methylnitronitrosoguanidine and 3-aminobenzamide

Centrosome amplification (also known as centrosome overduplication) is common in cancer cells and can be induced by DNA damaging agents. However, the mechanism and significance of centrosome amplification during carcinogenesis or after DNA damage are not clear. Previously, we showed that centrosome amplification could be induced by 3-aminobenzamide (3-AB), an inhibitor of poly(ADP-ribose) polymerases (PARPs) in mouse embryonic fibroblasts. In this paper, we determined if the effect of 3-AB on centrosome amplification was dependent on DNA damage in CHO-K1 cells. We used the well-known mutagen/carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Ten micromolar MNNG and 10 mM 3-AB induced significant centrosome amplification in 18.1 ± 1.1% and 19.4 ± 1.8% of CHO-K1 cells, respectively, compared to 7.0 ± 0.5% of untreated CHO-K1 cells. AG14361, another potent inhibitor of PARPs, also induced centrosome amplification. We then used γ-H2AX analysis and alkaline comet assays to show that 10 μM MNNG induced a significant number of DNA lesions and cell cycle arrest at the G(2) /M phase. However, 10 mM 3-AB neither induced DNA lesions nor altered cell cycle progression. In the umu test, 10 μM MNNG was mutagenic, but 10 mM 3-AB was not. In addition, 10 μM MNNG induced significant accumulation of ataxia telangiectasia mutated protein in the nuclei, but 10 mM 3-AB did not. Thus, we found no association between apparent DNA lesions and centrosome amplification after 3-AB treatment. Therefore, we propose the presence of a novel pathway for centrosome amplification that does not necessarily require DNA lesions but involves regulation of epigenetic changes or post-translational modifications including polyADP-ribosylation.

Topics: Animals; Ataxia Telangiectasia Mutated Proteins; Azulenes; Benzamides; Benzodiazepines; Cell Cycle; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Proliferation; Centrosome; CHO Cells; Cricetinae; DNA; DNA Damage; DNA-Binding Proteins; Histones; Methylnitronitrosoguanidine; Mitosis; Mutagenesis; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Protein Serine-Threonine Kinases; Tumor Suppressor Proteins

The formation of ATP produced from poly(ADP-ribose) [(ADP-R)n] has been suggested to be required to repair damaged DNA. Here we investigate whether this ATP is involved in DNA replication processes during DNA repair. Poly(ADP-ribosyl)ated mid-S phase cell nuclei, which were isolated from synchronized HeLa S3 cells followed by the treatment with a DNA damaging agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), were revealed to retain DNA replication synthesizing activity during preincubation for de-poly(ADP-ribosyl)ation only in the presence of pyrophosphate (PPi) before DNA synthesis was started by adding 3 mM ATP. This DNA replication activity was not maintained in the presence of a potent and specific inhibitor of poly(ADP-ribose) glycohydrolase (PARG), Oenothein B (Oen B) during the preincubation with PPi. In the preincubation with PPi, muM orders of ATP was produced from (ADP-R)n. These results point to an important function of ATP generated from (ADP-R)n in nuclei for the maintenance of replication apparatus during DNA repair.

Topics: Adenosine Triphosphate; Benzamides; Cell Nucleus; Chromatography, High Pressure Liquid; Chromosomal Proteins, Non-Histone; Diphosphates; DNA Damage; DNA Repair; DNA Replication; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glycoside Hydrolases; HeLa Cells; Humans; Hydrolyzable Tannins; Methylnitronitrosoguanidine; Models, Biological; NAD; Poly Adenosine Diphosphate Ribose; S Phase; Thymidine Monophosphate

The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) which was initially known for its role in the repair of oxidative stress-induced DNA damage, has also been reported to play a mediating role in the inflammatory response. Studies with PARP-1 knockout models have shown that PARP-1 is a co-activator of Nuclear Factor-kappa B (NF-kappaB), although this appears not to require its enzyme activity. In addition, drug-induced inhibition of the enzyme activity of PARP-1 was observed to reduce the production of pro-inflammatory mediators. In this study, the flavonoid compound flavone was demonstrated to significantly inhibit the enzyme activity of PARP-1. Further evaluation of flavone in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated human pulmonary epithelial and vascular endothelial cells revealed that both the decrease in NAD(+) levels, as well as the formation of PAR-polymers was dose-dependently attenuated by flavone. In addition, flavone was found to reduce the lipopolysaccharide (LPS)-induced interleukin (IL)-8 production in pulmonary epithelial cells, which was confirmed by transcription analysis. Furthermore, the transcription Inhibitor kappa B alpha (of IkappaBalpha) was significantly increased by flavone. The results of the present study indicate that the flavonoid flavone could be a potential candidate for application in treatment of chronic inflammatory diseases. PARP-1 inhibition could have beneficial effects in such diseases as Chronic Obstructive Pulmonary Disease (COPD) and diabetes, by preservation of cellular NAD(+) levels and attenuating inflammatory conditions.

