poly(ADP-ribose) Polymerase-1 Knockdown Research Articles

AKT-dependent nuclear localization of EPRS1 activates PARP1 in breast cancer cells

Glutamyl-prolyl-tRNA synthetase (EPRS1) is a bifunctional aminoacyl-tRNA-synthetase (aaRS) essential for decoding the genetic code. EPRS1 resides, with seven other aaRSs and three noncatalytic proteins, in the cytoplasmic multi-tRNA synthetase complex (MSC). Multiple MSC-resident aaRSs, including EPRS1, exhibit stimulus-dependent release from the MSC to perform noncanonical activities distinct from their primary function in protein synthesis. Here, we show EPRS1 is present in both cytoplasm and nucleus of breast cancer cells with constitutively low phosphatase and tensin homolog (PTEN) expression. EPRS1 is primarily cytosolic in PTEN-expressing cells, but chemical or genetic inhibition of PTEN, or chemical or stress-mediated activation of its target, AKT, induces EPRS1 nuclear localization. Likewise, preferential nuclear localization of EPRS1 was observed in invasive ductal carcinoma that were also P-Ser473-AKT+. EPRS1 nuclear transport requires a nuclear localization signal (NLS) within the linker region that joins the catalytic glutamyl-tRNA synthetase and prolyl-tRNA synthetase domains. Nuclear EPRS1 interacts with poly(ADP-ribose) polymerase 1 (PARP1), a DNA-damage sensor that directs poly(ADP-ribosyl)ation (PARylation) of proteins. EPRS1 is a critical regulator of PARP1 activity as shown by markedly reduced ADP-ribosylation in EPRS1 knockdown cells. Moreover, EPRS1 and PARP1 knockdown comparably alter the expression of multiple tumor-related genes, inhibit DNA-damage repair, reduce tumor cell survival, and diminish tumor sphere formation by breast cancer cells. EPRS1-mediated regulation of PARP1 activity provides a mechanistic link between PTEN loss in breast cancer cells, PARP1 activation, and cell survival and tumor growth. Targeting the noncanonical activity of EPRS1, without inhibiting canonical tRNA ligase activity, provides a therapeutic approach potentially supplementing existing PARP1 inhibitors.

Muscle PARP1 inhibition extends lifespan through AMPKα PARylation and activation in Drosophila.

Poly(ADP-ribose) polymerase-1 (PARP1) has been reported to play an important role in longevity. Here, we showed that the knockdown of the PARP1 extended the lifespan of Drosophila, with particular emphasis on the skeletal muscle. The muscle-specific mutant Drosophila exhibited resistance to starvation and oxidative stress, as well as an increased ability to climb, with enhanced mitochondrial biogenesis and activity at an older age. Mechanistically, the inhibition of PARP1 increases the activity of AMP-activated protein kinase alpha (AMPKα) and mitochondrial turnover. PARP1 could interact with AMPKα and then regulate it via poly(ADP ribosyl)ation (PARylation) at residues E155 and E195. Double knockdown of PARP1 and AMPKα, specifically in muscle, could counteract the effects of PARP1 inhibition in Drosophila. Finally, we showed that increasing lifespan via maintaining mitochondrial network homeostasis required intact PTEN induced kinase 1 (PINK1). Taken together, these data indicate that the interplay between PARP1 and AMPKα can manipulate mitochondrial turnover, and be targeted to promote longevity.

Influence of Poly(ADP-ribose)polymerase 1 Level on the Status of Base Excision Repair in Human Cells

