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.     

Do inhibitor studies demonstrate a role for poly(ADP-ribose) in DNA repair?

3-Aminobenzamide, an inhibitor of poly(ADP-ribose) synthesis, has been commonly used in attempts to demonstrate a regulatory role for the polymer during a late stage of repair. When a range of inhibitor concentrations was used paradoxical results were obtained. Up to 1 mM, 3-aminobenzamide appeared to reduce DNA break frequencies in cells damaged by methyl methane sulfonate; at doses of 2 mM and above, it appeared to increase break frequencies. In the high concentration range, many nonspecific side effects and cellular toxicity predominate. Evidence used to assert a role for poly(ADP-ribose) synthesis during ligation has usually been derived from experiments using high concentrations of 3-aminobenzamide, but these may be attributed to toxic side effects. 3-Aminobenzamide stimulates a large increase in repair replication which does not result from increased excision of damaged sites or an increased patch length but may be attributable to other cellular effects such as endogenous nuclease attack on DNA. The cellular effects of 3-aminobenzamide are therefore complicated by nonspecific effects over a commonly used concentration range and evidence for a specific regulatory role of poly(ADP-ribose) in DNA repair is weak.     

Disturbances in DNA precursor metabolism associated with exposure to an inhibitor of poly(ADP-ribose) synthetase

3-Aminobenzamide (3AB) is widely used as an inhibitor of poly(ADP-ribose) synthetase to study the effect of protein ribosylation on various cellular processes, but the specificity of its inhibition has not been demonstrated. We found that 3AB has a wide range of effects on DNA precursor metabolism, as determined by high-performance liquid chromatographic separation of deoxynucleosides derived from enzymatic digestion of cellular DNA. 3AB (10-20 mM) significantly reduced cell growth in human lymphoblastoid cells. Furthermore, the incorporation of [3H]deoxycytidine into DNA was significantly enhanced relative to incorporation of [3H]deoxythymidine, [3H]deoxyguanosine, and [3H]deoxyadenosine. Incorporation of fragments of [3H]glucose into the pyrimidine fraction of DNA was significantly inhibited relative to incorporation into the purine fraction. At only 1 mM, 3AB had a major inhibitory effect on the incorporation of the methyl group from [3H]methionine into deoxyguanosine, deoxyadenosine, and deoxycytidine, with 50% inhibition into deoxyguanosine and deoxyadenosine and 90% inhibition into deoxycytidine. The specificity of 3AB inhibition to poly(ADP-ribose) synthetase is therefore doubtful in view of this variety of metabolic effects, involving pyrimidine synthesis and de novo synthesis via the one-carbon pool.     

Effects of 3-aminobenzamide on the rejoining of DNA-strand breaks in mammalian cells exposed to methyl methanesulphonate; role of poly(ADP-ribose) polymerase

The effect of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) polymerase, on DNA-repair processes has been investigated after treating V79 hamster cells with methyl methanesulphonate (MMS). Repair activity was observed as changes in DNA-strand break levels. MMS induces transient strand breaks, the level of which slowly decreases with time. Addition of 3AB leads to a rapid increase in the number of breaks. The level of breaks increases linearly with time until it suddenly levels off. Increasing the concentration of 3AB does not change the slope of this curve, but the steady-state level of breaks increases. The incision-rejoining kinetics indicates that 3AB induces a delay in the strand-break rejoining process. In the absence of 3AB the breaks have a lifetime of 1-2 min and this is increased by a factor of 5 in the presence of 5 mM 3AB.     

