PARP-1 Research Articles

Protective Effects of PARP-1 Knockout on Dyslipidemia-Induced Autonomic and Vascular Dysfunction in ApoE−/− Mice: Effects on eNOS and Oxidative Stress

The aims of this study were to investigate the role of poly(ADP-ribose) polymerase (PARP)-1 in dyslipidemia-associated vascular dysfunction as well as autonomic nervous system dysregulation. Apolipoprotein (ApoE)−/− mice fed a high-fat diet were used as a model of atherosclerosis. Vascular and autonomic functions were measured in conscious mice using telemetry. The study revealed that PARP-1 plays an important role in dyslipidemia-associated vascular and autonomic dysfunction. Inhibition of this enzyme by gene knockout partially restored baroreflex sensitivity in ApoE−/− mice without affecting baseline heart-rate and arterial pressure, and also improved heart-rate responses following selective blockade of the autonomic nervous system. The protective effect of PARP-1 gene deletion against dyslipidemia-induced endothelial dysfunction was associated with preservation of eNOS activity. Dyslipidemia induced PARP-1 activation was accompanied by oxidative tissue damage, as evidenced by increased expression of iNOS and subsequent protein nitration. PARP-1 gene deletion reversed these effects, suggesting that PARP-1 may contribute to vascular and autonomic pathologies by promoting oxidative tissue injury. Further, inhibition of this oxidative damage may account for protective effects of PARP-1 gene deletion on vascular and autonomic functions. This study demonstrates that PARP-1 participates in dyslipidemia-mediated dysregulation of the autonomic nervous system and that PARP-1 gene deletion normalizes autonomic and vascular dysfunctions. Maintenance of eNOS activity may be associated with the protective effect of PARP-1 gene deletion against dyslipidemia-induced endothelial dysfunction.

LMTK2 and PARP-2 gene polymorphism and azoospermia secondary to meiotic arrest

To investigate whether the human LMTK2 and PARP-2 gene defects are associated with azoospermia by meiotic arrest, mutational analysis was performed on Japanese men with azoospermia. Via direct sequencing, mutational screening was carried out on the exon region of the genes, using genomic DNAs from 18 Japanese men. Statistical analysis was done on the detected single nucleotide polymorphisms (SNPs) in the patients and normal controls. Nine SNPs were detected in LMTK2 and five SNPs were detected in PARP-2. There were no significant differences in the genotype distribution and allele frequencies between the two groups in LMTK2. However, the genotype frequency of heterozygotes in SNP1 of PARP-2 was higher in the patient group. The haplotype analysis revealed that SNP1-SNP4 (T-A) of PARP-2 was significantly more frequent in the patient group. The PARP-2 gene might be associated with azoospermia by meiotic arrest in humans.

The Difference of Gene Expression between Morphine and Codeine on Ramos Cells

The purpose of this study was to investigate the discrepancy of gene expression of cells treated with heroin or morphine and codeine. Firstly, microarray was used to screen for the difference of genes, which are different in expression between heroin and non-drug users. Compared to non- drug user, 80 genes were distinctly induced in drug users. Only one gene, APOBEC3A gene was up-regulation, and the others were down-regulation. The expressions of seven out of the eighty affected genes, including CAMP, MPP1, OGFR, PARP10 RGS12, HES7, and, APOBEC3A were then validated by real-time RT-PCR amplification by using a cultured cell model. Ramos cells treated with morphine and codeine indicated that both drugs could down regulate CAMP gene expression. Ramos cells treated with morphine showed that the expression of MPP1, RGSI2, OGFR, P4RP10, and HES7 resulted in down-regulation, but cells treated with codeine revealed no induction. The APOBEC3A mRNA was not influenced in Ramos cells treated with morphine, but up-regulated in codeine-treated cells. According to these results, we hypothesize that there arc different gene expressions between cells exposed to morphine and codeine.

