Triple-negative breast cancer and poly(ADP-ribose) polymerase inhibitors

Recent gene profiling studies have identified at least 5 major subtypes of breast cancer, including normal type, luminal A type, luminal B type, human epidermal growth factor receptor (HER)-2 positive type, and basal-like type. Triple-negative breast cancer (TNBC), showing no or low expressions of estrogen receptor (ER), progesterone receptor (PgR), and HER2, considered important clinical biomarkers, accounts for 10% to 20% of all breast cancers. Hormonal therapy and molecular targeted therapy are not indicated for the management of TNBC, resulting in poor outcomes. Because TNBC lacks clear-cut therapeutic targets, effective treatment strategies remain to be established. However, TNBC is known to share similar biologic characteristics with basal-like type breast cancer and is often accompanied by loss of functional BRCA, a gene-modifying enzyme. Breast cancer with BRCA1 or BRCA2 mutations is accompanied by activation of the enzyme poly(ADP-ribose) polymerase (PARP). PARP, a DNA base-excision repair enzyme, is known to play a central role in gene repair, along with BRCA. Because some breast cancers with BRCA1 or BRCA2 mutations are TNBC, the suppression of PARP has attracted attention as a new treatment strategy for TNBC. In this article, we review the clinical characteristics of TNBC, discuss problems in treatment, and briefly summarize the international development status of PARP inhibitors.     

Inhibition of ADP-evoked platelet aggregation by selected poly(ADP-ribose) polymerase inhibitors

Pathologic platelet activation has been implicated in the pathogenesis of ischemic heart disease. Since cardiomyocytes can be protected from ischemia-reoxygenation injury by poly(ADP-ribose) polymerase (PARP) inhibitors mimicking the adenine/ADP part of NAD, their structural resemblance to ADP may also enable the blockade of platelet aggregation via binding to ADP receptors.Blood samples drawn from healthy volunteers were pre-incubated with different concentrations of PARP inhibitors: 4-hydroxyquinazoline, 2-mercapto-4(3 H)-quinazolinone, or HO-3089. ADP-, collagen- and epinephrine-induced platelet aggregation was evaluated according to the method described by Born. The effect of PARP inhibitors on thrombocyte aggregation was also examined when platelets were sensitized by heparin and in the presence of incremental concentrations of ADP.All examined PARP inhibitors reduced the ADP-induced platelet aggregation in a dose-dependent manner (significant inhibition at 20 microM for HO-3089 and at 500 microM for the other agents; P < 0.05), even if platelets were sensitized with heparin. However, their hindrance on platelet aggregation waned as the concentration of ADP rose (no effect at 40 microM ADP). PARP inhibitors had minimal effect on both collagen- and epinephrine-induced platelet aggregation.Our study first demonstrates the feasibility of a design for PARP inhibitors that does not only protect against ischemia-reperfusion-induced cardiac damage but may also prevent thrombotic events.     

Novel modulators of poly(ADP-ribose) polymerase

The nuclear enzyme poly(ADP-ribose) polymerase (PARP)-1 has an important role in regulating cell death and cellular responses to DNA repair. Pharmacological inhibitors of PARP have entered clinical testing as cytoprotective agents in cardiovascular diseases and as adjunct antitumor therapeutics. Initially, it was assumed that the regulation of PARP occurs primarily at the level of DNA breakage: recognition of DNA breaks was considered to be the primary regulator (activator) or the catalytic activity of PARP. Recent studies have provided evidence that PARP-1 activity can also be modulated by several endogenous factors, including various kinases, purines and caffeine metabolites. There is a gender difference in the contribution of PARP-1 to stroke and inflammatory responses, which is due, at least in part, to endogenous estrogen levels. Several tetracycline antibiotics are also potent PARP-1 inhibitors. In this article, we present an overview of novel PARP-1 modulators.     

