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Background: Neuroblastoma (NB), the most prevalent solid tumor in children, arises from sympathetic nervous system and accounts for 15% of pediatric cancer mortality. This malignancy exhibits substantial genetic and clinical heterogeneity, thus complicating treatment strategies. Poly(ADP-ribose) polymerase 1 (PARP1), a key enzyme catalyzing polyADP-ribosylation (PARylation), plays critical roles in various cellular processes, and contributes to tumorigenesis and aggressiveness. However, the functions and regulatory mechanisms of PARP1 in NB progression still remain to be determined., Methods: The association of PARP1 expression with NB patients' survival was analyzed by mining of R2 database. Western blotting, reverse transcription-polymerase chain reaction, MTT colorimetric, soft agar, and matrigel invasion assays were utilized to assess PARP1 expression and its effects on aggressiveness of NB cell lines. Chromatin immunoprecipitation (ChIP) sequencing and ChIP assays were employed to investigate the binding of Yin Yang 1 (YY1) to PARP1 promoter. Protein interactions were explored by BioGRID database analysis, molecular docking, and co-immunoprecipitation assay. RNA sequencing and crosslinking-immunoprecipitation high throughput sequencing datasets were used to identify precursor mRNA splicing targets of non-POU domain containing octamer binding protein (NONO)., Results: High PARP1 expression was associated with poor survival of NB patients. PARP1 over-expression enhanced the proliferation and invasion of NB cell lines, confirming its oncogenic roles. YY1 was identified as a key transcriptional regulator facilitating PARP1 expression. Additionally, PARP1 interacted with NONO to induce its PARylation, resulting in stabilization of NONO protein via preventing ubiquitin-mediated degradation. NONO facilitated the splicing and mRNA maturation of target genes a disintegrin and metalloproteinase domain 8 (ADAM8) and testis-expressed gene 14 (TEX14) in a PARylation-dependent manner. Rescue experiments indicated that YY1 facilitated PARP1-mediated PARylation of NONO and subsequent mRNA maturation of ADAM8 and TEX14 in NB cells. In clinical NB cases, high expression of YY1, PARP1, NONO, ADAM8, or TEX14 was associated with poor survival of patients., Conclusions: These findings indicate that YY1 drives PARP1 expression essential for PARylation of NONO in mRNA maturation during NB progression., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: All authors have agreed to publish this manuscript. Competing interests: The authors declare that they have no competing interests., (© 2024. The Author(s).)

Background: The histone variant macroH2A (mH2A), the most deviant variant, is about threefold larger than the conventional histone H2A and consists of a histone H2A-like domain fused to a large Non-Histone Region responsible for recruiting PARP-1 to chromatin. The available data suggest that the histone variant mH2A participates in the regulation of transcription, maintenance of heterochromatin, NAD + metabolism, and double-strand DNA repair., Results: Here, we describe a novel function of mH2A, namely its implication in DNA oxidative damage repair through PARP-1. The depletion of mH2A affected both repair and cell survival after the induction of oxidative lesions in DNA. PARP-1 formed a specific complex with mH2A nucleosomes in vivo. The mH2A nucleosome-associated PARP-1 is inactive. Upon oxidative damage, mH2A is ubiquitinated, PARP-1 is released from the mH2A nucleosomal complex, and is activated. The in vivo-induced ubiquitination of mH2A, in the absence of any oxidative damage, was sufficient for the release of PARP-1. However, no release of PARP-1 was observed upon treatment of the cells with either the DNA alkylating agent MMS or doxorubicin., Conclusions: Our data identify a novel pathway for the repair of DNA oxidative lesions, requiring the ubiquitination of mH2A for the release of PARP-1 from chromatin and its activation., (© 2024. The Author(s).)

