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E Kun

Publications and source records attributed to E Kun.

At least 19 recordsLinked to original sources
PubMed2001

Molecular interactions between poly(ADP-ribose) polymerase (PARP I) and topoisomerase I (Topo I): identification of topology of binding.

The molecular interactions of poly(ADP-ribose) polymerase I (PARP I) and topoisomerase I (Topo I) have been determined by the analysis of physical binding of the two proteins and some of their polypeptide components and by the effect of PARP I on the enzymatic catalysis of Topo I. Direct association of Topo I and PARP I as well as the binding of two Topo I polypeptides to PARP I are demonstrated. The effect of PARP I on the 'global' Topo I reaction (scission and religation), and the activation of Topo I by the 36 kDa polypeptide of PARP I and catalytic modifications by poly(ADP-ribosyl)ation are also shown. The covalent binding of Topo I to circular DNA is activated by PARP I similar to the degree of activation of the 'global' Topo I reaction, whereas the religation of DNA is unaffected by PARP I. The geometry of PARP I-Topo I interaction compared to automodified PARP I was reconstructed from direct binding assays between glutathione S-transferase fusion polypeptides of Topo I and PARP I demonstrating highly selective binding, which was correlated with amino acid sequences and with the 'C clamp' model derived from X-ray crystallography.

Binding Sites↗
PubMed2001

Inhibition of poly (ADP-ribose) synthetase by gene disruption or inhibition with 5-iodo-6-amino-1,2-benzopyrone protects mice from multiple-low-dose-streptozotocin-induced diabetes.

Activation of poly(ADP-ribose) synthetase (PARS, also termed polyADP-ribose polymerase or PARP) has been proposed as a major mechanism contributing to beta-cell destruction in type I diabetes. In the present study, we have investigated the role of PARS in mediating the induction of diabetes and beta-cell death in the multiple-low-dose-streptozotocin (MLDS) model of type I diabetes. Mice genetically deficient in PARS were found to be less sensitive to MLDS than wild type mice, with a lower incidence of diabetes and reduced hyperglycemia. A potent inhibitor of PARS, 5-iodo-6-amino-1,2-benzopyrone (INH(2)BP), was also found to protect mice from MLDS and prevent beta-cell loss, in a dose-dependent manner. Paradoxically, in the PARS deficient mice, the compound increased the onset of diabetes. In vitro the cytokine combination; interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma inhibited glucose-stimulated insulin secretion from isolated rat islets of Langerhans and decreased RIN-5F cell viability. The PARS inhibitor, INH(2)BP, protected both the rat islets and the beta-cell line, RIN-5F, from these cytokine-mediated effects. These protective effects were not mediated by inhibition of cytokine-induced nitric oxide formation. Inhibition of PARS by INH(2)BP was unable to protect rat islet cells from cytokine-mediated apoptosis. Cytokines, peroxynitrite and streptozotocin were all shown to induce PARS activation in RIN-5F cells, an effect suppressed by INH(2)BP. The present study provides evidence for in vivo PARS activation contributing to beta-cell damage and death in the MLDS model of diabetes, and indicates a role for PARS activation in cytokine-mediated depression of insulin secretion and cell viability in vitro.

Animals↗
PubMed2001

Selective augmentation of histone H1 phosphorylation sites by interaction of poly(ADP-ribose) polymerase and cdc2-kinase: comparison with protein kinase C.

The molecular interactions between PARP I, cdc2-kinase, PKC and histone H1 were determined with the aid of the common phosphate acceptor function of histone H1 to both kinases. PKC phosphorylates both histone H1 and PARP I and PARP I augments the acceptor function of histone H1. When both acceptors (PARP I and histone H1) are present an apparent distributive phosphorylation of both acceptors takes place. In contrast, cdc2-kinase only phosphorylates histone H1, and the activation of this reaction by PARP I does not involve PARP I-cdc2-kinase binding only PARP I-histone H1 association. Since the phosphorylation of histone H1 by PKC is a model reaction with no apparent physiologic consequences, the PARP I activated phosphorylation of histone H1 by cdc2-kinase, by contrast, reflects a physiologically meaningful regulation of the linker histone by a cyclin dependent kinase (cdc2-kinase). The increased phosphorylation of histone H1 by cdc2-kinase following PARP I-histone H1 binding results in the appearance of new phosphorylated histone H1 polypeptides as measured by proteolytic digestion and re-electrophoresis of cdc2-kinase phosphorylated polypeptides, indicating a probable conformational change in histone H1, following PARP I binding. The cell biologic significance of this reaction in PARP I ligand-induced enzyme induction is briefly analysed.

