Presence Of 3AB Research Articles

Poliovirus Protein 3AB Displays Nucleic Acid Chaperone and Helix-Destabilizing Activities

Poliovirus protein 3AB displayed nucleic acid chaperone activity in promoting the hybridization of complementary nucleic acids and destabilizing secondary structure. Hybridization reactions at 30 degrees C between 20- and 40-nucleotide RNA oligonucleotides and 179- or 765-nucleotide RNAs that contained a complementary region were greatly enhanced in the presence of 3AB. The effect was nonspecific as reactions between DNA oligonucleotides and RNA or DNA templates were also enhanced. Reactions were optimal with 1 mM MgCl(2) and 20 mM KCl. Analysis of the reactions with various 3AB and template concentrations indicated that enhancement required a critical amount of 3AB that increased as the concentration of nucleic acid increased. This was consistent with a requirement for 3AB to "coat" the nucleic acids for enhancement. The helix-destabilizing activity of 3AB was tested in an assay with two 42-nucleotide completely complementary DNAs. Each complement formed a strong stem-loop (DeltaG = -7.2 kcal/mol) that required unwinding for hybridization to occur. DNAs were modified at the 3' or 5' end with fluorescent probes such that hybridization resulted in quenching of the fluorescent signal. Under optimal conditions at 30 degrees C, 3AB stimulated hybridization in a concentration-dependent manner, as did human immunodeficiency virus nucleocapsid protein, an established chaperone. The results are discussed with respect to the role of 3AB in viral replication and recombination.

Peroxynitrite-induced oligodendrocyte toxicity is not dependent on poly(ADP-ribose) polymerase activation.

Oligodendrocyte loss is a characteristic feature of several CNS disorders, including multiple sclerosis (MS) and spinal cord injury. However, the mechanisms responsible for oligodendrocyte destruction remain undefined. As recent studies have implicated peroxynitrite in the pathogenesis of both spinal cord injury and MS, we have examined whether peroxynitrite may mediate at least some of the oligodendrocyte damage and demyelination observed in these conditions. Primary rat oligodendrocytes were exposed to authentic peroxynitrite in vitro and assessed for cytotoxicity. Mitochondrial function, measured by the reduction of MTT to formazan, and mitochondrial membrane potential were used as indicators of cell viability. Cell death was quantitated by measuring either the release of lactate dehydrogenase from, or the uptake of propidium iodide into, damaged and dying cells. Peroxynitrite dose-dependently reduced the viability of primary oligodendrocytes and induced cell death. Furthermore, peroxynitrite significantly increased DNA strand breakage and the activity of poly(ADP-ribose) polymerase (PARP) in oligodendrocyte cultures. To identify whether PARP activation plays a role in peroxynitrite-induced oligodendrocyte toxicity, we examined the effects of the PARP inhibitors 3-aminobenzamide (3AB) and 5-iodo-6-amino-1,2-benzopyrone (INH(2)BP) on mitochondrial function and cell death in oligodendrocytes. The presence of 3AB and INH(2)BP did not protect oligodendrocytes from peroxynitrite-induced cytotoxicity. However, both compounds significantly reduced PARP activity in these cells. Primary oligodendrocytes generated from PARP-deficient mice were also highly susceptible to peroxynitrite-induced cell death. Therefore, our results show that peroxynitrite exerts cytotoxic effects on oligodendrocytes in vitro independently of PARP activation.