The base excision repair (BER) system is aimed at repair of the largest group of DNA lesions, namely of damaged bases. The main steps in BER are: recognition and removal of the aberrant base, cutting the DNA sugar-phosphate backbone, gap processing (including dNMP insertion), and DNA ligation. The precise functioning of BER depends on the regulation of each step of the process by regulatory/accessory proteins, the most important of which is poly(ADP-ribose)polymerase 1 (PARP1). PARP1 plays an important role in various processes of DNA repair, maintenance of genome integrity, and regulation of mRNA stability and decay. In this regard, PARP1 can affect BER both at the level of proteins involved in the process and at the level of expression of the mRNAs encoding them. There are no systematic data on the effect of various amounts of PARP1 on the activity of key BER proteins and the levels of mRNAs encoding them in human cells. In our work, using whole-cell extracts and RNA preparations obtained from the parental HEK293T cell line and its derivative HEK293T/P1-KD cell line with reduced PARP1 expression (shPARP1-expressing cells, PARP1 knockdown), we assessed the levels of mRNA encoding BER proteins: PARP1, PARP2, uracil DNA glycosylase (UNG2), AP endonuclease 1 (APE1), DNA polymerase β (POLβ), DNA ligase III (LIG3), and XRCC1. In parallel, the catalytic activity of these enzymes was evaluated. No significant effect of the PARP1 amount of on the mRNA levels of UNG2, APE1, POLβ, LIG3, and XRCC1 was found. At the same time, in HEK293T/P1-KD cells, the amount of PARP2 mRNA was reduced by 2 times. The activities of these enzymes in whole-cell extracts of HEK293T and HEK293T/P1-KD cells also did not differ significantly. Under the conditions of poly(ADP-ribose) synthesis, the efficiency of the reactions catalyzed by UNG2, APE1, POLβ, and LIG3 also did not change significantly. In addition, it was shown that a reduced amount of PARP1 in the extract of HEK293T/P1-KD cells does not cause fundamental changes in the nature of DNA PARylation compared to the extract of the parental HEK293T cell line.

Treatment of PTEN-Null Breast Cancer by a Synthetic Lethal Approach Involving PARP1 Gene Silencing

The loss of the phosphatase and tensin homolog (PTEN) deleted from chromosome 10 is frequently observed in a variety of human cancers and appears to be an ideal target in synthetic lethality–based treatment. In this study, the synthetic lethal interaction between PTEN loss and the gene silencing of poly [ADP-ribose] polymerase 1 (PARP1) was examined in human triple-negative breast cancer cells (PTEN-null MDA-MB-468 and PTEN-positive MDA-MB-231 cells). Polycation liposomes previously developed by us were employed to deliver the small interfering ribonucleic acid (siRNA) targeted toward PARP1 (siPARP1) into the cancer cells. The silencing of the PARP1 gene exerted a cytocidal effect on the MDA-MB-468 cells but had no effect on the MDA-MB-231 cells and the human umbilical vein endothelial cells employed as normal cells. The simultaneous knockdown of PARP1 and PTEN in the MDA-MB-231 cells resulted in the significant inhibition of cell growth. The data suggest that the effects of the PARP1 knockdown on the cells were dependent on the PTEN status. A significant increase in the DNA breaks and the extent of apoptosis, possibly due to the failure of DNA repair, was observed upon PARP1 knockdown in the MDA-MB-468 cells compared with the case in the MDA-MB-231 cells. Our findings suggest that the synthetic lethal approach via PARP1 gene silencing holds promise for the treatment of patients with PTEN-null breast cancer.

PARP1 inhibition enhances reactive oxygen species on gut microbiota.

Poly(ADP-ribose) polymerase 1 (PARP1) plays a key role in genome stability by modulating DNA-damage responses. Activated by DNA interruptions through ultraviolet (UV) exposure, PARylation is synthesized by PARP1 and serves as a survival mechanism for cancer and metabolic diseases. Several strategies including ROS and antimicrobial peptides(AMPs) function in host defenses, while the targeted tissue and mechanism under DNA damage are unknown. Here, we show that DNA damage induces responses specifically in the gut tissue. The knockdown of PARP1 reduces the activation of PARylation. Parp1 knockdown under DNA damage results in over-accumulated ROS and secretion of AMPs through the regulation of Relish, a subunit of nuclear factor-κB(NF-κB). Double-knockdown of Parp1 and Relish specifically in the gut inhibits AMP secretion. In conclusion, the host defense is achieved through ROS accumulation rather than the AMPs under DNA damage. In contrast, the knockdown of PARP1 exacerbates ROS accumulation to a harmful level. Under this circumstance, NF-κb targeted AMP secretion is provoked for host defense. Microbiome and functional analysis provide evidence for the hazard of DNA damage and show variations in the metabolic pathways following Parp1 inhibition. Our findings suggest the notion that PARP1 inhibition contributes to ROS accumulation under DNA damage and its role in NF-κb activation for host defense.

Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage

BackgroundN-acetyltransferase 10 (NAT10), an abundant nucleolar protein with both lysine and RNA cytidine acetyltransferase activities, has been implicated in Hutchinson-Gilford progeria syndrome and human cancer. We and others recently demonstrated that NAT10 is translocated from the nucleolus to the nucleoplasm after DNA damage, but the underlying mechanism remains unexplored.MethodsThe NAT10 and PARP1 knockout (KO) cell lines were generated using CRISPR-Cas9 technology. Knockdown of PARP1 was performed using specific small interfering RNAs targeting PARP1. Cells were irradiated with γ-rays using a 137Cs Gammacell-40 irradiator and subjected to clonogenic survival assays. Co-localization and interaction between NAT10 and MORC2 were examined by immunofluorescent staining and immunoprecipitation assays, respectively. PARylation of NAT10 and translocation of NAT10 were determined by in vitro PARylation assays and immunofluorescent staining, respectively.ResultsHere, we provide the first evidence that NAT10 underwent covalent PARylation modification following DNA damage, and poly (ADP-ribose) polymerase 1 (PARP1) catalyzed PARylation of NAT10 on three conserved lysine (K) residues (K1016, K1017, and K1020) within its C-terminal nucleolar localization signal motif (residues 983–1025). Notably, mutation of those three PARylation residues on NAT10, pharmacological inhibition of PARP1 activity, or depletion of PARP1 impaired NAT10 nucleoplasmic translocation after DNA damage. Knockdown or inhibition of PARP1 or expression of a PARylation-deficient mutant NAT10 (K3A) attenuated the co-localization and interaction of NAT10 with MORC family CW-type zinc finger 2 (MORC2), a newly identified chromatin-remodeling enzyme involved in DNA damage response, resulting in a decrease in DNA damage-induced MORC2 acetylation at lysine 767. Consequently, expression of a PARylation-defective mutant NAT10 resulted in enhanced cellular sensitivity to DNA damage agents.ConclusionCollectively, these findings indicate that PARP1-mediated PARylation of NAT10 is key for controlling its nucleoplasmic translocation and function in response to DNA damage. Moreover, our findings provide novel mechanistic insights into the sophisticated paradigm of the posttranslational modification-driven cellular response to DNA damage.7RTHzWMYUwTGweyv2z3-hVVideo

Parthanatos participates in glutamate-mediated HT22 cell injury and hippocampal neuronal death in kainic acid-induced status epilepticus rats.

Epileptic seizures or status epilepticus (SE) can cause hippocampal neuronal death, which has detrimental effects. Parthanatos, a new form of programmed cell death, is characterized by hyperactivation of poly (ADP-ribose) polymerase-1 (PARP-1), excessive synthesis of poly ADP-ribose polymer, mitochondrial depolarization, and nuclear translocation of apoptosis-inducing factor, observed in various neurodegenerative disorders but rarely reported in epilepsy. We aimed to investigate whether parthanatos participates in the mechanism of seizure-induced hippocampal neuronal death. Glutamate-mediated excitotoxicity cell model was used to study the mechanism of seizure-induced cell injury. Injection of kainic acid into the amygdala was used to establish the epileptic rat model. Corresponding biochemical tests were carried out on hippocampal tissues and HT22 cells following indicated treatments. In vitro, glutamate time-dependently induced HT22 cell death, accompanied by parthanatos-related biochemical events. Pretreatment with PJ34 (PARP-1 inhibitor) or small interfering RNA-mediated PARP-1 knockdown effectively protected HT22 cells against glutamate-induced toxic effects and attenuated parthanatos-related biochemical events. Application of the antioxidant N-acetylcysteine (NAC) rescued HT22 cell death and reversed parthanatos-related biochemical events. In vivo, PJ34 and NAC afforded protection against SE-induced hippocampal neuronal damage and inhibited parthanatos-related biochemical events. Parthanatos participates in glutamate-induced HT22 cell injury and hippocampal neuronal damage in rats following epileptic seizures. ROS might be the initiating factor during parthanatos.

