Poly (ADP-ribose) Polymerase 1 mRNA Research Articles

Sevoflurane Preconditioning Rescues PKMζ Gene Expression from Broad Hypoxia-Induced mRNA Downregulation Correlating with Improved Neuronal Recovery

Hypoxia due to stroke is a major cause of neuronal damage, leading to loss of cognition and other brain functions. Sevoflurane preconditioning improves recovery after hypoxia. Hypoxia interferes with protein expression at the translational level; however, its effect on mRNA levels for neuronal protein kinase and anti-apoptotic genes is unclear. To investigate the link between sevoflurane preconditioning and gene expression, hippocampal slices were treated with 4% sevoflurane for 15 min, a 5 min washout, 10 min of hypoxia, and 60 min of recovery. We used quantitative PCR to measure mRNA levels in the CA1 region of rat hippocampi. The mRNA levels for specific critical proteins were examined, as follows: Protein kinases, PKCγ (0.22), PKCε (0.38), and PKMζ (0.55) mRNAs, and anti-apoptotic, bcl-2 (0.44) and bcl-xl (0.41), were reduced 60 min after hypoxia relative to their expression in tissue not subjected to hypoxia (set to 1.0). Sevoflurane preconditioning prevented the reduction in PKMζ (0.88 vs. 1.0) mRNA levels after hypoxia. Pro-apoptotic BAD mRNA was not significantly changed after hypoxia, even with sevoflurane preconditioning (hypoxia 0.81, sevo hypoxia 0.84 vs. normoxia 1.0). However, BAD mRNA was increased by sevoflurane in non-hypoxic conditions (1.48 vs. 1.0), which may partially explain the deleterious effects of volatile anesthetics under certain conditions. The DNA repair enzyme poly ADP-ribose polymerase 1 (PARP-1) was increased by sevoflurane in tissue not subjected to hypoxia (1.23). PARP-1 mRNA was reduced in untreated tissue after hypoxia (0.21 vs. 1.0); sevoflurane did not improve PARP-1 after hypoxia (0.27). Interestingly, the mRNA level of the cognitive kinase PKMζ, a kinase essential for learning and memory, was the only one protected against hypoxic downregulation by sevoflurane preconditioning. These findings correlate with previous studies that found that sevoflurane-induced improvement of neuronal survival after hypoxia was dependent on PKMζ. Maintaining mRNA levels for critical proteins may provide an important mechanism for preserving neuronal function after stroke.

Regulation of Alternative Splicing by PARP1 in HTR-8/Svneo Cells: Implications for Placental Development and Spontaneous Abortion.

Alternative splicing affects gene expression during placental development. The present study aimed to identify poly (ADP-ribose) polymerase 1 (PARP1)-regulated alternative splicing events in HTR-8/Svneo cells. Decidual tissues were collected from women with induced abortion and spontaneous abortion. PARP1 transcription was quantified by RT-qPCR. Small interfering RNA (siRNA) was used to knock down the PARP1 expression in HTR-8/Svneo cells. The transfection efficiency was verified by RT-qPCR and Western blotting. Total RNA was extracted, and the RNA-sequencing approach was used to identify alternative splicing events and transcriptomes. The PARP1 knockdown-induced differentially expressed genes with changes in alternative splicing events were quantified by RT-qPCR. Functional analysis, which included the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways, was performed. The PARP1 mRNA expression increased in decidual tissues in the spontaneous abortion group, when compared to the induced abortion group. However, the PARP1 knockdown significantly downregulated 1491 genes and upregulated 881 genes in HTR-8/Svneo cells. Furthermore, 227 genes that underwent alternative splicing were identified, and these were differentially expressed in siPARP1 cells, when compared to siNC cells. The functional analysis revealed that these alternative splicing genes affected the functional phenotypes of extravillous cytotrophoblasts. Furthermore, the PARP1 knockdown led to alterations in gene expression and specific alternative splicing patterns in extravillous trophoblasts.

Poly (ADP-Ribose) Polymerase 1 Induces Cyclic GMP-AMP Synthase-stimulator of Interferon Genes Pathway Dysregulation to Promote Immune Escape of Colorectal Cancer Cells.