Clinical poly(ADP-ribose) polymerase inhibitors for the treatment of cancer

Poly(ADP-ribose) polymerase (PARP) is believed to play a critical role in the detection of DNA damage and initiation of DNA repair. Although inhibition of PARP has received increasing attention for therapeutic application in a wide variety of acute and chronic diseases, most of the current clinical data surrounding PARP inhibition is in the field of oncology. At least eight different PARP inhibitors have been, or are expected to be evaluated in the clinical oncology setting in 2007 and 2008. This review summarizes the most recently presented or published data on these therapeutic molecules, and discusses how these drugs may continue to be developed in the future.     

Inhibition of poly(ADP-ribose)polymerase activity by nucleoside analogs of thymidine.

The poly ADP-ribosylation of proteins catalyzed by poly(ADP-ribose)polymerase (PARP) is involved in a number of important cellular metabolic activities. We evaluated various analogs of deoxythymidine and deoxyuridine as inhibitors of PARP. Most of these compounds have antiviral and/or anticancer activities. The structural requirements for these nucleoside analogs to be inhibitors of PARP were determined. The compounds evaluated had various substitutions on the 2-, 4- and/or 5-position of the pyrimidine ring, as well as on the 2'-, 3'- and/or 5'-position of the pentose moiety. Inhibition of PARP was strongly dependent on the size of the alkyl or halogen substituent on the 5-position of the pyrimidine ring. Whereas the 5-position of the pyrimidine ring could be varied, alteration of the 2- or 4-position drastically decreased the inhibition of PARP. Kinetic analysis was performed with concentrations of 1-10 microM NAD+. The Ki values for many compounds were five to seven times lower than the Ki for 3-aminobenzamide, a previously described potent inhibitor of PARP. Compounds with combined substituents at both the 5-position of the pyrimidine ring and the 3'- or 5'-position of deoxyribose generally were potent inhibitors of PARP, as for example 3'-amino-2', 3'-dideoxy-(E)-5-(2-bromovinyl)uridine (Ki = 0.7 microM), or 5'-azido-2',5'-dideoxy-5-ethyluridine (Ki = 0.8 microM). The 5-halogenated analogs had Ki values of 18, 35, 110 and greater than 1000 microM for 5-iodo-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, 5-chloro-2'-deoxyuridine, and 5-fluoro-2'-deoxyuridine, respectively, and the 5-alkyl analogs had Ki values of 45, 2.2, 7, 16 and 180 microM for 5-methyl-2'-deoxyuridine, 5-ethyl-2'-deoxyuridine, 5-propyl-2'-deoxyuridine, 5-butyl-2'-deoxyuridine and 5-pentyl-2'-deoxyuridine, respectively. Two other compounds with substituents in the 5-position of the pyrimidine moiety also had potent activities: (E)-5-(2-bromovinyl)-2'-deoxyuridine (Ki = 6 microM) and 5-trifluoromethyl-2'-deoxyuridine (Ki = 1.6 microM). Compounds substituted in the 2'-, 3'- and/or 5'-position of the deoxyribose moiety were investigated and 5'-azido-5'-deoxythymidine, 5'-amino-5'-deoxythymidine, 3'-azido-3'-deoxythymidine and 3'-deoxythymidine (d2T) and Ki values of 12, 16, 18 and 30 microM, respectively.     

New inhibitors of poly(ADP-ribose) polymerase (PARP)

Poly(ADP-ribose) polymerase-1 (PARP-1), the most prominent member of the PARP family, is a DNA-binding protein that is activated by nicks in DNA occurring during inflammation, ischaemia, neurodegeneration or cancer therapy. Activated PARP-1 consumes NAD⁺ that is cleaved into nicotinamide and ADP-ribose and polymerises the latter onto nuclear acceptor proteins. This highly energy consuming process is pivotal for the maintenance of genomic stability although over-activation can culminate in cell dysfunction and necrosis. Therefore, PARP-1 is regarded as a promising target for the development of drugs useful in various forms of inflammation, ischaemia–reperfusion injury and as an adjunct in cancer therapy. This review summarises the structural classes of known PARP-1 inhibitors, with a focus on new inhibitors published for this target, between 2002 and July 2004. The chemistry and biological data disclosed in these patent applications are discussed in light of new structural knowledge of the catalytic domain of the PARP family and recent work with potent inhibitors demonstrating the effects of PARP inhibition in various animal disease models.     