Hypoxia-inducible factor-1α and poly [ADP ribose] polymerase 1 cooperatively regulate Notch3 expression under hypoxia via a noncanonical mechanism

Upregulation of Notch3 expression has been reported in many cancers and is considered a marker for poor prognosis. Hypoxia is a driving factor of the Notch3 signaling pathway; however, the induction mechanism and role of hypoxia-inducible factor-1α (HIF-1α) in the Notch3 response are still unclear. In this study, we found that HIF-1α and poly [ADP-ribose] polymerase 1 (PARP-1) regulate Notch3 induction under hypoxia via a noncanonical mechanism. In the analyzed cancer cell lines, Notch3 expression was increased during hypoxia at both the mRNA and protein levels. HIF-1α knockdown and Notch3 promoter reporter analyses indicated that the induction of Notch3 by hypoxia requires HIF-1α and also another molecule that binds the Notch3 promoter’s guanine-rich region, which lacks the canonical hypoxia response element. Therefore, using mass spectrometry analysis to identify the binding proteins of the Notch3 promoter, we found that PARP-1 specifically binds to the Notch3 promoter. Interestingly, analyses of the Notch3 promoter reporter and knockdown of PARP-1 revealed that PARP-1 plays an important role in Notch3 regulation. Furthermore, we demonstrate that PARP inhibitors, including an inhibitor specific for PARP-1, attenuated the induction of Notch3 by hypoxia. These results uncover a novel mechanism in which HIF-1α associates with PARP-1 on the Notch3 promoter in a hypoxia response element–independent manner, thereby inducing Notch3 expression during hypoxia. Further studies on this mechanism could facilitate a better understanding of the broader functions of HIF-1α, the roles of Notch3 in cancer formation, and the insights into novel therapeutic strategies.

Positive Feedback Regulation of Poly(ADP-ribose) Polymerase 1 and the DNA-PK Catalytic Subunit Affects the Sensitivity of Nasopharyngeal Carcinoma to Etoposide.

Etoposide (VP-16) is used for the treatment of various cancers, including nasopharyngeal carcinoma (NPC); however, cancers develop resistance to this agent by promoting DNA repair. The DNA-PK (DNA-PKcs) catalytic subunit and poly(ADP-ribose) polymerase 1 (PARP1) mediate acquired resistance and poor survival in NPC cells exposed to DNA damaging agents. DNA repair can alter the sensitivity of NPC cells to DNA damaging agents, and these two enzymes function concomitantly in response to DNA damage in vivo. Therefore, we explored the relationship between DNA-PKcs and PARP1, which may affect NPC cell survival by regulating DNA repair after VP-16 treatment. We performed quantitative real-time polymerase chain reaction, western blotting, and enzyme-linked immunoassays and found that DNA-PKcs knockdown downregulated the PARP1 and PAR expression. Conversely, PARP1 knockdown reduced DNA-PKcs activity, indicating the mutual regulation between DNA-PKcs and PARP1 in VP-16-induced DNA repair. Moreover, a combination treatment with olaparib (a PARP1 inhibitor) and NU7441 (a DNA-PKcs inhibitor) sensitized NPC cells to VP-16 in vitro and in vivo, suggesting that the combined treatment of olaparib, NU7441, and a DNA-damaging agent may be a successful treatment regimen in patients with NPC.

PARP1-RNA interaction analysis: PARP1 regulates the expression of extracellular matrix-related genes in HK-2 renal proximal tubular epithelial cells.

Recent studies suggest that Poly(ADP-ribose) polymerase 1 (PARP1) acts as an RNA-binding protein in a majority of renal diseases with tubular cell injury. However, detailed knowledge of RNA targets and the RNA-binding regions for PARP1 is unknown. Herein, mapping of iRIP-seq reads in HK-2 renal tubular epithelial cells showed a biased distribution at coding sequence (CDS) and intron regions that is specific to these cells. A total of 1708 differentially expressed genes were identified after PARP1 knockdown using RNA-seq. Furthermore, transcriptome analysis also showed that selective variable splicing was globally regulated by PARP1 in HK-2 cells. By comparison of PARP1 RNA-seq and iRIP-seq data, we found 68 overlapping genes that are enriched in 'extracellular matrix' pathway. Follow-up identification of their interactions may contribute vital insights into the regulatory role of PARP1 as an RNA-binding protein in HK-2 cells.

