Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Apurinic endonuclease 1/redox effector factor-1 (Ape1/Ref-1 or Ape1) is an essential protein with two distinct functions. It is a DNA repair enzyme in the base excision repair (BER) pathway and a reduction-oxidation (redox) signaling factor maintaining transcription factors in an active reduced state. Our laboratory previously demonstrated that Ape1 is overexpressed in ovarian cancer and potentially contributes to resistance. Therefore, we utilized siRNA technology to knockdown protein levels of Ape1 in ovarian cancer cell line, SKOV-3x. Knocking Ape1 down had dramatic effects on cell growth in vitro but was not due to an increase in apoptosis and at least partially due to an extension in transit time through S-phase. Similarly, human ovarian tumor xenografts with reduced levels of Ape1 protein demonstrated a dramatic reduction in tumor volume (p<0.01) and also statistically significant (p=0.02) differences in (18)F-fluorodeoxyglucose (FDG) uptake indicating reduced glucose metabolism and cellular proliferation. Ape1's role in DNA repair and redox signaling is important to our basic understanding of ovarian cancer cell growth and these findings strongly support Ape1 as a therapeutic target.
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PMID:Knockdown of the DNA repair and redox signaling protein Ape1/Ref-1 blocks ovarian cancer cell and tumor growth. 1797 6

The aims of this study were to investigate mechanisms of action involved in H2AX phosphorylation by DNA interstrand crosslinking (ICL) agents and determine whether gammaH2AX could be a suitable pharmacological marker for identifying potential ICL cellular chemosensitivity. In normal human fibroblasts, after treatment with nitrogen mustard (HN2) or cisplatin, the peak gammaH2AX response was detected 2-3 h after the peak of DNA ICLs measured using the comet assay, a validated method for detecting ICLs in vitro or in clinical samples. Detection of gammaH2AX foci by immunofluorescence microscopy could be routinely detected with 6-10 times lower concentrations of both drugs compared to detection of ICLs using the comet assay. A major pathway for repairing DNA ICLs is the initial unhooking of the ICL by the ERCC1-XPF endonuclease followed by homologous recombination. HN2 or cisplatin-induced gammaH2AX foci persisted significantly longer in both, ERCC1 or XRCC3 (homologous recombination) defective Chinese hamster cells that are highly sensitive to cell killing by ICL agents compared to wild type or ionising radiation sensitive XRCC5 cells. An advantage of using gammaH2AX immunofluorescence over the comet assay is that it appears to detect ICL chemosensitivity in both ERCC1 and HR defective cells. With HN2 and cisplatin, gammaH2AX foci also persisted in chemosensitive human ovarian cancer cells (A2780) compared to chemoresistant (A2780cisR) cells. These results show that gammaH2AX can act as a highly sensitive and general marker of DNA damage induced by HN2 or cisplatin and shows promise for predicting potential cellular chemosensitivity to ICL agents.
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PMID:Histone H2AX phosphorylation as a molecular pharmacological marker for DNA interstrand crosslink cancer chemotherapy. 1850 35

CD59, belonging to membrane complement regulatory proteins (mCRPs), inhibits the cytolytic activity of complement and is over-expressed in solid cancers, including ovary cancer. The aim of the present study was to construct recombinant retrovirus encoding shRNA targeted human CD59 and infect A2780 cells in order to investigate the relationship between decreased CD59 expression and tumorigenesis of ovary cancer. siCD59 and siCD59-C were successfully constructed and identified by PCR, restriction endonuclease analyses and DNA sequencing, respectively. The siCD59 was able to efficiently infect A2780 cells, which was confirmed by Western blotting. When incubated with fresh normal human serum (8%, v/v) for 1 h at 37 degrees centigrade, the cell viability was decreased and cell damage was increased in siCD59 infected A2780 cells compared to siCD59-C infected cells. This led to the activation of caspase-3. The apoptosis in siCD59 infected cells was shown with hypercondensed nuclei using Hoechst staining. Meanwhile, the weight of ovary tumor graft in nude mice was significantly decreased in siCD59 group compared to that of siCD59-C group. And the expression of CD59 protein in tumor tissue in siCD59 group was significantly decreased. These results suggested that CD59 silencing in ovary cancer cells via retrovirus-mediated RNAi can enhance complement-mediated cell damage, inhibiting growth of ovary cancer. CD59 might be a potential target for gene therapy in ovary cancer.
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PMID:CD59 silencing via retrovirus-mediated RNA interference enhanced complement-mediated cell damage in ovary cancer. 1925 81