Topics: Alcohol Oxidoreductases; Antioxidants; Benzamides; Cell Line, Tumor; Cell Nucleus; Cyclic N-Oxides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ferrous Compounds; Flavones; Flavonoids; Gene Expression Regulation; Humans; Hydrogen Peroxide; Interleukin-8; Lipopolysaccharides; Methylnitronitrosoguanidine; Molecular Structure; NAD; NF-kappa B; Nucleotidases; Phenanthrenes; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Reverse Transcriptase Polymerase Chain Reaction; Spin Trapping; Transcription, Genetic

Polyadenosylation of nuclear enzymes is well known to regulate the cellular repair capacity after DNA damage. PARP mediates the transfer of poly-ADP-ribose moieties on itself and other nuclear proteins by the breakdown of NAD+. The present study investigated how modulation of PARP activity interferes with cell death induced by two different alkylating agents used in cancer chemotherapy. 1-methyl-3-nitro-1-nitrosoguanidinium (MNNG) decreased cellular reduction capacity (WST-1 assay) in HL60 and CCRF-CEM cells, accompanied by increased activity of PARP and depletion of intracellular NAD+ and ATP. Pretreatment with the PARP inhibitors 3-AB or 4-AN resulted in transient cell protection, which was associated with a switch from necrosis to apoptosis in CCRF-CEM cells and enhanced apoptosis in HL60 cells. Both PARP inhibitors delayed the drop in WST-1 reduction and retained NAD+ and ATP levels required for apoptosis. Furthermore, 3-AB or 4-AN prevented progressive DNA degradation in MNNG-treated CCRF-CEM cells. In contrast to MNNG, we did not observe early activation of PARP, decrease in WST-1 reduction, or wasteful consumption of NAD+ and ATP after treatment with melphalan. However, preincubation with 3-AB or 4-AN resulted in decreased HL60 cell membrane blebbing and reduced formation of apoptotic bodies. In conclusion, the cell death preventing effects of PARP inhibitors are mediated by their ability to maintain cellular energy metabolism, to inhibit the activation of endonucleolytic DNA degradation and to prevent cell blebbing. Surprisingly, these protective effects of PARP inhibitors on different cell functions seem to be independent of each other and are rather determined by the respective cytotoxic mechanisms implicated by different drugs. Our results support the hypothesis, that PARP activation and/or cleavage plays a regulatory role in the induction of apoptosis.

Topics: 1-Naphthylamine; Adenosine Triphosphate; Alkylating Agents; Apoptosis; Benzamides; Cell Death; Cell Line, Tumor; Cell Size; Enzyme Inhibitors; HL-60 Cells; Humans; Leukemia; Melphalan; Methylnitronitrosoguanidine; NAD; Naphthalimides; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Quinolones

Measurement of poly(ADP-ribose) levels was performed by a new method using a monoclonal antibody against poly(ADP-ribose) and flow cytometry from small amount of cultured cells without the need for isolation of poly(ADP-ribose) polymer. The increase of poly(ADP-ribose)-associated fluorescence intensity was observed in individual human leukemic HL-60 cells when treated with the carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and was blocked by the treatment with 3-aminobenzamide before MNNG treatment. It is easy and rapid to detect the time-dependent change of poly(ADP-ribose) levels in HL-60 cells after MNNG treatment. We easily found that the increase of the poly(ADP-ribose) level in nicotinic acid-treated lymphocytes after MNNG treatment was observed, but not in nicotinamide-treated lymphocytes. We investigated the change of poly(ADP-ribose) levels especially in the early phase of apoptosis. Our method is simple and rapid. It is suggested that the investigation of poly(ADP-ribosyl)ation in various fields is possible by using this new method.

Topics: Benzamides; Enzyme Inhibitors; Flow Cytometry; HL-60 Cells; Humans; Methylnitronitrosoguanidine; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerase Inhibitors

A cell line deficient in DNA ligase I and sensitive to poly(ADP-ribose) inhibitors, 46BR, was used to examine the relationship between DNA ligation and a large stimulation of repair replication that is seen in cells grown in a poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide (3AB), after exposure to alkylating agents. Repair replication was stimulated at least 10-fold by 3AB in both normal and ligase-deficient cells. Despite increased 3AB toxicity, repair replication in ligase I-deficient cells was unchanged from that in normal cells. This evidence is consistent with previous observations that the enhancement of repair replication by 3AB is not a direct function of DNA break frequencies. The stimulation may instead result from alkylation damage to other cellular organelles that release nucleases that cause additional damage to DNA, which cells attempt to repair.

Topics: Benzamides; Calcium; Cells, Cultured; DNA Ligases; DNA Repair; DNA Replication; Enzyme Inhibitors; Fibroblasts; Humans; Methylnitronitrosoguanidine; Poly(ADP-ribose) Polymerase Inhibitors

5-Hydroxymethyl-2'-deoxyuridine (hmdUrd) is incorporated into DNA as a thymidine analog resulting in extensive substitution of thymine residues with 5-hydroxymethyluracil (hmUra) residues. These hmUra residues are then subject to excision by action of hmUra-DNA glycosylase. 3-Aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthesis, is toxic to cells that incorporate and repair hmdUrd. To demonstrate that incorporation and repair of hmdUrd stimulates synthesis of poly(ADP-ribose) from intracellular NAD, V79 hamster cells were treated with hmdUrd and intracellular NAD levels were measured. Following hmdUrd treatment, NAD levels fell markedly (80-90%) within 4 h and remained low for at least 10 h, before partially recovering by 24 h. The degree of NAD lowering was dose dependent and paralleled net hmdUrd incorporation. The NAD lowering was largely prevented by concurrent treatment with 4 mM 3AB. No effects on NAD levels were seen following treatment with deoxythymidine or bromodeoxyuridine, which are incorporated into DNA but, in contrast to hmdUrd, are not repaired. When the incorporation of hmdUrd into DNA was blocked with hydroxyurea or aphidicolin, no NAD lowering was seen. HmdUrd also did not produce lowering of NAD concentrations in mutant cell strains deficient in the ability either to incorporate hmdUrd into DNA or to repair hmdUrd from DNA. These results demonstrate that synthesis of poly(ADP-ribose) resulted directly from the incorporation into DNA of the nucleoside hmdUrd and its subsequent repair. These results unequivocally demonstrate that the initiation of normal DNA base excision repair by itself, and not DNA damage per se, is a sufficient stimulus for the induction of poly(ADP-ribose) synthesis.