MTOR may interact with PARP-1 to regulate visible light-induced parthanatos in photoreceptors

BackgroundExcessive light exposure is a detrimental environmental factor that plays a critical role in the pathogenesis of retinal degeneration. However, the mechanism of light-induced death of retina/photoreceptor cells remains unclear. The mammalian/mechanistic target of rapamycin (mTOR) and Poly (ADP-ribose) polymerase-1 (PARP-1) have become the primary targets for treating many neurodegenerative disorders. The aim of this study was to elucidate the mechanisms underlying light-induced photoreceptor cell death and whether the neuroprotective effects of mTOR and PARP-1 inhibition against death are mediated through apoptosis-inducing factor (AIF).MethodsPropidium iodide (PI)/Hoechst staining, lentiviral-mediated short hairpin RNA (shRNA), Western blot analysis, cellular fraction separation, plasmid transient transfection, laser confocal microscopy, a mice model, electroretinography (ERG), and hematoxylin-eosin (H & E) staining were employed to explore the mechanisms by which rapamycin/3-Aminobenzamide (3AB) exert neuroprotective effects of mTOR/PARP-1 inhibition in light-injured retinas.ResultsA parthanatos-like death mechanism was evaluated in light-injured 661 W cells that are an immortalized photoreceptor-like cell line that exhibit cellular and biochemical feature characteristics of cone photoreceptor cells. The death process featured over-activation of PARP-1 and AIF nuclear translocation. Either PARP-1 or AIF knockdown played a significantly protective role for light-damaged photoreceptors. More importantly, crosstalk was observed between mTOR and PARP-1 signaling and mTOR could have regulated parthanatos via the intermediate factor sirtuin 1 (SIRT1). The parthanatos-like injury was also verified in vivo, wherein either PARP-1 or mTOR inhibition provided significant neuroprotection against light-induced injury, which is evinced by both structural and functional retinal analysis. Overall, these results elucidate the mTOR-regulated parthanatos death mechanism in light-injured photoreceptors/retinas and may facilitate the development of novel neuroprotective therapies for retinal degeneration diseases.ConclusionsOur results demonstrate that inhibition of the mTOR/PARP-1 axis exerts protective effects on photoreceptors against visible-light–induced parthanatos. These protective effects are conducted by regulating the downstream factors of AIF, while mTOR possibly interacts with PARP-1 via SIRT1 to regulate parthanatos.1tsUKNBnPvppEr1XWeshRGVideo Graphical Schematic diagram of mTOR interacting with PARP-1 to regulate visible light-induced parthanatos. Increased ROS caused by light exposure penetrates the nuclear membrane and causes nuclear DNA strand breaks. PARP-1 detects DNA breaks and synthesizes PAR polymers to initiate the DNA repair system that consumes a large amount of cellular NAD+. Over-production of PAR polymers prompts the release of AIF from the mitochondria and translocation to the nucleus, which leads to parthanatos. Activated mTOR may interact with PARP-1 via SIRT1 to regulate visible light-induced parthanatos.

Identification of probe-quality degraders for Poly(ADP-ribose) polymerase-1 (PARP-1)

Poly(ADP-ribose) polymerase-1 (PARP-1), a critical DNA repair enzyme in the base excision repair pathway, has been pursued as an attractive cancer therapeutic target. Intervention with PARP-1 has been proved to be more sensitive to cancer cells carrying BRCA1/2 mutations. Several PARP-1 inhibitors have been available on market for the treatment of breast, ovarian and prostatic cancer. Promisingly, the newly developed proteolysis targeting chimaeras (PROTACs) may provide a more potential strategy based on the degradation of PARP-1. Here we report the design, synthesis, and evaluation of a proteolysis targeting chimaera (PROTAC) based on the combination of PARP-1 inhibitor olaparib and the CRBN (cereblon) ligand lenalidomide. In SW620 cells, our probe-quality degrader compound 2 effectively induced PARP-1 degradation which results in anti-proliferation, cells apoptosis, cell cycle arresting, and cancer cells migratory inhibition. Thus, our findings qualify a new chemical probe for PARP-1 knockdown.

