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Poly(ADP-ribose) polymerase (PARP) knockout mice are resistant to murine models of human diseases such as cerebral and myocardial ischemia, traumatic brain injury, diabetes, Parkinsonism, endotoxic shock and arthritis, implicating PARP in the pathogenesis of these diseases. Potent selective PARP inhibitors are therefore being evaluated as novel therapeutic agents in the treatment of these diseases. Inhibition or depletion of PARP, however, increases genomic instability in cells exposed to genotoxic agents. We recently demonstrated the presence of a genomically unstable tetraploid population in PARP(-/-) fibroblasts and its loss after stable transfection with PARP cDNA. To elucidate whether the genomic instability is attributable to PARP deficiency or lack of PARP activity, we investigated the effects of PARP inhibition on development of tetraploidy. Immortalized wild-type and PARP(-/-) fibroblasts were exposed for 3 weeks to 20 microM GPI 6150 (1,11b-dihydro-[2H:]benzopyrano[4,3,2-de]isoquinolin-3-one), a novel small molecule specific competitive inhibitor of PARP (K(i) = 60 nM) and one of the most potent PARP inhibitors to date (IC(50) = 0.15 microM). Although GPI 6150 initially decreased cell growth in wild-type cells, there was no effect on cell growth or viability after 24 h. GPI 6150 inhibited endogenous PARP activity in wild-type cells by approximately 91%, to about the residual levels in PARP(-/-) cells. Flow cytometric analysis of unsynchronized wild-type cells exposed for 3 weeks to GPI 6150 did not induce the development of tetraploidy, suggesting that, aside from its catalytic function, PARP may play other essential roles in the maintenance of genomic stability.

An early transient burst of poly(ADP-ribosyl)ation of nuclear proteins was recently shown to be required for apoptosis to proceed in various cell lines (Simbulan-Rosenthal, C., Rosenthal, D., Iyer, S., Boulares, H., and Smulson, M. (1998) J. Biol. Chem. 273, 13703-13712) followed by cleavage of poly(ADP-ribose) polymerase (PARP), catalyzed by caspase-3. This inactivation of PARP has been proposed to prevent depletion of NAD (a PARP substrate) and ATP, which are thought to be required for later events in apoptosis. The role of PARP cleavage in apoptosis has now been investigated in human osteosarcoma cells and PARP -/- fibroblasts stably transfected with a vector encoding a caspase-3-resistant PARP mutant. Expression of this mutant PARP increased the rate of staurosporine and tumor necrosis factor-alpha-induced apoptosis, at least in part by reducing the time interval required for the onset of caspase-3 activation and internucleosomal DNA fragmentation, as well as the generation of 50-kilobase pair DNA breaks, thought to be associated with early chromatin unfolding. Overexpression of wild-type PARP in osteosarcoma cells also accelerated the apoptotic process, although not to the same extent as that apparent in cells expressing the mutant PARP. These effects of the mutant and wild-type enzymes might be due to the early and transient poly(ADP-ribose) synthesis in response to DNA breaks, and the accompanying depletion of NAD apparent in the transfected cells. The accelerated NAD depletion did not seem to interfere with the later stages of apoptosis. These results indicate that PARP activation and subsequent cleavage have active and complex roles in apoptosis.

We have investigated the association of human topoisomerase I with poly(ADP-ribosylated) domains of chromatin and the effects of this modification on the enzyme activity. In vitropoly(ADP-ribosylation) assays demonstrated that this enzyme was one of the major acceptors for this chromatin-dependent post-translational modification. Western blotting procedures using antibody to topoisomerase I indicated that under extensive poly(ADP-ribosylation) conditions, where a majority of poly(ADP-ribose) acceptor molecules form aggregates, the major population of the topoisomerase I associated with chromatin was apparently non-aggregated. The catalytic activity of the topoisomerase I associated with the poly(ADP-ribosylated) chromatin was 3–5-fold inhibited. Additionally, antibody to poly(ADP-ribose) was used to immunofractionate selectively the modified domains of chromatin. Our data suggests the presence of topoisomerase I, both adjacent and distal to the poly(ADP-ribosylated) sites of chromatin. Unmodified and a significant portion of the modified species of enzyme migrated as approximately 100-kDa proteins. However, the modified form of topoisomerase was noted to be catalytically less active as compared to the enzyme bound to the non-poly(ADP-ribosylated) nucleosomes. These results provide evidence, at the cellular level, for the poly(ADP-ribosylation)-mediated regulation of human topoisomerase I and suggest a functional significance for poly(ADP-ribosylation) in topoisomerase-related processes (replication, transcription, and recombination) in eukaryotes.