Colorectal cancer (CRC) ranks third globally in cancer incidence and mortality, posing a significant human concern. Recent advancements in immunotherapy are noteworthy. This study explores immune modulation for CRC treatment. Initially targeting poly (ADP-ribose) polymerase 1 (PARP-1), a gene overexpressed in CRC tissues per The Cancer Genome Atlas, we examined its correlation with immune cell infiltration using the Tumor Immune Estimation Resource tool. Quantitative reverse transcription polymerase chain reaction assessed PARP-1 mRNA and inflammation-related gene expression in tumor tissues and cells. Assessing CD8 + T-cell proliferation and cytotoxicity towards HCT116 cells involved carboxyfluorescein diacetate succinimidyl ester and lactate dehydrogenase kits. Chemotaxis was gauged using a Transwell system in a CD8 + T-cell coculture setup, with immunofluorescence revealing cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) levels in HCT116 cells. Enzyme-linked immunosorbent assay kits measured CD8 + T-cell cytokine secretion. The findings suggested that PARP-1 was overexpressed in CRC tissues and cells and this overexpression was positively correlated with Treg cell infiltration. Overexpression of PARP-1 could significantly reduce the proportion of cGAS and STING-positive cells in HCT116 cells, dampen the proliferation, tumor-killing capacity, and chemotaxis of CD8 + T cells, and inhibit the secretion of related cytokines. The introduction of STING agonists could reverse the effects caused by overexpressed PARP-1. In vivo experiments affirmed the independent anti-tumor effects of PARP-1 inhibitors and STING agonists, synergistically inhibiting tumor growth. Silencing PARP-1 in HCT116 cells potentially boosts CD8 + T-cell activity against these cells through the cGAS-STING pathway.

Study on the molecular mechanism of autophagy and apoptosis induced by ultrafine carbon black in human bronchial epithelial cells and the intervention effect of N-acetylcysteine

Objective: To investigate the molecular mechanism of autophagy and apoptosis induced by ultrafine carbon black in human bronchial epithelial cells (BEAS-2B cells), and to study the intervention effect and mechanism of N-acetylcysteine (NAC) on ultrafine carbon black-induced oxidative damage in BEAS-2B cells. Methods: In March 2023, BEAS-2B cells were used as research object, an in vitro airway model exposed to ultrafine carbon black was constructed. A control group and three carbon black exposure groups (50, 100, 200 μg/ml) were set up, and the cells were treated with corresponding concentrations of ultrafine carbon black for 24 hours. In addition, the experiment was divided into control group, NAC+ control group, 100 μg/ml carbon black exposure group and NAC+ exposure group. The corresponding groups were treated with 2 mmol/L NAC for 1 h and 100 μg/ml ultrafine carbon black for 24 h, respectively. Cell viability was measured by CCK-8 assay. Intracellular reactive oxygen species (ROS) level was detected by chemical fluorescence method. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), as well as the content of malondialdehyde (MDA) were detected by colorimetry. The mRNA and protein expressions of autophagy-related genes[Atg5, Atg7, Beclin1, microtubule-associated protein light chain 3B (LC3B), p62 and lysosome-associated membrane protein 2 (LAMP2) ] and apoptosis-related genes [B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase3, Caspase9 and poly (ADP-ribose) polymerase 1 (PARP1) ] were determined by fluorescence quantitative PCR and Western blot. Cell apoptosis was determined by flow cytometry. Results: Compared with the control group, the relative survival rates of BEAS-2B cells in 50, 100, 200 μg/ml carbon black exposure groups were significantly decreased, the levels of ROS and MDA were significantly increased, and the activities of SOD, GSH-Px and CAT were significantly decreased (P<0.05). The relative survival rate, ROS and MDA levels, SOD, GSH-Px and CAT activities were significantly correlated with the exposure dose of ultrafine carbon black (r(s)=-0.755, 0.826, 0.934, -0.810, -0.880, -0.840, P<0.05). Compared with the control group, the relative expression levels of Atg5, Atg7, Beclin1, LC3B, p62, LAMP2, Bax, Caspase3, Caspase9, PARP1 mRNA and Atg5, Atg7, Beclin1, LC3BⅡ, p62, LAMP2, Bax, cleaved Caspase3 (C-Caspase3), cleaved Caspase9 (C-Caspase9), cleaved PARP1 (C-PARP1) protein and the ratio of LC3BⅡ/LC3BⅠ in 50, 100 and 200 μg/ml carbon black exposure groups were significantly increased, while the relative expression levels of Bcl-2 mRNA and protein were significantly decreased (P<0.05). The changes of the above indexes were significantly correlated with the exposure dose of carbon black (r(s)=0.892, 0.879, 0.944, 0.892, 0.828, 0.880, 0.814, 0.794, 0.931, 0.918, 0.813, 0.866, 0.774, 0.695, 0.918, 0.761, 0.794, 0.944, 0.833, 0.866, 0.905, -0.886, -0.748, P<0.05). Compared with 100 μg/ml carbon black exposure group, the relative survival rate, the activities of SOD, GSH-Px and CAT in NAC+exposure group were significantly increased, while the levels of ROS and MDA were significantly decreased, and the relative expression levels of LC3B, p62 and Caspase3 mRNA and protein as well as the ratio of LC3BⅡ/LC3BⅠ were significantly decreased, and the differences were statistically significant (P<0.05). Compared with the control group, the apoptosis rates of BEAS-2B cells in 50, 100, 200 μg/ml carbon black exposure groups were significantly increased (P<0.05), and there was a significant positive correlation between ultrafine carbon black exposure dose and cell apoptosis rate (r(s)=0.944, P<0.05). While compared with 100 μg/ml carbon black exposure group, the apoptosis rate of NAC+exposure group was significantly decreased, and the difference was statistically significant (P<0.05) . Conclusion: Cell autophagy and apoptosis may be important pathophysiological mechanisms of ultrafine carbon black-induced oxidative damage in BEAS-2B cells. NAC can alleviate the occurrence of BEAS-2B cell damage caused by ultrafine carbon black by regulating oxidative stress and the cascading autophagy and apoptosis pathways.