SIRT1 Promotes Cell Survival under Stress by Deacetylation-Dependent Deactivation of Poly(ADP-Ribose) Polymerase 1

Poly(ADP-ribose) polymerase 1 (PARP1) and SIRT1 deacetylase are two NAD-dependent enzymes which play major roles in the decision of a cell to live or to die in a stress situation. Because of the dependence of both enzymes on NAD, cross talk between them has been suggested. Here, we show that PARP1 is acetylated after stress of cardiomyocytes, resulting in the activation of PARP1, which is independent of DNA damage. SIRT1 physically binds to and deacetylates PARP1. Increased acetylation of PARP1 was also detected in hearts of SIRT1(-/-) mice, compared to that detected in the hearts of SIRT1(+/+) mice, confirming a role of SIRT1 in regulating the PARP1 acetylation in vivo. SIRT1-dependent deacetylation blocks PARP1 activity, and it protects cells from PARP1-mediated cell death. We also show that SIRT1 negatively regulates the activity of the PARP1 gene promoter, thus suggesting that the deacetylase controls the PARP1 activity at the transcriptional level as well. These data demonstrate that the activity of PARP1 is under the control of SIRT1, which is necessary for survival of cells under stress conditions.

Apoptosis induced by celecoxib combinated with cisplatin in human osteosarcoma cell line MG-63 via down-regulation of PI3K/Akt

Objective To identify the anti-proliferation of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, and the combination of celecoxib and cisplatin in MG-63 cells, and to explore the poten-tial molecular mechanisms involved. Methods MG-63 cells were treated with the combination of celecoxib (50 μmol/L or 100 μmol/L) and/or cisplatin (10 μg/ml) for 48 h in serum-supplemented medium. Cell viabil-ity was measured by MTT assay; apoptosis was determined by electronmicroscope and flow cytometry (FCM); gene transcription and protein expression were detected by RT-PCR and/or Western blot analysis. Results MG-63 cells were significantly inhibited by celecoxib at G1 phase. The cisplatin-induced apoptosis was 5.93%, and potentiated to 6.66% and 37.15% while combining with celecoxib (50 μmol/L and 100 μmol/L) respectively. There was significant synergetic effect between celecoxib and cisplatin. The protein expression of COX-2 did not occur in cells treated with celecoxib. PI3K/Akt, survivin, Bcl-2 were significantly down-regulated in cells treated with the combination of celecoxib and cisplatin. Moreover, the decreased expres-sions of pro-caspase-9, pro-caspase-3 and cleaved PARP-1 were detected by Western blot analysis. And pAkt (Thr308), survivin and Bcl-2 levels down-regulated in cells treating with Wortmannin for 48 h, a spe-cific PI3K inhibitor. Conclusion Celecoxib exerts its anti-tumor activities through COX-2 independent mechanisms, which may be PI3K/Akt-dependent, and survivin and Bcl-2-related. PI3K may be at the center of the celecoxib effects, which play an essential role in the regulation of survivin and Bcl-2. Key words: Osteosarcoma; Cisplatin; Apoptosis; Caspases

Micronucleus Occurrence Related to Base Excision Repair Gene Polymorphisms in Chinese Workers Occupationally Exposed to Vinyl Chloride Monomer

To evaluate whether base excision repair gene polymorphisms contributed to susceptibility of chromosomal damage induced by vinyl chloride monomer (VCM). Cytokinesis block micronucleus test was performed on 185 VCM-exposed workers to detect chromosomal damage in peripheral lymphocytes. The PCR-restriction fragment length polymorphism technique was applied to detect polymorphisms of TDG, PARP1, and APE1 genes. Univariate and multivariate Poisson regression analysis was performed. There was a 1.198-fold increased micronucleus frequency for individuals carrying TDG 199Gly/Ser + Ser/Ser genotypes compared with those carrying Gly/Gly genotype (P < 0.05). Individuals with the combined genotypes of TDG 199Gly/Ser + Ser/Ser and APE1 64Ile/Val + Val/Val had a significantly increased micronucleus frequency compared with those carrying wild-type genotypes for both genes. VCM-exposed workers carrying TDG 199Gly/Ser + Ser/Ser genotypes were susceptible to chromosomal damage. A combination of TDG Gly199Ser and APE1 Ile64Val polymorphisms may modify the risk of chromosomal damage.