A transient burst of poly(ADP-ribosyl)ation of nuclear proteins occurs early, prior to commitment to death, in human osteosarcoma cells undergoing apoptosis, followed by caspase-3-mediated cleavage of poly(ADP-ribose) polymerase (PARP). The generality of this early burst of poly(ADP-ribosyl)ation has now been investigated with human HL-60 cells, mouse 3T3-L1, and immortalized fibroblasts derived from wild-type mice. The effects of eliminating this early transient modification of nuclear proteins by depletion of PARP protein either by antisense RNA expression or by gene disruption on various morphological and biochemical markers of apoptosis were then examined. Marked caspase-3-like PARP cleavage activity, proteolytic processing of CPP32 to its active form, internucleosomal DNA fragmentation, and nuclear morphological changes associated with apoptosis were induced in control 3T3-L1 cells treated for 24 h with anti-Fas and cycloheximide but not in PARP-depleted 3T3-L1 antisense cells exposed to these inducers. Similar results were obtained with control and PARP-depleted human Jurkat T cells. Whereas immortalized PARP +/+ fibroblasts showed the early burst of poly(ADP-ribosyl)ation and a rapid apoptotic response when exposed to anti-Fas and cycloheximide, PARP -/- fibroblasts exhibited neither the early poly (ADP-ribosyl)ation nor any of the biochemical or morphological changes characteristic of apoptosis when similarly treated. Stable transfection of PARP -/- fibroblasts with wild-type PARP rendered the cells sensitive to Fas-mediated apoptosis. These results suggest that PARP and poly(ADP-ribosyl)ation may trigger key steps in the apoptotic program. Subsequent degradation of PARP by caspase-3-like proteases may prevent depletion of NAD and ATP or release certain nuclear proteins from poly(ADP-ribosyl)ation-induced inhibition, both of which might be required for late stages of apoptosis.

The possibility that chromatin domains containing acetylated histones are proximal to domains containing chromosomal high mobility group protein 17 (HMG-17) has been investigated. Oligonucleosomes containing [3H]acetate-labeled histones have been immunofractionated on anti-HMG-17 IgG-Sepharose columns. Ninety-one per cent of the 3H counts present in the oligonucleosomes specifically bound to the anti-HMG-17 column. Extraction of HMG-17 from chromatin by treatment with 0.4 M NaCl abolished the specific binding of acetylated chromatin to the Sepharose columns. Autoradiographic analysis of polyacrylamide gels of the bound fraction revealed that it contained all the major acetylated histone species. We conclude that acetylated histones are present on or near nucleosomes containing protein HMG-17.

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 (Cuvillier, O., Pirianov, G, Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, J. S., and Spiegel, S. (1996) Nature 381, 800-803). Here, we investigated the role of SPP and the protein kinase C activator, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), in the caspase cascade leading to the proteolysis of poly(ADP-ribose) polymerase (PARP) and lamins. In Jurkat T cells, Fas ligation or addition of exogenous C2-ceramide induced activations of caspase-3/CPP32 and caspase-7/Mch3 followed by PARP cleavage, effects that can be blocked either by SPP or TPA. Furthermore, both SPP and TPA inhibit the activation of caspase-6/Mch2 and subsequent lamin B cleavage. Ceramide, in contrast to Fas ligation, did not induce activation of caspase-8/FLICE and neither SPP nor TPA were able to prevent this activation. Thus, SPP, likely generated via protein kinase C-mediated activation of sphingosine kinase, suppresses the apoptotic pathway downstream of FLICE but upstream of the executioner caspases, caspase-3, -6, and -7.

When HeLa cell nuclei were treated with micrococcal nuclease (nucleate 3-oligonucleotidohydrolase, EC 3.1.4.7), lysed, and centrifuged, the supernatant from early digests contained two predominant classes of polynucleosomes of repeat size 8N and 16N. With increasing digestion time, the 16 N polynucleosome appeared to be cleaved to the 8N species and finally to the basic subunit of chromatin. The size of the polynucleosomes has been determined by DNA analysis and on polyacrylamide electrophoretic gels of native chromatin particles. The 16N polynucleosome appears to be a unique higher ordered structural component of HeLa cell chromatin. Our recent report, showing that the nuclear protein-modifying enzyme poly(ADP-ribose) polymerase increases in specific activity progressively with increasing nucleosome repeat size up to 8-10N, has been extended in the present study. Activity was also elevated in the polynucleosomes of the 16N structure preferentially cleaved by micrococcal nuclease, although specific activity of the enzyme was highest in octanucleosomes. Acceptors for poly(ADP-ribose) have also been determined in these particles.

