Relationship between poly(ADP-ribose) polymerase activity and DNA synthesis in cultured hepatocytes

Previous studies have demonstrated that an increase in poly(ADP-ribose) polymerase activity could be closely related to DNA replication during liver regeneration and to DNA repair synthesis in different experimental systems. This relationship was further investigated by studying the time course of endogenous and total poly(ADP-ribose) polymerase activity in cultured rat hepatocytes stimulated by epidermal growth factor. This mitogen has been shown to stimulate DNA synthesis in liver cells both in vivo and in vitro. A 6-fold increase in endogenous activity was observed early after epidermal growth factor addition, just before DNA synthesis. A subsequent 4-fold increment in total enzyme activity, concomitant with DNA synthesis, was detected. Orotic acid, which has recently shown mitoinhibitory effect, abolished the epidermal-growth-factor-induced increase in endogenous and total poly(ADP-ribose) polymerase activity, as well as DNA synthesis. On the contrary, 3-aminobenzamide inhibitor of poly(ADP-ribose) polymerase completely suppressed the endogenous activity but only partially modified the increase in total catalytic level and the overall pattern of thymidine incorporation. Taken together, these data indicate that, in cultured hepatocytes, the induction of DNA synthesis is supported by an increased poly(ADP-ribose) polymerase activity.     

Effect of epidermal growth factor on cultured adult rat hepatocytes

When adult rat hepatocytes were cultured in plastic Petri dishes in a medium containing insulin and glucagon, supplementation with epidermal growth factor (EGF) had a pronounced effect on their viability, morphology, and biochemical integrity. Transmission and scanning electron microscopic studies showed that after 1 week cells denied EGF accumulated numerous non-electron-dense bodies and filamentous whorls, had irregular nuclei, and exhibited atypical cell surfaces. In contrast, cells grown for 2-3 weeks in the presence of EGF had well-preserved cellular organelles and remained as an epithelial-like monolayer. After 3 weeks EGF-exposed cultures were still inducible for liver-specific tyrosine aminotransferase, and both rat albumin and rat transferrin were recoverable from the culture medium. Virtually no viable cells were present at 3 weeks in EGF-deprived cultures.     

Effect of simulated microgravity conditions on poly(ADP-ribose) polymerase activity in primary cultures of adult rat hepatocytes

The possible involvement of poly(ADP-ribose) polymerase [PARP; E.C. 2.4.2.30] in the adaptive response to low-g conditions was studied in cultured adult rat hepatocytes exposed to simulated microgravity produced by the random positioning machine (RPM-3D-clinostat). Four different poly(ADP-ribose) polymerases (PARPs) have been identified recently. The best-studied member of this family is PARP-1, a highly conserved, multimodular 113 kDa protein. In multicellular organisms PARPs catalyze poly(ADP-ribose) synthesis from NAD+ to a number of structural and catalytic proteins. Moreover, PARP-1 can control its protein and DNA interactions by catalyzing its automodification with poly(ADP-ribose) molecules that can include up to 200 ADP-ribose residues and several branching points; by these polymers, PARP-1 may nocovalently interact with other proteins and alter their functions. PARP-1 binds to DNA and is activated by free ends interacting with several other DNA damage checkpoint proteins. Thus, PARPs may target specific signal network proteins via poly(ADP-ribose) and regulate their domain functions. Poly(ADP-ribosyl)ation plays a central role in genome stability and is involved in DNA replication and repair, gene expression, cell differentiation and transformation. We have shown that a loss of PARP-1 activity is a critical event in the early molecular steps of the hepatocarcinogenesis process. Moreover, a prompt increase in this enzymatic activity is linked not only to the presence of DNA free ends but is linked also to the start of DNA synthesis. More recently, we have reported that PARP-1 is involved in hormone-mediated gene expression in vitro and in vivo during rat liver regeneration.     

Effect of epidermal growth factor on ornithine decarboxylase activity and urea synthesis in isolated rat hepatocytes.

Ornithine decarboxylase (ODC) activity is induced by protein-synthesis independent mechanisms in freshly isolated rat hepatocytes, incubated either without or with a mixture of amino acids in the incubation medium. Urea synthesis rates were two- to three-fold higher in those hepatocytes incubated in the presence of amino acids that in those lacking amino acids in the medium. Epidermal growth factor (EGF) delayed ODC induction, but only in the presence of amino acids. EGF significantly decreased ureagenesis when hepatocytes were incubated in the presence of amino acids and only endogenous substrates were available. No evidence of any link between ODC induction and urea synthesis was found.     