A Knockout of Poly(ADP-Ribose) Polymerase 1 in a Human Cell Line: An Influence on Base Excision Repair Reactions in Cellular Extracts.

Base excision repair (BER) is the predominant pathway for the removal of most forms of hydrolytic, oxidative, and alkylative DNA lesions. The precise functioning of BER is achieved via the regulation of each step by regulatory/accessory proteins, with the most important of them being poly(ADP-ribose) polymerase 1 (PARP1). PARP1's regulatory functions extend to many cellular processes including the regulation of mRNA stability and decay. PARP1 can therefore affect BER both at the level of BER proteins and at the level of their mRNAs. Systematic data on how the PARP1 content affects the activities of key BER proteins and the levels of their mRNAs in human cells are extremely limited. In this study, a CRISPR/Cas9-based technique was used to knock out the PARP1 gene in the human HEK 293FT line. The obtained cell clones with the putative PARP1 deletion were characterized by several approaches including PCR analysis of deletions in genomic DNA, Sanger sequencing of genomic DNA, quantitative PCR analysis of PARP1 mRNA, Western blot analysis of whole-cell-extract (WCE) proteins with anti-PARP1 antibodies, and PAR synthesis in WCEs. A quantitative PCR analysis of mRNAs coding for BER-related proteins-PARP2, uracil DNA glycosylase 2, apurinic/apyrimidinic endonuclease 1, DNA polymerase β, DNA ligase III, and XRCC1-did not reveal a notable influence of the PARP1 knockout. The corresponding WCE catalytic activities evaluated in parallel did not differ significantly between the mutant and parental cell lines. No noticeable effect of poly(ADP-ribose) synthesis on the activity of the above WCE enzymes was revealed either.

B-cell lymphoma-extra large is a promising drug target in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is an aggressive skin tumour with neuroendocrine differentiation. Immunotherapies are effective in the treatment of patients with advanced-stage MCC, but for patients whose tumours cannot be controlled by the immune system, alternative approaches are urgently needed. To identify overexpressed oncogenes as potential drug targets for MCC. NanoString platform, digital droplet polymerase chain reaction (ddPCR) and fluorescence in situ hybridization (FISH) assays were used to determine copy number variations (CNVs); BCL2L1 and PARP1 mRNA expression levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR), B-cell lymphoma extra-large (Bcl-xL) and poly (ADP-ribose) polymerase 1 (PARP1) protein by immuno-blot. Specific Bcl-xL inhibitors and a PARP1 inhibitor were used alone or in combination to test their antitumour effect. Screening for CNVs in 13 classic Merkel cell polyomavirus (MCPyV)-positive and MCPyV-negative MCC cell lines revealed BCL2L1 gains and amplifications, confirmed by ddPCR in 10 cell lines. By ddPCR and FISH, we demonstrated that BCL2L1 gains are present in tumour tissue. BCL2L1 copy number gains were associated with increased Bcl-xL mRNA and protein expression. However, high Bcl-xL expression was not restricted to MCC cells harbouring a BCL2L1 gain/amplification, suggesting additional epigenetic means of regulation. The functional relevance of Bcl-xL in MCC cells was demonstrated by the fact that specific Bcl-xL inhibitors (A1331852 and WEHI-539) led to the induction of apoptosis. Owing to the strong expression and activation of PARP1 in MCC cell lines, we next tested the combination of Bcl-xL inhibitors with the PARP1 inhibitor olaparib, which showed synergistic antitumour effects. Bcl-xL, which is highly expressed in MCC, appears to be an attractive therapeutic target for the treatment of this tumour, especially as the effect of specific Bcl-xL inhibitors is synergistically enhanced by simultaneous PARP inhibition.