One of the major products of the poly(ADP-Rib) polymerase reaction is the H1 dimer, formed by cross-linking two H1 molecules with a 15-unit poly(ADP-Rib) chain. In the present study we have attempted to characterize those regions of H1 molecules which participate in cross-linking, and in particular, to determine whether the reaction involves two NH2-terminal domains, two COOH-terminal domains, or a combination of these two. When we used reconstituted chromatin, formed by the addition of various H1 peptides to H1-depleted chromatin, we observed poly(ADP-ribosylation) in those complexes containing fragments of the NG-H1 domain (i.e. NH2-terminal tail plus the globular (G) region). Furthermore, with this material, increasing NAD concentration and incubation time led to a progressive elongation of poly(ADP-Rib) on the peptide approaching a chain length of 11-12 units. Reconstituted chromatin that contained other H1-derived peptides, such as C-H1 and G-H1 (i.e. only carboxyl or only globular domains respectively), were not poly(ADP-ribosylated). CG-H1 was noted to be a substrate in the reconstituted system. However, in contrast to NG-H1, where extensive chain elongation was observed, only very short chain, probably mono(ADP-ribosylation) occurred on this H1 peptide. In a complementary approach, the H1 dimer complex or its precursors were cleaved with N-bromosuccinimide, and peptide fragments isolated. This analysis confirmed that both the NH2- and COOH-terminal domains of H1 are poly(ADP-ribosylated) and that elongation of the ADP-ribose chain occurred primarily at the NH2 terminus during H1 dimer synthesis.

Poly(ADP-ribose) polymerase (PADPRP) is biologically significant in the rejoining of DNA strand breaks. Post confluent cultures of 3T3-L1 preadipocytes showed marked increases in PADPRP protein and activity when the cells were induced to differentiate into adipocytes. When this increase in PADPRP expression was prevented in stably transfected 3T3-L1 cells by induction of PADPRP antisense RNA synthesis, the cells did not differentiate nor undergo the two or three rounds of DNA replication that are required for initiation of the differentiation process. 3T3-L1 cells expressing PADPRP antisense RNA under differentiation conditions were easily detached from plates and in some cases eventually died. When newly expressed PADPRP protein and DNA synthesis was assessed in cells at zero time or at 24 h after induction of differentiation by incorporation of bromodeoxyuridine or [3H]thymidine into DNA, significant incorporation was shown to occur in control cells after 24 h, but not in antisense cells. Furthermore, during the first 24 h, the co-immunoprecipitation of PADPRP and DNA polymerase alpha was observed in control cells, whereas no such complex formation was noted in the induced antisense cells, nor in uninduced control cells.

The poly(ADP-ribosylation) of histones, which occurs within a limited and functionally specific domain of chromatin, is a novel post-translational modification. However, in the past it has been difficult to study this process in living cells because the substrate of the reaction (NAD) does not permeate the plasma membrane. In the current study, antibodies specific for histone H1 and poly(ADP-ribose) were used to study the occurrence of poly(ADP-ribose)+ species of H1 in vivo. Perchloric acid-extracted proteins from synchronously growing HeLa cells were fractionated by electrophoresis and transferred to nitrocellulose, and the transferred moieties were allowed to react with the specific antibodies and then with 125I-labeled protein A. The results conclusively demonstrate the natural occurrence of poly(ADP-ribose)-crosslinked complexes of histone H1 (i.e., H1 dimer), at the S/G2 phase transition of the cell cycle.

Antibody to poly(ADP-ribose) has been covalently coupled to Sepharose and utilized to isolate selectively oligonucleosomes undergoing the poly(ADP-ribosyl)ation reaction from the bulk of chromatin. Approximately 12% of the unfractionated oligonucleosomes were bound to the immunoaffinity column and these represented essentially 100% of the original poly(ADP-ribosyl)ated nucleosomal species in the unfractionated chromatin. Poly(ADP-ribosyl)ated chromatin was not bound by preimmune IgG columns. KSCN eluted the modified nucleosomes in the form of nucleoprotein complexes. The eluted chromatin components were shown to contain poly(ADP-ribosyl)ated histones as well as automodified poly(ADP-ribose) polymerase. By using [3H]lysine- and [3H]arginine-labeled chromatin, it was shown that the poly-(ADP-ribosyl)ated histones, attached to stretches of oligonucleosomes bound to the column, had a 6-fold enrichment of the modification compared to histones of the unfractionated chromatin. This indicated that non-poly(ADP-ribosyl)ated nucleosomes, connected and proximal to the modified regions, were copurified by this procedure. This allowed characterization of the oligonucleosomal DNA around poly(ADP-ribosyl)ated chromatin domains to be compared with the unbound bulk chromatin. The data indicated that immunofractionated poly(ADP-ribosyl)ated oligonucleosomal DNA contained significant amounts of internal single-strand breaks compared with bulk chromatin. The bound nucleo-protein complexes were found to be enzymatically active for poly(ADP-ribose) polymerase after elution from the antibody column. In contrast, the unbound nucleosomes, representing 90% of the unfractionated chromatin, were totally inactive in the poly(ADP-ribosyl)ation reaction.