Background: Endometriosis is a gynecological disease characterized by the presence of endometrial tissue in abnormal locations, leading to severe symptoms, inflammation, pain, organ dysfunction, and infertility. Surgical removal of endometriosis lesions is crucial for improving pain and fertility outcomes, with the goal of complete lesion removal. This study aimed to analyze the location and expression patterns of poly (ADP-ribose) polymerase 1 (PARP-1), epithelial cell adhesion molecule (EpCAM), and folate receptor alpha (FRα) in endometriosis lesions and evaluate their potential for targeted imaging., Methods: Gene expression analysis was performed using the Turku endometriosis database (EndometDB). By immunohistochemistry, we investigated the presence and distribution of PARP-1, EpCAM, and FRα in endometriosis foci and adjacent tissue. We also applied an ad hoc platform for the analysis of images to perform a quantitative immunolocalization analysis. Double immunofluorescence analysis was carried out for PARP-1 and EpCAM, as well as for PARP-1 and FRα, to explore the expression of these combined markers within endometriosis foci and their potential simultaneous utilization in surgical treatment., Results: Gene expression analysis revealed that PARP-1, EpCAM, and FOLR1 (FRα gene) are more highly expressed in endometriotic lesions than in the peritoneum, which served as the control tissue. The results of the immunohistochemical study revealed a significant increase in the expression levels of all three biomarkers inside the endometriosis foci compared to the adjacent tissues. Additionally, the double immunofluorescence analysis consistently demonstrated the presence of PARP-1 in the nucleus and the expression of EpCAM and FRα in the cell membrane and cytoplasm., Conclusion: Overall, these three markers demonstrate significant potential for effective imaging of endometriosis. In particular, the results emphasize the importance of PARP-1 expression as a possible indicator for distinguishing endometriotic lesions from adjacent tissue. PARP-1, as a potential biomarker for endometriosis, offers promising avenues for further investigation in terms of both pathophysiology and diagnostic-therapeutic approaches., (© 2024. The Author(s).)

Background: Poly (ADP-ribose) polymerase inhibitor (PARPi) resistance poses a significant challenge in ovarian carcinoma (OC). While the role of DOT1L in cancer and chemoresistance is acknowledged, its specific role in PARPi resistance remains unclear. This study aims to elucidate the molecular mechanism of DOT1L in PARPi resistance in OC patients., Methods: This study analyzed the expression of DOT1L in PARPi-resistant cell lines compared to sensitive ones and correlated it with clinical outcomes in OC patients. Comprehensive in vitro and in vivo functional experiments were conducted using cellular and mouse models. Molecular investigations, including RNA sequencing, chromatin immunoprecipitation (ChIP) and Cleavage Under Targets and Tagmentation (CUT&Tag) assays, were employed to unravel the molecular mechanisms of DOT1L-mediated PARPi resistance., Results: Our investigation revealed a robust correlation between DOT1L expression and clinical PARPi resistance in non-BRCA mutated OC cells. Upregulated DOT1L expression in PARPi-resistant tissues was associated with diminished survival in OC patients. Mechanistically, we identified that PARP1 directly binds to the DOT1L gene promoter, promoting transcription independently of its enzyme activity. PARP1 trapping induced by PARPi treatment amplified this binding, enhancing DOT1L transcription and contributing to drug resistance. Sequencing analysis revealed that DOT1L plays a crucial role in the transcriptional regulation of PLCG2 and ABCB1 via H3K79me2. This established the PARP1-DOT1L-PLCG2/ABCB1 axis as a key contributor to PARPi resistance. Furthermore, we discovered that combining a DOT1L inhibitor with PARPi demonstrated a synergistic effect in both cell line-derived xenograft mouse models (CDXs) and patient-derived organoids (PDOs)., Conclusions: Our results demonstrate that DOT1L is an independent prognostic marker for OC patients. The PARP1-DOT1L/H3K79me2-PLCG2/ABCB1 axis is identified as a pivotal contributor to PARPi resistance. Targeted inhibition of DOT1L emerges as a promising therapeutic strategy for enhancing PARPi treatment outcomes in OC patients., (© 2024. The Author(s).)