Electroporation of cultured adult rat hepatocytes with the c-myc gene potentiates DNA synthesis in response to epidermal growth factor

The human c-myc gene was introduced and transiently expressed in adult rat hepatocyte cultures by the technique of electroporation and its effect on DNA synthesis was examined. Epidermal growth factor (EGF) has been found to stimulate a wave of DNA synthesis in electroporated rat hepatocytes. Hepatocyte cultures electroporated with the c-myc gene showed a potentiation of this EGF effect exhibiting rates of DNA synthesis up to 50% greater than those of control electroporated cultures, as determined by [3H]thymidine labeling of cell nuclei. This potentiation was dependent on the amount of c-myc DNA transfected. The potentiation was due neither to an alteration in the dose-response of the stimulatory effect of EGF nor to a change in the time course of the DNA synthesis wave.     

The effects of putative DNA repair inhibitors on DNA adduct levels and unscheduled DNA synthesis in rat hepatocytes exposed to 2-acetylaminofluorene

The enzymology of DNA repair is currently under active investigation. The purpose of the present study was to examine the involvement of a number of enzymes (DNA polymerase alpha and beta, DNA topoisomerase II and ribonucleotide reductase) in the repair of chemically induced DNA damage in a mammalian cell system. This was done by studying the effects of inhibitors of these enzymes on the levels of 2-acetylaminofluorene (2-AAF)-DNA adducts and on the induction of UDS in primary cultures of rat hepatocytes exposed to the carcinogen in vitro. The results obtained with aphidicolin (an inhibitor of DNA polymerase alpha) show that the binding of 2-AAF to cellular DNA was significantly higher in samples exposed to this compound. Moreover, induction of UDS by 2-AAF was completely blocked in the presence of this compound. Dideoxythymidine, a DNA polymerase beta inhibitor, led to complex results. It produced a reduced DNA-specific activity due to [3H]2-AAF adduct formation as well as a diminished but still detectable UDS response in the presence of 2-AAF. Inhibitors of DNA topoisomerase II (nalidixic acid) and ribonucleotide reductase (hydroxyurea) did not cause any statistically significant change in the accumulation of 2-AAF adducts nor did they affect the induction of UDS. The data clearly suggest that DNA polymerase alpha participates in the repair of 2-AAF adducts in hepatocytes. In addition, neither DNA topoisomerase II activity, nor limitations in the precursor nucleotide pools appear to be critical factors in this process.     

Ethanolamine modulates DNA synthesis through epidermal growth factor receptor in rat primary hepatocytes

Summary Ethanolamine (Etn) stimulates hepatocyte proliferation in vivo and in vitro; however, the physiological function of Etn in hepatocytes has yet to be elucidated. In the present study, we examined the effect of Etn using a primary culture of rat hepatocytes. The level of membrane phosphatidylethanolamine (PE) significantly decreased when the hepatocytes were cultured without Etn but increased to the level found in the liver when the culture medium was supplemented with 20–50 μM Etn. Moreover, Etn stimulated DNA synthesis in a dose-dependent manner and had a synergistic effect with epidermal growth factor (EGF). A binding assay and Western blotting showed that the number of EGF receptors was 22–30% lower in cells grown in the absence of Etn compared to those grown in its presence, but the respective Kd values were almost the same. Furthermore, tyrosine phosphorylation of the EGF receptor was significantly lower in cells grown without Etn. Phosphatidylcholine (PC) synthesis in the liver is unique in that it occurs via stepwise methylation of PE. We found that without Etn supplementation, bezafibrate-induced inhibition of PE methylation increased the level of PE by decreasing its conversion to PC and stimulated DNA synthesis. Moreover, the function of EGF in stimulating DNA synthesis was significantly enhanced under Etn-sufficient conditions. These data suggest that Etn is a nutritional factor required for synthesis of adequate PE, levels of which are important for hepatocyte proliferation.     

Influence of poly(ADP-ribose) polymerase on the enzymatic synthesis of SV40 DNA.

The influence of poly(ADP-ribose) polymerase (PARP) on the replication of DNA containing the SV40 origin of replication has been examined. Extensive replication of SV40 DNA can be carried out in the presence of T antigen, topoisomerase I, the multimeric human single strand DNA-binding protein (HSSB), and DNA polymerase alpha-DNA primase (pol alpha-primase) complex (the monopolymerase system). In the monopolymerase system, both small products (Okazaki fragments), arising from lagging strand synthesis, and long products, arising from leading strand synthesis, are formed. The synthesis of long products requires the presence of relatively high levels of pol alpha-primase complex. In the presence of PARP, the synthesis of long products was blocked and only small Okazaki fragments accumulated, arising from the replication of the lagging strand template. The inhibition of leading strand synthesis by PARP can be effectively reversed by supplementing the monopolymerase system with the multimeric activator 1 protein (A1), the proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA polymerase delta (the dipolymerase system). The inhibition of leading strand synthesis in the monopolymerase system was caused by the binding of PARP to the ends of DNA chains, which blocked their further extension by pol alpha. The selective accumulation of Okazaki fragments was shown to be due to the coupled synthesis of primers by DNA primase and their immediate extension by pol alpha complexed to primase. PARP had little effect on this coupled reaction, but did inhibit the subsequent elongation of products, presumably after pol alpha dissociated from the 3'-end of the DNA fragments. PARP inhibited several other enzymatic reactions which required free ends of DNA chains. PARP inhibited exonuclease III, DNA ligase, the 5' to 3' exonuclease, and the elongation of primed DNA templates by pol alpha. In contrast, PARP only partly competed with the elongation of primed DNA templates by the pol delta elongation system which required SSB, A1, and PCNA. These results suggest that the binding of PARP at the ends of nascent DNA chains can be displaced by the binding of A1 and PCNA to primer ends. HSSB can be poly(ADP-ribosylated) in vivo as well as in vitro. However, the selective effect of PARP in blocking leading strand synthesis in the monopolymerase system was shown to depend primarily on its DNA binding property rather than on its ability to synthesize poly(ADP-ribose).     