Resistance to platinum is a major limitation for the treatment of ovarian cancer. In an effort to overcome the platinum resistance problem in ovarian cancer treatment, we explored the correlation between cisplatin resistance and the human AP endonuclease (APE1 or Ref-1). APE1/Ref-1 is a multifunctional protein that is not only an essential enzyme in base excision repair pathway, but also acts as a major redox-signaling factor that has a wide variety of important cellular functions including transcription factor regulation, oxidative signaling and cell cycle control. In this study, we examined APE1/Ref-1 expression by immunohistochemistry in sections of ovarian cancers from 78 patients who were administered standard adjuvant chemotherapy based on platinum post-operatively. Altered levels and subcellular APE1/Ref-1 expression was found in patients not responding to platinum-based chemotherapy comparing with those who responded to platinum-based chemotherapy. Meanwhile, we detected the APE1/Ref-1 expression in A2780 and CP70 cell lines which have different sensitivity to cisplatin. We found similar altered APE1/Ref-1 expression in them. We hypothesized that the APE1/Ref-1 expression is responsible in part for the cisplatin resistance. To answer this hypothesis, we decreased the APE1/Ref-1 level by silencing RNA targeting technology in A2780 and CP70 cell lines. The A2780 cells treated with APE1-siRNA had IC50 values ranging from 6.70 to 1.74 microM cisplatin compared with 15.81 microM for control A2780 cells. The CP70 cells treated with APE1-siRNA had 1.62-4.63-fold enhancement in cisplatin sensitivity. The apoptosis assays using TUNEL analysis showed that decreased APE1/Ref-1 level resulted in increased apoptosis levels in A2780 and CP70 cell lines compared with the control-treated cells. These data suggest that APE1/Ref-1 levels play an important role in the sensitization of ovarian cancer cells to apoptosis. In vitro studies revealed that it is possible to substantially enhance the cisplatin cytotoxicity by decreasing APE1/Ref-1 level in cisplatin-resistant cell lines.
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PMID:Alterations in the expression of the apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) in human ovarian cancer and indentification of the therapeutic potential of APE1/Ref-1 inhibitor. 1978 61

This paper describes an associated analysis method of DNA methylation for the detection of cancer using an optically amplifying cationic conjugated polymer (CCP, poly{(1,4-phenylene)-2,7-[9,9-bis(6'-N,N,N-trimethyl ammonium)-hexyl fluorene] dibromide)}. Genomic DNA is digested by methylation-sensitive restriction endonuclease, followed by PCR amplification to incorporate fluorescein-labeled dNTP. Only methylated DNA can be amplified by PCR, and the methylation level is detected through fluorescence resonance energy transfer (FRET) between CCP and fluorescein that is incorporated into the PCR product. The methylation levels of RASSF1A, OPCML, and HOXA9 promoters of 35 ovarian cancer samples and 11 normal samples were assayed. In accordance with the degree of methylation levels, they are clustered to three sections and assigned a value. Through an associated analysis, we acquired a threshold for cancer detection with a sensitivity of 85.7%. The assay takes about 20 h to obtain the detection results and shows great potential as a useful tool for diagnostic and screening of cancer.
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PMID:Associated analysis of DNA methylation for cancer detection using CCP-based FRET technique. 2432 47