DNA topoisomerase II inhibition and gene amplification in V79/B7 cells

Topoisomerase II inhibitors such as etoposide (VP16) are able to stabilize the enzyme—DNA complex by trapping the topoisomerase on DNA without affecting its strand-break activity. To test if this inhibition resulting in chromosomal breakage via double-strand breaks could underlie gene amplification, we performed VP16 treatments followed by selection for PALA resistance in V79/B7 Chinese hamster cells. We found that VP16 induced PALA-resistant cells very efficiently, and in a dose-dependent manner. On the other hand VP16 in combination with 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) polymerase involved in DNA repair, reduced the frequency of PALA-resistant cells. Cytogenetic analysis revealed a higher number of chromosomal aberrations in VP16-treated cells than in cells treated with VP16 plus 3AB. These results suggest a correlation between frequency of chromosomal aberrations and frequency of PALA-resistant cells, and are consistent with models which consider chromosomal breakage as an important step in initiating gene amplification.     

Effects of 3-aminobenzamide on cellular ribosomal RNA content and cell cycle progression in inhibitor resistant and sensitive L1210 cells

The poly(ADP-ribosyl)ation inhibitor 3 aminobenzamide (3AB) is used extensively to probe the involvement of post-translational modifications of proteins in the control of DNA repair and cell cycle progression. However, 3AB appears to lack specificity for the synthetase, and the use of excessive concentrations of the inhibitor may adversely affect the potential responsiveness of cells to DNA-damaging agents. Here we address the concentration dependency of the cellular impact of 3AB alone by using flow cytometry to analyze the cell cycle phase-dependent, anti-proliferative effects of 3AB on mouse L1210 cells together with fluctuations in RNA (predominantly ribosomal) levels. We report that 3AB, at cytostatic concentrations, does not block cells in G2 committed to mitosis but imposes an immediate G1 and S phase arrest. Eventually cells arrested in G1 and S phase can reenter cycle but become irreversibly blocked in G2 and are incapable either of progression to mitosis or of the reinitiation of DNA synthesis when cytokinesis is blocked by colcemid exposure. 3AB exposure rapidly reduced RNA levels in all phases of the cell cycle with recovery from depletion apparent only at nontoxic concentrations (5 mM). The responses of a 3AB-resistant subline, capable of sustained culture growth in a normally cytostatic concentration of inhibitor (25 mM), suggest a close association between the sensitivity to RNA depletion and cell cycle arrest.     

Effects of sodium azide on replicative and repair DNA synthesis in barley embryos

Sodium azide inhibited protein synthesis and replicative DNA synthesis at doses used for mutation induction (0.1–1 mM) in barley embryos. During subsequent treatment for 24 h using lower doses of azide, partial recovery from this inhibition was observed, especially in the case of replicative DNA synthesis. Contrary to expectation, cysteine acted as a weak enhancer of the inhibitory effect of azide on DNA replication. Under conditions of minimal replicative DNA synthesis, DNA-repair synthesis was detected after the azide action as measured by the BND-cellulose method. Benazzmide — an inhibitor of poly(ADP-ribose)-polymerase — stimulated the azide-dependent repair synthesis approximately 2-fold.     

Effect of inhibitors of poly(ADP-ribose)polymerase on the radiation response of HeLa S3 cells

The purpose of this study was to investigate possible involvement of poly(ADP-ribosyl)ation reactions in X-ray-induced cell killing, repair of potentially lethal damage (PLD), and formation and repair of radiation-induced DNA damage. As tools we used the inhibitors of poly(ADP-ribose)polymerase, 3-aminobenzamide (3AB), and 4-aminobenzamide (4AB). Both drugs inhibited PLD repair equally well but did not increase radiation-induced cell killing when cells were plated immediately after irradiation. 3AB affected repair of radiation-induced DNA damage, while 4AB had no effect. When 3AB was combined with aphidicolin (APC), it was found that the amount of DNA damage increased during the postirradiation incubation period. This means that the presence of 3AB stimulates the formation of DNA damage after X-irradiation. It is concluded that 3AB and 4AB sensitize HeLaS3 cells for radiation-induced cell killing by inhibiting repair of PLD. Because of the different effects of both inhibitors on repair of PLD and repair of radiation-induced DNA damage (a process known to be affected by inhibition of poly(ADP-ribosyl)ation), it is concluded that the observed inhibition of PLD repair is not caused by inhibition of poly(ADP-ribose)polymerase, and that the inhibitors affect repair of PLD and repair of DNA damage through independent mechanisms.     