Background: Esophageal cancer is one of the 10 most common cancers worldwide and its incidence is dramatically increasing. Despite some improvements, the current surveillance protocol with white light endoscopy and random untargeted biopsies collection (Seattle protocol) fails to diagnose dysplastic and cancerous lesions in up to 50% of patients. Therefore, new endoscopic imaging technologies in combination with tumor-specific molecular probes are needed to improve early detection. Herein, we investigated the use of the fluorescent Poly (ADP-ribose) Polymerase 1 (PARP1)-inhibitor PARPi-FL for early detection of dysplastic lesions in patient-derived organoids and transgenic mouse models, which closely mimic the transformation from non-malignant Barrett's Esophagus (BE) to invasive esophageal adenocarcinoma (EAC)., Methods: We determined PARP1 expression via immunohistochemistry (IHC) in human biospecimens and mouse tissues. We also assessed PARPi-FL uptake in patient- and mouse-derived organoids. Following intravenous injection of 75 nmol PARPi-FL/mouse in L2-IL1B (n = 4) and L2-IL1B/IL8Tg mice (n = 12), we conducted fluorescence molecular endoscopy (FME) and/or imaged whole excised stomachs to assess PARPi-FL accumulation in dysplastic lesions. L2-IL1B/IL8Tg mice (n = 3) and wild-type (WT) mice (n = 2) without PARPi-FL injection served as controls. The imaging results were validated by confocal microscopy and IHC of excised tissues., Results: IHC on patient and murine tissue revealed similar patterns of increasing PARP1 expression in presence of dysplasia and cancer. In human and murine organoids, PARPi-FL localized to PARP1-expressing epithelial cell nuclei after 10 min of incubation. Injection of PARPi-FL in transgenic mouse models of BE resulted in the successful detection of lesions via FME, with a mean target-to-background ratio > 2 independently from the disease stage. The localization of PARPi-FL in the lesions was confirmed by imaging of the excised stomachs and confocal microscopy. Without PARPi-FL injection, identification of lesions via FME in transgenic mice was not possible., Conclusion: PARPi-FL imaging is a promising approach for clinically needed improved detection of dysplastic and malignant EAC lesions in patients with BE. Since PARPi-FL is currently evaluated in a phase 2 clinical trial for oral cancer detection after topical application, clinical translation for early detection of dysplasia and EAC in BE patients via FME screening appears feasible., (© 2024. The Author(s).)

Background: Mechanisms of development and progression of high-grade serous ovarian cancer (HGSOC) are poorly understood. EVI1 and PARP1, part of TGF-ß pathway, are upregulated in cancers with DNA repair deficiencies with DNA repair deficiencies and may influce disease progression and survival. Therefore we questioned the prognostic significance of protein expression of EVI1 alone and in combination with PARP1 and analyzed them in a cohort of patients with HGSOC., Methods: For 562 HGSOC patients, we evaluated EVI1 and PARP1 expression by immunohistochemical staining on tissue microarrays with QuPath digital semi-automatic positive cell detection., Results: High EVI1 expressing (> 30% positive tumor cells) HGSOC were associated with improved progression-free survival (PFS) (HR = 0.66, 95% CI: 0.504-0.852, p = 0.002) and overall survival (OS) (HR = 0.45, 95% CI: 0.352-0.563, p < 0.001), including multivariate analysis. Most interestingly, mutual high expression of both proteins identifies a group with particularly good prognosis. Our findings were proven technically and clinically using bioinformatical data sets for single-cell sequencing, copy number variation and gene as well as protein expression., Conclusions: EVI1 and PARP1 are robust prognostic biomarkers for favorable prognosis in HGSOC and imply further research with respect to their reciprocity., (© 2023. The Author(s).)