The Effect of H2O2 and Tertiary Butyl Hydroperoxide Upon a Murine Immortal Lens Epithelial Cell Line, αTN4-1

The effect of comparable concentrations of H2O2 and tertiary butyl hydroperoxide (TBHP) upon an immortal murine lens epithelial cell line was examined as part of an ongoing effort to delineate differences in the mechanism by which these peroxides cause cell death. Both compounds result in cell death of normal, unconditioned cells within 24hr. It was found that with similar conditions, TBHP conditioned αTN4-1 cells survive H2O2 stress while H2O2 conditioned cells are killed by TBHP. To better understand how these peroxides act, their effect upon unconditioned cells has been investigated. Both peroxides cause a rapid loss of GSH and disruption of pump activity as illustrated by 14C-choline transport and 86Rb uptake. While H2O2 exposure resulted in extensive DNA damage, TBHP had a minimal effect. DNA damage caused by H2O2 was shown to activate polyADP-ribosyl polymerase (PARP), leading to depletion of NAD and ATP. H2O2 induced cell death could be delayed by addition of 3-aminobenzamide (3AB), an inhibitor of PARP. ATP levels in cells subjected to H2O2 were also maintained by the presence of 3AB. H2O2 stress also disrupted glycolysis and mitochondrial activity but these parameters were not affected by TBHP. TBHP induced cell death, under the relatively mild conditions used in this work, appears to be caused by membrane disruption and loss of a reducing environment.

Stability and reactivity of low-spin ferric hydroperoxo and alkylperoxo complexes with bipyridine and phenantroline ligands

In this work the first-order rate constants of self-decomposition of hydroperoxo and alkylperoxo complexes [Fe(bpy) 2(OOH)Py](NO 3) 2 ( 2a-Py), [Fe(phen) 2(OOH)Py](NO 3) 2 ( 2b-Py) and [Fe(bpy) 2(OO tBu)CH 3CN](NO 3) 2 ( 3a-CH 3CN) were determined in the presence of various substrates and at various temperatures. It was observed, that the alkylperoxo species are far less stable than corresponding hydroperoxo intermediates, k=1.2×10 −2 s −1 ( 3a-CH 3CN in CH 3CN at −10°C) and k=2×10 −4 s −1 ( 2a-Py in CH 3CN at −10°C). The sixth ligand (Py in 2a-Py and 2b-Py; CH 3CN in 3a-CH 3CN) can be replaced by other donor molecules B in appropriate solvent systems. Using d 9- tBuOOH, 2D NMR signals of tBuOO moieties of complexes 3a-CH 3CN, 3a-CH 3OH and 3a-H 2O were observed. The rate of decomposition of hydroperoxo complexes [Fe(bpy) 2(OOH)B](NO 3) 2 ( 2a-B), where B are derivatives of Py (3-Br-Py, 3-Me-Py, 4-Me-Py and 4-Me 2N-Py) increases with the growth of basisity of B (push effect). Such effect is markedly smaller for alkylperoxo species [Fe(bpy) 2(OO tBu)B](NO 3) 2 ( 3a-B). The addition of organic substrates (cyclohexane, cyclohexene, methyl phenyl sulfide) in concentrations up to 3 M at −10°C to +20°C does not noticeably change the rate of self-decomposition of 2a-B, [Fe(phen) 2(OOH)B](NO 3) 2 ( 2b-B) and 3a-B. Thus the intermediates concerned do not directly react with organic substrates. The reactivity patterns of 2a-B, 2b-B and 3a-B were characteristic for free radical oxidation. OH · and HO 2 · radicals were trapped in solution containing 2a-Py, and tBuOO · free radicals were detected in solution in the presence of 3a-B. The determined rates of self-decomposition of complexes 2a-B, 2b-B and 3a-B can be used for evaluation of the upper limit for their reactivity towards organic substrates.

Apoptosis and necrosis following exposure of U937 cells to increasing concentrations of hydrogen peroxide: the effect of the poly(ADP-ribose)polymerase inhibitor 3-aminobenzamide.