Differential gene expression in gamma-irradiated BALB/3T3 fibroblasts under the influence of 3-aminobenzamide,an inhibitior of parp enzyme

3-Aminobenzamide (3AB) is an inhibitor of poly (ADP-ribose) polymerase (PARP), an enzyme implicated in the maintenance of genomic integrity, which is activated in response to radiation-induced DNA strand breaks. cDNA macroarray membranes containing 1536 clones were used to characterize the gene expression profiles displayed by mouse BALB/3T3 fibroblasts (A31 cell line) in response to ionizing irradiation alone or in combination with 3AB. A31 cells in exponential growth were pre-treated with 3AB 4mM 1h before gamma-irradiation (4Gy), remaining in culture during 6h until harvesting time. A31 cells treated with 3AB alone presented a down-regulation in genes involved in protein processing and cell cycle control, while an up-regulation of genes involved in apoptosis and related to DNA/RNA synthesis and repair was verified. A31 cells irradiated with 4Gy displayed 41 genes differentially expressed, being detected a down-regulation of genes involved in protein processing and apoptosis, and genes controlling the cell cycle. Concomitantly, another set of genes for protein processing and related to DNA/RNA synthesis and repair were found to be up-regulated. A positive or negative interaction effect between 3AB and radiation was verified for 29 known genes. While the combined treatment induced a synergistic effect on the expression of LCK proto-oncogene and several genes related to protein synthesis/processing, a negative interaction effect was found for the expression of genes related to cytoskeleton and extracellular matrix assembly (SATB1 and Anexin III), cell cycle control (tyrosine kinase), and genes participating in DNA/RNA synthesis and repair (RNA helicase, FLAP endonuclease-1, DNA-3 glycosylase methyladenine, splicing factor SC35 and Soh1). The present data open the possibility to investigate the direct participation of specific genes, or gene products acting in concert in the mechanism underlying the cell response to radiation-induced DNA damage under the influence of PARP inhibitor.     

3-Aminobenzamide does not affect radiation-induced inhibition of DNA synthesis in human cells

3-Aminobenzamide (3AB), a potent inhibitor of poly(ADP-ribose) synthesis, does not affect the dose response for ionizing radiation-induced inhibition of DNA synthesis in human fibroblasts. If the radioresistant DNA synthesis observed in fibroblasts from patients with ataxia-telangiectasia (A-T) were due to reduced poly(ADP-ribose) synthesis after irradiation, as has been proposed, the response in normal cells incubated with 3AB would have been similar to that observed in A-T cells. Therefore, altered poly(ADP-ribose) synthesis in A-T cells is not solely responsible for their radioresistant DNA synthesis.     

Chromodomain helicase DNA-binding protein 4 (CHD4) regulates homologous recombination DNA repair, and its deficiency sensitizes cells to poly(ADP-ribose) polymerase (PARP) inhibitor treatment

To ensure genome stability, cells have evolved a robust defense mechanism to detect, signal, and repair damaged DNA that is generated by exogenous stressors such as ionizing radiation, endogenous stressors such as free radicals, or normal physiological processes such as DNA replication. Homologous recombination (HR) repair is a critical pathway of repairing DNA double strand breaks, and it plays an essential role in maintaining genomic integrity. Previous studies have shown that BRIT1, also known as MCPH1, is a key regulator of HR repair. Here, we report that chromodomain helicase DNA-binding protein 4 (CHD4) is a novel BRIT1 binding partner that regulates the HR repair process. The BRCA1 C-terminal domains of BRIT1 are required for its interaction with CHD4. Depletion of CHD4 and overexpression of the ATPase-dead form of CHD4 impairs the recruitment of BRIT1 to the DNA damage lesions. As a functional consequence, CHD4 deficiency sensitizes cells to double strand break-inducing agents, reduces the recruitment of HR repair factor BRCA1, and impairs HR repair efficiency. We further demonstrate that CHD4-depleted cells are more sensitive to poly(ADP-ribose) polymerase inhibitor treatment. In response to DNA damage induced by poly(ADP-ribose) polymerase inhibitors, CHD4 deficiency impairs the recruitment of DNA repair proteins BRIT1, BRCA1, and replication protein A at early steps of HR repair. Taken together, our findings identify an important role of CHD4 in controlling HR repair to maintain genome stability and establish the potential therapeutic implications of targeting CHD4 deficiency in tumors.     