The host nuclear enzyme poly(ADP-ribose) polymerase has been shown to be associated with the replicative intermediate and mature forms of polyoma virus minichromosomes. Minichromosome-associated histones H2A and H2B as well as several nonhistone proteins were poly ADP-ribosylated by endogenous poly(ADP-ribose) polymerase. In addition, minichromosome fractions catalyzed the formation in vitro of dimers of endogenous histone H1 linked by poly(ADP-ribose). Poly ADP-ribosylated polyoma virus minichromosome chromatin labeled in vivo with [3H]thymidine could be retained and eluted from anti-poly(ADP-ribose) immunoglobulin G-Sepharose. Pulse-labeled replicative intermediate minichromosomes were retained better on the antibody columns than were mature minichromosomes labeled for 2.5 h. The possible role of poly ADP-ribosylation of viral nucleosomes during polyoma replication or transcription is discussed.

cDNAs encoding poly(ADP-ribose) polymerase from a human hepatoma lambda gt11 cDNA library were isolated by immunological screening. One insert of 1.3 kilobases (kb) consistently hybridized on RNA gel blots to an mRNA species of 3.6-3.7 kb, which is consistent with the size of RNA necessary to code for the polymerase protein (116 kDa). This insert was subsequently used in both in vitro hybrid selection and hybrid-arrested translation studies. An mRNA species from HeLa cells of 3.6-3.7 kb was selected that was translated into a 116-kDa protein, which was selectively immunoprecipitated with anti-poly (ADP-ribose) polymerase. To confirm that the 1.3-kb insert from lambda gt11 encodes for poly(ADP-ribose) polymerase, the insert was used to screen a 3- to 4-kb subset of a transformed human fibroblast cDNA library in the Okayama-Berg vector. One of these vectors [pcD-p(ADPR)P; 3.6 kb] was tested in transient transfection experiments in COS cells. This cDNA insert contained the complete coding sequence for polymerase as indicated by the following criteria: A 3-fold increase in in vitro activity was noted in extracts from transfected cells compared to mock or pSV2-CAT transfected cells. A 6-fold increase in polymerase activity in pcD-p(ADPR)P transfected cell extracts compared to controls was observed by "activity gel" analysis on gels of electrophoretically separated proteins at 116 kDa. A 10- to 15-fold increase in newly synthesized polymerase was detected by immunoprecipitation of labeled transfected cell extracts. Using pcD-p(ADPR)P as probe, it was observed that the level of poly(ADP-ribose) polymerase mRNA was elevated at 5 and 7 hr of S phase of the HeLa cell cycle, but was unaltered when artificial DNA strand breaks are introduced in HeLa cells by alkylating agents.

Recently we described a full-length cDNA for the human nuclear enzyme poly(ADP-ribose) polymerase. Here, we report the chromosomal localization and partial map of the human gene for this enzyme as well as the complete coding sequence for this protein. The nucleotide sequence reveals a single 3042-base open reading frame encoding a protein with a predicted Mr of 113,135. A comparison of this deduced amino acid sequence with the amino acid sequence of three peptides derived from human poly(ADP-ribose) polymerase revealed a match of 27 amino acid residues. A computer-derived structural analysis of the enzyme and a search for similarities with other proteins confirmed that the polymerase belongs to a subfamily of DNA/NAD-binding proteins and DNA-repair proteins. Possible Zn2+-binding "fingers," a nucleotide-binding fold, and a nuclear transport signal were noted. Additionally, chromosomal mapping has identified polymerase-hybridizing sequences on human chromosomes 1 (the active gene), 13, and 14 (processed pseudogenes). Using the polymerase cDNA as a probe, we also have detected several DNA restriction fragment length polymorphisms in normal humans.