Background: Prostate cancer (PCa) is a prevalent malignant disease affecting a significant number of males globally. Elevated expression of the Bloom's syndrome protein (BLM) helicase has emerged as a promising cancer biomarker, being associated with the onset and progression of PCa. Nevertheless, the precise molecular mechanisms governing BLM regulation in PCa remain elusive., Methods: The expression of BLM in human specimens was analyzed using immnohistochemistry (IHC). A 5'-biotin-labeled DNA probe containing the promoter region of BLM was synthesized to pull down BLM promoter-binding proteins. Functional studies were conducted using a range of assays, including CCK-8, EdU incorporation, clone formation, wound scratch, transwell migration, alkaline comet assay, xenograft mouse model, and H&E staining. Mechanistic studies were carried out using various techniques, including streptavidin-agarose-mediated DNA pull-down, mass spectrometry (MS), immunofluorescence (IF), dual luciferase reporter assay system, RT-qPCR, ChIP-qPCR, co-immunoprecipitation (co-IP), and western blot., Results: The results revealed significant upregulation of BLM in human PCa tissues, and its overexpression was associated with an unfavorable prognosis in PCa patients. Increased BLM expression showed significant correlations with advanced clinical stage (P = 0.022) and Gleason grade (P = 0.006). In vitro experiments demonstrated that BLM knockdown exerted inhibitory effects on cell proliferation, clone formation, invasion, and migration. Furthermore, PARP1 (poly (ADP-ribose) polymerase 1) was identified as a BLM promoter-binding protein. Further investigations revealed that the downregulation of PARP1 led to increased BLM promoter activity and expression, while the overexpression of PARP1 exerted opposite effects. Through mechanistic studies, we elucidated that the interaction between PARP1 and HSP90AB1 (heat shock protein alpha family class B) enhanced the transcriptional regulation of BLM by counteracting the inhibitory influence of PARP1 on BLM. Furthermore, the combination treatment of olaparib with ML216 demonstrated enhanced inhibitory effects on cell proliferation, clone formation, invasion, and migration. It also induced more severe DNA damage in vitro and exhibited superior inhibitory effects on the proliferation of PC3 xenograft tumors in vivo., Conclusions: The results of this study underscore the significance of BLM overexpression as a prognostic biomarker for PCa, while also demonstrating the negative regulatory impact of PARP1 on BLM transcription. The concurrent targeting of BLM and PARP1 emerges as a promising therapeutic approach for PCa treatment, holding potential clinical significance., (© 2023. The Author(s).)

Background: Radiotherapy is an important treatment for lung cancer, mainly by triggering DNA double-strand breaks to induce cell death. Blocking DNA damage repair can increase the radiosensitivity of tumor cells. Recent studies have identified long noncoding RNAs as key regulators in DNA damage repair. The lncRNA ANRIL was previously shown to be involved in homologous recombination (HR) repair, but its specific mechanism has not been fully elucidated., Methods: The downstream interacting miRNAs of ANRIL were predicted according to miRanda software. Fluorescence quantitative PCR was used to detect the expression levels of ANRIL and candidate miRNAs. Clone formation experiment and cell viability assays detect cell viability after ionizing radiation. Apoptosis assay was used to detect the apoptosis of cells after 8 h of ionizing radiation. Western blot analysis and immunofluorescence assays verified the protein expression levels of the downstream target molecule PARP1 of miR-7-5p and key molecules in the HR pathway. Fluorescent reporter gene experiments were used to verify the interaction between ANRIL and miR-7-5p and between miR-7-5p and PARP1., Results: Bioinformatics analysis and qPCR validation suggested that miR-7-5p might be a downstream molecule of ANRIL. The expression of miR-7-5p was up-regulated after knockdown of ANRIL, and the expression of miR-7-5p was down-regulated after overexpression of ANRIL. Meanwhile, there was a negative correlation between ANRIL and miR-7-5p expression changes before and after ionizing radiation. The luciferase reporter gene assay confirmed the existence of ANRIL binding site with miR-7-5p, and found that transfection of miR-7-5p inhibitor can reduce the radiation sensitivity of ANRIL-KD cells. A downstream target molecule of miR-7-5p related to HR repair, PARP1, was screened through website prediction. Subsequently, it was confirmed by Western blot and luciferase reporter assays that miR-7-5p could down-regulate the expression of PARP1, and there was a miR-7-5p binding site on the 3'UTR of PARP1 mRNA. This suggests that ANRIL may act as a competitive endogenous RNA to bind miR-7-5p and upregulate the expression of PARP1. Western blot and immunofluorescence staining were used to detect the expression changes of HR repair factors in ANRIL-KD cells after ionizing radiation, and it was found that knockdown of ANRIL can inhibit the expression of PARP1, BRCA1 and Rad51, hinder radiation-induced HR repair, and eventually result in resensitizing ANRIL-KD cells to ionizing radiation., Conclusions: Our findings provide evidence that ANRIL targets the miR-7-5p/PARP1 axis to exert its regulatory effect on HR repair, suggesting that altering ANRIL expression may be a promising strategy to overcome radiation resistance., (© 2023. The Author(s).)