Novel Injury Scoring Tool for Assessing Brain Injury following Neonatal Hypoxia-Ischemia in Mice

The variability of severity in hypoxia-ischemia (HI)-induced brain injury among research subjects is a major challenge in developmental brain injury research. Our laboratory developed a novel injury scoring tool based on our gross pathological observations during hippocampal extraction. The hippocampi received scores of 0–6 with 0 being no injury and 6 being severe injury post-HI. The hippocampi exposed to sham surgery were grouped as having no injury. We have validated the injury scoring tool with T2-weighted MRI analysis of percent hippocampal/hemispheric tissue loss and cell survival/death markers after exposing the neonatal mice to Vannucci’s rodent model of neonatal HI. In addition, we have isolated hippocampal nuclei and quantified the percent good quality nuclei to provide an example of utilization of our novel injury scoring tool. Our novel injury scores correlated significantly with percent hippocampal and hemispheric tissue loss, cell survival/death markers, and percent good quality nuclei. Caspase-3 and Poly (ADP-ribose) polymerase-1 (PARP1) have been implicated in different cell death pathways in response to neonatal HI. Another gene, sirtuin1 (SIRT1), has been demonstrated to have neuroprotective and anti-apoptotic properties. To assess the correlation between the severity of injury and genes involved in cell survival/death, we analyzed caspase-3, PARP1, and SIRT1 mRNA expressions in hippocampi 3 days post-HI and sham surgery, using quantitative reverse transcription polymerase chain reaction. The ipsilateral (IL) hippocampal caspase-3 and SIRT1 mRNA expressions post-HI were significantly higher than sham IL hippocampi and positively correlated with the novel injury scores in both males and females. We detected a statistically significant sex difference in IL hippocampal caspase-3 mRNA expression with comparable injury scores between males and females with higher expression in females.

Pharmacological targets and molecular mechanisms of plumbagin to treat colorectal cancer: A systematic pharmacology study

Plumbagin (PL) pharmacologically plays the anti-proliferative effects in cancer cells, including effective suppression of colorectal cancer (CRC). However, the exact molecular mechanism of PL to treat CRC remains unclear. Using available SwissTargetPrediction and SuperPred databases, the anti-cancer biotargets of PL were identified, and the CRC-diseased targets were obtained through a DisGeNET database. The biological processes, and signaling pathways of PL to treat CRC were identified and visualized. Further, clinical and cell culture data were used to validate some bioinformatic findings. As shown in bioinformatics findings, 64 predictive biotargets of PL to treat CRC were collected, and 7 most important biotargets of tumor protein p53 (TP53), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), mitogen-activated protein kinase 1 (MAPK1), E1A-associated protein p300 (EP300), poly (ADP-ribose) polymerase 1 (PARP1), nuclear factor kappa p65 protein (RELA), Bcl-2 like protein 1 (BCL2L1) were identified respectively. In addition, top 20 functional biological processes, signaling pathways of PL to treat CRC were screened and prioritized. In human study, CRC samples showed elevated expressions of neoplastic MAPK1, PARP1 mRNAs and reduced EP300 mRNA level. In cell culture study, PL-treated CRC cells resulted in down-regulated MAPK1, PARP1 mRNA expressions and up-regulation of EP300 mRNA level, characterized with suppressed cell proliferation. Taken together, the therapeutic biotargets and molecular mechanisms of PL to treat CRC were screened and identified by using a systematic pharmacology analysis, and some bioinformatic findings were validated in clinical and cell line experiments. Potentially, these hub biotargets may be the biomarkers for CRC detection and treatment.

Parthanatos-associated proteins are stimulated intraocularly during development of experimental murine cytomegalovirus retinitis in mice with retrovirus-induced immunosuppression.

The mechanisms that contribute to retinal tissue destruction during the onset and progression of AIDS-related human cytomegalovirus (HCMV) retinitis remain unclear. Evidence for the stimulation of multiple cell death pathways including apoptosis, necroptosis, and pyroptosis during the pathogenesis of experimental murine cytomegalovirus (MCMV) retinitis in mice with retrovirus-induced immunosuppression (MAIDS) has been reported. Parthanatos is a caspase-independent cell death pathway mediated by rapid overactivation of poly (ADP-ribose) polymerase-1 (PARP-1) and distinct from other cell death pathways. Using the MAIDS model of MCMV retinitis, studies were performed to test the hypothesis that intraocular MCMV infection of mice with MAIDS stimulates parthanatos-associated messenger RNAs (mRNAs) and proteins within the eye during the development of retinal necrosis that takes place by 10 days after MCMV infection. MCMV-infected eyes of MAIDS mice exhibited significant stimulation of PARP-1 mRNA and proteins at 3 days after infection but declined thereafter at 6 and 10 days after infection. Additional studies showed the intraocular stimulation of mRNAs or proteins before MCMV retinitis development for two additional participants in parthanatos, polymer of ADP-ribose and poly (ADP-ribose) glycohydrolase. These results provide new evidence for a role for parthanatos during MAIDS-related MCMV retinitis that may also extend to AIDS-related HCMV retinitis.