Topics: Animals; Antineoplastic Agents; Benzamides; Bromodeoxyuridine; Carcinogens; Cell Line; Cell Survival; Cricetinae; Cricetulus; DNA Repair; Kinetics; Methylnitronitrosoguanidine; NAD; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerase Inhibitors; Thymidine; Time Factors

Previous studies showed that S-(1,2-dichlorovinyl)-L-cysteine perturbs intracellular Ca2+ homeostasis [Vamvakas et al., Mol Pharmacol 38: 455-461, 1990]. The objective of the present study was to investigate the cellular events that precede and that follow S-(1,2-dichlorovinyl)-L-cysteine-induced mitochondrial Ca2+ release. In incubations with isolated kidney mitochondria, S-(1,2-dichlorovinyl)-L-cysteine-induced Ca2+ efflux is preceded by increased oxidation of mitochondrial pyridine nucleotides and is prevented by ATP, an inhibitor of the hydrolysis of pyridine nucleotides, and by meta-iodobenzylguanidine, an acceptor of ADP-ribose moieties. In LLC-PK1 cells, elevation in the cytosolic Ca2+ concentration is followed by a several-fold increase in DNA double-strand breaks which is attributed to the activation of Ca2+- and Mg(2+)-dependent endonucleases. The formation of DNA double-strand breaks is followed by increased poly(ADP-ribosylation) of nuclear proteins. S-(1,2-Dichlorovinyl)-L-cysteine-induced cytotoxicity in LLC-PK1 cells is blocked by chelation of cytosolic Ca2+ with Quin-2, by inhibition of DNA fragmentation with aurintricarboxylic acid and by inhibition of increased poly(ADP-ribosyl)transferase activity by 3-aminobenzamide. These findings indicate that S-(1,2-dichlorovinyl)-L-cysteine bioactivation in renal cells may initiate the following cascade of events: increased oxidation and hydrolysis of mitochondrial pyridine nucleotides resulting in the modification of mitochondrial membrane proteins by pyridine nucleotide-derived ADP-ribose moieties, followed by Ca2+ release. Elevated Ca2+ concentrations may activate Ca(2+)-dependent endonucleases, which leads to DNA fragmentation followed by increased poly(ADP-ribosylation) of nuclear proteins and, finally, cytotoxicity.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Benzamides; Biotransformation; Calcium; Cell Division; Cell Line; Cysteine; DNA Damage; Down-Regulation; Kidney Cortex; Methylnitronitrosoguanidine; Mitochondria; Nuclear Proteins; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Swine

1. The results of this study have contributed to the definition of three categories of chemical inhibitors of DNA replication in mammalian cells. 2. Inhibitors of replicon cluster initiation [4-nitroquinoline-N-oxide (4-NQO), etoposide (VP-16), teniposide (VM-26), amsacrine (m-AMSA), N-methyl-N'-nitro-N-nitrozoguanidine (MNNG), cis-Pt(II)diammine dichloride (cis-PDD)], which needed similar doses to produce a slow and persistent (up to 4 hr) inhibition of DNA synthesis, followed by significant cell killing. 3. Inhibitors of DNA replication by indirect action [3-aminobenzamide [correction of 3-aminobezamide] (3-AB), cycloheximide (CHX), puromycin (PRC), bisbenzimide Hoechst No. 33258 (H-33258]), that showed reduced cytotoxic effects, and caused a slow (60 min) and reversible inhibition of DNA synthesis. 4. Inhibitors of formation and/or polymerization of deoxyribonucleotides [5-aminouracil (5-AU), bisbenzimide Hoechst No. 33342 (H-33342)], which induced a fast (20 min) and reversible suppression of DNA replication, associated with limited cell killing.

Topics: 4-Nitroquinoline-1-oxide; Amsacrine; Animals; Benzamides; Bisbenzimidazole; Cell Division; Cell Line; Cisplatin; Cricetinae; Cricetulus; Cycloheximide; DNA Replication; Etoposide; Gene Expression Regulation; In Vitro Techniques; Kinetics; Methylnitronitrosoguanidine; Puromycin; Teniposide; Transcription, Genetic; Uracil

Alkylating agents cause a marked depletion of cellular NAD+ levels by activating nuclear ADP-ribosyl transferase (ADPRT), which utilizes NAD+ as a substrate in the synthesis of poly(ADP-ribose). As a consequence of NAD+ depletion, it is possible that cellular ATP pools could be depleted. Because of this, exogenously supplied NAD+ had been proposed as a way to counteract some of the effects of an alkylator. We found that exogenously supplied NAD+ significantly increased intracellular levels of NAD+ in MMS- and MNNG-treated V79 Chinese hamster cells. Cytotoxicity was not changed by the exogenously supplied NAD+, however. 3-Aminobenzamide (3-ABA), an ADPRT inhibitor, prevented the depletion of intracellular NAD+ by MMS or MNNG treatment and potentiated cytotoxicity. As was the case without 3-ABA, exogenously supplied NAD+ plus 3-ABA did not change the cytotoxicity, even though NAD+ levels were increased. Intracellular ATP levels were also measured and were found to be unaffected following MMS treatment, and only slightly depleted following MNNG treatment. Exogenously supplied NAD+ raised these levels above those for their respective controls. Because survival was unaffected by elevated levels of NAD+ and ATP, our results suggest that depletion of cellular NAD+ pools following MMS and MNNG treatment is not a critical factor in determining cytotoxicity for these V79 cells. The energy reserves of V79 cells, at doses of MMS or MNNG which kill 99% of the cells, are apparently adequate to maintain normal levels of ATP.