Sex differences in the response to activation of the poly (ADP-ribose) polymerase pathway after experimental stroke

It is increasingly recognized that histological and functional outcomes after stroke are shaped by biologic sex. Emerging data suggests that ischemic cell death pathways are sexually dimorphic (Hurn, P., Vannucci, S., Hagberg, H. (2005) Adult or perinatal brain injury: does sex matter?. Stroke 36, 193–195 ; Lang, J.T., McCullough, L.D. (2008) Pathways to ischemic neuronal cell death: are sex differences relevant?. J. Transl. Med. 6). Reducing neuronal nitric oxide (NO) or poly-ADP-ribose polymerase (PARP1) activation protects only the male brain (Hagberg, H., et al. PARP-1 gene disruption in mice preferentially protects males from perinatal brain injury. J. Neurochem. 90, 1068–1075 (2004)), and paradoxically enhances ischemic injury in females (McCullough, L.D., et al. Ischemic nitric oxide and poly (ADP-ribose) polymerase-1 in cerebral ischemia: male toxicity, female protection. J. Cereb. Blood Flow Metab. 25, 502–512 (2005)). In this study, we examined downstream mediators of NO/PARP activation to investigate possible mediators of ischemic sexual dimorphism. Nuclear translocation of Apoptosis Inducing Factor (AIF) was equivalent in wild type males and females after stroke and was unaffected by estrogen exposure. Deletion of PARP1 led to a dramatic reduction in stroke-induced poly (ADP-ribose) polymerase (PAR) formation and AIF translocation in both sexes, yet ischemic damage was reduced only in males. Subsequent examination of AIF-deficient Harlequin mice demonstrated that male Harlequin mice had less PAR formation, reduced AIF translocation and less ischemic damage than male wild type mice. In contrast, female Harlequin mice had no neuroprotective effect of gene deletion despite robust reductions in PAR formation and AIF translocation. Although equivalent activation of this cell death pathway occurs in both sexes after ischemia, detrimental effects are only present in males. AIF translocation and PAR formation do not mediate ischemic injury in the female brain, therefore agents designed to reduce PARP1 activation are unlikely to benefit females.

Inflammation and stress-related candidate genes, plasma interleukin-6 levels, and longevity in older adults

Interleukin-6 (IL-6) is an inflammatory cytokine that influences the development of inflammatory and aging-related disorders and ultimately longevity. In order to study the influence of variants in genes that regulate inflammatory response on IL-6 levels and longevity, we screened a panel of 477 tag SNPs across 87 candidate genes in >5000 older participants from the population-based Cardiovascular Health Study (CHS). Baseline plasma IL-6 concentration was first confirmed as a strong predictor of all-cause mortality. Functional alleles of the IL6R and PARP1 genes were significantly associated with 15%–20% higher baseline IL-6 concentration per copy among CHS European–American (EA) participants (all p<10−4). In a case/control analysis nested within this EA cohort, the minor allele of PARP1 rs1805415 was nominally associated with decreased longevity (p=0.001), but there was no evidence of association between IL6R genotype and longevity. The PARP1 rs1805415 – longevity association was subsequently replicated in one of two independent case/control studies. In a pooled analysis of all three studies, the “risk” of longevity associated with the minor allele of PARP1 rs1805415 was 0.79 (95%CI 0.62–1.02; p=0.07). These findings warrant further study of the potential role of PARP1 genotype in inflammatory and aging-related phenotypes.

Triple negative breast cancer: a phase 2, multi-center, open-label, randomized trial of gemcitabine/carboplatin (G/C), with or without BSI-201, a PARP inhibitor.

Abstract Abstract #2120 Background: PARP is a critical enzyme of cell proliferation and DNA repair and BSI-201 has been shown to be a potent inhibitor of PARP-1 in humans. Triple negative breast cancer (TNBC) shares important features with BRCA1-related breast cancer, a validated target for PARP inhibition. Our studies demonstrate that PARP-1 gene expression is statistically significantly upregulated in TNBC compared with normal breast tissue. The primary objective of this study is to assess the Clinical Benefit Rate (CBR=CR+PR+SD &amp;gt; 6 months) of Gemcitabine/Carboplatin with or without BSI-201 in patients with TNBC.&amp;#x2028; Methods: Eligible subjects are ≥ 18 years old who had received ≤ 2 prior chemotherapies for metastatic disease with histologically documented breast cancer that is ER-negative, PR-negative, and HER2-negative. Patients were randomized (1:1 ratio) to one of 2 study arms: a) arm 1: G/C alone; b) arm 2: BSI-201 + G/C. G/C was given on days 1 and 8; G = 1000 mg/m2, C at an AUC=2. In study arm 2, BSI-201 was administered I.V. twice weekly (days 1, 4, 8 and 11) at a dose of 5.6 mg/kg. Cycles were defined as being 21 days in duration. Modified RECIST criteria are used to assess tumor response every 6 weeks (every 2 cycles). Subjects who have progressive disease may crossover to receive BSI-201 and continue G/C. Archived tissue samples were retrieved for the assay of cancer related genes including PARP-1 by multiplex quantitative RT-PCR.&amp;#x2028; Results: To date, 50 subjects have been enrolled and treated for up to 8 cycles of therapy. Overall 39 of 50 (78%) subjects experienced at least 1 adverse event. The frequency and nature of the AE's do not differ between the two treatment groups. Gene expression profiling results from the first 28 patients enrolled confirm that the cancers of patients enrolled have uniformly low hormone receptor expression, variable HER2 receptor expression and significant upregulation of PARP-1 compared with normal breast tissue.&amp;#x2028; Conclusions: This is the first randomized study of a PARP inhibitor in cancer patients. BSI-201 is safe and well tolerated when given in combination with G/C and adverse events observed were consistent with the known safety profiles of G / C regimens. An updated evaluation of toxicity profile and PARP-1 expression analysis will be presented. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2120.