A 3-hr exposure of U937 cells to hydrogen peroxide (H2O2) followed by a 6-hr posttreatment incubation in fresh culture medium promotes apoptosis or necrosis, depending on the oxidant concentration. Addition of 3-aminobenzamide (3AB) during the recovery phase prevented necrosis and caused apoptosis. 3AB did not, however, affect the apoptotic response of cells treated with apogenic concentrations of H2O2. Cells exposed for 3 hr to 1.5 mM H2O2, while showing some signs of suffering, maintained a normal nuclear organization and good organelle morphology. At the biochemical level, the oxidant promoted the formation of Mb-sized DNA fragments and rapidly depleted both the adenine nucleotide and non-protein sulphydryl pools, which did not recover during posttreatment incubation in the absence or presence of 3AB. These results allow a novel interpretation of the concentration-dependent switch from apoptosis to necrosis. We propose that H2O2 activates the apoptotic response at the early times of peroxide exposure and that this process can be completed, or inhibited, during the posttreatment incubation phase. Inhibition of apoptosis leads to necrosis and can be prevented by 3AB via a mechanism independent of inhibition of poly(ADP-ribose)polymerase. As a corollary, the necrotic response promoted by high concentrations of H2O2 in U937 cells appears to be the result of specific inhibition of the late steps of apoptosis.

Poly(ADP-ribosyl)ation is required for p53-dependent signal transduction induced by radiation.

p53 and poly(ADP-ribose) polymerase (PARP) are both DNA damage recognition proteins and can be functionally activated by DNA strand breaks. To understand the functional interaction between these two proteins, the effects of a PARP inhibitor, 3-aminobenzamide (3AB), on the p53 pathway were investigated in human glioblastoma cells with different p53 status. Consistent with previous studies, irradiation with gamma-rays induced both p53 and WAF1 accumulation in A-172 cells (wtp53) but not in T98G cells (mp53). However, the presence of 3AB but not its analog suppressed radiation-induced accumulation of wtp53 and the expression of WAF1 and MDM2. Similar results were also obtained from U87MG, another human glioblastoma cell line with wtp53 status. Northern blotting analysis showed that 3AB inhibited the gamma-ray-induced WAF1 gene expression. Moreover, 3AB but not its analog inhibited irradiation-induced activation of sequence-specific DNA binding of wtp53 as detected using 32P-labeled or biotin-labeled p53 consensus sequence (p53CON). However, immunoblotting with an anti-poly(ADP-ribose) antibody showed that p53 proteins of the p53CON-bound fraction did not contain poly(ADP-ribose) (PAR). These findings suggested that poly(ADP-ribosyl)ation is required for rapid accumulation of p53, activation of p53 sequence-specific DNA binding and its transcriptional activity after DNA damage.

Studies with poliovirus polymerase 3Dpol. Stimulation of poly(U) synthesis in vitro by purified poliovirus protein 3AB.

The synthesis in vitro of poly(U) on a poly(A) template with oligo(dT)15 primer by poliovirus RNA polymerase 3Dpol (280 ng/ml) is strongly stimulated (50-100 fold) by the addition of purified poliovirus polypeptide 3AB. The synthesis of product continues linearly with time for up to 90 min. The reaction with 3Dpol alone can be reactivated and similarly enhanced by the addition of 3AB at 30 min of incubation. Optimal stimulation is achieved under conditions where the concentration of 3Dpol and of template is low, when the molar ratio of 3AB to 3Dpol is about 100:1 and that of 3AB to poly(A) is about 25:1. In the presence of 3AB, the yield of product made by 3Dpol is much increased but its size is unchanged. From a number of basic proteins and peptides tested, a few were found which also exhibited limited enhancement of polymerase activity. The stimulatory effect of 3AB is probably related to its ability to bind both the template-primer, poly(A).oligo(dT)15, and 3Dpol (Molla, A., Harris, K. S., Paul, A. V., Shin, S. H., Mugavero, J., and Wimmer, E. J. (1994) J. Biol. Chem. 269, 27015-27020). RNA synthesis on purified poliovirus RNA with oligo(dT)15 primer is enhanced by 3AB about 5-10 fold, and this reaction is highly sensitive to detergent.

