Tannins elevate the level of poly(ADP-ribose) in HeLa cell extracts

Phenolic phytochemicals such as tannins, which are natural constituents of green tea, red wine, and other plant products, are considered to have cancer-preventive properties. An important endogenous mediator of tumorigenesis is the nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP-1). PARP-1 synthesizes polymers of ADP-ribose (PAR), which, in turn, are degraded by the catabolic enzyme poly(ADP-ribose) glycohydrolase (PARG). In the present study, we investigated the effects of tannins on the level of PAR in HeLa nuclear extracts. The addition of tannins to nuclear extracts led to a 40-fold elevation of PAR-levels. The observed increased PAR-levels resulted from inhibition of the catalytic activity of PARG. Additionally, the human PARG cDNA was cloned and the recombinant enzyme was overexpressed and isolated. Recombinant PARG was immobilized using an affinity column composed of tannins covalently linked to Sepharose beads. Finally, an interaction between immobilized PARG and endogenous PARP-1 from HeLa cell extracts is demonstrated.     

Inhibitory effect of tannin on poly(ADP-ribose) glycohydrolase from human placenta

Tannin, a naturally occurring polyphenolic compound, was found to inhibit the activity of purified poly(ADP-ribose) glycohydrolase from human placenta. The inhibition was dose-dependent and half maximal with 2.8 micrograms/ml of tannin. The inhibitory effect of tannin was two and three orders of magnitude more than those of ADP-ribose and cAMP, respectively. Kinetic analysis revealed that the inhibition by tannin was competitive with respect to the substrate poly(ADP-ribose).     

Effect of DNA on poly (ADP-ribose) glycohydrolase and the degradation of histone H1-poly (ADP-ribose) complex from HeLa cell nuclei.

A poly(ADP-ribose)-H1 histone complex has been isolated from HeLa cell nuclei incubated with NAD. The rate of poly(ADP-ribose) glycohydrolase catalyzed hydrolysis of the polymer in the complex is only 1/9 that of free poly(ADP-ribose), indicating that the polymer is in a protected environment within the complex. Comparison of the rate of hydrolysis of free poly(ADP-ribose) in the presence or absence of H1 to that in the complex synthesized de novo indicates a specific mode of packaging of the complex. This is further indicated by the fact that alkaline dissociation of the complex followed by neutralization markedly exposes the associated poly(ADP-ribose) to the glycohydrolase. The complex also partially unfolds when it binds to DNA as evidenced by a 2-fold increase in the rate of glycolytic cleavage of poly(ADP-ribose). This effect of DNA is not due to a stimulation of the glycohydrolase per se since hydrolysis of free polymer by the enzyme is strongly inhibited by DNA, especially single-stranded DNA. Inhibition of glycohydrolase by DNA results from the binding of the enzyme to DNA and conditions which decrease this binding (increased ionic strength or addition of histone H1 which competes for DNA binding) relieve the DNA inhibition.     

Poly(ADP-ribosyl)ation of chromatin in an in-vitro poly(ADP-ribose)-turnover system.

This paper describes the effect of an in-vitro poly(ADP-ribose) turnover system on the poly(ADP-ribosyl)ation of chromatin. Both poly(ADP-ribose)polymerase and poly(ADP-ribose)glycohydrolase were highly purified and used in 4 different turnover systems: non-turnover, slow, medium and fast turnover. These turnover systems were designed to reflect possible turnover conditions in intact cells. The major protein acceptors for poly(ADP-ribose) are histones and the polymerase itself, a process referred to as automodification. The level of poly(ADP-ribose) modification of polymerase, histone H1 and core histones has been measured. The size of the polymer for each of the 3 groups of acceptor proteins has been determined by gel electrophoresis. After many turnover cycles at medium and fast turnover, the histones (H1 and core) become the main poly(ADP-ribose) acceptor proteins. The rate at which steady-state polymer levels are reached and the total accumulation of polymer in a given turnover system are both inversely proportional to the amount of glycohydrolase present. Furthermore, increasing amounts of glycohydrolase in the turnover systems reduces average polymer size. The polymer synthesized in the medium and fast turnover systems is degraded by glycohydrolase in a biphasic fashion and in these systems the half-life of polymer agreed with results found in intact cells. Our results show that the relative levels of polymerase and glycohydrolase activities can regulate the proportional poly(ADP-ribose) distribution on chromatin-associated acceptor proteins during steady-state turnover conditions. The patterns of modification of polymerase and histones under turnover conditions agree with in vivo observations.     