Poly(ADP-ribose) Polymerase-1 Down-regulates BRCA2 Expression through the BRCA2 Promoter

Expression of the BRCA2 tumor suppressor gene is tightly linked to its roles in DNA damage repair and maintenance of chromosomal stability and genomic integrity. Three transcription factors that activate (USF, NF-kappaB, and Elf1) and a single factor that represses (SLUG) BRCA2 promoter activity have been reported. In addition, a 67-bp region (-582 to -516) associated with inhibition of promoter activity has been identified. However, it remains unclear how the 67-bp region contributes to regulation of BRCA2 expression. Here, we describe the affinity purification of a 120-kDa protein that binds to a silencer-binding region within the 67-bp repression region of the BRCA2 promoter. Mass spectrometry revealed the identity of the protein as poly-(ADP-ribose) polymerase-1 (Parp-1). Gel shift, antibody super-shift, and chromatin immunoprecipitation (ChIP) assays demonstrated that Parp-1 is associated with the BRCA2 promoter both in vitro and in vivo. Furthermore, Parp-1 inhibitors (either 3-AB or NU1025) and Parp-1 gene specific siRNA resulted in increased levels of endogenous BRCA2 expression. Inhibition of Parp-1 activity (by 3-AB) reduced histone 3 lysine 9 acetylation and blocked Parp-1 binding to the BRCA2 promoter. These results indicate that Parp-1 down-regulates BRCA2 expression through an interaction with a repression region of the BRCA2 promoter.

Abstract 311: Poly(ADP-ribose) Polymerase-1 Inhibition Prevents High Fat Diet-induced Cardiac Hypertrophy by Modulating Matrix-destabilizing Proteases and Oxidative Stress

Background: We recently showed that PARP-1 is activated within atherosclerotic plaques and that inhibition of this enzyme, exerts protective effects against high fat diet-induced atherogenesis. Given the tight association between atherosclerosis and cardiac pathologies, we examined, whether PARP-1 is an active participant in high fat diet-induced cardiac hypertrophy and whether PARP-1 gene knockout prevents such pathological abnormalities. Methods: Apolipoprotein (ApoE)−/− mice fed a high-fat diet were used as a model of atherosclerosis. C57BL/6 ApoE−/− mice that are deficient in PARP-1 (ApoE-PARP-1d−/−) were generated in our laboratory. Hemodynamic parameters were obtained by a small animal-adapted ultrasound imaging system. Results: Hemodynamic parameters revealed that ApoE−/− mice on regular diet exhibited markers of cardiac hypertrophy. PARP-1 gene knockout markedly protected against dyslipidemia-induced cardiac hypertrophy. An increase in dyslipidemia burden further exacerbated the latter parameters in ApoE−/− mice. Inhibition of PARP-1 by gene deletion provided a significant protection against the parameters of cardiac hypertrophy. Histology showed enlarged hypertrophied cardiac myocytes in ApoE−/− mice with typical “boxcar-shaped” nuclei and high-fat diet caused separation of the myocardial fibers suggestive of dilated cardiomyopathy in these mice. PARP-1 gene deletion maintained the structural integrity of cardiac myocytes. MMP activity, measured by zymography, was dramatically increased in heart and aorta of ApoE−/− mice on high-fat diet compared to ApoE-PARP-1d−/− mice on similar diet regimen, which was accompanied by increased collagen degradation and mast cell degranulation. PARP-1 gene knockout was associated with increased TIMP-2 expression in various cells of vascular origin which seemed to neutralize the degenerative effects of MMP activity. Discussion: Our results demonstrate that PARP-1 gene deletion protects against dyslipidemia-induced cardiac hypertrophy and dilatations. We also provide evidence that PARP-1 inhibition protects against these pathologies by increasing expression of TIMP-2 and subsequent inhibition of tissue-degrading proteases in a mouse model of atherosclerosis.