Ionizing radiations in Caenorhabditis elegans induce poly(ADP-ribosyl)ation, a conserved DNA-damage response essential for survival

Poly(ADP-ribosyl)ation is one of the first responses to DNA damage in mammals. Although it is involved in base excision repair, its exact role has not been ascertained yet. Poly(ADP-ribose) polymerase-1 (PARP-1) and PARP-2 mediate most of the poly(ADP-ribosyl)ation response in mammals and are well conserved in evolution. Their respective homologues PME-1 and PME-2 are found in the nematode Caenorhabditis elegans, a well-known genetically tractable model currently used in DNA damage response research. Here we report the functional analysis of PME-1 and PME-2 in presence of DNA damage. Worms irradiated with high doses of ionizing radiations displayed a sharp drop in their NAD(+) content immediately after treatment, and a biphasic increase in poly(ADP-ribose). The physiological importance of the poly(ADP-ribosyl)ation response was highlighted when worms were preincubated with mammalian PARP inhibitors (3AB, DHQ, PJ34) and irradiated. The embryonic survival rate of the progeny was significantly decreased in a dose-dependent manner. The inhibitor 3AB had a weak effect on embryonic survival, followed closely by DHQ. However, PJ34, a member of the phenantridinone family, was very effective even when used at low concentration (100nM). In vitro PARP assay using recombinant PME-1 and PME-2 showed a similar pattern of inhibition where 3AB and DHQ were weak inhibitors, and PJ34 a stronger one. Inhibitors affect mostly the poly(ADP-ribose) polymers elongation at high concentrations. These results suggest that poly(ADP-ribosyl)ation in response to DNA damage is an ancient and very important biochemical process protecting DNA from deleterious modification.     

3-Aminobenzamide does not affect X-ray-induced cytogenetic damage in G0 human lymphocytes

The inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide (3AB) has been reported to have very different effects on X-ray-induced chromosome aberrations in G0 human lymphocytes. One group of investigators observed a 2-3-fold increase in the yield of rings, dicentrics and chromosome breaks after X-irradiation and 3AB treatment, whereas another group found that 3AB had no effect on X-ray-induced chromosome aberrations. To resolve this discrepancy, we repeated the experiments as described by both groups and found no effect of 3 mM or 5 mM 3AB on the frequency of chromosome aberrations induced by either 1 Gy or 2 Gy of X-rays. Furthermore, we found no effect of 3AB on X-ray-induced aberration yields in C-banded prematurely condensed chromosome preparations from unstimulated human lymphocytes. These results indicate that poly(ADP-ribose) polymerase is not involved in the repair of cytogenetic damage in G0 human lymphocytes.     

Yield of SCEs and translocations produced by 3 aminobenzamide in cultured Chinese hamster cells

Different concentrations of 3-aminobenzamide (3AB), a strong inhibitor of poly(ADP-ribose) polymerase (PARP), were used to study their effect on the BrdU-substituted DNA of the Chinese hamster AA8 cell line. The frequencies of sister chromatid exchanges (SCEs) and translocations were determined using the fluorescence plus Giemsa (FPG) and fluorescence in situ hybridization (FISH) techniques, respectively. The results indicate that 3AB effectively induced a dose-dependent increase in the frequency of SCEs, but this enhancement in the yield of SCEs was not paralleled by an increase in translocations. These results are discussed in terms of the as yet poorly understood molecular mechanisms of action of the enzyme PARP.     