Binding Sites↗
PubMed2000

Cancer cell selectivity of 5-iodo-6-aminobenzopyrone (INH2BP) and methyl-3,5-diiodo-4(4'-methoxyphenoxy) benzoate (DIME).

The cellular pharmacologic actions, as measured by cell killing, of INH2BP, DIME and INO2BA (+ BSO) were determined in three types of cancer cells and compared to their action on quiescent confluent human foreskin fibroblast (HSF) and pre-confluent growing fibroblasts. The confluent HSF cells were completely refractory to the action of INH2BP and DIME, but were killed by INO2BA (+ BSO). Proliferating HSF and all three tumor cell types were killed by all three drugs. The apparent in vivo tumor specificity of INH2BP and DIME is explained by preferential cell cycle dependent selective drug uptake into tumor cells and by drug metabolism that reverses drug action in less vigorously cycling normal cells. The covalent binding of iodonitrosobenzamide (formed from INO2BA) and its toxicity are regulated by the concentration of GSH, and exhibit no cell cycle selectivity.

Antineoplastic Agents↗
PubMed2000

Activation of topoisomerase I by poly [ADP-ribose] polymerase.

Poly(ADP-ribose) polymerase (PARP I) and Topoisomerase I (Topo I) were reisolated from calf thymus to eliminate cross contamination as tested by immunotransblots. The specific activity of Topo I was greatly increased by added PARP I, following saturation kinetics. Recombinant PARP I and isolated PARP I at final purity were indistinguishable in terms of their activation of Topo I. There was a coincidence of experimentally obtained binding constants and computer generated values based on the kinetic model, indicating that the association of PARP I and Topo I is rate limiting in the catalytic activation of Topo I by PARP I. Polypeptide domains of PARP I that are required for protein-protein binding and protein-DNA binding also activate Topo I. Fluorescence resonance energy transfer between fluorophor-labeled PARP I and Topo I was demonstrated. The binding of Topo I to circular SV40 DNA, assayed either by the formation of a) the sum of non-covalently and covalently attached Topo I to DNA or b) by the covalently bound transient intermediate in the presence of camptothecin, was augmented when PARP I protein was bound to SV40 DNA. These binding experiments provide a molecular basis for the kinetic activation of Topo I by PARP I inasmuch as the increased superhelicity of SV40 DNA induced by PARP I may facilitate the formation of a more Topo I-DNA complex that increases the rate of the DNA breakage-reunion cycle of Topo I catalysis.

Animals↗
PubMed2000

Binding of topo I to PARP I - antibody immunocomplex.

It is frequently quoted in the literature that the cellular role of PARP I is its participation in the recognition of single strand breaks of genomic DNA (l.c.1). Although there is little doubt that PARP I can be made to respond powerfully as an factor in the recognition of DNA damage, it seems unlikely that this auxilliary, or telelogically defined, role of this highly abundant nuclear protein exhausts its physiologic cellular function. We have reported that Topo I is greatly activated by its association with PARP I (J Mol Med 5: 533-540, 2000). Translation of this in vitro model experiments to physiologic conditions was accomplished by the demonstration of the quantitative binding of Topo I to a PARP I - antibody complex, as reported here. This experiment demonstrates for the first time that the colligative action of PARP I can regulate a highly significant cellular process, the control of readability of genomic DNA, i.e., gene expression, without the artificiality of induced DNA damage.

Antigen-Antibody Complex↗
PubMed1999

Interaction of cytocidal drugs and the inhibition of caspase-3 by 3-nitrosobenzamide.

The effect of 3-nitrosobenzamide (NOBA) on the etoposide, staurosporine and dexamethason induced rapid (4-6 hr), caspase-dependent apoptosis was investigated in thymocytes and lymphoma cells by flow cytometric assay of DNA fragmentation. When NOBA (ED(50) = 4 microM) was added to these cell systems, the rapid onset of apoptosis was prevented. Such apparent protection by NOBA was related to the inactivation of caspase-3, by s-nitrosylation of 1.3 mol -SH per enzyme molecule out of 7 -SH groups. Since NOBA by itself induces DNA fragmentation within 18 hr in lymphoma cells, our results indicate that at least two active cell death pathways exist with apparent dissimilar kinetics and molecular mechanisms.