PARP-1 Val762Ala polymorphism, CagA+ H. pylori infection and risk for gastric cancer in Han Chinese population

PARP-1 plays important role in the BER (base excision repair) and maintenance of genomic integrity. Previous study found the Val762Ala genetic variant in the PARP-1 gene contributed to susceptibility of some cancers and decreased PARP-1 enzyme activity in response to oxidative damage. Helicobacter pylori (H. pylori) infection was thought to be one of the major causes of gastric cancer. In this study, we investigated the association between the PARP-1 Val762Ala polymorphism, CagA(+) H. pylori infection, and the risk for gastric cancer. This hospital-based, case-control study was performed involving 556 individuals (236 cases with gastric cancer and 320 controls without evidence of neoplasm and gastrointestinal disease) using a PCR-RFLP method. Chi-square test and logistic regression analysis were used to count OR and 95% CI. 762Ala/Ala genotype was overrepresented in the cases (16.9%) compared with controls (10.3%), (OR, 1.942; 95% CI, 1.157-3.257, P = 0.011). Multivariate analysis showed that two factors were significantly associated with risk of gastric cancer, including CagA(+) H. pylori infection (OR, 2.562; 95% CI, 1.174-5.240, P = 0.037), PARP-1 762AA genotype (OR, 1.772; 95% CI, 1.065-3.867; P = 0.042). Stratification analysis indicated that among Cag(+) H. pylori positive subjects, 762Ala/Ala carriers had higher risk for developing gastric cancer compared with 762Val/Val carrier (OR, 2.337; 95% CI, 1.148-4.758; P = 0.017). PARP-1 762Ala/Ala could be a risk factor for gastric cancer in Han Chinese population; PARP-1 762Val/Ala polymorphism and Cag(+) H. pylori infection jointly contribute to higher risk for gastric cancer.

Induction of apoptosis by the inhibitors of poly(ADP-ribose)polymerase in HeLa cells

To investigate the role of poly(ADP-ribose)polymerase (PARP) in the physiological condition of cell growth, we studied the ability of PARP inhibitors to induce apoptosis. Benzamide (BA) and 4-amino-1,8-naphthalimide (NAP), two well-known inhibitors of PARP, treatment increased nuclear fragmentation and caspase-3 activity in HeLa (Human cervical cancer cell line) cells. The increase of cellular NAD(+) level was observed in HeLa cells treated with BA in comparison with untreated control cells. For unrevealing the specific PARP family member responsible for such induction of apoptosis we knocked down and over-expressed PARP-1 gene in HeLa cells. PARP-1 knock down cells were sensitive to BA induced nuclear fragmentation and caspase-3 activation while exogenous expression of PARP-1 rendered cells resistant to BA induced apoptosis. This result indicated that inhibition of PARP-1 resulted in induction of apoptosis.

Leu54Phe and Val762Ala polymorphisms in the poly(ADP-ribose)polymerase-1 gene are associated with diabetic polyneuropathy in Russian type 1 diabetic patients

Poly(ADP-ribose) polymerase-1 (PARP-1) is an ubiquitous DNA-binding protein involved in the cellular response to various genotoxic agents. Excessive PARP-1 activation is known to lead to the depletion of intracellular NAD+ and ATP pools and hence to threat cell survival. Therefore, PARP-1 could be involved in neuronal death and contribute to the development of diabetic polyneuropathy (DPN). This study addressed the association of Leu54Phe and Val762Ala polymorphisms of PARP-1 with DPN in Russian type 1 diabetic (T1D) patients. Eighty-six T1D patients with severe DPN and 93 T1D patients with no clinical signs of DPN have been studied by a polymerase chain reaction restriction fragment length polymorphism approach. Using Fisher's exact test revealed the association of the Phe54 and Val762 variants of PARP-1 (odds ratio (OR), 1.66 and 2.88, respectively) with increased risk of DPN in T1D. These results suggest that the PARP1 gene is involved in the pathogenesis of diabetic neuropathy in a Russian population. Additionally, a logistic regression analysis revealed a significant association between the neurological variances such as vibration detection threshold (OR, 2.08), vibration and temperature perception thresholds (OR, 1.32 and 1.67, respectively), and sensory and motor nerve conduction velocities (OR, 2.34 and 2.58, respectively), with DPN.

