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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Angiogenesis is a critical step in tumor growth and metastatic invasion. We here report the study of the vascular status of 10 benign and 9 malignant pheochromocytomas. We examined the vascular architecture after immunostaining endothelial cells (CD34) and vascular smooth muscle cells (alpha-actin) and identified a vascular pattern characteristic of malignant lesions. To define a gene expression profile indicative of the invasive phenotype, we studied by in situ hybridization the expression of genes encoding several pro- and anti-angiogenic factors [hypoxia-inducible factor (HIF-1 alpha), EPAS1, vascular endothelial growth factor (VEGF), VEGF receptors, angiopoietins and their receptor Tie2, five genes of the endothelin system, and thrombospondin 1]. A semiquantitative evaluation of the labeling revealed an induction of genes encoding EPAS1, VEGF, VEGFR-1, VEGFR-2, endothelin receptor, type B (ETB) and endothelin receptor, type A (ETA) in malignant pheochromocytomas as compared to benign tumors. These differences were observed in tumor cells, in endothelial cells, or in both. Quantification by real-time reverse-transcriptase polymerase chain reaction showed an increase of EPAS1, VEGF, and ETB transcripts of 4.5-, 3.5-, and 10-fold, respectively, in malignant versus benign tumors. Furthermore, we observed a strong correlation between the expression of EPAS1 and VEGF in tumoral tissue and between EPAS1 and ETB in endothelial cells. Altogether, our observations show that analysis of angiogenesis provides promising new criteria for the diagnosis of malignant pheochromocytomas.
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PMID:Angiogenesis and vascular architecture in pheochromocytomas: distinctive traits in malignant tumors. 1236 97

Locked nucleic acids (LNA) are novel high-affinity DNA analogs that can be used as genotype-specific drugs. The LNA oligonucleotides (LNA PO ODNs) are very stable in vitro and in vivo without the need for a phosphorothiolated backbone. In this study we tested the biological fate and the efficacy in tumor growth inhibition of antisense oligonucleotides directed against the gene of the large subunit of RNA polymerase II (POLR2A) that are completely synthesized as LNA containing diester backbones. These full LNA oligonucleotides strongly reduce POLR2A protein levels. Full LNA PO ODNs appeared to be very stable compounds when injected into the circulation of mice. Full LNA PO ODNs were continuously administered for 14 days to tumor-bearing nude mice. Tumor growth was inhibited sequence specifically at dosages from 1 mg/kg/day. LNA PO ODNs appeared to be non-toxic at dosages <5 mg/kg/day. Biodistribution studies showed the kidneys to have the highest uptake of LNA PO ODNs and urinary secretion as the major route of clearance. This report shows that LNA PO ODNs are potent genotype-specific drugs that can inhibit tumor growth in vivo.
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PMID:In vivo tumor growth inhibition and biodistribution studies of locked nucleic acid (LNA) antisense oligonucleotides. 1256 Apr 91

Approximately 60% of all breast tumors are estrogen-responsive and chemicals that show estrogenic or anti-estrogenic properties are able to interact with breast tumor growth. In a breast tumor, adipose stromal cells (fibroblasts) surrounding the epithelial tumor contain the aromatase enzyme, which converts androgens into estrogens. Exposure to aromatase inducers can therefore lead to increased estrogen levels and possibly to accelerated breast tumor growth. Subsequently, breast tumor cells synthesize and secrete elevated levels of factors such as prostaglandin E2 (PGE2), interleukin-6 (IL-6), and IL-6 soluble receptor (IL-6sR), which in turn have the ability to stimulate aromatase gene transcription in fibroblasts, establishing a positive feedback loop. In this study, a technique that allows for culturing MCF-7 epithelial breast tumor cells and healthy primary human mammary fibroblasts together in one compartment was developed. To establish the positive feedback loop, the co-culture was exposed to estrogenic compounds. RNA was isolated and reverse-transcriptase polymerase chain reaction (RT-PCR) was performed on the aromatase and pS2 genes. Exposure of the co-culture to estradiol (E2), diethylstilbestrol (DES), and bisphenol-A (BPA), resulted in a three- to seven-fold increase of pS2 transcription levels. Furthermore, pS2 transcription levels increased even more when the aromatase substrate testosterone (20 nM) was present in the co-culture medium. Exposure of the co-culture to the aromatase inducer dexamethasone (DEX) resulted in increased pS2 transcription levels, as well as increased aromatase transcription levels. Simultaneous exposure to DEX and the synthetic anti-estrogen ICI 182,780 almost completely blocked the pS2 response. The aromatase induction response was not altered by ICI 182,780 treatment. Simultaneous exposure to DEX and the non-steroidal aromatase inhibitor fadrozole, abolished the effect of the presence of testosterone in the co-culture medium, but did not result in pS2 gene transcription levels as low as seen after exposure to ICI 182,780. These observations indicate the presence of a positive feedback loop in our co-culture system. This co-culture provides a more sophisticated and sensitive system to detect direct and indirect estrogenic effects of compounds and their possible effects on breast tumor promotion.
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PMID:Co-culture of primary human mammary fibroblasts and MCF-7 cells as an in vitro breast cancer model. 1552 92