Poly(ADP-ribose) polymerase-1 promotes expression of miR-155 by the up-regulation of methyl-CpG binding domain protein 2 in TK6 cells exposed to hydroquinone

Hydroquinone (HQ), one of the major metabolites of benzene, can induce aberrant gene expression. MiR-155, a tumor activator, participates in various biological processes, including DNA damage response. However, the molecular mechanism of aberrant miR-155 expression is still not completely elucidated. Here, we investigated the mechanism of abnormal expression of miR-155 induced by poly(ADP-ribose)polymerase-1 (PARP-1) expression in HQ-treated TK6 lymphoblastoid cells. We examined the expression of genes related to abnormal expression of miR-155 to explore the reason for this phenomenon. The results of the present study showed that miR-155 was significantly increased and reactive oxygen species (ROS) were decreased in cells treated with HQ for 72 h compared with PBS-treated cells. Meanwhile, E4F1, PARP-1 and PARP-1 related co-regulators (NF-κB, HDAC1, and HDAC2), acetylated histone H3 (H3Ac) were increased in a concentration-dependent manner. Experiments for treatment with 5-AzaC (DNMTs inhibitor), TSA (HDACs inhibitor), DOX (to activate PARP-1) or MG132 (proteasome inhibitor) revealed that the MBDs and PARP-1 was positively associated with miR-155 expression. Moreover, in cells treated with HQ in conjunction with PARP-1 knockdown, expression of miR-155, H3Ac and MBD2 protein were decreased, compared with negative control. In conclusion, PARP-1 activates expression of miR-155 via acetylation by regulating MBD2 in TK6 cells exposed to HQ.

Poly(ADP-ribose) polymerase 1 (PARP1) promotes oxidative stress–induced association of Cockayne syndrome group B protein with chromatin

Cockayne syndrome protein B (CSB) is an ATP-dependent chromatin remodeler that relieves oxidative stress by regulating DNA repair and transcription. CSB is proposed to participate in base-excision repair (BER), the primary pathway for repairing oxidative DNA damage, but exactly how CSB participates in this process is unknown. It is also unclear whether CSB contributes to other repair pathways during oxidative stress. Here, using a patient-derived CS1AN-sv cell line, we examined how CSB is targeted to chromatin in response to menadione-induced oxidative stress, both globally and locus-specifically. We found that menadione-induced, global CSB-chromatin association does not require CSB's ATPase activity and is, therefore, mechanistically distinct from UV-induced CSB-chromatin association. Importantly, poly(ADP-ribose) polymerase 1 (PARP1) enhanced the kinetics of global menadione-induced CSB-chromatin association. We found that the major BER enzymes, 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1), do not influence this association. Additionally, the level of γ-H2A histone family member X (γ-H2AX), a marker for dsDNA breaks, was not increased in menadione-treated cells. Therefore, our results support a model whereby PARP1 localizes to ssDNA breaks and recruits CSB to participate in DNA repair. Furthermore, this global CSB-chromatin association occurred independently of RNA polymerase II-mediated transcription elongation. However, unlike global CSB-chromatin association, both PARP1 knockdown and inhibition of transcription elongation interfered with menadione-induced CSB recruitment to specific genomic regions. This observation supports the hypothesis that CSB is also targeted to specific genomic loci to participate in transcriptional regulation in response to oxidative stress.

Gold nanorods/siRNA complex administration for knockdown of PARP-1: a potential treatment for perinatal asphyxia.

BackgroundPerinatal asphyxia interferes with neonatal development, resulting in long-term deficits associated with systemic and neurological diseases. Despite the important role of poly (ADP-ribose) polymerase 1 (PARP-1) in the regulation of gene expression and DNA repair, overactivation of PARP-1 in asphyxia-exposed animals worsens the ATP-dependent energetic crisis. Inhibition of PARP-1 offers a therapeutic strategy for diminishing the effects of perinatal asphyxia.MethodsWe designed a nanosystem that incorporates a specific siRNA for PARP-1 knockdown. The siRNA was complexed with gold nanorods (AuNR) conjugated to the peptide CLPFFD for brain targeting.ResultsThe siRNA was efficiently delivered into PC12 cells, resulting in gene silencing. The complex was administered intraperitoneally in vivo to asphyxia-exposed rat pups, and the ability of the AuNR-CLPFFD/siRNA complex to reach the brain was demonstrated.ConclusionThe combination of a nanosystem for delivery and a specific siRNA for gene silencing resulted in effective inhibition of PARP-1 in vivo.