Simulated microgravity conditions affect poly(ADP-ribose) polymerase activity in cultured human lymphocytes

Human peripheral blood lymphocytes (PBL), activated with concanavalin A (ConA), were used to determine the effects of simulated microgravity on poly(ADP-ribose) polymerase (PARP) activity. Results indicate that the ConA stimulation of human cultured PBL induces a partial but signitficant inhibition of PARP-1 acitvity (-30%). In control PBL, not exposed to ConA, after 24 hours, there was a clear decrease in PARP-1 acitivty (-40%). In PBL exposed to ConA and simulated weightlessness, activity decreased by -37%.     

Effect of amyloid beta peptide on poly(ADP-ribose) polymerase activity in adult and aged rat hippocampus

It is suggested that the fibrillar amyloid beta peptide (A beta) in brain plays a direct role in neurodegeneration in Alzheimer's disease, probably through activation of reactive oxygen species formation. Free radicals and numerous neurotoxins elicit DNA damage that subsequently activates poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30). In this study the effect of neurotoxic fragment (25-35) of full length A beta peptide on PARP activity in adult and aged rat hippocampus was investigated. In adult (4 month old) rat hippocampus the A beta 25-35 peptide significantly enhanced PARP activity by about 80% but had no effect on PARP activity in cerebral cortex and in hippocampus from aged (24-27 month old) rats. The effect of A beta peptide was reduced by half by the nitric oxide synthase inhibitor N-nitro-L-arginine. Stimulation of glutamate receptor(s) itself enhanced PARP activity by about 80% in adult hippocampus. However, A beta 25-35 did not exert any additional stimulatory effect. These results indicate that A beta, through NO and probably other free radicals, induces activation of DNA bound PARP activity exclusively in adult but not in aged hippocampus.     

PARP-2, A novel mammalian DNA damage-dependent poly(ADP-ribose) polymerase.

Poly(ADP-ribosylation) is a post-translational modification of nuclear proteins in response to DNA damage that activates the base excision repair machinery. Poly(ADP-ribose) polymerase which we will now call PARP-1, has been the only known enzyme of this type for over 30 years. Here, we describe a cDNA encoding a 62-kDa protein that shares considerable homology with the catalytic domain of PARP-1 and also contains a basic DNA-binding domain. We propose to call this enzyme poly(ADP-ribose) polymerase 2 (PARP-2). The PARP-2 gene maps to chromosome 14C1 and 14q11.2 in mouse and human, respectively. Purified recombinant mouse PARP-2 is a damaged DNA-binding protein in vitro and catalyzes the formation of poly(ADP-ribose) polymers in a DNA-dependent manner. PARP-2 displays automodification properties similar to PARP-1. The protein is localized in the nucleus in vivo and may account for the residual poly(ADP-ribose) synthesis observed in PARP-1-deficient cells, treated with alkylating agents or hydrogen peroxide.     

Transforming growth factor beta 1 inhibits epidermal growth factor receptor endocytosis and down-regulation in cultured fetal rat hepatocytes.

Incubation of fetal rat hepatocytes (FRH) with transforming growth factor beta 1 (TGF-beta 1) resulted in growth arrest and a biphasic effect on epidermal growth factor (EGF) receptor. After 2 h of exposure, EGF receptor (EGFR) was reduced by 43%. From 6 to 24 h, TGF-beta 1 exposure resulted in progressive increase in EGFR up to 74% over control. The increased binding was due to increase in high affinity EGF binding sites. FRH grown in medium containing EGF exhibited down-regulated EGFR with loss of high affinity EGF binding sites. With TGF-beta 1 exposure, high affinity EGFR was not down-regulated by EGF. Since down-regulation of EGFR involves internalization, the kinetics of EGF receptor-mediated endocytosis were examined. In TGF-beta 1-exposed FRH, EGF endocytosis was inhibited, with a reduction in the first order rate constant for the process from 0.078 to 0.043 min-1. Despite inhibition of growth, receptor down-regulation, and EGF endocytosis after TGF-beta 1 exposure, EGF-induced receptor autophosphorylation was preserved as demonstrated by [32P]phosphate-labeling of immunoprecipitated EGFR. These observations provide direct evidence that TGF-beta 1 regulates growth of fetal cells. Further, they suggest that TGF-beta 1 regulates endocytosis of EGF and possibly of other ligands.     