聚腺苷二磷酸核糖聚合酶抑制剂的临床研究进展

聚腺苷二磷酸核糖聚合酶(PARP)在癌症治疗中是一个非常重要的新靶点,通过碱基切除修复方式对单股DNA进行修复。近年来,新的协同放疗或化疗的PARP抑制剂已经进入了I、II或III期临床试验。众多的试验数据表明PARP抑制剂不仅可以作为化疗和放疗的增敏剂,而且在BRCA1和BRCA2基因突变的乳腺癌中可单独使用,选择性杀死DNA修复缺陷的癌细胞。本文综述了PARP抑制剂的作用机制和临床研究结果,评估了其不良反应和潜在药效,并提出了临床策略中可能存在的问题以及未来发展方向。     

PARP抑制剂用于肿瘤治疗的研究进展

聚腺苷酸二磷酸核糖转移酶(poly(ADP-ribose)polymerase,PARP)是当今癌症治疗的一个新靶点,其能够催化ADP-核糖单元从烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD+)转移至各种受体蛋白。PARP参与DNA修复和转录调控,不但在调节细胞存活和死亡过程中具有关键作用,同时也是肿瘤发展和炎症发生过程中的主要转录因子。PARP在碱基切除修复的DNA单链缺口(SSBs)修复中具有关键作用,抑制其活性能够增强放疗和DNA损伤类化疗药物的效果。目前已有至少8个PARP抑制剂进入临床,最新的体内外实验表明PARP抑制剂不但能够作为放化疗增敏剂,单独使用也能选择性杀伤DNA修复缺陷的肿瘤细胞,如BRCA1和BRCA2缺陷的乳腺癌细胞。大量的临床试验证明:该类药物毒副作用小、效果明确且短期耐受性良好,对于癌症治疗前景广阔。本文主要对PARP抑制剂的原理及其研究进展进行综述。     

Inhibition of poly(ADP-ribose) polymerase activation attenuates beta-lapachone-induced necrotic cell death in human osteosarcoma cells

beta-Lapachone, a novel anticancer drug, induces various human carcinoma cells to undergo apoptotic cell death. However, we report here that, in human osteocarcinoma (U2-OS) cells, beta-lapachone induces necrosis rather than apoptosis. beta-Lapachone-induced necrotic cell death in U2-OS cells was characterized by propidium iodide uptake, cytochrome c release, a decreased mitochondrial membrane potential, and ATP depletion. The mitochondrial potential transition (MPT), including the reduction of the mitochondrial transmembrane potential and the release of mitochondrial cytochrome c, occurred in beta-lapachone-treated cells; cotreatment of these cells with cyclosporin A, an inhibitor of MPT pore, failed to prevent necrotic cell death. This indicates that the MPT transition does not play a crucial role in this process. Furthermore, beta-lapachone-induced necrosis was independent of oxidative stress and caspase activation. However, excessive poly(ADP-ribose) polymerase (PARP) activation and subsequent depletion of intracellular NAD(+) and ATP were seen in beta-lapachone-treated U2-OS cells. Cotreatment with a PARP inhibitor, 3-aminobenzamide, decreased beta-lapachone-induced PARP activation and provided significant protection from necrosis by preventing depletion of intracellular NAD(+) and ATP. Taken together, our results suggest that PARP plays an important role in the signaling pathway for beta-lapachone-induced necrosis in U2-OS cells.     

Cardioprotective effects of poly(ADP-ribose) polymerase inhibition.