We established a variant of MIAPaCa-2 human pancreatic cancer cells that is resistant to 2',2'-difluorodeoxycytidine (gemcitabine, dFdCyd), MIAPaCa-2/dFdCyd, and elucidated the biochemical characteristics and mechanism of dFdCyd-resistance in these cells. We also evaluated 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106, RNA polymerase inhibitor), a new anticancer ribonucleoside, for antitumor activity against the resistant cells in vitro and in vivo. MIAPaCa-2/dFdCyd cells were 2541-fold more resistant to dFdCyd than parental MIAPaCa-2 cells, and the major mechanism of the dFdCyd-resistance was found to be a decrease in the intracellular pool of dFdCyd and its active metabolites, which would result in a decrease in incorporation of dFdCyd triphosphate into DNA. This finding was confirmed by the discovery of decreased deoxycytidine kinase activity, increased cytidine deaminase and ribonucleotide reductase activity, and increased 5'-nucleotidase mRNA expression in the MIAPaCa-2/dFdCyd cells. The cytotoxicity of TAS-106 as an antitumor nucleoside analog was similar in both parental and dFdCyd-resistant cells, with IC(50) values of 6.25 and 6.27 nM, respectively, and this finding was supported by similar intracellular uptake and metabolism of TAS-106 in both cell lines. We also evaluated the in vivo antitumor activity of TAS-106 against MIAPaCa-2 and dFdCyd-resistant MIAPaCa-2/dFdCyd tumors implanted into nude mice. The tumor growth inhibition rate of weekly additions of TAS-106 (7 mg/kg, iv) against parental and dFdCyd-resistant tumors was 73% and 76%, respectively, while that of dFdCyd administered twice a week (240 mg/kg, iv) was 84% and 34%, respectively. These results suggest that TAS-106 would contribute to the treatment of patients with advanced pancreatic carcinomas in whom dFdCyd-based chemotherapy has failed.
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PMID:Possible antitumor activity of 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106) against an established gemcitabine (dFdCyd)-resistant human pancreatic cancer cell line. 1590 71

Vascular endothelial growth factor (VEGF) activity is correlated with a progressive tumor disease in patients with hepatocellular carcinoma (HCC). In spite of the well-recognized role of VEGF in HCC, there are few data available regarding therapeutic strategies to block VEGF activity. Therefore, we employed a recombinant adenoviral vector encoding a soluble dominant negative fragment of VEGF receptor 2 (Flk-1), AdsFlk-1, to control pre-established murine orthotopic and metastatic hepatomas. Vector function was confirmed via reverse-transcriptase polymerase chain reaction and ELISA, and angiostatic effects were analyzed in vitro and in vivo. Antitumoral effects of systemic AdsFlk-1 application were studied in a subcutaneous and orthotopic Hepa129 HCC model. Cell supernatant containing the truncated form of Flk-1 had no direct effect on cell proliferation of Hepa129 cells in vitro but reduced endothelial tube formation on matrigel matrix by approximately 80% in vitro. Endothelial-like cell infiltration into matrigel plugs in vivo was also decreased by 80%. Systemic treatment of tumor-bearing mice inhibited tumor growth by 84% compared with the corresponding control group within 16 days after vector application. Likewise, the survival rate was significantly improved in the AdsFlk-1 group compared with control. Orthotopic tumor growth was reduced by 82%, and development of malignant ascites was also retarded. In conclusion, systemic adenoviral-mediated gene transfer of an Flk-1 fragment significantly inhibited tumor growth in orthotopic and metastatic murine HCC. The data support the value of VEGF blockade as an effective target for HCC treatment.
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PMID:Effective angiostatic treatment in a murine metastatic and orthotopic hepatoma model. 1591 56