Poly(ADP-ribose) polymerase activity in mouse cells which exhibit temperature-sensitive DNA synthesis

The poly(ADP-ribose) polymerase activity of wild-type mouse L cells and of Balb/C-3T3 mouse fibroblasts remained relatively unchanged (at approx. 400 nmol substrate utilized/mg DNA per h) in actively-growing cells incubated at 34 degrees C or at 38.5 degrees C for at least 72 h. A similar result was obtained with the following temperature-sensitive cells grown at the permissive temperature (34 degrees C): ts A1S9 mouse L cells, ts C1 mouse L cells and Balb/C-3T3 ts mouse fibroblasts. The poly(ADP-ribose) polymerase activity of the temperature-sensitive cells was little affected during incubation for 20-24 h at the non-permissive temperature of 38.5 degrees C under which conditions temperature-inactivation of DNA replication was complete. Thereafter, this enzyme activity was found to increase some 2-fold, at a time when normal semi-conservative DNA synthesis was totally suppressed and replaced by repair replication (Sheinin, R. and Guttman, S. (1977) Biochim. Biophys. Acta 479, 105-118; Sheinin, R., Dardick, I. and Doane, F.W. (1980) Exp. Cell. Res., in the press).     

Changes in activity and mRNA levels of poly(ADP-ribose) polymerase during rat liver regeneration

ADP-ribosylation of nuclear proteins, catalysed by the enzyme poly(ADP-ribose) polymerase, is involved in the regulation of different cellular processes of DNA metabolism. To further clarify the role of the enzyme during proliferating activity of mammalian cells, we have studied the control of gene expression in regenerating rat liver. The changes in activity and mRNA levels were analysed during the early and late phases of the compensatory model. When enzyme activity was measured in isolated liver nuclei obtained at different times after hepatectomy, two different phases were observed: an early wave occurring before the onset of DNA synthesis, and a second one, starting several hours after the onset of DNA synthesis and returning to control values at later times. The evaluation of the enzymatic level in nuclear extracts and by activity gel analysis showed a more gradual increase starting 1 day after hepatectomy, in concomitance with the peak of DNA synthesis. By using a specific murine cDNA probe, a significant enhancement of mRNA levels for poly(ADP-ribose) polymerase was observed during liver regeneration, slightly preceding the onset of DNA synthesis. The results obtained show that changes in poly(ADP-ribose) polymerase activity, during liver regeneration, are associated both to early events preceding the increase in DNA synthesis and to later phases of the cell proliferation process.     

Inhibition of poly(ADP-ribose)polymerase binding to DNA by thymidine dimer

The ability of poly(ADP-ribose)polymerase to bind damaged DNA was assessed by electrophoretic mobility shift assay. DNA binding domain of poly(ADP-ribose)polymerase (PARPDBD) binds to synthetic deoxyribonucleotide duplex 10-mer. However, the synthetic deoxyribonucleotide duplex containing cys-syn thymidine dimer which produces the unwinding of DNA helix structure lost its affinity to PARPDBD. It was shown that the binding of PARPDBD to the synthetic deoxyribonucleotide duplex was not affected by O6-Me-dG which causes only minor distortion of DNA helix structure. This study suggests that the stabilized DNA helix structure is important for poly(ADP-ribose)polymerase binding to DNA breaks, which are known to stimulate catalytic activity of poly(ADP-ribose)polymerase.     

The B subunit of cholera toxin enhances DNA synthesis in rat hepatocytes induced by insulin and epidermal growth factor.

The B subunit of cholera toxin, which binds to ganglioside GM1, enhanced DNA synthesis in rat hepatocytes in primary culture induced by insulin and/or epidermal growth factor. The effect was dose-dependent, and whole cholera toxin, activating adenylate cyclase, showed a higher effect than the B subunit alone. The B subunit acted additively with other agents that also increase cyclic AMP levels. A competitive antagonist of cyclic AMP could not suppress the effect of the B subunit completely. These data suggest that the effect is independent of the cyclic AMP signal pathway, and that GM1 plays a role in hepatocyte proliferation.     

Inhibition of poly(ADP-ribose) polymerase activity is insufficient to induce tetraploidy

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 µM 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 (IC₅₀ = 0.15 µM). 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 ~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.