P57 The PARP1 gene is over-expressed in triple negative breast cancer

P57 The PARP1 gene is over-expressed in triple negative breast cancer

Poly(ADP-Ribose) Polymerase Inhibition Reduces Atherosclerotic Plaque Size and Promotes Factors of Plaque Stability in Apolipoprotein E–Deficient Mice

Poly(ADP-ribose) polymerase (PARP) was suggested to play a role in endothelial dysfunction that is associated with a number of cardiovascular diseases. We hypothesized that PARP may play an important role in atherogenesis and that its inhibition may attenuate atherosclerotic plaque development in an experimental model of atherosclerosis. Using a mouse (apolipoprotein E [ApoE](-/-)) model of high-fat diet-induced atherosclerosis, we demonstrate an association between cell death and oxidative stress-associated DNA damage and PARP activation within atherosclerotic plaques. PARP inhibition by thieno[2,3-c]isoquinolin-5-one reduced plaque number and size and altered structural composition of plaques in these animals without affecting sera lipid contents. These results were corroborated genetically with the use of ApoE(-/-) mice that are heterozygous for PARP-1. PARP inhibition promoted an increase in collagen content, potentially through an increase in tissue inhibitor of metalloproteinase-2, and transmigration of smooth muscle cells to intima of atherosclerotic plaques as well as a decrease in monocyte chemotactic protein-1 production, all of which are markers of plaque stability. In PARP-1(-/-) macrophages, monocyte chemotactic protein-1 expression was severely inhibited because of a defective nuclear factor-kappaB nuclear translocation in response to lipopolysaccharide. Furthermore, PARP-1 gene deletion not only conferred protection to foam cells against H2O2-induced death but also switched the mode of death from necrosis to apoptosis. Our results suggest that PARP inhibition interferes with plaque development and may promote plaque stability, possibly through a reduction in inflammatory factors and cellular changes related to plaque dynamics. PARP inhibition may prove beneficial for the treatment of atherosclerosis.

PARP-1 and iNOS relationship in allergen-induced lung inflammation: dependence of iNOS expression on PARP-1 and its dispensability for eosinophil recruitment post-IL-5 production (98.1)

Abstract The inducible nitric oxide synthase (iNOS) has been suggested to play an important role in the pathogenesis of asthma by contributing to eosinophilia and the ensuing oxidative damage after allergen exposure. IL-5 was shown to induce iNOS expression and that a reduction in iNOS expression reduces airway recruitment of eosinophils in response to allergen exposure. We recently showed that PARP-1 also plays a critical role in the pathogenesis of the disease by promoting eosinophil recruitment by participating in the cascade leading to IL-5 production. Here, we investigated the interrelationship between PARP-1 and iNOS in inducing eosinophilia. Our results show that iNOS gene deletion reduces eosinophil recruitment but to lesser degree as compared to PARP-1 gene deletion, suggesting a partial role for this protein in airway inflammation. PARP-1 gene deletion severely reduced iNOS expression in the airways of ovalbumin (allergen)-challenged mice and consequent nitration of target proteins and this phenomenon was closely associated with an impairment of p65 NF-κB nuclear translocation and phosphorylation of its inhibitor, I-κBα. IL-5-intranasal administration restored the eosinophilia in ovalbumin-challenged PARP-1−/− mice but did not reverse iNOS expression, suggesting that iNOS may be dispensable for eosinophil recruitment after IL-5 production. Altogether, our results further support the role of PARP-1 in the process of eosinophil recruitment during allergen-induced lung inflammation and provide evidence for a potential specific relationship between PARP-1 and iNOS. Support: NIH-HL072889 and 1P20RR18766 to HB.