Background: Small nucleolar RNAs (snoRNAs) are dysregulated in many cancers, although their exact role in tumor genesis and progression remains unclear., Methods: The expression profiles of snoRNAs in endometrial cancer (EC) tissues were analyzed using data from The Cancer Genome Atlas, and SNORD104 was identified as an upregulated snoRNA in EC. The tumorigenic role of SNORD104 in EC was established in CCK8, colony formation, EdU, apoptosis, Transwell, and in vivo xenograft experiments. The molecular mechanisms of SNORD104 were analyzed by RNA immunoprecipitation (RIP), Nm-seq, RTL-P assay, RNA stability assay, qRT-PCR, and western blotting., Results: Antisense oligonucleotide (ASO)-mediated knockdown of SNORD104 in Ishikawa cells significantly inhibited their proliferation, colony formation ability, migration, and invasion in vitro and increased apoptosis. On the other hand, overexpression of SNORD104 promoted EC growth in vivo and in vitro. RIP assay showed that SNORD104 binds to the 2'-O-methyltransferase fibrillarin (FBL), and according to the results of Nm-seq and RTL-P assay, SNORD104 upregulated PARP1 (encoding poly (ADP-ribose) polymerase 1) 2'-O-methylation. The binding of FBL to PARP1 mRNA was also verified by RIP assay. Furthermore, SNORD104 expression was positively correlated with PARP1 expression in EC tissues. In the presence of actinomycin D, SNORD104 increased the stability of PARP1 mRNA and promoted its nuclear localization. Finally, silencing FBL or PARP1 in the HEC1B cells overexpressing SNORD104 inhibited their proliferative and clonal capacities and increased apoptosis rates., Conclusions: SNORD104 enhances PARP1 mRNA stability and translation in the EC cells by upregulating 2'-O-methylation and promotes tumor growth., (© 2022. The Author(s).)

Background: N-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., Methods: The 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 137 Cs 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., Results: Here, 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., Conclusion: Collectively, 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. Video Abstract., (© 2022. The Author(s).)

Background: NONO-TFE3 translocation renal cell carcinoma (NONO-TFE3 tRCC) is one subtype of RCCs associated with Xp11.2 translocation/TFE3 gene fusions RCC (Xp11.2 tRCCs). Long non-coding RNA (lncRNA) has attracted great attention in cancer research. The function and mechanisms of TRAF3IP2 antisense RNA 1 (TRAF3IP2-AS1), a natural antisense lncRNA, in NONO-TFE3 tRCC remain poorly understood., Methods: FISH and qRT-PCR were undertaken to study the expression, localization and clinical significance of TRAF3IP2-AS1 in Xp11.2 tRCC tissues and cells. The functions of TRAF3IP2-AS1 in tRCC were investigated by proliferation analysis, EdU staining, colony and sphere formation assay, Transwell assay and apoptosis analysis. The regulatory mechanisms among TRAF3IP2-AS1, PARP1, PTEN and miR-200a-3p/153-3p/141-3p were investigated by luciferase assay, RNA immunoprecipitation, Western blot and immunohistochemistry., Results: The expression of TRAF3IP2-AS1 was suppressed by NONO-TFE3 fusion in NONO-TFE3 tRCC tissues and cells. Overexpression of TRAF3IP2-AS1 inhibited the proliferation, migration and invasion of UOK109 cells which were derived from cancer tissue of patient with NONO-TFE3 tRCC. Mechanistic studies revealed that TRAF3IP2-AS1 accelerated the decay of PARP1 mRNA by direct binding and recruitment of N 6 -methyladenosie methyltransferase complex. Meanwhile, TRAF3IP2-AS1 competitively bound to miR-200a-3p/153-3p/141-3p and prevented those from decreasing the level of PTEN., Conclusions: TRAF3IP2-AS1 functions as a tumor suppressor in NONO-TFE3 tRCC progression and may serve as a novel target for NONO-TFE3 tRCC therapy. TRAF3IP2-AS1 expression has the potential to serve as a novel diagnostic and prognostic biomarker for NONO-TFE3 tRCC detection.