Abstract 3921: Poly(ADP-ribose) polymerase 1 (PARP-1) targeting radionuclides are highly cytotoxic across a panel of 20 neuroblastoma cell lines

Abstract Introduction: Neuroblastoma is the most common extracranial solid malignancy in childhood, with only up to 50% 5-year survival in high-risk cases. It overexpresses the nuclear enzyme PARP-1, which can be targeted for specific delivery of radionuclides to DNA. Two such novel radiolabeled small molecule therapeutics are [211At]MM4, containing an alpha particle-emitting astatine-211, and [125I]KX1, containing an Auger electron-emitting iodine-125. Methods: Twenty human neuroblastoma cell lines were screened in vitro for sensitivity to [211At]MM4 and [125I]KX1, with non-targeted radionuclides and non-radioactive PARP-1 inhibitors as controls. Next, one sensitive and one resistant cell line was chosen for dosimetry based on radiopharmacology and cellular geometry properties measured specific to each therapeutic and cell line. Monte Carlo simulation was then performed to calculate the radiation dose to the cell nucleus at 50% cell kill. Relative biological effectiveness (RBE) of alpha and Auger particles against gamma irradiation was determined. Finally, the mechanism responsible for differential radiosensitivity across the twenty cell lines was examined by correlation with PARP-1 mRNA level. Results: [211At]MM4 and [125I]KX1 showed closely correlated (R2=0.93) cytotoxicity across the twenty neuroblastoma cell lines. [211At]MM4 demonstrated 102-103 times and 108-109 times greater cytotoxic potency compared to unbound 211At and its non-radioactive PARP-1 inhibitor analog respectively. More sensitive cell lines demonstrated greater sensitivity to [211At]MM4 even when normalized against their sensitivity to alpha radiation (R2=0.40, p&amp;lt;0.01). [125I]KX1 also showed 104-106 times greater potency than its non-radioactive analog. No cell kill by biochemical inhibition of PARP-1 was seen at therapeutic doses of [211At]MM4 and [125I]KX1.The sensitive and resistant cell lines had similar PARP-1 binding of [211At]MM4 and [125I]KX1, but only the sensitive cell line had significantly higher RBE of alpha (9.2) and Auger (4.2) radiation compared to external gamma irradiation. RBE of alpha against Auger therapy was similar in both sensitive (2.2) and insensitive (1.9) cell lines. Cytotoxic sensitivity to PARP-1 targeted radiopharmaceutical was not correlated with PARP-1 mRNA level (R2=0.10, p=0.17). Conclusion: Delivery of alpha and Auger emitters directly to neuroblastoma nuclear DNA by radiolabeled PARP-1 inhibitors results in enhanced cytotoxicity without requiring enzymatic PARP-1 inhibition. Sensitivity to radiolabeled PARP-1 inhibitors is characterized by increased RBE against gamma irradiation and only partially explained by inherent radiosensitivity, warranting further investigation of underlying biological factors. This promising efficacy in vitro can be examined in vivo and in other cancer types for broader application. Citation Format: Hwan Lee, Adam Mansfield, Minu Samanta, Sean W. Reilly, Kuiying Xu, Sean D. Carlin, John M. Maris, Robert H. Mach, Mehran Makvandi, Daniel A. Pryma. Poly(ADP-ribose) polymerase 1 (PARP-1) targeting radionuclides are highly cytotoxic across a panel of 20 neuroblastoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3921.

PARP1 expression in soft tissue sarcomas is a poor‐prognosis factor and a new potential therapeutic target

Soft tissue sarcomas (STSs) are aggressive tumors with few efficient systemic therapies. Poly(ADP‐ribose) polymerase‐1 (PARP1) inhibitors represent an emerging therapeutic option in tumors with genomic instability. The genomics of STSs is complex in more than half of cases, suggesting a high level of inherent DNA damage and genomic instability. Thus, STSs could be efficiently targeted with PARP inhibitors. Promising preclinical results have been reported, but few data are available regarding PARP1 expression in clinical samples. We examined PARP1 mRNA expression in 1464 clinical samples of STS, including 1432 primary tumors and 32 relapses, and searched for correlations with clinicopathological features, including metastasis‐free survival (MFS). Expression was heterogeneous across the samples, not different between primary and secondary tumors, and was correlated to PARP1 DNA copy number. In the 1432 primary tumors, the ‘PARP1‐high’ samples were associated with younger patients, more frequent locations at the extremities, superficial trunk and head and neck, more leiomyosarcomas and other STSs and less liposarcomas and myxofibrosarcomas, more grade 3, more high‐risk CINSARC tumors, and more ‘chromosomically instable’ tumors. They were associated with shorter MFS, independently of other significant prognostic features, including the CINSARC signature. We found a strong involvement of genes overexpressed in the ‘PARP1‐high’ samples in cell cycle, DNA replication, and DNA repair. PARP1 expression refines the prediction of MFS in STSs, and similar expression exists in secondary and primary tumors, supporting the development of PARP1 inhibitors.