Bimodal induction of sister-chromatid exchanges by luminol,an inhibitor of poly(ADP-ribose) synthetase,during the S-phase of the cell cycle

The cell cycle dependence of sister chromatid exchanges (SCEs) induced by luminol, a new potent inhibitor of poly(ADP-ribose) synthetase, was studied in Chinese hamster V79 cells. Continuous treatment with luminol during two whole cell cycles in the presence of 5-bromo-2-deoxyuridine (BrdUrd), or in the first or second cycle induced SCEs very efficiently in a linear dosedependent manner. However, no enhancement of SCE levels was observed after luminol treatment in a cycle preceding BrdUrd treatment, in contrast to results found with other strong SCE inducers such ascis-diammine-dichloroplatinum (II) (CDDP) and mitomycin C (MMC). Luminol was about ten times as effective in inducing SCEs as 3-aminobenzamide (3AB), an inhibitor of the NAD⁺ site of poly(ADP-ribose) synthetase. The induction of SCEs by luminol was restricted to the Sphase of the cell cycle with peaks at an early and a late stage, corresponding to the biphasic replication of DNA. The mechanism of SCE appears to be the same at the early and late stages of S-phase for luminol-induced SCE formation.     

Potentiation of cell killing by inhibitors of poly(ADP-ribose) polymerase in four rodent cell lines exposed to N-methyl-N-nitrosourea or UV light

The sensitivities (Do-values) of the cytotoxic effect of MNU on four rodent cell lines were: mouse L1210, 0.07 mM; rat Yoshida sarcoma, 0.52 mM; Chinese hamster V79A, 0.70 mM and the UV sensitive, X-ray sensitive V79/79, 0.35 mM. The abilities of maximum non-toxic doses of the poly-(ADP-ribose) polymerase inhibitors, 5-methyl nicotinamide (5MeN), 3-methoxybenzamide (3MBA) and caffeine to potentiate this cytotoxicity and that of UV light in V79A and V79/79 was measured. The degree of potentiation (ratio Do without inhibitor/Do with inhibitor) was both agent and cell line dependent. In general the lymphoid cell lines L1210 and YS showed greater potentiation, up to 4-fold, than did the fibroblast lines V79A and V79/79. The use of inhibitors in pairs suggested that 5MeN and 3MBA affect one process whereas caffeine affects additional processes. The data provide further support for a role for poly(ADP-ribose) in DNA repair, but indicate that metabolic factors may modify the effectiveness of individual inhibitors of poly(ADP-ribose) polymerase in different cell lines.     

Effects of 3-aminobenzamide on Chinese hamster cells treated with thymidine analogues and DNA-damaging agents. Chromosomal aberrations, mutations and cell-cycle progression

The nuclear enzyme, poly(ADP-ribose) synthetase is involved in the repair of damaged DNA. We report here the results obtained with 3-aminobenzamide (3AB), an inhibitor of this enzyme, on induced biological effects. 3AB increases the frequency of chromosomal aberrations induced by DMS, EMS, ENU, bleomycin and CldUrd. The magnitude of the effect is dependent on the type of chemical used, the combinations with DMS and EMS being the most potent ones. No potentiation was observed after treatment of cells with MMC. Mutation frequencies were determined on the HPRT locus and showed that 3AB did not increase the frequency of gene mutations induced by EMS, ENU and CldUrd. Cell-cycle progression is affected when cells are grown in medium containing CldUrd and 3AB, primarily when the inhibitor is present during the second cell cycle when substituted DNA becomes replicated. The extent of the effect depends on the amount of analogue incorporated and is independent of the presence of the analogue in the medium during the second cell cycle. Analysis of chromosomal aberrations in delayed G2 cells with the aid of the premature chromosome-condensation technique revealed numerous aberrations after incorporation of CldUrd and treatment with 3AB.     