Background: Acute myeloid leukemia (AML) is a heterogenic lethal disorder characterized by the accumulation of abnormal myeloid progenitor cells in the bone marrow which results in hematopoietic failure. Despite various efforts in detection and treatment, many patients with AML die of this cancer. That is why it is important to develop novel therapeutic options, employing strategic target genes involved in apoptosis and tumor progression., Methods: The aim of the study was to evaluate PARP1, PARP2, PARP3, and TRPM2 gene expression at mRNA level using qPCR method in the cells of hematopoietic system of the bone marrow in patients with acute myeloid leukemia, bone marrow collected from healthy patients, peripheral blood of healthy individuals, and hematopoietic stem cells from the peripheral blood after mobilization., Results: The results found that the bone marrow cells of the patients with acute myeloid leukemia (AML) show overexpression of PARP1 and PARP2 genes and decreased TRPM2 gene expression. In the hematopoietic stem cells derived from the normal marrow and peripheral blood after mobilization, the opposite situation was observed, i.e. TRPM2 gene showed increased expression while PARP1 and PARP2 gene expression was reduced. We observed positive correlations between PARP1, PARP2, PARP3, and TRPM2 genes expression in the group of mature mononuclear cells derived from the peripheral blood and in the group of bone marrow-derived cells. In AML cells significant correlations were not observed between the expression of the examined genes. In addition, we observed that the reduced expression of TRPM2 and overexpression of PARP1 are associated with a shorter overall survival of patients, indicating the prognostic significance of these genes expression in AML., Conclusions: Our research suggests that in physiological conditions in the cells of the hematopoietic system there is mutual positive regulation of PARP1, PARP2, PARP3, and TRPM2 genes expression. PARP1, PARP2, and TRPM2 genes at mRNA level deregulate in acute myeloid leukemia cells.

Background: Poly (ADP-ribosyl) ation (PARylation) is an important posttranslational modification that regulates DNA repair, gene transcription, stress responses and developmental processes in multicellular organisms. Poly (ADP-ribose) polymerase (PARP) catalyzes PARylation by consecutively adding ADP-ribose moieties from NAD + to the amino acid receptor residues on target proteins. Arabidopsis has three canonical PARP members, and two of these members, AtPARP1 and AtPARP2, have been demonstrated to be bona fide poly (ADP-ribose) polymerases and to regulate DNA repair and stress response processes. However, it remains unknown whether AtPARP3, a member that is highly expressed in seeds, has similar biochemical activity to that of AtPARP1 and AtPARP2. Additionally, although both the phylogenetic relationships and structural similarities indicate that AtPARP1 and AtPARP2 correspond to animal PARP1 and PARP2, respectively, two previous studies have indicated that AtPARP2, and not AtPARP1, accounts for most of the PARP activity in Arabidopsis, which is contrary to the knowledge that PARP1 is the predominant PARP in animals., Results: In this study, we obtained both in vitro and in vivo evidence demonstrating that AtPARP3 does not act as a typical PARP in Arabidopsis. Domain swapping and point mutation assays indicated that AtPARP3 has lost NAD + -binding capability and is inactive. In addition, our results showed that AtPARP1 was responsible for most of the PARP enzymatic activity in response to the DNA damage-inducing agents zeocin and methyl methanesulfonate (MMS) and was more rapidly activated than AtPARP2, which supports that AtPARP1 remains the predominant PARP member in Arabidopsis. AtPARP1 might first become activated by binding to damaged sites, and AtPARP2 is then poly (ADP-ribosyl) ated by AtPARP1 in vivo., Conclusions: Collectively, our biochemical and genetic analysis results strongly support the notion that AtPARP3 has lost poly (ADP-ribose) polymerase activity in plants and performs different functions from those of AtPARP1 and AtPARP2. AtPARP1, instead of AtPARP2, plays the predominant role in PAR synthesis in both seeds and seedlings. These data bring new insights into our understanding of the physiological functions of plant PARP family members.

Background: Recently, we showed that PARP1 is involved in cotranscriptional splicing, possibly by bridging chromatin to RNA and recruiting splicing factors. It also can influence alternative splicing decisions through the regulation of RNAPII elongation. In this study, we investigated the effect of PARP1-mediated chromatin changes on RNAPII movement, during transcription and alternative splicing., Results: We show that RNAPII pauses at PARP1-chromatin structures within the gene body. Knockdown of PARP1 abolishes this RNAPII pausing, suggesting that PARP1 may regulate RNAPII elongation. Additionally, PARP1 alters nucleosome deposition and histone post-translational modifications at specific exon-intron boundaries, thereby affecting RNAPII movement. Lastly, genome-wide analyses confirmed that PARP1 influences changes in RNAPII elongation by either reducing or increasing the rate of RNAPII elongation depending on the chromatin context., Conclusions: These studies suggest a context-specific effect of PARP1-chromatin binding on RNA polymerase movement and provide a platform to delineate PARP1's role in RNA biogenesis and processing.