Bone marrow PARP1 mRNA levels predict response to treatment with 5-azacytidine in patients with myelodysplastic syndrome.

Poly (ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme that participates in the DNA repair of malignant cells, with various consequences on their survival. We have recently shown that PARP1 mRNA levels in the bone marrow of patients with myelodysplastic syndrome (MDS) are correlated to prognosis. To evaluate PARP1 as a biomarker of response to 5-azacytidine in patients with MDS, we measured PARP1 mRNA levels by a quantitative real-time PCR in diagnostic bone marrow samples of 77 patients with MDS treated with 5-azacytidine. Patients with higher PARP1 mRNA levels had a better response to 5-azacytidine per the IWG criteria (p = 0.006) and a longer median survival after 5-azacytidine initiation (p = 0.033). Multivariate analysis revealed that PARP1 mRNA level was the only factor affecting response to treatment and survival after treatment with 5-azacytidine. A next-generation sequencing for 40 genes of interest in MDS and quantification of the methylation levels of the PARP1 promoter were also carried out in a subset of samples (16 and 18 samples respectively). It is the first time that a single, easily measurable biomarker shows a clear correlation with response to treatment and survival in a patient population consisting of previously untreated patients with MDS homogeneously treated with 5-azacytidine. The fact that PARP1 is also a treatment target in several malignancies underscores the importance of our finding for the potential use of PARP1 inhibitors in MDS.

Dysregulation of PARP1 is involved in development of Barrett's esophagus.

AIMTo investigate the potential role of poly(ADP-ribose) polymerase 1 (PARP1) in the development of Barrett’s esophagus (BE).METHODSA BE mouse model was established to examine the esophageal morphological changes and molecular changes. Microarray analysis was performed to compare the gene expression profiles between BE patients and healthy controls. qPCR was used to examine the PARP1 expression in cell lines after treatment with H2O2 and bile acids (pH 4). Immunofluorescence staining, comet assay, and annexin V staining were used to evaluate the impact of PARP1 activity on cell survival and DNA damage response after oxidative stress.RESULTSThe gene expression profile in normal and BE esophageal epithelial cells showed that PARP1, the major poly(ADP-ribose) polymerase, was overexpressed in BE. In the mouse model of BE, positive staining for NF-κB, γH2AX, and poly(ADP-ribose) (PAR) was observed. H2O2 and bile acids (pH 4) increased the PARP1 mRNA expression level in normal esophageal epithelial cells. Using shRNA-PARP1 to suppress PARP1 activity decreased the cell viability after treatment with H2O2 and bile acids (pH 4), and increased the oxidative damage as demonstrated by an increase in the levels of H2O2, intracellular reactive oxygen species (ROS), oxidative DNA damage, double-strand breaks, and apoptosis (P < 0.01).CONCLUSIONThe dysfunction of PARP1 in esophageal epithelial cells increases the levels of ROS and oxidative DNA damage, which could be common risk factors for BE and esophageal adenocarcinoma.

Cancer Imprints an Increased PARP-1 and p53-Dependent Resistance to Oxidative Stress on Lymphocytes of Patients That Later Develop Alzheimer's Disease.

We have proposed that a common biological mechanism deregulated in opposite directions might explain the inverse epidemiological association observed between Alzheimer's disease (AD) and cancer. Accordingly, we showed that lymphocytes from AD patients have an increased susceptibility, whereas those from survivors of a skin cancer, an increased resistance to oxidative death induced by hydrogen peroxide (H2O2), compared to healthy controls (HC). We investigated the susceptibility to H2O2-induced death of lymphocytes in survivors of any type of cancer and in cancer survivors who later developed AD (Ca&AD). We also explored the involvement of Poly [ADP-ribose] polymerase-1 (PARP-1) and p53 pathways in the process, since both are involved in the increased susceptibility to death of AD lymphocytes. Lymphocytes from 11 cancer and 13 Ca&AD patients, and 12 HC were submitted to increasing concentrations of H2O2 for 20 h. Cell death was determined by flow cytometry, in the presence or absence of PARP-1 inhibition (3-aminobenzamide, 3-ABA), or p53 inhibition (pifithrin-α) or stabilization (Nut-3). PARP-1 and p53 mRNA levels were determined by Real-Time PCR. Lymphocytes from cancer and Ca&AD patients showed increased survival compared to HC, without differences between them, opposite to the increased susceptibility to death previously shown in AD. PARP-1 inhibition provided marked protection from H2O2-induced death in the two groups of patients, significantly greater than in HC. Pharmacological inhibition of p53 increased lymphocyte survival in Ca&AD patients, contrary to the effect previously reported in HC and AD. PARP-1 and p53 mRNA levels were elevated in Ca&AD lymphocytes compared with controls. In all, these results show that cancer imprints an increased resistance to H2O2-induced death in lymphocytes that persists after AD development, and is dependent on both PARP-1 and p53. p53 inhibition showed a differential role in cancer and Ca&AD compared to HC and AD lymphocytes, that could explain the inverse susceptibility to oxidative death in cancer and AD. These results are in agreement with the hypothesis of a common biological mechanism in AD and cancer. The similar cell death susceptibility and cell death pattern observed in cancer and Ca&AD lymphocytes suggests that cancer history leaves long term effects on lymphocyte cell death susceptibility.