Poly(ADP-ribose) synthesis is involved in the toxic effects of alkylating agents but does not regulate DNA repair

Poly(ADP-ribose) is a nuclear polymer that is synthesized in response to DNA-strand breaks and covently modifies numerous nuclear proteins. Inhibition of poly(ADP-ribose) polymerase by 3-amino-benzamide in cells exposed to DNA-damaging agents has a variety of cellular effects, including increases in cell killing, frequency of single-strand breaks, reapir replication, and sister-chromatid exchange. These increases have been interpreted as an indication that poly(ADP-ribose) polymerization regulates the rate of ligation. Because of slow ligation, continued repair polymerization should therefore generate longer repair patches. Direct measurement of the rate of ligation of intracellular repair patches and of the size of repair patches indicates that they are unchanged when poly(ADP-ribose) polymerization is inhibited. We therefore conclude that poly(ADP-ribose) does not regulate the ligation stage of repair but instead may regulate the activity of intracellular nucleases and other enzymes that can cause additional DNA damage and changes in chromatin struture.     

Antagonistic action of a tumor promoter and a poly(adenosine diphosphoribose) synthesis inhibitor in radiation-induced transformation in vitro

Transformation of mouse C3H 10T1/2 cells by X-irradiation in vitro was blocked by the addition of 1 mM 3-aminobenzamide, an inhibitor of polyadenosine diphosphoribose (poly[ADP-ribose]) synthesis immediately after irradiation. 3-Aminobenzamide also inhibited an increase in the frequency of transformants caused by the addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, 7 days after irradiation. These results demonstrate a role for poly(ADP-ribose) synthesis during the initiation and promotion stages of transformation. From previous studies it is known that poly(ADP-ribose) synthesis is stimulated by the DNA damage caused by X rays during initiation. During promotion, however, 12-O-tetradecanoylphorbol-13-acetate acted as a mitogen but did not induce detectable DNA damage, and we could detect no stimulation of poly(ADP-ribose) synthetase. The roles of poly(ADP-ribose) during initiation and during promotion must, therefore, be significantly different.     

Poly(ADP-ribose) synthesis and inhibition of DNA synthesis in Chinese hamster cells treated with methylating agents and thymidine

A possible role of poly(ADP-ribose) synthesis in modulating the response of V79 cells to DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS) was investigated. Inhibition of [3H]thymidine (dThd) incorporation into DNA and lowering of NAD+ levels in intact cells were employed as parameters of DNA-synthesis inhibition and poly(ADP-ribose) synthesis, respectively. Dose responses of these parameters were studied in cells 2 and 24 h after treatment with the methylating agents in medium with or without dThd. The initial inhibition of DNA synthesis was uniformly associated with stimulation of poly(ADP-ribose) synthesis whether the cells were treated with MNNG or MMS, incubated with or without 20 microM dThd which did not inhibit poly(ADP-ribose) synthesis, or incubated with 3 mM dThd which did inhibit the latter synthesis. By contrast, the DNA-synthesis inhibition detected 24 h after treatment with MNNG was not associated with poly(ADP-ribose) synthesis. These data suggest that (i) the mechanism of this later inhibition of DNA synthesis is different from that of the initial inhibition, (ii) DNA-synthesis inhibition does not stimulate poly(ADP-ribose) synthesis, and (iii) single-strand breaks, resulting from N-methylation of the DNA, stimulate poly(ADP-ribose) synthesis, which may produce the initial inhibition of DNA synthesis. The initial inhibition of DNA synthesis was not uniformly associated with mutagenesis and dThd facilitation of MNNG-induced cytotoxicity and mutagenesis. This indicates that O-methylation of DNA does not stimulate poly(ADP-ribose) synthesis. Our data suggest that, in V79 cells treated with methylating agents, poly(ADP-ribose) synthesis is stimulated by single-strand breaks, inhibits DNA synthesis, and thereby serves to allow time for repair of the DNA prior to replication.