Array comparative genomic hybridization analysis of chromosomal imbalances and their target genes in gastrointestinal stromal tumors

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract. The tumors characteristically harbor KIT or PDGFRA mutations, and mutant tumors respond to imatinib mesylate (Glivectrade mark). Chromosomal imbalances resulting in altered gene dosage are known to have a role in the molecular pathogenesis of these tumors, but the target genes remain to be identified. The present study aimed to identify some of these genes. In total, 35 GIST samples were screened for chromosomal imbalances by array-based comparative genomic hybridization. A cDNA array was used to define the minimal common overlapping areas of DNA copy number change. Eight confirmative, replicate hybridizations were performed using an oligonucleotide array. The most recurrent copy number losses were localized to 14q, 22q, and 1p. Gains were less common with 8q being the most recurrent. Two recurrent deleted regions of 14q were 14q11.2 harboring the PARP2, APEX1, and NDRG2 genes and 14q32.33 harboring SIVA. Additional target candidates were NF2 at chromosome 22, CDKN2A/2B at 9p, and ENO1 at 1p for copy number losses, and MYC at 8q for copy number gains. Array CGH proved to be an effective tool for the identification of chromosome regions involved in the development and progression of GISTs.

Regulatory Cross-talk in the Double par Locus of Plasmid pB171

The double par locus of Escherichia coli virulence factor pB171 consists of two adjacent and oppositely oriented par loci of different types, called par1 and par2. par1 encodes an actin ATPase (ParM), and par2 encodes an oscillating, MinD-like ATPase (ParA). The par loci share a central cis-acting region of approximately 200 bp, called parC1, located between the two par loci. An additional cis-acting region, parC2, is located downstream of the parAB operon of par2. Here we show that ParR of par1 and ParB of par2 bind cooperatively to unrelated sets of direct repeats in parC1 to form the cognate partition and promoter repression complexes. Surprisingly, ParB repressed transcription of the noncognate par operon, indicating cross-talk and possibly epistasis between the two systems. The par promoters, P1 and P2, affected each other negatively. The DNA binding activities of ParR and ParB correlated well with the observed transcriptional regulation of the par operons in vivo and in vitro. Integration host factor (IHF) was identified as a novel factor involved in par2-mediated plasmid partitioning.

Expression of the Gene Encoding Poly(ADP-ribose) Polymerase-1 Is Modulated by Fibronectin during Corneal Wound Healing

Poly(ADP-ribose) polymerase (PARP)-1 is a nuclear enzyme essential in several cellular functions such as DNA repair, DNA transcription, carcinogenesis, and apoptosis. Expression of the PARP-1 gene is mainly dictated by the transcription factor Sp1. Fibronectin (FN), a component from the extracellular matrix transiently expressed at high levels during wound healing of the corneal epithelium, was reported to exert a positive influence on expression of the alpha5 integrin subunit gene promoter by altering the state of Sp1 phosphorylation, a process that depended on the activation of the ERK signaling pathway. The present study was undertaken to investigate whether PARP-1 gene expression might be similarly regulated by FN through the same signaling pathways and attempted to link expression of this gene to corneal wound healing in vitro. Expression of PARP-1, Sp1/Sp3, ERK1/2, phospho-ERK1/2, P38 and phospho-P38 was monitored by Western blot in cultures of rabbit corneal epithelial cells (RCECs) grown on FN in the presence of inhibitors of the MAPK, PI3K, and P38 signaling pathways. Electrophoretic mobility shift assays (EMSAs) were conducted to assess the binding of Sp1 and Sp3 in nuclear extracts from RCECs grown on FN in the presence of inhibitors. Plasmids bearing the PARP-1 promoter fused to the CAT reporter gene were also transfected into RCECs grown under similar culture conditions to assess the influence of these inhibitors on PARP-1 promoter activity. Expression of PARP-1, Sp1, and Sp3 increased considerably in RCECs grown on FN and translated into increased binding of Sp1 and Sp3 to their DNA target sites. In addition, FN increased PARP-1 promoter activity in a cell-density-dependent manner. Inhibition of both the MAPK and the PI3K pathways entirely abolished these properties. PARP-1 gene expression was strongly activated by FN through alterations in the phosphorylation state of Sp1 and Sp3 that resulted from the activation of the MAPK and PI3K signaling pathways, thereby suggesting that PARP-1 may play a critical function during the highly proliferative phase that characterizes wound healing of the corneal epithelium.