Modulation of chromatin superstructure induced by poly(ADP-ribose) synthesis and degradation

It has been demonstrated recently by Poirier et al. (Poirier, G. G., de Murcia, G., Jongstra-Bilen, J., Niedergang, C., and Mandel, P. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3423-3427) that poly(ADP-ribosyl)ation of pancreatic nucleosomes causes relaxation of the chromatin superstructure through H1 modification. The in vitro effect of poly(ADP-ribose) synthesis and degradation on calf thymus chromatin was investigated by the time course incorporation of ADP-ribose, electron microscopy, analytical ultracentrifugation, and autoradiography of the protein acceptors. Purified calf thymus poly(ADP-ribose) polymerase and partially purified bull testis poly(ADP-ribose) glycohydrolase were used. Degradation of ADP-ribose units on hyper(ADP-ribosyl)ated H1 by poly(ADP-ribose) glycohydrolase restores the native condensed chromatin superstructure. This reversible conformational change induced by poly(ADP-ribosyl)ation on nucleosomal arrangement could be one of the mechanisms by which the accessibility of DNA polymerases and/or excision-repair enzymes is favored, the native structure being fully restorable.     

Isolation and purification of poly(ADP-ribose) glycohydrolase from pig thymus

Poly(ADP-ribose) glycohydrolase has been purified about 12 300-fold from pig thymus with a recovery of 8.5%. The specific activity of the purified enzyme is 13.8 mumol min -1 mg protein -1. The molecular weight was estimated to be 59 000 by gel filtration through Sephadex G-100 in a non-denaturing solvent. Analysis of the final preparation by sodium dodecyl sulphate gel electrophoresis reveals two protein bands of molecular weight, 61 500 and 67 500. The Km value for poly(ADP-ribose) is estimated to be 1.8 microM monomer units. The enzyme preparation is free from phosphodiesterase, NADase and ADP-ribosyltransferase activities. The purified enzyme is inhibited by cyclic AMP, ADP-ribose, naphthylamine, histones H1, H2A, H2B, H3, polylysine, polyarginine, polyornithine and protamine. The inhibition by histone is relieved by an equal mass of DNA. Single-stranded DNA, poly(A), poly(I) and polyvinyl sulphate were inhibitory, but double-stranded DNA was not inhibitory.     

Cytoplasmic poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase in AEV-transformed chicken erythroblasts

Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase activities were both investigated in chicken erythroblasts transformed by Avian Erythroblastosis Virus. Respectively 21% and 58% of these activities were found to be present in the post-mitochondrial supernatant (PMS). Fractionation of the PMS on sucrose gradients and poly(A⁺) mRNA detection by hybridization to [³H] poly(U) show that cytoplasmic poly(ADP-ribose) polymerase is exclusively localized in free mRNP. The glycohydrolase activity sedimented mostly in the 6 S region but 1/3 of the activity was in the free mRNP zone. Seven poly(ADP-ribose) protein acceptors were identified in the PMS in the Mr 21000–120000 range. The Mr 120000 protein corresponds to automodified poly(ADP-ribose) polymerase. A Mr 21000 protein acceptor is abundant in PMS and a Mr 34000 is exclusively associated with ribosomes and ribosomal subunits. The existence of both poly(ADP-ribose) polymerase and glycohydrolase activities in free mRNP argues in favour of a role of poly(ADP-ribosylation) in mRNP metabolism. A possible involvement of this post translational modification in the mechanisms of repression-derepression of mRNA is discussed.Abbreviations ADP-ribose adenosine (5) diphospho(5)--D ribose - poly(ADP-ribose) polymer of ADP-ribose - mRNP messenger ribonucleoprotein particles - PMSF phenylmethylsulfonyl fluoride - LDS lithium dodecyl sulfate - TCA trichloroacetic acid     

Characterization of two forms of poly(ADP-ribose) glycohydrolase in guinea pig liver