Free radical and oxidant production in cardiac myocytes during ischemia/reperfusion, cardiomyopathy, cardiotoxic drug exposure and ageing leads to DNA strand-breakage which activates the nuclear enzyme poly(ADP-ribose) polymerase (PARP) and initiates an energy consuming, inefficient cellular metabolic cycle with transfer of the ADP-ribosyl moiety of NAD+ to protein acceptors. These processes lead to the functional impairment of the myocytes and promote myocyte death. During the last decade a growing number of experimental studies demonstrated the beneficial effects of PARP inhibition in cell cultures through rodent models and more recently in pre-clinical large animal models of regional and global ischemia/reperfusion injury and various forms of heart failure. The current article provides an overview of the experimental evidence implicating PARP as a pathophysiological modulator of cardiac myocyte injury in vitro and in vivo.     

Role of poly(ADP-ribose) polymerase in sulfur mustard toxicity

Sulfur mustard (SM) is a chemical warfare agent leading to severe blistering of skin and mucosal surfaces, and as a long-term effect, to an increased risk for malignancies. At the molecular level, SM acts as a bifunctional alkylating agent, leading to DNA mono-adducts and di-adducts. This review is focussed on the role of poly(ADP-ribosyl)ation in the cell and tissue responses to SM-induced damage and potential role of inhibitors of poly(ADP-ribosyl)ation as therapeutic agents for SM injury.     

Inhibition of poly(ADP-ribose) polymerase prevents vascular hyporesponsiveness induced by lipopolysaccharide in isolated rat aorta

Recent studies clearly show that there is a relationship between endotoxemia and impaired vascular responsiveness. The aim of this study was to investigate whether treatment with the new potent PARP inhibitor PJ34 could prevent the vascular hyporesponsiveness induced by lipopolysaccharide (LPS). Endotoxemia was induced in rats by LPS injection (20 mgkg-1, i.p.). Administration of LPS caused a decrease in mean blood pressure and an increase in heart rate. In endothelium-denuded rings of thoracic aorta from untreated rats, contractile responses to KCl and phenylephrine decreased after LPS injection. Furthermore, there was a significant loss of endothelium-dependent vasodilatation in response to acetylcholine in LPS-treated rats. The animals pretreated with PJ34 (10 mgkg-1, i.p., 30 min before LPS injection), the effect of LPS on vascular responsiveness was lower than the untreated ones. Pretreating the animals with PJ34 before the LPS challenge prevented the decline in mean blood pressure. However, this did not result in significant changes to the heart rate. The inhibitory effect of LPS treatment on both KCl- and phenylephrine-induced contraction responses was significantly antagonized by PJ34. Additionally, pretreatment of the rats with PJ34 attenuated the LPS-induced endothelial dysfunction in endothelium-intact aorta rings. This study demonstrates that PARP activation in the vascular system is an important contributory factor to the impaired vascular responsiveness associated with endotoxic shock. Hence, the pharmacological inhibition of PARP pathway might be an effective intervention to prevent endotoxin-induced vascular hyporesponsiveness.     

Inhibition of poly(ADP-ribose) polymerase attenuates lung tissue damage after hind limb ischemia-reperfusion in rats.