Background: MicroRNAs (miRNAs) posttranscriptionally regulate gene expression and thereby contribute to the modulation of numerous complex and disease-relevant cellular processes, including cell proliferation, cell motility, apoptosis and stress response. miRNA-31-5p is encoded on a genomic fragile site, 9p21.3, which is reportedly lost in many hepatocellular carcinoma (HCC) tumors. Based on previous findings, we hypothesized that miR-31-5p alters chemosensitivity and that miR-31-5p mimics may influence sensitivity to chemotherapeutics in HCC as well as in a variety of other cancers., Methods: MiR-31-5p and PARP1 in HCC tissues were tested by RT-PCR and histological analysis, respectively. Next, clonogenic assay and western blot were used to detect miR-31-5p and PARP1 to modulate sensitivity to OXA-based chemotherapy. The distribution of OXA in the nuclear and intracellular was detected by ICP-MS. Coimmunoprecipitation was used to characterize the protein-protein interaction between PARP1 and ABCB9. A xenograft nude mouse model was used to examine the in vivo effects of miR-31-5p., Results: Reintroduction of miR-31-5p into miR-31-5p-null Hep3B cells significantly enhanced clonogenic resistance to oxaliplatin. Although miR-31-5p re-expression increased chemoresistance, it paradoxically increased the relative intracellular accumulation of oxaliplatin. This effect was coupled with a significantly decreased intranuclear concentration of oxaliplatin by ICP-MS. miR-31-5p prevents the nuclear location of PARP1 detected by immunofluorescence, histological analysis and Western blotting analysis. We subsequently identified an indirect miR-31-5p-mediated upregulation of ABCB9, which is a transporter associated with drug accumulation in lysosomes, along with an increased uptake of oxaliplatin to lysosomes; these phenomena were associated with a downregulation of PARP1, a bipotential transcriptional regulator with multiple miR-31-5p binding sites. However, the indirect overexpression of ABCB9 promoted cellular chemosensitivity, suggesting that miR-31-5p promotes chemoresistance largely via an ABCB9-independent mechanism., Conclusions: Overall, our data suggest that the loss of miR-31-5p from HCC tumors promotes chemosensitivity, and this knowledge may be prognostically beneficial in the context of therapeutic sensitivity.

Background: Nicotinamide adenine dinucleotide (NAD + ) is a critical molecule involved in various biological functions. Poly (ADP-ribose) polymerase 1 (PARP1) and sirtuin 1 (SIRT1) affect cellular NAD + levels and play essential roles in regulating metabolism. However, there has been little research on the effects of PARP1 and SIRT1 crosstalk during senescence., Methods: We isolated endothelial progenitor cells (EPCs) from human umbilical cord blood and treated them with a PARP1 inhibitor (PJ34)., Results: Using a stress-induced premature aging model built by H 2 O 2 , transfection with adenoviral vectors, and Western blot analysis, we observed that PJ34 treatment preserved intracellular NAD + levels, increased SIRT1 activity, decreased p53 acetylation, and improved the function of stress-induced premature aging EPCs., Conclusions: Our results suggest that PJ34 improves the function of aging-induced EPCs and may contribute to cellular therapies for atherosclerosis.