Molecular characterization of mutations at the hprt locus in V79 Chinese hamster cells induced by bleomycin in the presence of inhibitors of DNA repair

The molecular basis of bleomycin (BLM)-induced mutations in the absence and presence of inhibitors of DNA repair was investigated in V79 cells with Southern hybridization techniques. 43% of the BLM-induced thioguanine-resistant mutants suffer from large alterations of hrpt DNA sequences. To understand the role of DNA repair in the process of mutagenesis, the effect of inhibitors of DNA repair on the frequency and types of BLM-induced mutations was tested. The inhibitors used were arabinofuranosyl cytosine (araC), didesoxythymidine (ddThd) and 3-aminobenzamide (3AB), which inhibit different steps of excision repair. Only 3AB caused a comutagenic effect. The increased mutation frequency was mainly due to additionally induced gene deletions. In the presence of 3AB, 70% of the HPRT-deficient mutants revealed partial or total deletions of the hprt coding sequences. Thus, it could be shown that BLM induces a broad range of types of mutation and that inhibited repair of BLM-induced DNA damage leads to specific types of mutations.

The effect of 3-aminobenzamide on X-ray induction of chromosome aberrations in Down syndrome lymphocytes

Human lymphocytes from normal and Down syndrome (DS) subjects were examined to determine the effect of 3-aminobenzamide (3AB) on X-ray-induced chromosome aberrations. Lymphocytes were treated with 150 or 300 rad of X-rays in the presence of 3 mM 3AB for various times after irradiation, and then the cells were analyzed for the presence of chromosome aberrations in mitotic cells. 3-Aminobenzamide had no effect on the frequency of chromosome aberrations produced by X-rays in G 0 lymphocytes from normal subjects. In contrast, lymphocytes from DS patients displayed an increase in the frequency of chromosome aberrations as a result of treatment with X-rays in the presence of 3AB. These observations indicate that DS lymphocytes are more sensitive to the inhibition of poly(ADP)ribose synthetase than normal lymphocytes.

Effect of Inhibitors of Poly(ADP-Ribose) Polymerase on the Heat Response of HeLa S3 Cells

The purpose of this study was to investigate a possible involvement of poly(ADP-ribosyl)ation reactions in hyperthermic cell killing and hyperthermic DNA strand-break induction and repair in HeLa S3 cells. The inhibitors of poly(ADP-ribose) polymerase, 3-aminobenzamide (3AB) and 4-aminobenzamide (4AB), were used as tools in this study. Both inhibitors could sensitize the cells for hyperthermic cell killing equally well, although 3AB is known to be a more effective enzyme inhibitor. The heat sensitization at the level of cell killing could be reversed when the compounds were still present during a 4-h postincubation at 37 degrees C. More heat-induced DNA strand breaks were formed in the presence of 3AB and 4AB. Repair of strand breaks was inhibited during the postincubation at 37 degrees C. Thus the effect of 3AB and 4AB on DNA strand-break repair was different from the cited effect on cell survival. It is concluded that the sensitizing effect of 3AB and 4AB on hyperthermic cell killing is not caused by inhibition of poly(ADP-ribose) polymerase and is also not related to repair of DNA strand breaks.

Inhibition of poly(ADP-ribose) synthesis may affect DNA repair prior to ligation.

The effects of modification of poly(ADP-ribosyl)ation reactions have been examined in normal (F107) and ataxia telangiectasia (AT23) fibroblasts following damage by methyl methanesulphonate (MMS) and u.v. light. The technique of benzoylated DEAE (BD)-cellulose chromatography was utilized to estimate both the extent and nature of the damage to DNA induced by these agents and to examine the effects of an inhibitor of poly(ADP-ribose) synthetase, 3-aminobenzamide (3AB), on these parameters. Single strand breakage, determined by nucleoid sedimentation, and levels of poly ADP(ribose) synthesis were monitored. Increase in the proportion of DNA containing single-stranded regions, as measured by stepwise elution from BD-cellulose, was observed following MMS damage in both cell types. In the presence of 3AB, a further accumulation of DNA containing single-stranded regions occurred, with the effect being more prominent in AT23 fibroblasts. U.v. light damage did not induce increased binding to BD-cellulose in normal cells, and the increase observed in AT23 cells was much less than that seen following alkylation damage. Examination of the nature of single-stranded damage by caffeine gradient elution from BD-cellulose following MMS treatment revealed discrete structural lesions, which were enhanced in the presence of 3AB. A similar effect was exerted by arabinofuranosyl cytosine. The behaviour of these intermediates, which could be associated with repair, was not in accord with the suggestion that 3AB inhibits only the ligation stage of the repair process. Our results suggest that specific intermediate stages in DNA repair are sensitive to 3AB, and it seems likely that these stages occur prior to ligation.