The objective of this study was to investigate the effects of 3-aminobenzamide (3-AB) on tissue damage in lung after hind limb ischemia-reperfusion (I/R), by assessing blood biochemical assay and histopathological analysis. Thirty-five adult Wistar rats were divided into five groups. After application of anaesthesia both hind limbs were occluded with tourniquets. Following ischemia period for 60 min, the tourniquets were removed allowing reperfusion for 120 min. The IR group received 0.5 ml of saline while the IR+AB group received 3-AB (10 mgkg(-1) intraperitoneally). The IR+DMSO group was given 0.5 ml 10% DMSO 30 min before the removal of the tourniquets. The control group received 0.5 ml saline and the AB group received 0.5 ml 3-AB (10 mgkg(-1)) intraperitoneally. At the end of the reperfusion period, mid-line sternotomy was performed. Blood samples were taken with cardiac puncture. Bronchoalveolar lavage (BAL) of the left lung was performed with saline. Right lung was preserved for histopathological evaluation and biochemical examination. Lung tissue malondialdehyde (MDA) and 3-nitrotyrosine levels, myeloperoxidase and Na+/K+ ATP-ase activities, wet to dry weight ratios, and plasma and BAL fluid MDA levels were determined. Histopathological evaluation was performed, too. Hind limb IR caused significant increase in the lung tissue 3-NT to total tyrosine ratio (p = 0.014), wet to dry weight ratio (p = 0.000), MPO activity (p = 0.000), and MDA levels (p = 0.000). The animals treated with 3-AB showed a statistically significant decrease in these values (p < 0.05). Na+/K+ ATP-ase activity which was found to be decreased significantly with IR, returned to near normal levels with 3-AB treatment. Additionally, lung tissue injury in IR group characterized with moderate interstitial congestion and neutrophil infiltration, showed remarkable amelioration following 3-AB treatment. Our results strongly support the view that poly(ADP-ribose) polymerase (PARP) plays an important role in the inflammatory process in hind limb I/R-induced lung injury and as a PARP inhibitor, 3-AB seems to have a potential to treat this inflammatory injury.     

Potential clinical applications of poly(ADP-ribose) polymerase (PARP) inhibitors.

Poly(ADP-ribose) polymerases (PARPs) are defined as cell signaling enzymes that catalyze the transfer of ADP-ribose units from NAD(+)to a number of acceptor proteins. PARP-1, the best characterized member of the PARP family, that presently includes six members, is an abundant nuclear enzyme implicated in cellular responses to DNA injury provoked by genotoxic stress (oxygen radicals, ionizing radiations and monofunctional alkylating agents). Due to its involvement either in DNA repair or in cell death, PARP-1 is regarded as a double-edged regulator of cellular functions. In fact, when the DNA damage is moderate, PARP-1 participates in the DNA repair process. Conversely, in the case of massive DNA injury, elevated PARP-1 activation leads to rapid NAD(+)/ATP consumption and cell death by necrosis. Excessive PARP-1 activity has been implicated in the pathogenesis of numerous clinical conditions such as stroke, myocardial infarction, shock, diabetes and neurodegenerative disorders. PARP-1 could therefore be considered as a potential target for the development of pharmacological strategies to enhance the antitumor efficacy of radio- and chemotherapy or to treat a number of clinical conditions characterized by oxidative or NO-induced stress and consequent PARP-1 activation. Moreover, the discovery of novel functions for the multiple members of the PARP family might lead in the future to additional clinical indications for PARP inhibitors.     

Role of the poly(ADP-ribose)polymerase activity in vancomycin-induced renal injury

The aim of the present study was to investigate the role of poly(ADP-ribose)polymerase (PARP) activity in vancomycin (VCM)-induced renal injury and to determine whether 1,5-isoquinelinediol (ISO), a PARP inhibitor agent, could be offered as an alternative therapy in VCM-induced renal impairment. Rats were divided into four groups as follows: (i) control (Group 1); (ii) VCM-treated (Group 2); (iii) VCM plus ISO-treated (Group 3); and (iv) ISO-treated (Group 4). VCM (200 mg/kg, i.p., twice daily) was administered to Groups 2 and 3 for 7 days. ISO (3 mg/kg/day, i.p.) treatment was started 24 h before the first administration of VCM and continued for 8 days. After the 14th VCM injection, the animals were placed in metabolic cages to collect urine samples. All the rats were sacrificed by decapitation, blood samples were taken in tubes and kidneys were excised immediately. Blood urea nitrogen (BUN) and plasma creatinine, and urinary N-acetyl-β-d-glucosaminidase (NAG, a marker of renal tubular injury) were used as markers of VCM-induced renal injury in rats. Light microscopy was used to evaluate semi-quantitative analysis of the kidney sections. Poly(ADP-ribose) (PAR, the product of activated PARP) and PARP-1 expressions in renal tissues were demonstrated by immunohistochemistry and Western blot. VCM administration increased BUN levels from 8.07 ± 0.75 mg/dL to 53.87 ± 10.11 mg/dL. The plasma creatinine levels were 0.8 ± 0.04 mg/dL and 3.38 ± 0.51 mg/dL for the control and VCM-treated groups, respectively. Also, urinary excretion of NAG was increased after VCM injection. Besides, there was a significant dilatation of the renal tubules, eosinophilic casts within some tubules, desquamation and vacuolization of renal tubule epithelium, and interstitial tissue inflammation in VCM-treated rats. In VCM-treated rats, both PAR and PARP-1 expressions were increased in renal tubular cells. ISO treatment attenuated VCM-induced renal injury, as indicated by BUN and plasma creatinine levels, urinary NAG excretion, and renal histology. PARP inhibitor treatment also decreased PAR and PARP-1 protein expressions similar to that of controls. Herewith, the overactivation of the PARP pathway may have a role in VCM-induced renal impairment and pharmacological inhibition of this pathway might be an effective intervention to prevent VCM-induced acute renal injury.     