Topics: Adenosine Triphosphate; Animals; Benzamides; Cell Survival; Cells, Cultured; Cricetinae; Fibroblasts; Intracellular Fluid; Kinetics; Methyl Methanesulfonate; Methylnitronitrosoguanidine; NAD; Nucleoside Diphosphate Sugars; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerase Inhibitors

Poly(ADP-ribosyl)ation of nuclear proteins is an immediate response of most eukaryotic cells to DNA strand breaks, as induced by carcinogen treatment. DNA amplification, on the other hand, can be induced in cell culture systems by chemical or physical carcinogens, too, reaching peak levels a few days after induction treatment. We have previously shown that 3-aminobenzamide, an inhibitor of poly(ADP-ribosyl)ation, potentiates carcinogen-induced simian virus 40 DNA amplification in hamster cells which served as a short-term model system (Bürkle et al., Cancer Res., 47: 3632-3636, 1987). Here we report that those results can be extended to the development of methotrexate (MTX) resistance associated with dihydrofolate reductase (DHFR) gene amplification in a different hamster cell line. (a) Treatment with the alkylating carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) 3 days before selection with 350 nM MTX induced the MTX resistance frequency by 17- to 100-fold, as expected. Addition of 3-aminobenzamide (0.1 to 1 mM) before MNNG treatment further potentiated the frequency of MTX resistance by up to 5-fold in a dose-dependent manner, parallel to a potentiation of cytotoxicity. MTX resistance frequency was potentiated not only relative to the decrease in cell survival but also in absolute terms. The same potentiation occurred after cotreatment with benzamide (1 mM), another poly(ADP-ribosyl)ation inhibitor, under conditions which precluded direct drug interactions. Benzoic acid, a noninhibitory analogue, had no effect on the MNNG-induced MTX resistance frequency. (b) Neither 3-aminobenzamide, nor benzamide, nor benzoic acid at 1 mM, respectively, had any effect on the spontaneous frequency of MTX resistance. (c) Individual MTX-resistant colonies were expanded to determine their DHFR gene copy number. The relative frequency of DHFR gene amplification was similar (14% versus 22%) whether clones were derived from cultures induced with MNNG alone or MNNG in the presence of 1 mM 3-aminobenzamide. We conclude that poly(ADP-ribosyl)ation should act as a negative regulatory factor in the induction of DNA amplification, since inhibition of poly(ADP-ribose) polymerase potentiates both MNNG-induced simian virus 40 DNA amplification, as shown previously, and MNNG-induced MTX resistance associated with DHFR gene amplification, as shown in this paper.

Topics: Animals; Benzamides; Cricetinae; DNA Damage; Drug Resistance; Drug Synergism; Gene Amplification; Methotrexate; Methylnitronitrosoguanidine; Poly(ADP-ribose) Polymerase Inhibitors; Proto-Oncogenes; Tetrahydrofolate Dehydrogenase

The O6-methylguanine-DNA-methyltransferase (transferase) activity in a rat hepatoma cell line (H4 cells) is enhanced as a response to DNA damaging agents. To study whether poly (ADP-ribosylation) is involved in this induction, the cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) that induces the transferase activity and stimulates poly (ADP-ribose) synthesis. Addition of poly (ADP-ribose) polymerase inhibitors enhanced the transferase increase induced by MNNG. The influence of the inhibitors on the transferase induction was dose and time-dependent. The results suggest that poly (ADP-ribose) is involved in the induction of this protein.

Topics: Animals; Benzamides; DNA Damage; DNA Repair; Liver Neoplasms, Experimental; Methylnitronitrosoguanidine; Methyltransferases; O(6)-Methylguanine-DNA Methyltransferase; Poly(ADP-ribose) Polymerase Inhibitors; Rats; Tumor Cells, Cultured

DNA damage inflicted by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine, or by UV254nm, stimulated the catabolism of protein-bound poly(ADP-ribose) in the chromatin of cultured hepatocytes. The stimulation was highest at the largest doses of DNA-damaging treatment. As a consequence, the half-life of ADP-ribosyl polymers may drop to less than 41 s. This rapid turnover contrasts with the slow catabolism of a constitutive fraction of polymers exhibiting a half-life of 7.7 h. Our data suggest that post-incisional stimulation of poly(ADP-ribose) biosynthesis in DNA-excision repair is coupled with an adaptation of poly(ADP-ribose) catabolism in mammalian cells.

Topics: Animals; Benzamides; Cells, Cultured; Chromatin; DNA Damage; Kinetics; Liver; Male; Methylnitronitrosoguanidine; Nucleoside Diphosphate Sugars; Poly Adenosine Diphosphate Ribose; Radiation-Sensitizing Agents; Rats; Rats, Inbred Strains; Ultraviolet Rays

Hepatoma tissue culture (HTC) cells were incubated in the presence of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) to study the variations in the bisnucleosides polyphosphates (Ap4X) pool size. A transient but sensitive accumulation of these compounds is observed; if 3-aminobenzamide (3AB) which is a potent inhibitor of the ADP-ribosyltransferase (ADPRT) is added after the MNNG treatment, a more pronounced and persistent accumulation of Ap4X can be seen. A moderate heat-shock (30 min at 43 degrees C) results also in a small accumulation of Ap4X but the shape of the accumulation curve is quite different and the increase of the Ap4X pool is not sensitive to the presence of 3AB. However, both MNNG treatment and hyperthermia cause a marked inhibition of protein synthesis. On the other hand, the ADPRT activity is enhanced in the presence of MNNG whereas hyperthermia has little or a slightly inhibitory effect on this activity. These results suggest that MNNG treatment triggers an Ap4X accumulation in eukaryotic cells different from that observed after heat-shock and it seems likely that these compounds are involved in the DNA excision repair system in which the ADPRT enzyme is also implicated.