Animals↗
PubMed1998

Protective effects of 5-iodo-6-amino-1,2-benzopyrone, an inhibitor of poly(ADP-ribose) synthetase against peroxynitrite-induced glial damage and stroke development.

Peroxynitrite triggers DNA single-strand breakage, which activates the nuclear enzyme poly(ADP-ribose) synthetase (PARS). Activation of PARS depletes its substrate, NAD+, slowing the rate of glycolysis, electron transport, and ATP formation, resulting in cell necrosis. Here, we demonstrate that inhibition of PARS with the novel, potent PARS inhibitor 5-iodo-6-amino-1,2-benzopyrone (INH2BP) protects against peroxynitrite-induced cell death (as measured by measurement of mitochondrial respiration and release of lactate dehydrogenase) in C6 glioma cells in vitro, and in a murine stroke model in vivo. Inhibition of PARS with INH2BP may represent a novel approach for the experimental therapy of stroke.

Animals↗
PubMed1998

Protection against peroxynitrite-induced fibroblast injury and arthritis development by inhibition of poly(ADP-ribose) synthase.

Peroxynitrite, a cytotoxic oxidant formed from nitric oxide (NO) and superoxide, induces DNA strand breakage, which activates the nuclear enzyme poly(ADP-ribose) synthase (PARS; EC 2.4.2.30). The cellular function of PARS was determined in fibroblast lines from PARS knockout animals (PARS-/-) and corresponding wild-type animals (PARS+/+), with the aid of the lipophilic PARS inhibitor 5-iodo-6-amino-1,2-benzopyrone (INH2BP). We investigated the role of PARS in peroxynitrite-induced fibroblast injury in vitro and also in the development of arthritis in vivo. Exposure of embryonic fibroblasts from the PARS+/+ animals to peroxynitrite caused DNA single-stand breakage and PARS activation and caused an acute suppression of mitochondrial respiration. INH2BP protected the PARS+/+ cells against the suppression of mitochondrial respiration in response to peroxynitrite (50-100 microM). Similarly to PARS inhibition with INH2BP, the PARS-/- cells were protected against peroxynitrite-induced injury. The protection against cellular injury by PARS-/- phenotype or INH2BP waned when cells were challenged with higher concentrations of the oxidant. Inhibition of PARS by INH2BP or by PARS-/- phenotype reduced inducible nitric-oxide synthase (iNOS; EC 1.14.13.39) mRNA levels and inhibited production of NO in immunostimulated cells. INH2BP had no peroxynitrite scavenging or hydroxyl radical scavenging effects, and it exerted no additional (nonspecific) effects in the PARS-/- cells. In collagen-induced arthritis, significant staining for nitrotyrosine, a marker of peroxynitrite formation, was found in the inflamed joints. Oral treatment with INH2BP (0.5 g/kg, daily), starting at the onset of arthritis (day 25), delayed the development of the clinical signs at days 26-35 and improved histological status in the knee and paw. Our data demonstrate that deletion of PARS by genetic manipulation or pharmacological inhibition of PARS protects against oxidant-induced cellular injury in vitro and exhibits anti-inflammatory effects in vivo.

Animals↗
PubMed1998

Poly(ADP-ribose) polymerase, a potential target for drugs: Cellular regulatory role of the polymer and the polymerase protein mediated by catalytic and macromolecular colligative actions (Review).

The cellular coenzymatic role of NAD, being a pleiotropic cofactor for diverse cellular reactions, is extended to poly(ADP-ribose) and to the highly abundant nuclear protein, poly(ADP-ribose) polymerase, with special focus on the pharmacological action of ligands on the latter. The polymer is defined to possess a helical configuration. From direct analyses of the polymer under physiological conditions, it is concluded that the polymerase is dormant in normal tissues, but is activated under certain pathological conditions: malignancy, retroviral integrate containing cells, and in a variety of inflammatory states. The interaction of poly(ADP-ribose) polymerase ligands with the DNA component of the active poly (ADP-ribose) polymerase - DNA complex is shown. A major cellular function of the poly(ADP-ribose) polymerase protein is its binding capacity to a large number of nuclear proteins and DNA sites, an effect which is induced by drugs that inhibit the polymerase activity. The malignancy-reverting effect of poly(ADP-ribose) polymerase ligand drugs is illustrated in chemically and oncovirally transformed cancer cells. The poly(ADP-ribose) polymerase ligand-induced cessation of HIV replication is analyzed. Peroxynitrite-induced DNA damage-initiated pathological responses are shown to be inhibited by a specific poly(ADP-ribose) polymerase ligand. The irreversibly acting C-NO drugs oxidize asymmetric zinc fingers [poly(ADP-ribose) polymerase, HIV gag-precursor protein] and act as anti-cancer and anti-HIV agents, an effect that is regulated by cellular concentration of GSH.