Poly (ADP-Ribose) Polymerase-1 (PARP-1) Induction by Cocaine Is Post-Transcriptionally Regulated by miR-125b.

Cocaine exposure alters gene expression in the brain via methylation and acetylation of histones along with methylation of DNA. Recently, poly (ADP-ribose) polymerase-1 (PARP-1) catalyzed PARylation has been reported as an important regulator of cocaine-mediated gene expression. In this study, we report that the cellular microRNA “miR-125b” plays a key role for cocaine-induced PARP-1 expression. Acute and chronic cocaine exposure resulted in the downregulation of miR-125b concurrent with upregulation of PARP-1 in dopaminergic neuronal cells and nucleus accumbens (NAc) of mice but not in the medial prefrontal cortex (PFC) or ventral tegmental area (VTA). In silico analysis predicted a binding site of miR-125b in a conserved 3’-untranslated region (3’UTR) of the PARP-1 mRNA. Knockdown and overexpression studies showed that miR-125b levels negatively correlate with PARP-1 protein expression. Luciferase reporter assay using a vector containing the 3’UTR of PARP-1 mRNA confirmed regulation of PARP-1 by miR-125b. Specific nucleotide mutations within the binding site abrogated miR-125b’s regulatory effect on PARP-1 3’UTR. Finally, we established that downregulation of miR-125b and concurrent upregulation of PARP-1 is dependent on binding of cocaine to the dopamine transporter (DAT). Collectively, these results identify miR-125b as a post-transcriptional regulator of PARP-1 expression and establish a novel mechanism underlying the molecular effects of cocaine action.

Adaptive response in mouse bone marrow stromal cells exposed to 900 MHz radiofrequency fields: Impact of poly (ADP-ribose) polymerase (PARP)

This study examined whether non-ionizing radiofrequency fields (RF) exposure is capable of inducing poly (ADP-ribose) polymerase-1 (PARP-1) in bone marrow stromal cells (BMSCs) and whether it plays a role in RF-induced adaptive response (AR). Bone marrow stromal cells (BMSCs) were exposed to 900MHz RF at 120μW/cm2 power flux density for 3h/day for 5days and then challenged with a genotoxic dose of 1.5Gy gamma-radiation (GR). Some cells were also treated with 3-aminobenzamide (3-AB, 2mM final concentration), a potent inhibitor of PARP-1. Un-exposed and sham (SH)-exposed control cells as well as positive control cells exposed to gamma radiation (GR) were included in the experiments. The expression of PARP-1 mRNA and its protein levels as well as single strand breaks in the DNA and the kinetics of their repair were evaluated at several times after exposures. The results indicated the following. (a) Cells exposed to RF alone showed significantly increased PARP-1 mRNA expression and its protein levels compared with those exposed to SH- and GR alone. (b) Treatment of RF-exposed cells with 3-AB had diminished such increase in PARP-1. (c) Cells exposed to RF+GR showed significantly decreased genetic damage as well as faster kinetics of repair compared with those exposed to GR alone. (d) Cells exposed to RF+3-AB+GR showed no such decrease in genetic damage. Thus, the overall date suggested that non-ionizing RF exposure was capable of inducing PARP-1 which has a role in RF-induced AR.