The dual role of poly(ADP-ribose) polymerase-1 in modulating parthanatos and autophagy under oxidative stress in rat cochlear marginal cells of the stria vascularis

Oxidative stress is reported to regulate several apoptotic and necrotic cell death pathways in auditory tissues. Poly(ADP-ribose) polymerase-1 (PARP-1) can be activated under oxidative stress, which is the hallmark of parthanatos. Autophagy, which serves either a pro-survival or pro-death function, can also be stimulated by oxidative stress, but the role of autophagy and its relationship with parthanatos underlying this activation in the inner ear remains unknown. In this study, we established an oxidative stress model in vitro by glucose oxidase/glucose (GO/G), which could continuously generate low concentrations of H2O2 to mimic continuous exposure to H2O2 in physiological conditions, for investigation of oxidative stress-induced cell death mechanisms and the regulatory role of PARP-1 in this process. We observed that GO/G induced stria marginal cells (MCs) death via upregulation of PARP-1 expression, accumulation of polyADP-ribose (PAR) polymers, decline of mitochondrial membrane potential (MMP) and nuclear translocation of apoptosis-inducing factor (AIF), which all are biochemical features of parthanatos. PARP-1 knockdown rescued GO/G-induced MCs death, as well as abrogated downstream molecular events of PARP-1 activation. In addition, we demonstrated that GO/G stimulated autophagy and PARP-1 knockdown suppressed GO/G-induced autophagy in MCs. Interestingly, autophagy suppression by 3-Methyladenine (3-MA) accelerated GO/G-induced parthanatos, indicating a pro-survival function of autophagy in GO/G-induced MCs death. Taken together, these data suggested that PARP-1 played dual roles by modulating parthanatos and autophagy in oxidative stress-induced MCs death, which may be considered as a promising therapeutic target for ameliorating oxidative stress-related hearing disorders.

Bcl-2 protects TK6 cells against hydroquinone-induced apoptosis through PARP-1 cytoplasm translocation and stabilizing mitochondrial membrane potential.

B cell leukemia/lymphoma-2 (Bcl-2) suppresses apoptosis by binding the BH3 domain of proapoptotic factors and thereby regulating mitochondrial membrane potential (MMP). This study aimed to investigate the role of Bcl-2 in controlling the mitochondrial pathway of apoptosis during hydroquinone (HQ)-induced TK6 cytotoxicity. In this study, HQ, one metabolite of benzene, decreased the MMP in a concentration-dependent manner and induced the generation of reactive oxygen species (ROS), the activation of the DNA damage marker γ-H2AX, and production of the DNA damage-responsive enzyme poly(ADP-ribose)polymerase-1 (PARP-1). Exposure of TK6 cells to HQ leads to an increase in Bcl-2 and co-localization with PARP-1 in the cytoplasm. Inhibition of Bcl-2 using the BH3 mimetic, ABT-737, suppressed the PARP-1 nuclear to cytoplasm translocation and sensitized TK6 cells to HQ-induced apoptosis through depolarization of the MMP. Western blot analysis indicated that ABT-737 combined with HQ increased the levels of cleaved PARP and γ-H2AX, but significantly decreased the level of P53. Thus, ABT-737 can influence PARP-1 translocation and induce apoptosis via mitochondria-mediated apoptotic pathway, independently of P53. In addition, we found that knockdown of PARP-1 attenuated the HQ-induced production of cleaved PARP and P53. These results identify Bcl-2 as a protective mediator of HQ-induced apoptosis and show that upregulation of Bcl-2 helps to localize PARP-1 to the cytoplasm and stabilize MMP. Environ. Mol. Mutagen. 59:49-59, 2018. © 2017 Wiley Periodicals, Inc.

PARP1 controls KLF4-mediated telomerase expression in stem cells and cancer cells.