Journal Article↗
PubMed1998

Molecular pharmacology of methyl-3,5-diiodo-4 (4'methoxyphenoxy) benzoate (DIME) and its non-hydrolyzible ethanone analog (DIPE) (Review).

A molecular structural relationship of thyroid hormones to methyl-3,5-diiodo-4-(4'-methoxy-phenoxy) benzoate (DIME) and 1-[3,5-diiodo-4-(4'-methoxyphenoxy)-phenyl]-ethanone) (DIPE) and to apoptosis-mediated metamorphogenic mechanisms is postulated. DIME disrupts microtubule assembly already in anaphase, preparing cells for G2/M block, chromosome aggregation and caspase-3 mediated apoptosis. Cooperative action of DIME and vincristine, defining mutually exclusive cellular sites, identifies microtubules as primary drug targets followed by downstream cellular consequences, leading to cell death. Absence of in vivo toxicity of DIME appears to be related to impermeability to DIME of normal cells, but not of tumor cells in vivo. Normal tissue cells hydrolyze DIME but most tumor cells, except lung cancer cells, do not. DIPE, being resistant to enzymatic hydrolysis, is equally effective in all tumor cells.

Animals↗
PubMed1998

Unusual potentiation by vinca alkaloids of the cytostatic and cytocidal action of methyl-3,5-diiodo-4-(4'-methoxyphenoxy) benzoate (DIME) and its nonhydrolyzable ethanone analog (DIPE) on MDA-MB-231 human mammary cancer cells.

Drug interaction between DIME or DIPE ¿1-[3, 5-diiodo-4-(4'-methoxyphenoxy)-phenyl]-ethanone¿ with vincristine and vinblastine on the growth rate of MDA-MB-231 human mammary cancer cells was determined by the median effect kinetic method. Mutually exclusive cellular binding sites were identified kinetically and isobologram analyses showed potentiation. The combind effect of 0.75 MICROM DIME and 2 nM vincristine demonstrated a nearly type of mutual activation. It was shown that the nonhydrolyzable DIME derivative DIPE is equivalent to DIME, but because of its biological stability is a preferred drug candidate. Vinblastine-DIME cooperative action is similar to that of vincristine-DIME (or DIPE). Activation of caspase 3 by both DIME and vincristine is greatly potentiated when both drugs are added simultaneously in a given proportion. We propose that following a primary binding of DIME and vinca alkaloids to microtubules, an as yet unrecognized mutual activation of caspase 3 apoptotic path is initiated, explaining DNA fragmentation and cell death. A subpopulation of cancer cells, capable of slow growth at 1.5 microM DIME was identified. This cell type was also killed by the DIME-vincristine drug combination.

Antineoplastic Agents↗
PubMed1997

Isolation and identification of a proteinase from calf thymus that cleaves poly(ADP-ribose) polymerase and histone H1.

A proteinase was isolated from calf thymus that degraded pADPRT, histone H1 and alpha-casein in a Ca(2+)-dependent manner. In a five-step procedure, a homogenous proteinase was obtained with a subunit structure of 80 and 30 kDa. The amino-acid homology of an internal sequence as well as kinetic and inhibitor assays identified the proteinase as calpain I. It is suggested that even though the general substrate alpha-casein is widely used for the assaying of calpains, more appropriately physiological cellular components (pADPRT and histone H1) specify the thymus proteinase.

Amino Acid Sequence↗
PubMed1995

Potential chemotherapeutic activity of 4-iodo-3-nitrobenzamide. Metabolic reduction to the 3-nitroso derivative and induction of cell death in tumor cells in culture.