A poly(ADP-ribose) glycohydrolase from guinea pig liver cytoplasm has been purified approximately 45,000-fold to apparent homogeneity. The cytoplasmic poly(ADP-ribose) glycohydrolase designated form II differed in several respects from the nuclear poly(ADP-ribose) glycohydrolase I (Mr = 75,500) previously purified from the same tissue (Tanuma et al., 1986a). The purified glycohydrolase II consists of a single polypeptide with Mr of 59,500 estimated by a sodium dodecyl sulfate-polyacrylamide gel. A native Mr of 57,000 was determined by gel permeation. Peptide analysis of partial proteolytic degradation of glycohydrolases II and I with Staphylococcus aureus V8 protease revealed that the two enzymes were structurally different. Amino acid analysis showed that glycohydrolase II had a relatively low proportion of basic amino acid residues as compared with glycohydrolase I. Glycohydrolase II and I were acidic proteins with isoelectric points of 6.2 and 6.6, respectively. The optimum pH for glycohydrolases II and I were around 7.4 and 7.0, respectively. The Km value for (ADP-ribose)n (average chain length n = 15) and the Vmax for glycohydrolase II were 4.8 microM and 18 mumol of ADP-ribose released from (ADP-ribose)n.min-1.(mg of protein)-1, respectively. The Km was about 2.5 times higher, and Vmax 2 times lower, than those observed with glycohydrolase I. Unlike glycohydrolase I, glycohydrolase II was inhibited by monovalent salts. ADP-ribose and cAMP inhibited glycohydrolase II more strongly than glycohydrolase I. These results suggest that eukaryotic cells contain two distinct forms of poly(ADP-ribose) glycohydrolase exhibiting differences in properties and subcellular localization.     

Lignin inhibits (ADP-ribose)n glycohydrolase activity

(ADP-ribose)n glycohydrolase activity was inhibited in vitro by lignin, a naturally occurring polymethoxyphenolic compound. However, coniferyl alcohol, which is a main component of lignin, was not inhibitory even at 100 micrograms/ml. Lignin caused competitive inhibition with respect to the substrate (ADP-ribose)n and its Ki value was 18 micrograms/ml. These results suggest that lignin with a polymerized structure has a functional domain that interacts with the (ADP-ribose)n glycohydrolase molecule at the same site as (ADP-ribose)n.     

Strategy for selection of cell variants deficient in poly(ADP-ribose) polymerase

A selection strategy to obtain cells deficient in poly(ADP-ribose) polymerase was developed based on the fact that treatment with high levels of N-methyl-N'-nitro-N-nitrosoguanidine results in sufficient activation of poly(ADP-ribose) polymerase to cause NAD and ATP depletion leading to cessation of all energy-dependent processes and rapid cell death. In contrast, cells with low levels of poly(ADP-ribose) polymerase should not consume their NAD and might therefore be more likely to survive the DNA damage. Using this approach, we have cloned a number of cell lines containing 37-82% enzyme activity. The apparent decrease in poly(ADP-ribose) polymerase activity is not due to increases in NAD glycohydrolase, poly(ADP-ribose) glycohydrolase, or phosphodiesterase activities. Further characterization of the poly(ADP-ribose) polymerase-deficient cells indicates that they have prolonged generation times and increased rates of spontaneous sister chromatid exchanges.     

Cellular regulation of ADP-ribosylation of proteins. IV. Conversion of poly(ADP-ribose) polymerase activity to NAD-glycohydrolase during retinoic acid-induced differentiation of HL60 cells.

Two enzymatic activities of the nuclear enzyme poly(ADP-ribose) polymerase or transferase (ADPRT, EC 2.4.2.30), a DNA-associating abundant nuclear protein with multiple molecular activities, have been determined in HL60 cells prior to and after their exposure to 1 microM retinoic acid, which results in the induction of differentiation to mature granulocytes in 4-5 days. The cellular concentration of immunoreactive ADPRT protein molecules in differentiated granulocytes remained unchanged compared to that in HL60 cells prior to retinoic acid addition (3.17 +/- 1.05 ng/10(5) cells), as did the apparent activity of poly(ADP-ribose) glycohydrolase of nuclei. On the other hand, the poly(ADP-ribose) synthesizing capacity of permeabilized cells or isolated nuclei decreased precipitously upon retinoic acid-induced differentiation, whereas the NAD glycohydrolase activity of nuclei significantly increased. The nuclear NAD glycohydrolase activity was identified as an ADPRT-catalyzed enzymatic activity by its unreactivity toward ethenoadenine NAD as a substrate added to nuclei or to purified ADPRT. During the decrease in in vitro poly(ADP-ribose) polymerase activity of nuclei following retinoic acid treatment, the quantity of endogenously poly(ADP-ribosylated) ADPRT significantly increased, as determined by chromatographic isolation of this modified protein by the boronate affinity technique, followed by gel electrophoresis and immunotransblot. When homogenous isolated ADPRT was first ADP-ribosylated in vitro, it lost its capacity to catalyze further polymer synthesis, whereas the NAD glycohydrolase function of the automodified enzyme was greatly augmented. Since results of in vivo and in vitro experiments coincide, it appears that in retinoic acid-induced differentiated cells (granulocytes) the autopoly(ADP-ribosylated) ADPRT performs a predominantly, if not exclusively, NAD glycohydrolase function.     