Topics: Adenine Nucleotides; Alkylation; Benzamides; Carcinoma, Hepatocellular; Dinucleoside Phosphates; DNA; DNA Repair; Hot Temperature; Kinetics; Liver Neoplasms; Methylnitronitrosoguanidine; NAD; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Protein Biosynthesis; Tumor Cells, Cultured

We studied the potentiation by 3-aminobenzamide (3AB) of killing of nine human cell lines exposed to alkylating agents. Cell lines included normal, transformed and DNA repair-proficient and -deficient phenotypes. 3AB potentiated cell killing by the methylating agents methylmethanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in all lines tested. The degree of potentiation ranged from 1.7- to 3.8-fold, based on the LD99. The average potentiation observed with MMS (2.7-fold) was greater than with MNNG (2.2-fold). On average the potentiation of MMS and MNNG killing of repair-deficient Mer- lines (2.4-fold) was similar to that of repair-proficient Mer+ lines. The degree of 3AB potentiation of MNNG killing (2.0-fold) was similar in Mer+ Rem- lines and in Mer+ Rem+ lines. Mer+ Rem+, Mer+ Rem-, Mer- Rem+, and Mer- Rem- strains all appeared proficient in a 3AB-sensitive DNA repair pathway. Within experimental error, 20 mM 3AB did not inhibit the removal of the MNNG-induced methylpurines 7-methylguanine, O6-methylguanine and 3-methyladenine from the DNA of repair-proficient Mer+ Rem+ HT29 cells, consistent with evidence that 3AB inhibits the ligation step of excision repair. 3AB potentiated cell killing by the bifunctional alkylating agents 1-(2-chlorethyl)-1-nitrosourea or busulfan, two anti-neoplastic drugs, by only 0.9- to 1.5-fold. These drugs therefore produce DNA damage which is not efficiently repaired by the pathways that repair methylated bases.

Topics: Antineoplastic Agents; Benzamides; Busulfan; Cell Line; Cell Survival; DNA Repair; Drug Synergism; Ethylnitrosourea; Humans; Kinetics; Methyl Methanesulfonate; Methylation; Methylnitronitrosoguanidine; Tumor Cells, Cultured; Tumor Stem Cell Assay

NAD is the substrate of a novel chromatin-associated enzyme-ADP-ribosyl transferase (ADPRT). In this study, the cell-cycle dependent change in cellular NAD content was observed in a line of human amnion FL cells. It was found that the cellular NAD content of FL cells was highest in G1 and lowest in S/G2-G2. 3AB, a potent ADPRT inhibitor, can inhibit the cell cycle dependent change in cellular NAD content and also inhibit DNA synthesis in the S phase and extend the S phase. The results indicate that ADP-ribosylation may be involved in DNA replication and cell cycle progression. It was also found that the DNA-damaging agents, MNNG, MMS and 4NQO could lower cellular NAD content in a dose-dependent way. This DNA-damage-induced NAD lowering could be partially or completely prevented by the ADPRT inhibitors, 3AB or nicotinamide, which were shown to exert no influence on either the cellular NAD content of normal quiescent FL cells or the metabolic blocking agent, 2,4-DNP-induced cellular NAD lowering. The possibility of establishing a simple and specific method to detect DNA-damaging agents by measuring cellular NAD content in the presence or absence of ADPRT inhibitor is explored.

Topics: 4-Nitroquinoline-1-oxide; Benzamides; Cell Cycle; Cell Line; DNA Damage; DNA Replication; Humans; Interphase; Methyl Methanesulfonate; Methylnitronitrosoguanidine; NAD; Niacinamide; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases

Adriamycin caused significant interphase death in HL-60 cells during six hours of incubation, which was abolished by the poly(ADP-ribose) polymerase inhibitors, 3-aminobenzamide or nicotinamide. Neither agent changed adriamycin uptake by HL-60 cells. Although 3-aminobenzamide did not alter the number of DNA strand breaks caused by adriamycin, it prevented adriamycin-induced depletion of intracellular NAD+ and ATP, and maintained energy charge. These findings suggest that the activation of poly(ADP-ribose) synthesis plays an important role in the adriamycin-induced interphase death of proliferating HL-60 cells.

Topics: Benzamides; Cell Line; Cell Survival; DNA Damage; Doxorubicin; Humans; Interphase; Kinetics; Leukemia, Myeloid, Acute; Methylnitronitrosoguanidine; Niacinamide

The effect of DNA damage caused by N-methyl-N'-nitro-nitrosoguanidine (MNNG) on poly(ADP-ribose) synthesis, NAD levels, and purine nucleotide metabolism was studied in human T-lymphoblasts. Excessive DNA breaks caused by MNNG activated poly(ADP-ribose) polymerase and rapidly consumed intracellular NAD. NAD depletion was followed by rapid catabolism of ATP as well as induction of total purine nucleotide catabolism leading to excretion of purine catabolic products. MNNG-treated cells were not able to replenish the intracellular nucleotide pools due to the depletion of intracellular ATP and phosphoribosylpyrophosphate pools which are required for de novo purine biosynthesis. Inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide prevented both the depletion of NAD pools and the associated changes in purine nucleotide metabolism.