Telomerase is highly expressed in cancer and embryonic stem cells (ESCs) and implicated in controlling genome integrity, cancer formation and stemness. Previous studies identified that Krüppel-like transcription factor 4 (KLF4) activates telomerase reverse transcriptase (TERT) expression and contributes to the maintenance of self-renewal in ESCs. However, little is known about how KLF4 regulates TERT expression. Here, we discover poly(ADP-ribose) polymerase 1 (PARP1) as a novel KLF4-interacting partner. Knockdown of PARP1 reduces TERT expression and telomerase activity not only in cancer cells, but also in human and mouse ESCs. Recruitment of KLF4 to TERT promoter is reduced in PARP1-suppressed cells. The poly(ADP-ribose) polymerase activity is dispensable, while the oligo(ADP-ribose) polymerase activity is required for the PARP1- and KLF4-mediated TERT activation. Repression of Parp1 in mouse ESCs decreases expression of pluripotent markers and induces differentiation. These results suggest that PARP1 recruits KLF4 to activate telomerase expression and stem cell pluripotency, indicating a positive regulatory role of the PARP1–KLF4 complex in telomerase expression in cancer and stem cells.

Early Passage Mesenchymal Stem Cells Display Decreased Radiosensitivity and Increased DNA Repair Activity.

Cell therapies using human mesenchymal stem cells (MSCs) have received much attention in the past decade. In pursuit of the therapeutic potential of MSCs, cell expansion is required to generate a great number of cells with desired phenotype and functionality. Long‐term expansion in vitro, however, can lead to altered functions. To explore the changes in DNA damage responses (DDR) in MSCs expanded, DDR pathways following irradiation were characterized in early‐ and late‐passage bone marrow MSCs. Seventy‐two hours after irradiation, the percentage of sub‐G1 cells in early‐passage MSCs did not change significantly. Reduced TUNEL staining was observed in early‐passage MSCs compared to late‐passage MSCs 4 h after irradiation. Comet assay also revealed that early‐passage MSCs were more resistant to irradiation or DNA damages induced by genotoxic agents than late‐passage MSCs. ATM phosphorylation and γ‐H2AX and phospho‐p53 increased in early‐passage MSCs while decreased in late‐passage MSCs. Through inhibition by KU55933, DDR pathway in early‐passage MSCs was shown to be ATM‐dependent. Higher levels of poly (ADP‐ribose) polymerase‐1 (PARP‐1) and PAR synthesis were observed in early‐passage MSCs than in late‐passage MSCs. Knockdown of PARP‐1 in early‐passage MSCs resulted in sensitization to irradiation‐induced apoptosis. Overexpression of PARP‐1 in late passage MSCs could render irradiation resistance. Lower activity of DDR in late‐passage MSCs was associated with rapid proteasomal degradation of PARP‐1. In conclusion, early‐passage MSCs are more irradiation‐resistant and have increased DDR activity involving PARP‐1, ATM and their downstream signals. Stem Cells Translational Medicine 2017;6:1504–1514

PARP-1 may be involved in angiogenesis in epithelial ovarian cancer.

Poly (ADP-ribose) polymerase 1 (PARP-1) is involved in DNA repair and has been implicated in chemoresistance. The present study investigated whether PARP-1 promotes angiogenesis in ovarian cancer. PARP-1 and vascular endothelial growth factor A (VEGF-A) expression and CD34+ microvascular density (MVD) were assessed using immunohistochemistry in 60 human epithelial ovarian cancer specimens. PARP-1 was stably knocked-down in SKOV3 cells using a specific small interfering RNA (siRNA); angiogenic capacity was assessed using the human umbilical vein endothelial cell (HUVEC) tubule formation assay; and PARP-1 and VEGF-A expression were examined by reverse transcription-quantitative polymerase chain reaction, western blotting and ELISA. PARP-1 was found to be expressed in 73.3% (44/60) of the human epithelial ovarian cancer specimens and was significantly associated with VEGF-A, MVD, tumor size, histological grade and lymphatic metastasis (P<0.05). Compared with cells transfected with a negative control siRNA, knockdown of PARP-1 significantly suppressed the ability of SKOV3 cell-conditioned media to promote HUVEC tubule formation on Matrigel in vitro. Knockdown of PARP-1 in SKOV3 cells also significantly reduced VEGF-A mRNA and protein expression and secretion. In summary, PARP-1 is overexpressed and may enhance angiogenesis in epithelial ovarian cancer by upregulating VEGF-A.