Characteristics of the inhibition of poly(ADP-ribose) glycohydrolase by homopolypurines

Poly(ADP-ribose) glycohydrolase partially purified from rat testis was markedly inhibited by the homopolypurines polyG, polyI and polyA. The inhibition was competitive with respect to poly(ADP-ribose) and the Ki for polyG and polyA was 2.8 uM and 5.5 uM, respectively. This inhibitory effect of the homopolypurines was practically eliminated when 250 mM KCl was present in the reaction mixture. Moreover, the inhibition exerted by polyI or polyA was markedly diminished after hybridization with polyC or polyT, respectively.     

NAD Glycohydrolase Activities and ADP-Ribose Uptake in Erythrocytes From Normal Subjects and Cancer Patients

Erythrocytes from cancer patients exhibited up to fivefold higher NAD glycohydrolase activities than control erythrocytes from normal subjects and also similarly increased [14C] ADP-ribose uptake values. When [adenosine-14C] NAD was used instead of free [14C] ADP-ribose, the uptake was dependent on ecto-NAD glycohydrolase activity. This was reflected in the inhibition of ADP-ribose uptake from [adenosine-14C] NAD by Cibacron Blue. ADP-ribose uptake in erythrocytes appeared to be complex: upon incubation with free [14C] ADP-ribose, the radiolabel associated with erythrocytes was located in nearly equal parts in cytoplasm and plasma membrane. Part of [14C] ADP-ribose binding to the membrane was covalent, as indicated by its resistance to trichloroacetic acid-treatment. A preincubation with unlabeled ADP-ribose depressed subsequent erythrocyte NAD glycohydrolase activity and binding of [14C] ADP-ribose to erythrocyte membrane; but it failed to inhibit the transfer of labeled ADP-ribose to erythrocyte cytoplasm. On the other hand, incubation with [adenosine-14C] NAD did not result in a similar covalent binding of radiolabel to erythrocyte membrane. In line with this finding, a preincubation with unlabeled NAD was not inhibitory on subsequent NAD glycohydrolase reaction and ADP-ribose binding. ADP-ribose binding and NAD glycohydrolase activities were found also in solubilized erythrocyte membrane proteins and, after size fractionation, mainly in a protein fraction of around 45kDa-molecular weight.     

Histone shuttling by poly ADP-ribosylation

The enzymes poly(ADP-ribose)polymerase and poly(ADP-ribose) glycohydrolase may cooperate to drive a histone shuttle mechanism in chromatin. The mechanism is triggered by binding of the N-terminal zinc-finger domain of the polymerase to DNA strand breaks, which activates the catalytic activities residing in the C-terminal domain. The polymerase converts into a protein carrying multiple ADP-ribose polymers which displace histones from DNA by specifically targeting the histone tails responsible for DNA condensation. As a result, the domains surrounding DNA strand breaks become accessible to other proteins. Poly(ADP0ribose) glycohydrolase attacks ADP-ribose polymers in a specific order and thereby releases histones for reassociation with DNA. Increasing evidence from different model systems suggests that histone shuttling participates in DNA repairin vivo as a catalyst for nucleosomal unfolding.     

Poly(ADP-ribose) accessibility to poly(ADP-ribose) glycohydrolase activity on poly(ADP-ribosyl)ated nucleosomal proteins

Hydrolysis of protein-bound 32P-labelled poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase shows that there is differential accessibility of poly(ADP-ribosyl)ated proteins in chromatin to poly(ADP-ribose) glycohydrolase. The rapid hydrolysis of hyper(ADP-ribosyl)ated forms of histone H1 indicates the absence of an H1 dimer complex of histone molecules. When the pattern of hydrolysis of poly(ADP-ribosyl)ated histones was analyzed it was found that poly(ADP-ribose) attached to histone H2B is more resistant than the polymer attached to histone H1 or H2A or protein A24. Polymer hydrolysis of the acceptors, which had been labelled at high substrate concentrations (greater than or equal to 10 microM), indicate that the only high molecular weight acceptor protein is poly(ADP-ribose) polymerase and that little processing of the enzyme occurs. Finally, electron microscopic evidence shows that hyper(ADP-ribosyl)ated poly(ADP-ribose) polymerase, which is dissociated from its DNA-enzyme complex, binds again to DNA after poly(ADP-ribose) glycohydrolase action.     