NF90 regulates PARP1 mRNA stability in hepatocellular carcinoma

Poly (ADP-ribose) polymerase 1 (PARP1) is an ADP- ribosylation enzyme and plays important roles in a variety of cellular processes, including DNA damage response and tumor development. However, the post-transcriptional regulation of PARP1 remains largely unknown. In this study, we identified that the mRNA of PARP1 is associated with nuclear factor 90 (NF90) by RNA immunoprecipitation plus sequencing (RIP-seq) assay. The mRNA and protein levels of PARP1 are dramatically decreased in NF90-depleted cells, and NF90 stabilizes PARP1's mRNA through its 3'UTR. Moreover, the expression levels of PARP1 and NF90 are positively correlated in hepatocellular carcinoma (HCC). Finally, we demonstrated that NF90-depleted cells are sensitive to PARP inhibitor Olaparib (AZD2281) and DNA damage agents. Taken together, these results suggest that NF90 regulates PARP1 mRNA stability in hepatocellular carcinoma cells, and NF90 is a potential target to inhibit PARP1 activity.

LncRNA-SLC6A9-5:2: A potent sensitizer in 131I-resistant papillary thyroid carcinoma with PARP-1 induction.

Recent studies have indicated that long non-coding RNAs play crucial roles in numerous cancers, including thyroid cancer, while their function in the mechanism of thyroid cancer 131I resistance has not been elucidated to date. The present study identified a functional long non-coding RNA, SLC6A9-5:2, which was involved in the radioactive therapy resistance of thyroid cancer. We demonstrated that SLC6A9-5:2 was remarkably downregulated in 131I-resistant thyroid cancer cell lines and 131I-insensitive patients and was positively correlated with Poly (ADP-ribose) polymerase 1 (PARP-1) expression and its activation. After downregulating SLC6A9 or blocking PARP-1 artificially, the sensitive thyroid cancer cells mostly displayed a tolerant phenotype under 131I exposure. Furthermore, SLC6A9-5:2 overexpression was positively correlated with PARP-1 mRNA and protein levels, which restored the sensitivity of resistant thyroid cancer cells. The present study further revealed that cancer cell death was primarily caused by ATP exhaustion in excessive DNA repair with high PARP-1 activity. In patients with thyroid cancer, a positive correlation between SLC6A9-5:2 and PARP-1 was identified, and low SLC6A9-5:2 expression was associated with a worse prognosis of papillary thyroid carcinoma. Hence, our data provide a new lncRNA-mediated regulatory mechanism implying that SLC6A9-5:2 can be used as a novel therapeutic target for 131I-resistant thyroid cancer.

MicroRNAs Modulate Oxidative Stress in Hypertension through PARP-1 Regulation.

Oxidative stress is thought to contribute to aging and age-related diseases, such as cardiovascular and neurodegenerative diseases, and is a risk factor for systemic arterial hypertension. Previously, we reported differential mRNA and microRNA (miRNA) expression between African American (AA) and white women with hypertension. Here, we found that the poly-(ADP-ribose) polymerase 1 (PARP-1), a DNA damage sensor protein involved in DNA repair and other cellular processes, is upregulated in AA women with hypertension. To explore this mechanism, we identified two miRNAs, miR-103a-2-5p and miR-585-5p, that are differentially expressed with hypertension and were predicted to target PARP1. Through overexpression of each miRNA-downregulated PARP-1 mRNA and protein levels and using heterologous luciferase reporter assays, we demonstrate that miR-103a-2-5p and miR-585-5p regulate PARP1 through binding within the coding region. Given the important role of PARP-1 in DNA repair, we assessed whether overexpression of miR-103a-2-5p or miR-585-5p affected DNA damage and cell survival. Overexpression of these miRNAs enhanced DNA damage and decreased both cell survival and colony formation. These findings highlight the role for PARP-1 in regulating oxidative DNA damage in hypertension and identify important new miRNA regulators of PARP-1 expression. These insights may provide additional avenues to understand hypertension health disparities.

MiR-335 regulates the chemo-radioresistance of small cell lung cancer cells by targeting PARP-1

The role of miR-335 in the regulation of chemosensitivity and radiosensitivity of small cell lung cancer (SCLC) was investigated. miR-335 was significantly downregulated in multi-drug-resistant SCLC H69AR and H446DDP cells compared with parental cells as detected by qRT-PCR. Then, we demonstrated the negative correlation between miR-335 expression and the chemo-radiosensitivity of SCLC cells, including cell proliferation, cell clonality and cell apoptosis. In addition, miR-335 overexpression inhibited cell migration in vitro and tumor growth in vivo, whereas inhibition of miR-335 promoted cell migration and tumor growth. The underlying mechanism was further studied. Poly [ADP-ribose] polymerase 1 (PARP-1) was identified as a direct target gene of miR-335 in SCLC by bioinformatics analysis and validated via luciferase reporter assay. Overexpression of miR-335 decreased the expression of PARP-1 mRNA and protein, and NF-κB protein levels were correspondingly downregulated, thus regulating the chemo-radiosensitivity of SCLC. Taken together, these findings indicate that miR-335 may serve as a critical regulator of chemo-radiotherapy resistance in SCLC and a new potential therapeutic target.