A C-nitroso prodrug, 4-iodo-3-nitrobenzamide, was synthesized, and its action on a variety of tumor cells of human and animal origin tested. This prodrug was reduced transiently by tumor cells to 4-iodo-3-nitrosobenzamide at a very low rate, which was, however, sufficient to kill tumor cells. The final reduction product was 4-iodo-3-aminobenzamide, and no intermediates accumulated. No toxicity could be observed in hamsters even at 200 mg/kg, given i.p. daily for 7 days. The chemical reactivity of both 4-iodo-3-nitrosobenzamide and its noniodinated homolog with reduced ascorbate yielded the hydroxylamines. With glutathione, 4-iodo-3-aminobenzamide was formed, suggesting glutathione sulfinic acid formation. Confirming earlier studies, 4-iodo-3-nitrosobenzamide inactivated poly(ADP-ribose) polymerase by zinc ejection from the first zinc finger of this nuclear protein. The iodinated nitroso compound was more effective than its iodine-free analog. Selective tumoricidal action appeared to correlate with the reduction of the nitro group to nitroso in tumor cells, and with the previously described subsequent induction of tumor apoptosis by the C-nitroso intermediate. These processes were accelerated by buthionine sulfoximine, which diminishes cellular GSH.

Animals↗
PubMed1995

Identification of domains of poly(ADP-ribose) polymerase for protein binding and self-association.

Cellular proteins extracted from normal and cancer cells bind polymerizing ADP-ribose transferase (pADPRT) on nitrocellulose membrane transblots. Histones at 1 mg/ml concentration completely prevent the binding of pADPRT to cellular proteins, indicating that the binding of histones to pADPRT sites competitively blocks the association of pADPRT to proteins other than histones. The direct binding of pADPRT to histones is shown by cross-linking with glutaraldehyde. The COOH-terminal basic histone H1 tail binds to the basic polypeptide domain of pADPRT. The basic domain present in the NH2-terminal part of core histones is the probable common structural feature of all core histones that accounts for their binding to pADPRT. Two polypeptide domains of pADPRT were identified, by way of CNBr fragments, to bind histones. These two domains are located within the 64-kDa fragment of pADPRT and are contiguous with the polypeptide domains that were shown to participate in self-association of pADPRT, ending at the 606th amino acid residue. The polypeptide domains of pADPRT which participate in DNA binding are thus shown to associate also with other proteins. Intact pADPRT binds to both the zinc-free or zinc-reconstituted basic polypeptide fragments of pADPRT. Histones activate auto-poly(ADP)-ribosylation of pADPRT by increasing the number of short oligomers on pADPRT. This reaction is also dependent in a biphasic manner on the concentration of pADPRT. Histones in solution are only marginally poly(ADP)-ribosylated but are good polymer acceptors when incorporated into artificial nucleosome structures.

3T3 Cells↗
PubMed1995

Reversion of malignant phenotype by 5-iodo-6-amino-1,2-benzopyrone a non-covalently binding ligand of poly(ADP-ribose) polymerase.

A non-covalently binding inhibitory ligand of poly(ADP-ribose) polymerase, 5-iodo-6-amino-1,2-benzopyrone, when incubated at 5-600 microM external concentration with an E-ras-transformed tumorigenic cell line or with human prostatic carcinoma cells for 40 to 60 days converts both cancer cells to a non-tumorigenic phenotype that is characterized by drastic changes in cell morphology, absence of tumorigenicity in nude mice, and a high rate of aerobic glycolysis.

Animals↗
PubMed1995

Specific disulfide formation in the oxidation of HIV-1 zinc finger protein nucleocapsid p7.

In vitro oxidation of the HIV-1 nucleocapsid protein p7 by the C-nitroso compound 3-nitrosobenzamide (NOBA) has been investigated. When reconstituted p7 was incubated with NOBA, three disulfide bonds were formed per molecule of p7, Cys 15-Cys 18, Cys 28-Cys 36, and Cys 39-Cys 49. These were identified using the proteolytic enzyme endoproteinase Lys-C and mass spectrometry. When the denatured protein (Apo-p7) was incubated with NOBA, a more random pattern of multiple S-S linkages was found. Oxidation of reconstituted p7 also occurred on treatment with cupric ions (Cu2+), and the same three major disulfide bonds were formed as in the reaction with NOBA. These results suggest the interpretation that the oxidation reaction occurs at the zinc-binding centers while zinc cations are still bound and that the two zinc fingers are not identical in their chemical properties. This latter point is consistent with the independent biological roles reported previously for the two fingers in the viral infection cycle.

Amino Acid Sequence↗