PCB-induced endothelial cell dysfunction: Role of poly(ADP-ribose) polymerase

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants implicated in the development of pro-inflammatory events critical in the pathology of atherosclerosis and cardiovascular disease. PCB exposure of endothelial cells results in increased cellular oxidative stress, activation of stress and inflammatory pathways leading to increased expression of cytokines and adhesion molecules and ultimately cell death, all of which can lead to development of atherosclerosis. To date no studies have been performed to examine the direct effects of PCB exposure on the vasculature relaxant response which if impaired may predispose individuals to hypertension, an additional risk factor for atherosclerosis. Overactivation of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) following oxidative/nitrosative stress in endothelial cells and subsequent depletion of NADPH has been identified as a central mediator of cellular dysfunction. The aim therefore was to investigate whether 2,2′,4,6,6′-pentachlorobiphenyl (PCB 104) directly causes endothelial cell dysfunction via increased oxidative stress and subsequent overactivation of PARP. Exposure of ex vivo rat aortic rings to PCB 104 impaired the acetylcholine-mediated relaxant response, an effect that was dependent on both concentration and exposure time. In vitro exposure of mouse endothelial cells to PCB 104 resulted in increased cellular oxidative stress through activation of the cytochrome p450 enzyme CYP1A1 with subsequent overactivation of PARP and NADPH depletion. Pharmacological inhibition of CYP1A1 or PARP protected against the PCB 104-mediated endothelial cell dysfunction. In conclusion, the environmental contaminants, PCBs, can activate PARP directly impairing endothelial cell function that may predispose exposed individuals to development of hypertension and cardiovascular disease.     

Inhibitors of poly (ADP-ribose) polymerase modulate signal transduction pathways in colitis

During inflammatory bowel diseases, oxidative and nitrosative stress induces DNA damage and activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP), resulting in depletion of intracellular energetics, intestinal barrier dysfunction and cellular death. The aim of our study was to evaluate the therapeutic efficacy of in vivo inhibition of PARP in experimental colitis, which was induced by rectal instillation of trinitrobenzene sulfonic acid (TNBS) in rats. In vehicle-treated rats, TNBS treatment resulted in colonic erosion and ulceration. Neutrophil infiltration (indicated by myeloperoxidase activity in the colon) was associated with formation of nitrotyrosine and marked apoptosis. Elevated levels of plasma nitrate/nitrite, metabolites of nitric oxide (NO), were also found. These inflammatory events were associated with the activation of nuclear factor-kappa B (NF-kappa B) and activator protein-1 (AP-1) in the colon; NF-kappa B was maximally activated at 3 and 7 days, whereas AP-1 increased 1 day after TNBS administration and declined thereafter. Treatment of the rats with the PARP inhibitors, 3-aminobenzamide or 1,5-dihydroxyisoquinoline, resolved colonic damage and reduced plasma levels of NO metabolites. Resolution of the damage was associated with reduction of neutrophil infiltration, nitrotyrosine formation and apoptosis. Treatment with PARP inhibitors also reduced DNA binding of NF-kappa B and AP-1 in the colon. These data demonstrate that pharmacological inhibition of PARP ameliorates colitis. Reduction of the inflammatory process is associated with modification of the activation of signal transduction pathways.     