Replication fork stalling can promote genomic instability, predisposing to cancer and other diseases. Stalled replication forks may be processed by sister chromatid recombination (SCR), generating error-free or error-prone homologous recombination (HR) outcomes. In mammalian cells, a long-standing hypothesis proposes that the major hereditary breast/ovarian cancer predisposition gene products, BRCA1 and BRCA2, control HR/SCR at stalled replication forks. Although BRCA1 and BRCA2 affect replication fork processing, direct evidence that BRCA gene products regulate homologous recombination at stalled chromosomal replication forks is lacking, due to a dearth of tools for studying this process. Here we report that the Escherichia coli Tus/Ter complex can be engineered to induce site-specific replication fork stalling and chromosomal HR/SCR in mouse cells. Tus/Ter-induced homologous recombination entails processing of bidirectionally arrested forks. We find that the Brca1 carboxy (C)-terminal tandem BRCT repeat and regions of Brca1 encoded by exon 11-two Brca1 elements implicated in tumour suppression-control Tus/Ter-induced homologous recombination. Inactivation of either Brca1 or Brca2 increases the absolute frequency of 'long-tract' gene conversions at Tus/Ter-stalled forks, an outcome not observed in response to a site-specific endonuclease-mediated chromosomal double-strand break. Therefore, homologous recombination at stalled forks is regulated differently from homologous recombination at double-strand breaks arising independently of a replication fork. We propose that aberrant long-tract homologous recombination at stalled replication forks contributes to genomic instability and breast/ovarian cancer predisposition in BRCA mutant cells.
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PMID:BRCA1 controls homologous recombination at Tus/Ter-stalled mammalian replication forks. 2477 1

The apurinic/apyrimidinic endonuclease 1 (APE1) is a protein central to the base excision DNA repair pathway and operates in the modulation of gene expression through redox-dependent and independent mechanisms. Aberrant expression and localization of APE1 in tumors are recurrent hallmarks of aggressiveness and resistance to therapy. We identified and characterized the molecular association between APE1 and nucleophosmin (NPM1), a multifunctional protein involved in the preservation of genome stability and rRNA maturation. This protein-protein interaction modulates subcellular localization and endonuclease activity of APE1. Moreover, we reported a correlation between APE1 and NPM1 expression levels in ovarian cancer, with NPM1 overexpression being a marker of poor prognosis. These observations suggest that tumors that display an augmented APE1/NPM1 association may exhibit increased aggressiveness and resistance. Therefore, targeting the APE1/NPM1 interaction might represent an innovative strategy for the development of anticancer drugs, as tumor cells relying on higher levels of APE1 and NPM1 for proliferation and survival may be more sensitive than untransformed cells. We set up a chemiluminescence-based high-throughput screening assay in order to find small molecules able to interfere with the APE1/NPM1 interaction. This screening led to the identification of a set of bioactive compounds that impair the APE1/NPM1 association in living cells. Interestingly, some of these molecules display anti-proliferative activity and sensitize cells to therapeutically relevant genotoxins. Given the prognostic significance of APE1 and NPM1, these compounds might prove effective in the treatment of tumors that show abundant levels of both proteins, such as ovarian or hepatic carcinomas.
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PMID:Inhibitors of the apurinic/apyrimidinic endonuclease 1 (APE1)/nucleophosmin (NPM1) interaction that display anti-tumor properties. 2586 59