Poly(ADP-ribose) degradation by glycohydrolase starts with an endonucleolytic incision

We have recently shown that poly(ADP-ribose) polymerase forms poly(ADP-ribose) by adding ADP-ribose residues to the polymerase-proximal end of an enzyme-bound nascent chain. In this light we have reexamined the mode of hydrolysis of enzyme-bound poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase. When the substrate has been labeled by a pulse-chase protocol, soluble glycohydrolase releases a significant amount of labeled oligomer which can only come from the enzyme-distal (2') end of the polymer. This constitutes additional evidence for the proximal growth of chains. Oligomer is infrequently released from the proximal (1") end of enzyme-bound chains. Rather, the bulk of the poly(ADP-ribose) is digested directly to ADP-ribose monomers. We conclude that poly(ADP-ribose) glycohydrolase starts digestion with an endonucleolytic incision and then removes ADP-ribose residues processively in the 2'----1" direction. Therefore, in contrast to earlier models of polymer growth and hydrolysis, a single poly(ADP-ribose) chain may be extended at one end and simultaneously degraded at the other end. The balance between synthesis and degradation may control the quantity and distribution of polymer around the DNA break which occasions its synthesis.     

Poly(ADP-ribose) degradation by post-nuclear extracts from human cells

The nuclear metabolism of poly(ADP-ribose) is mainly regulated by poly(ADP-ribose) polymerase-1 (PARP-1) and by poly(ADP-ribose) glycohydrolase (PARG). A PARP-like enzyme, V-PARP, and a PARG isoform are present in the extra-nuclear compartment of mammalian cells, even if poly(ADP-ribose) has never been detected therein. In this work, we demonstrate the ability of post-nuclear extracts from HeLa and HL60 cells to degrade synthetic 32P-polymers of ADP-ribose to ADP-ribose and, further, to AMP. This reaction implies the combined action of PARG and of an ADP-ribose-degrading activity, possibly corresponding to a phosphodiesterase and/or to an ADP-ribose pyrophosphatase. The inhibition of PARG or ADP-ribose-degrading enzymes allowed the demonstration that in vitro synthesized 32P-poly(ADP-ribose) is first digested to ADP-ribose monomers by a typical PARG reaction, and that ADP-ribose is further rapidly converted into AMP by an Mg(2+)-dependent activity. Collectively, our results demonstrate the ability of the human cell post-nuclear fraction to convert synthetic poly(ADP-ribose) into utilizable AMP units by the concerted action of PARG and ADP-ribose-degrading activities.     

Effects of tannins and related polyphenols on superoxide-induced histamine release from rat peritoneal mast cells.

The effects of tannins and related polyphenols on KO2- and compound 48/80-induced histamine release from rat peritoneal mast cells were examined. Pretreatment with hydrolyzable tannins (1-100 microM) significantly inhibited KO2-induced histamine release. Dimeric ellagitannins, which have hexahydroxydiphenoyl (HHDP) and valoneoyl residues and/or a valoneoyl-related acyl unit in the molecule, showed more potent inhibitory effects than monomeric hydrolyzable tannins. The most effective inhibition was exhibited by agrimoniin and euphorbin C (IC50 0.68 and 0.80 microM), which have dehydrodigalloyl and euphorbinoyl groups, respectively, as well as the HHDP group. However, procyanidins, flavonoids and related polyphenols with small molecular weights, except for epigallocatechin gallate, exhibited negligible effects. Although clinically used antiallergic drugs, azelastine, astemizole, ketotifen and epinastine have been shown to prevent KO2-induced histamine release, their potencies were all less than those of ellagitannins. An inhibitory effect on compound 48/80-induced histamine release was also exhibited by higher molecular weight tannins. The inhibitory effect on histamine release caused by different stimulants suggested that ellagitannins act as cell membrane stabilizers as well as radical scavengers.