies (Simbulan-Rosenthal et al, 1998b). Although the presence of a novel activity capable of synthesizing ADP-ribose polymers has been shown recently in PARP7/7 cells, this activity, which is induced by treatment with MNNG, is considerably less than that in wild-type cells and may not fully compensate for PARP depletion (Shieh et al, 1998).…”

“…DNA histograms were derived at various times after release from aphidicolin block and the cell cycle phase distribution was quanti®ed and summarized in a ®gure showing per cent of wild-type (closed circles) and PARP7/7 (open circles) cells in S phase of the cell cycle PARP7/7 ®broblasts were stably transfected with pCD12, a plasmid encoding wild-type PARP, and grown in selective medium. A stable clone was selected for further study on the basis of its ability to express PARP protein as revealed by immunoblot analysis with antibodies to PARP as well as by PARP activity assays (Simbulan-Rosenthal et al, 1998b). These PARP7/ 7(+PARP) cells were synchronized by serum deprivation, released into the cell cycle by addition of serum, and analysed by¯ow cytometry.…”

“…Stable transfectants were selected in growth medium containing zeocin (500 mg/ml). Expression of PARP was con®rmed by RNA, DNA, and immunoblot analysis of control and stably transfected cell lines; these cell lines were recently used in a study investigating the role of PARP in the early stages of apoptosis (Simbulan-Rosenthal et al, 1998b).…”

DNA transitions induced by binding of PARP‐1 to cruciform structures in supercoiled plasmids

“…Figure 2a shows the measured volume distributions of PARP-1 molecules bound to the free ends in the three-way DNA junction model. On the basis of Jarque-Bera statistical analyses, the volumes show a normal distribution, with a mean volume of 255 6 63 nm 3 , which roughly corresponds to the size of a monomer. The theoretical molecular volume calculated from the protein's molecular weight is 213.7 nm 3 .…”

“…PARP-1 has been implicated in the DNA damage response as well as in cell growth, regulation, and apoptosis (2)(3)(4). The structure of the 113 kDa PARP-1 protein consists of three functional domains: the DNA binding domain, the central automodification domain, and the catalytic domain (5,6).…”