ERCC1-XPF is a structure-specific endonuclease that is required for the repair of DNA lesions, generated by the widely used platinum-containing cancer chemotherapeutics such as cisplatin, through the Nucleotide Excision Repair and Interstrand Crosslink Repair pathways. Based on mouse xenograft experiments, where ERCC1-deficient melanomas were cured by cisplatin therapy, we proposed that inhibition of ERCC1-XPF could enhance the effectiveness of platinum-based chemotherapy. Here we report the identification and properties of inhibitors against two key targets on ERCC1-XPF. By targeting the ERCC1-XPF interaction domain we proposed that inhibition would disrupt the ERCC1-XPF heterodimer resulting in destabilisation of both proteins. Using in silico screening, we identified an inhibitor that bound to ERCC1-XPF in a biophysical assay, reduced the level of ERCC1-XPF complexes in ovarian cancer cells, inhibited Nucleotide Excision Repair and sensitised melanoma cells to cisplatin. We also utilised high throughput and in silico screening to identify the first reported inhibitors of the other key target, the XPF endonuclease domain. We demonstrate that two of these compounds display specificity in vitro for ERCC1-XPF over two other endonucleases, bind to ERCC1-XPF, inhibit Nucleotide Excision Repair in two independent assays and specifically sensitise Nucleotide Excision Repair-proficient, but not Nucleotide Excision Repair-deficient human and mouse cells to cisplatin.
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PMID:Inhibition of the ERCC1-XPF structure-specific endonuclease to overcome cancer chemoresistance. 2595 41

DNA repair pathways present in all cells serve to preserve genome stability, but in cancer cells they also act reduce the efficacy of chemotherapy. The endonuclease ERCC1-XPF has an important role in the repair of DNA damage caused by a variety of chemotherapeutic agents and there has been intense interest in the use of ERCC1 as a predictive marker of therapeutic response in non-small cell lung carcinoma, squamous cell carcinoma and ovarian cancer. We have previously validated ERCC1 as a therapeutic target in melanoma, but all small molecule ERCC1-XPF inhibitors reported to date have lacked sufficient potency and specificity for clinical use. In an alternative approach to prevent the repair activity of ERCC1-XPF, we investigated the mechanism of ERCC1 ubiquitination and found that the key region was the C-terminal (HhH)₂ domain which heterodimerizes with XPF. This ERCC1 region was modified by non-conventional lysine-independent, but proteasome-dependent polyubiquitination, involving Lys33 of ubiquitin and a linear ubiquitin chain. XPF was not polyubiquitinated and its expression was dependent on presence of ERCC1, but not vice versa. To our surprise we found that ERCC1 can also homodimerize through its C-terminal (HhH)₂ domain. We exploited the ability of a peptide containing this C-terminal domain to destabilise both endogenous ERCC1 and XPF in human melanoma cells and fibroblasts, resulting in reductions of up to 85% in nucleotide excision repair and near two-fold increased sensitivity to DNA damaging agents. We suggest that the ERCC1 (HhH)₂ domain could be used in an alternative strategy to treat cancer.
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PMID:Disruption of DNA repair in cancer cells by ubiquitination of a destabilising dimerization domain of nucleotide excision repair protein ERCC1. 2890 17

Clustered regularly interspaced short palindromic repeats (CRISPR)-caspase 9 (Cas9) genome editing technology holds great promise for the field of human gene therapy. However, a lack of safe and effective delivery systems restricts its biomedical application. Here, a folate receptor-targeted liposome (F-LP) was used to deliver CRISPR plasmid DNA co-expressing Cas9 and single-guide RNA targeting the ovarian cancer-related DNA methyltransferase 1 (DNMT1) gene (gDNMT1). F-LP efficiently bound the gDNMT1 plasmid and formed a stable complex (F-LP/gDNMT1) that was safe for injection. F-LP/gDNMT1 effectively mutated endogenous DNMT1 in vitro, and then expressed the Cas9 endonuclease and downregulated DNMT1 in vivo. The tumor growth of both paclitaxel-sensitive and -resistant ovarian cancers were inhibited by F-LP/gDNMT1, which shows fewer adverse effects than paclitaxel injection. Therefore, CRISPR-Cas9-targeted DNMT1 manipulation may be a potential therapeutic regimen for ovarian cancer, and lipid-mediated delivery systems represent promising delivery vectors of CRISPR-Cas9 technology for precise genome editing therapeutics.
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PMID:In Vivo Ovarian Cancer Gene Therapy Using CRISPR-Cas9. 2933 33


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