Potentiation of alkylation-induced sister chromatid exchange frequency by 3-aminobenzamide is mediated by intracellular loss of NAD+.

3-Aminobenzamide (3AB) is a competitive inhibitor of poly-(ADP-ribose) polymerase. It will interact synergistically with certain monofunctional alkylating agents to increase the frequency of sister chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. 3AB will also increase the baseline SCE frequency in exposed cells. The extent of interaction between 3AB and monofunctional alkylating agents varies depending on the alkylating agent used and appears to be due to the different amounts of membrane damage produced by the alkylating agents. In this study, exogenously added beta-NAD+ was found to reduce substantially SCE frequency in cells that had been treated with combinations of 3AB and methyl methanesulfonate (MMS) but not in cells treated with 3AB and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MMS produces more cell membrane damage than MNNG at equitoxic doses. beta-NAD+ is the substrate for ADP-ribosylation and normally does not freely diffuse into cells. beta-NAD+ had no significant effect on SCE induction in intact cells or in cells treated with either 3AB or alkylating agent alone. In contrast to beta-NAD+, exogenously added alpha-NAD+, which is an inhibitor of poly(ADP-ribose) polymerase, increased SCE frequency in MMS-treated cells. Thus the interaction between 3AB and certain monofunctional alkylating agents in SCE formation is apparently due to cell membrane permeabilization and the loss of intracellular NAD+ which in turn probably results in a greater inhibition of ADP-ribosylation in the presence of 3AB.

3-Aminobenzamide sensitizes cultured Chinese hamster cells with bromodeoxyuridine-substituted DNA to genetic damage induced by long-wave UV

Chinese hamster cells, containing BrdUrd-substituted DNA, were irradiated in the presence of 3-aminobenzamide with short-wave UV, long-wave UV or X-rays and analyzed for induced SCEs or chromosomal aberrations. The data presented in this paper show that when BrdUrd-substituted cells are irradiated with lw-UV in the presence of 3-aminobenzamide, genetic damage is increased. Biochemical analysis shows that the molecular weight of BrdUrd-substituted DNA is reduced by this treatment. The sensitization is due to the combined action of lw-UV, incorporated BrdUrd and 3-aminobenzamide, without any involvement of inhibition of poly(ADP-ribose) synthetase. No potentiation occurs when cells are irradiated with X-rays and genetic damage is decreased when cells are irradiated with UV light of 254 nm in the presence of 3AB. This decrease coincides with a reduction in the amount of induced pyrimidine dimers, detected as T 4 endonuclease-sensitive sites in DNA.

3-Aminobenzamide does not increase repair patch size in mammalian cells.

Several groups of investigators have reported that the extent of repair replication following treatment of mammalian cells with dimethyl sulfate is increased by 3-aminobenzamide (3AB) over that occurring in its absence. Two plausable explanations for this phenomenon were tested. The first is that the number of nucleotides inserted per repair site, that is the repair patch size, is increased and the second is that the rate of repair is increased. Human T98G cells were treated with dimethyl sulfate and allowed to repair their DNA in the presence or absence of 3AB. It was found that the presence of 3AB did not increase the repair patch size nor the rate of removal of methylation products from the DNA, which is assumed to equal the rate of repair. The results of further experiments suggested that the 3AB effect can be explained by changes in nucleotide precursor pools.