Topics: Adenine; Adenosine Triphosphate; Benzamides; Cell Line; Cell Survival; DNA Damage; Glycine; Humans; Methylnitronitrosoguanidine; NAD; Niacinamide; Nucleoside Diphosphate Sugars; Poly Adenosine Diphosphate Ribose; Purines; T-Lymphocytes

A Simian virus 40-transformed Chinese hamster cell line (CO 60) amplifies integrated viral DNA sequences as a response to treatment with a variety of carcinogens. To study a possible involvement of poly(ADP-ribose) synthesis, DNA amplification was induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), an alkylating carcinogen that strongly stimulates poly(ADP-ribose) synthesis. In the presence of 3-aminobenzamide (3AB) (2 mM), a competitive inhibitor of poly(ADP-ribose) polymerase, MNNG-induced amplification was increased two to six times the level induced by MNNG alone. Concomitantly, 3AB reduced cellular poly(ADP-ribose) levels and increased MNNG-induced cytotoxicity, as expected. The effect of 3AB on MNNG-induced amplification depended both on the concentration of 3AB and the duration of its presence after MNNG treatment. By contrast, 3-aminobenzoic acid, a noninhibitory structural analogue of 3AB, had no influence on amplification induced by MNNG. These data strongly suggest an involvement of poly(ADP-ribose) in the process of DNA amplification, as it is shown that inhibition of carcinogen-stimulated poly(ADP-ribose) synthesis by 3AB is correlated with an enhancement of inducible DNA amplification in this cell line.

Topics: Animals; Benzamides; Cell Transformation, Viral; Cells, Cultured; Cricetinae; Cricetulus; DNA, Viral; Gene Amplification; Methylnitronitrosoguanidine; Poly Adenosine Diphosphate Ribose; Simian virus 40

The ribonucleotide reductase inhibitors deferoxamine and hydroxyurea induce monocyte-macrophage cell differentiation in the leukemic cell line HL-60 as judged by the expression of cell surface antigens, nonspecific esterase activity, and morphological changes. Treatment of HL-60 cells with deferoxamine results in inhibition of DNA synthesis and irreversible loss of colony-forming ability. In addition, both deferoxamine and hydroxyurea caused an increase in the number of DNA strand breaks in HL-60 cells. A DNA methylating agent, N-methyl-N'-nitro-N-nitrosoguanidine, also caused cellular differentiation in HL-60 cells associated with DNA strand breaks. These observations are consistent with a role for DNA damage or for inhibition of DNA synthesis and repair in the differentiation process of HL-60 cells.

Topics: Antigens, Surface; Benzamides; Cell Cycle; Cell Differentiation; Deferoxamine; DNA Repair; DNA, Neoplasm; Humans; Hydroxyurea; Leukemia, Myeloid, Acute; Methylnitronitrosoguanidine; Neoplasm Proteins; Ribonucleotide Reductases; RNA, Neoplasm; Tumor Cells, Cultured

When cells were exposed simultaneously for a 24-h period to the poly(ADP-ribose) synthetase inhibitor 3-aminobenzamide (3AB) (1 or 3 mM) plus aflatoxin B1 (AfB1), no increase in toxicity and limited enhancement of transformation frequency (less than 2 X) was observed. Similarly, simultaneous treatment of cell with 3AB plus methylcholanthrene (MCA) had limited effects, slightly decreasing both toxicity and transformation. In contrast, simultaneous treatment with non-toxic doses of 3AB together with the alkylating agents N-methyl-N'-N-nitro-nitrosoguanidine (MNNG) or ethyl methanesulfonate (EMS) resulted in substantial enhancement of the toxicity and transforming effects of both short-chain alkylating agents. When EMS and varying doses of 3AB (0.1-3 mM) were administered simultaneously for 24 h, increasing levels of 3AB were found to cause a dose-dependent enhancement in toxicity and transformation. To explore the relationship of MNNG- and 3AB-induced effects, two further experiments were performed. First, cells were treated with MNNG plus 3AB for varying lengths of time (4, 24, 72 h). Although exposure for as little as 4 h enhanced toxicity and transformation, these effects were even more profound following 24 or 72 h exposure. Second, cells were exposed to 3AB for varing times prior to or after MNNG exposure. Under these conditions the addition of 3AB up to 6 h post MNNG exposure caused profound enhancement of toxicity and transformation, whereas addition of 3AB 24 h post exposure had minimal effects. Thus the co-carcinogenic effect of 3AB is agent-specific, time-specific and dose-dependent.

Topics: Aflatoxin B1; Aflatoxins; Animals; Benzamides; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Ethyl Methanesulfonate; Kinetics; Methylcholanthrene; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C

Inhibition of poly(ADP-Rib) by benzamide (BA) or 3-amino-benzamide (3AB) for a limited period (i.e., when ADP-ribosylation is elevated) during and shortly following X-ray or MNNG-induced DNA damage of BALB/3T3 cells significantly (3- to 30-fold) enhanced transformation frequency by these agents. Individual Type III foci isolated from benzamide, X-ray, or X-ray plus benzamide treated cultures were established and characterized for growth in soft agar and for tumor induction in nude mice. DNA isolated from representative transformed lines established as a result of BA, X-ray or X-ray and BA treatments was transfected onto NIH/3T3 cells. Transformation efficiencies ranging from 0.17 to 0.28 foci/micrograms of DNA were observed suggesting the possibility that dominant transforming gene(s) were responsible for the oncogenic phenotype of radiation and benzamide transformed DNA.