Inhibition of poly(ADP-ribose) polymerase (PARP) influences the mode of sulfur mustard (SM)-induced cell death in HaCaT cells

Sulfur mustard (SM) is a bifunctional alkylating agent. Its primary toxic consequence is severe skin damage with blisters, occurring after skin contact. These vesicant properties of SM have been linked to cell death of proliferating keratinocytes in the basal layer of the skin. Catalytic activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP-1) has been demonstrated to be a major event in response to high levels of DNA damage, and PARP-1 activation may be part of apoptotic signaling. In other contexts, overstimulation of PARP-1 triggers necrotic cell death because of rapid consumption of its substrate, beta-nicotinamide adenine dinucleotide (NAD+) and the consequent depletion of ATP. These findings prompted us to evaluate whether SM induces apoptosis in keratinocytes like HaCaT cells and to determine whether blocking of PARP enzyme activity with 3-aminobenzamide (3AB) can influence the mode of cell death. HaCaT cells were exposed to SM (10-1,000 microM; 30 min) and then cultivated in SM-free medium with or without 3AB for up to 48 h. This treatment resulted in a time and SM dose-dependent increase of apoptotic cell death characterized by PARP-1 cleavage and DNA fragmentation during the experimental period. After just 45 min of exposure to 1 mM SM, we observed a significant increase in PARP-1 activity in HaCaT cells. About 6 h after exposure, intracellular ATP levels were diminished by 22%, which seemed to be completely prevented by the addition of 3AB directly after exposure. However, 18 h later, this 3AB effect on the SM concentration-dependent loss of ATP was no longer detectable. Interestingly, the effect of SM on total cell viability was not changed by 3AB. However, the mode of cell death was influenced by 3AB exhibiting an increase of apoptotic cells and a concomitant decrease of necrotic HaCaT cells during the first 24 h after SM exposure. Our results indicate that SM concentrations of 1 mM or higher induce a prominent PARP activation leading to ATP depletion and necrosis. In contrast, lower concentrations of SM cause minor PARP activation and, especially, PARP-1 cleavage by caspase 3 without ATP depletion. Because ATP is required for apoptosis, we suggest that ATP acts as an early molecular switch from apoptotic to necrotic modes of SM-induced cell death, at least at high concentrations (> or =1 mM). Thus, the observed early proapoptotic effect of 3AB at lower SM concentrations may point to the influence of ATP-independent cell-death regulating mechanisms.     

Role of poly(ADP-ribose) polymerase activation in endotoxin-induced cardiac collapse in rodents

Reactive oxygen and nitrogen species are overproduced in the cardiovascular system during circulatory shock. Oxidant-induced cell injury involves the activation of poly(ADP-ribose) polymerase (PARP). Using a dual approach of PARP-1 suppression, by genetic deletion or pharmacological inhibition with the new potent phenanthridinone PARP inhibitor PJ34 [the hydrochloride salt of N-(oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide], we studied whether the impaired cardiac function in endotoxic shock is dependent upon the PARP pathway. Escherichia coli endotoxin (lipopolysaccharide, LPS) at 55 mg/kg, i.p., induced a severe depression of the systolic and diastolic contractile function, tachycardia, and a reduction in mean arterial blood pressure in both rats and mice. Treatment with PJ34 significantly improved cardiac function and increased the survival of rodents. In addition, LPS-induced depression of left ventricular performance was significantly less pronounced in PARP-1 knockout mice (PARP(-/-)) as compared with their wild-type littermates (PARP(+/+)). Thus, PARP activation in the cardiovascular system is an important contributory factor to the cardiac collapse and death associated with endotoxin shock.