Background: Enhancing the antitumor activity of the DNA-damaging drugs is an attractive strategy to improve current treatment options. Trabectedin is an isoquinoline alkylating agent with a peculiar mechanism of action. It binds to minor groove of DNA inducing single- and double-strand-breaks. These kinds of damage lead to the activation of PARP1, a first-line enzyme in DNA-damage response pathways. We hypothesized that PARP1 targeting could perpetuate trabectedin-induced DNA damage in tumor cells leading finally to cell death., Methods: We investigated trabectedin and PARP1 inhibitor synergism in several tumor histotypes both in vitro and in vivo (subcutaneous and orthotopic tumor xenografts in mice). We searched for key determinants of drug synergism by comparative genomic hybridization (aCGH) and gene expression profiling (GEP) and validated their functional role., Results: Trabectedin activated PARP1 enzyme and the combination with PARP1 inhibitors potentiated DNA damage, cell cycle arrest at G2/M checkpoint and apoptosis, if compared to single agents. Olaparib was the most active PARP1 inhibitor to combine with trabectedin and we confirmed the antitumor and antimetastatic activity of trabectedin/olaparib combination in mice models. However, we observed different degree of trabectedin/olaparib synergism among different cell lines. Namely, in DMR leiomyosarcoma models the combination was significantly more active than single agents, while in SJSA-1 osteosarcoma models no further advantage was obtained if compared to trabectedin alone. aCGH and GEP revealed that key components of DNA-repair pathways were involved in trabectedin/olaparib synergism. In particular, PARP1 expression dictated the degree of the synergism. Indeed, trabectedin/olaparib synergism was increased after PARP1 overexpression and reduced after PARP1 silencing., Conclusions: PARP1 inhibition potentiated trabectedin activity in a PARP1-dependent manner and PARP1 expression in tumor cells might be a useful predictive biomarker that deserves clinical evaluation.

Background: Poly(ADP-ribose) polymerase 1 (PARP-1), which catalyzes poly(ADP-ribosyl)ation of proteins by using NAD + as a substrate, plays a key role in several nuclear events, including DNA repair, replication, and transcription. Recently, PARP-1 was reported to participate in the somatic cell reprogramming process. Previously, we revealed a role for PARP-1 in the induction of neural apoptosis in a cellular model of cerebral ischemia and suggested the possible use of PARP inhibitors as a new therapeutic intervention. In the present study, we examined the effects of PARP inhibitors on neural stem/progenitor cells (NSPCs) of the mouse brain., Results: PARP-1 was more abundant and demonstrated higher activity in NSPCs than in mouse embryonic fibroblasts. Treatment with PARP inhibitors suppressed the formation of neurospheres by NSPCs through the suppression of cell cycle progression and the induction of apoptosis. In order to identify the genes responsible for these effects, we investigated gene expression profiles by microarray analyses and found that several genes in the p53 signaling pathway were upregulated, including Cdkn1a, which is critical for cell cycle control, and Fas, Pidd, Pmaip1, and Bbc3, which are principal factors in the apoptosis pathway. Inhibition of poly(ADP-ribosyl)ation increased the levels of p53 protein, but not p53 mRNA, and enhanced the phosphorylation of p53 at Ser18. Experiments with specific inhibitors and also shRNA demonstrated that PARP-1, but not PARP-2, has a role in the regulation of p53. The effects of PARP inhibitors on NSPCs were not observed in Trp53 -/- NSPCs, suggesting a key role for p53 in these events., Conclusions: On the basis of the finding that PARP inhibitors facilitated the p53 signaling pathway, we propose that poly(ADP-ribosyl)ation contributes to the proliferation and self-renewal of NSPCs through the suppression of p53 activation.

Background: Alzheimer's disease (AD) is a progressive, multifactorial neurodegenerative disorder that is the main cause of dementia globally. AD is associated with increased oxidative stress, resulting from imbalance in production and clearance of reactive oxygen species (ROS). ROS can damage DNA and other macromolecules, leading to genome instability and disrupted cellular functions. Base excision repair (BER) plays a major role in repairing oxidative DNA lesions. Here, we compared the expression of BER components APE1, OGG1, PARP1 and Polβ in blood and postmortem brain tissue from patients with AD, mild cognitive impairment (MCI) and healthy controls (HC)., Results: BER mRNA levels were correlated to clinical signs and cerebrospinal fluid biomarkers for AD. Notably, the expression of BER genes was higher in brain tissue than in blood samples. Polβ mRNA and protein levels were significantly higher in the cerebellum than in the other brain regions, more so in AD patients than in HC. Blood mRNA levels of OGG1 was low and PARP1 high in MCI and AD., Conclusions: These findings reflect the oxidative stress-generating energy-consumption in the brain and the importance of BER in repairing these damage events. The data suggest that alteration in BER gene expression is an event preceding AD. The results link DNA repair in brain and blood to the etiology of AD at the molecular level and can potentially serve in establishing novel biomarkers, particularly in the AD prodromal phase.