Topics: Animals; Base Sequence; Benzamides; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; DNA; DNA, Neoplasm; Methylnitronitrosoguanidine; Mice; NAD; Oncogenes; Poly(ADP-ribose) Polymerase Inhibitors

Treatment of L1210 cells with increasing concentrations of MNNG produces heterogeneous perturbations of cellular deoxynucleoside triphosphate pools, with the magnitude and direction of the shift depending on the deoxynucleotide and on the concentration and time of exposure of the DNA damaging agent. 5 microM MNNG stimulated an increase in dATP, dCTP and dTTP but dGTP pools remained constant. These increases were not affected by 3-aminobenzamide, indicating that the pool size increases were produced by poly(ADP-ribose) polymerase independent reactions. 30 microM MNNG caused a time dependent decrease in dATP, dGTP, dTTP and dCTP. The dGTP pool was most drastically affected, becoming totally depleted within 3 hours. The fall in all 4 dNTP pools was substantially prevented by 3-aminobenzamide, suggesting that the decrease in dNTPs following DNA damage is mediated by a poly(ADP-ribose) polymerase dependent reaction. Severe depression of dGTP pools consequent to NAD and ATP depletion may provide a metabolic pathway for rapidly stopping DNA synthesis as a consequence of DNA damage and the activation of poly(ADP-ribose) polymerase.

Topics: Animals; Benzamides; Cell Line; Deoxyribonucleotides; DNA; DNA Repair; Enzyme Activation; Leukemia L1210; Methylnitronitrosoguanidine; Mice; NAD; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases

Hepatocytes were found to be remarkably resistant to suicidal NAD+ depletion due to consumption for chromatin-associated poly(ADP-ribose) biosynthesis, which normally follows infliction of DNA damage in mammalian cells. N-methyl-N'-nitro-N-nitrosoguanidine treatment, which depleted NAD+ levels of confluent fibroblasts to about 40% of controls, did not reduce hepatocellular NAD+ pools, although poly(ADP-ribose) concentrations were concomitantly elevated by 21-fold. This differential behavior, demonstrable also with other carcinogens, can be attributed to the different NAD+ biosynthetic capacities of these cells.

Topics: Animals; Benzamides; Carcinogens; Cell Line; Cells, Cultured; DNA Repair; Liver; Methylnitronitrosoguanidine; Mice; NAD; Nucleoside Diphosphate Sugars; Poly Adenosine Diphosphate Ribose; Rats; Sulfuric Acid Esters; Ultraviolet Rays

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.

Topics: Adenosine Triphosphate; Alkylation; Animals; Benzamides; Cell Line; Cell Membrane Permeability; Clone Cells; Cricetinae; Cricetulus; Drug Synergism; Female; Methyl Methanesulfonate; Methylnitronitrosoguanidine; NAD; Ovary; Sister Chromatid Exchange

Natural killer (NK) activity of human mononuclear cells is sensitive to inhibition by radiation, under the control of polymorphic X linked genes. In order to define the mechanism of this inhibition, we have evaluated the ability of treatments known to damage DNA to inhibit NK activity. The alkylating agents streptozotocin (SZ) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were potent inhibitors of NK activity. Further, a specific competitive inhibitor of adenosine diphosphoribosyl polymerase (ADPRP), 3-aminobenzamide, was able to prevent inhibition by gamma-radiation, UV radiation, and the two alkylating drugs, SZ and MNNG, suggesting the ADPRP, known to be activated by DNA strand breakage, mediates the inhibition by these treatments. NK activity of radioresistant subjects was somewhat more resistant to inhibition by SZ or UVR when compared to radiosensitive NK activity but neither of these treatments gave the clear phenotypic distinction of gamma-radiation, suggesting that chemical strand breakage does not precisely model gamma-radiation and also that the mechanism of UVR inhibition may differ from that of gamma-radiation. These results indicate a role for activation of ADPRP in the inhibitory effect of UV and gamma-radiation on human NK activity and suggest that the biochemical basis for polymorphism in the sensitivity of NK activity to gamma-radiation will be found in the sensitivity to ADPRP activation or the level of activation of this enzyme, known to be the key to DNA repair.

Topics: Benzamides; DNA Repair; Female; Humans; Immunity, Innate; Killer Cells, Natural; Male; Methylnitronitrosoguanidine; Poly(ADP-ribose) Polymerase Inhibitors; Polymorphism, Genetic; Streptozocin

Inhibition of poly (ADP-ribose) synthesis by agents such as 3-aminobenzamide (3-AB) potentiates the cytotoxic, carcinogenic, and clastogenic effects of certain DNA-damaging agents. Experiments were carried out in Chinese hamster ovary cells to compare chromosome aberration production and cytotoxicity with the induction of somatic mutations at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and sodium-potassium ATPase loci after treatment with 3-AB in combination with certain monofunctional alkylating agents. On its own, 1 to 10 mM concentrations of 3-AB were not mutagenic, reduced plating efficiencies only slightly, and produced a small elevation in the frequency of chromatid aberrations. In combination with ethyl methanesulfonate (EMS), 3-AB increased cytotoxicity and the frequency of alkylation-induced chromatid aberrations. 3-AB also increased the frequency of EMS and N-methyl-N'-nitro-N-nitrosoguanidine-induced 6-thioguanine-resistant cells (a marker for the HGPRT- phenotype). It had no effect on the frequency of EMS-induced ouabain-resistant cells (a marker for ATPase mutations). All the effects were dose dependent. Larger absolute increases were found with 10 mM 3-AB as compared with 1 mM 3-AB and with 2 mM EMS as compared to 1 mM EMS. The 3-AB-mediated increases in 6-thioguanine-resistant cells, which are often deletion mutations, and the lack of any increase in the frequency of ouabain-resistant cells, which can only arise through point mutation induction, along with the increases in chromosome aberration frequency, suggests that 3-AB increases the frequency of deletion mutations by increasing the frequency and duration of DNA strand breaks.

Topics: Animals; Benzamides; Cell Survival; Cells, Cultured; Chromosome Aberrations; Cricetinae; Cricetulus; DNA; Drug Synergism; Ethyl Methanesulfonate; Female; Methylnitronitrosoguanidine; Mutagens; Mutation; Ovary

The effects of post-treatments with caffeine in G2 on the frequency of chromosomal aberrations induced by thiotepa, mitomycin C and N-methyl-N-nitro-N'-nitrosoguanidine were studied in human lymphocytes. Caffeine was found to potentiate the frequency of chromatid aberrations induced by all 3 S-dependent agents tested; the most striking enhancement being obtained when caffeine was present during the last 1.5 h before harvesting. Post-treatments in G2 with 3-aminobenzamide had no influence on the aberration frequency induced by thiotepa and N-methyl-N-nitro-N'-nitrosoguanidine.

Topics: Benzamides; Caffeine; Cell Cycle; Cells, Cultured; Chromosome Aberrations; DNA Repair; Drug Synergism; Humans; Lymphocytes; Methylnitronitrosoguanidine; Mitomycins; Thiotepa

CB 1954 potentiates the cytotoxic action of the bifunctional alkylating agent melphalan (L-PAM). In vitro, this potentiation does not require the preincubation in hypoxia normally needed for other nitroaromatic compounds such as misonidazole. Chemopotentiation is observed when cells are held in CB 1954 in air after treatment with L-PAM. This may reflect an inhibition of DNA repair process(es). Structural considerations suggested that CB 1954 might be acting as an inhibitor of poly(ADP-ribosylation). However, an inhibition of the drop in NAD levels consequent on exposure to melphalan was not obtained. Furthermore, unlike the known poly(ADP-ribose) inhibitor, 3-aminobenzamide, CB 1954 does not potentiate the cytotoxicity of the monofunctional alkylator N-methyl-N nitro N-nitrosoguanidine, or inhibit NAD depletion caused by this agent. Therefore the evidence suggests that CB 1954 is not an inhibitor of poly(ADP ribosylation).

Topics: Animals; Aziridines; Azirines; Benzamides; Cell Line; Cell Survival; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; Drug Synergism; Melphalan; Methylnitronitrosoguanidine; Poly(ADP-ribose) Polymerase Inhibitors; Radiation-Sensitizing Agents

Transformation of mouse C3H 10T1/2 cells by various alkylating carcinogens can be modulated by inhibiting poly(ADP-ribose) synthesis with a low concentration of 3-amino-benzamide, which induces no additional toxicity or reported side effects. Transformation by methylating agents was decreased by 3-aminobenzamide, whereas transformation by ethylating agents was increased. These results confirm earlier work on transformation by methylating agents, X-rays and u.v. light. Transformation by ethylating agents, however, appears to proceed by a different mechanism.

Topics: Alkylating Agents; Animals; Benzamides; Carcinogens; Cell Transformation, Neoplastic; DNA Repair; Methylation; Methylnitronitrosoguanidine; Mice; Mice, Inbred C3H; Nucleoside Diphosphate Sugars; Poly Adenosine Diphosphate Ribose

3-Aminobenzamide (3-AB), an inhibitor of poly(ADP-ribosylation), is lethal to human fibroblasts with damaged DNA. Its cytotoxicity was determined relative to a number of factors including the types of lesions, the kinetics of repair, and the availability of alternative repair systems. A variety of alkylating agents, UV or gamma irradiation, or antimetabolites were used to create DNA lesions. 3-AB enhanced lethality with monofunctional alkylating agents only. Within this class of compounds, methylmethanesulfonate (MMS) treatments made cells more sensitive to 3-AB than did treatment with methylnitrosourea (MNU) or methylnitronitrosoguanidine (MNNG). 3-AB interfered with a dynamic repair process lasting several days, since human fibroblasts remained sensitive to 3-AB for 36-48 hours following MMS treatment. During this same interval, 3-AB caused these cells to arrest in G2 phase. Alkaline elution analysis also revealed that this slow repair was delayed further by 3-AB. Human mutant cells defective in DNA repair differed in their responses to 3-AB. Among mutants sensitive to monofunctional alkylating agents, ataxia telangiectasia cells were slightly more sensitive to 3-AB than control cells, while Huntington's disease cells had a near-normal response. Among UV-sensitive strains, xeroderma pigmentosum variant (XPV) cells were more sensitive to 3-AB after MMS than were XP complementation group A (A) cells, which responded normally. Greater lethality with 3-AB could be dependent on inability of the mutant cells to repair damage by other processes.

Topics: Benzamides; Caffeine; Cell Cycle; Cell Division; Cell Survival; DNA Repair; Fibroblasts; Humans; Male; Methyl Methanesulfonate; Methylnitronitrosoguanidine; Methylnitrosourea; Poly Adenosine Diphosphate Ribose; Time Factors

3-Aminobenzamide, an inhibitor of polyadenosine diphosphoribose polymerase, produced rapid reversible changes in single-strand break frequencies in DNA from primary human fibroblasts damaged by alkylating agents, but it did not cause such changes in the DNA of cells damaged by ultraviolet light. The increase in single-strand peak frequencies was not due to an accumulation of blocked repair sites, such as occurs with DNA polymerase inhibitors, but to a delay in the rejoining of induced breaks. 3-Aminobenzamide increases the net break frequency that results from a dynamic balance between excision and ligation. This balance appears to be regulated at the ligation step by adenosine diphosphate ribosylation, which is rapidly altered by addition or removal of 3-aminobenzamide. The rapidity with which strand break frequencies change in the presence of 3-aminobenzamide implies that individual strand breaks resulting from excision at any time after exposure have a lifetime of no more than about 30 min in the cell.

Topics: Benzamides; Cells, Cultured; DNA Repair; DNA, Single-Stranded; Fibroblasts; Humans; Methyl Methanesulfonate; Methylnitronitrosoguanidine; Methylnitrosourea; NAD+ Nucleosidase; Poly(ADP-ribose) Polymerases; Time Factors; Ultraviolet Rays; X-Rays