Seliciclib (CYC202, R-roscovitine) is a cyclin-dependent kinase (CDK) inhibitor that competes for the ATP binding site on the kinase. It has greatest activity against CDK2/cyclin E, CDK7/cyclin H, and CDK9/cyclin T. Seliciclib induces apoptosis from all phases of the cell cycle in tumor cell lines, reduces tumor growth in xenografts in nude mice and is currently in phase II clinical trials. This study investigated the mechanism of cell death in multiple myeloma cells treated with seliciclib. In myeloma cells treated in vitro, seliciclib induced rapid dephosphorylation of the carboxyl-terminal domain of the large subunit of RNA polymerase II. Phosphorylation at these sites is crucial for RNA polymerase II-dependent transcription. Inhibition of transcription would be predicted to exert its greatest effect on gene products where both mRNA and protein have short half-lives, resulting in rapid decline of the protein levels. One such gene product is the antiapoptotic factor Mcl-1, crucial for the survival of a range of cell types including multiple myeloma. As hypothesized, following the inhibition of RNA polymerase II phosphorylation, seliciclib caused rapid Mcl-1 down-regulation, which preceded the induction of apoptosis. The importance of Mcl-1 was confirmed by short interfering RNA, demonstrating that reducing Mcl-1 levels alone was sufficient to induce apoptosis. These results suggest that seliciclib causes myeloma cell death by disrupting the balance between cell survival and apoptosis through the inhibition of transcription and down-regulation of Mcl-1. This study provides the scientific rationale for the clinical development of seliciclib for the treatment of multiple myeloma.
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PMID:Seliciclib (CYC202, R-Roscovitine) induces cell death in multiple myeloma cells by inhibition of RNA polymerase II-dependent transcription and down-regulation of Mcl-1. 1595 89

Osteopontin (OPN) is a highly hydrophilic and negatively charged sialoprotein of approximately 298 amino acids with diverse regulatory functions, including cell adhesion and migration, tumor growth and metastasis, atherosclerosis, aortic valve calcification, and repair of myocardial injury. OPN is unique as an endogenous negative feedback inhibitor of NO expression. However, the specific cis- and trans-regulatory elements that determine the extent of endotoxin (LPS)- and NO-mediated induction of OPN synthesis are unknown. We have previously shown that LPS-induced S-nitrosylation of heterogeneous nuclear ribonucleoprotein (hnRNP)-A/B inhibits its activity as a constitutive trans-repressor of the OPN transcription by significantly decreasing its DNA binding activity. hnRNPs were originally described as chromatin-associated RNA-binding proteins that form complexes with RNA polymerase II transcripts. The hnRNP family is comprised of >20 proteins that contribute to the complex around nascent pre-mRNA and are thus able to modulate RNA processing. In this subsequent study, again using RAW 264.7 murine macrophages and COS-1 cells, we demonstrate that hnRNP-A/B and hnRNP-U proteins serve antagonistic transcriptional regulatory functions for OPN expression in the setting of LPS-stimulated NO synthesis. In the presence of NO, hnRNP-A/B dissociates from its OPN promoter site with subsequent derepression of OPN promoter activity. Subsequently, hnRNP-U binds to the same site to further augment OPN promoter activation. This has not been previously described for the hnRNP proteins. Our results represent a unique transcriptional regulatory mechanism which involves interplay between members of the hnRNP protein family.
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PMID:Transcriptional regulatory functions of heterogeneous nuclear ribonucleoprotein-U and -A/B in endotoxin-mediated macrophage expression of osteopontin. 1597 88

Lung cancer has emerged as a leading cause of cancer death in the world. Non-small cell lung cancer (NSCLC) accounts for 75-80% of all lung cancers. Current therapies are ineffective, thus new approaches are needed to improve the therapeutic ratio. Double stranded RNA (dsRNA)-mediated RNA interference (RNAi) has shown promise in gene silencing, the potential of which in developing new methods for the therapy of NSCLC needs to be tested. We report here RNAi induced effective silencing of the epidermal growth factor receptor (EGFR) gene, which is over expressed in NSCLC. NSCLC cell lines A549 and SPC-A1 were transfected with sequence- specific dsRNA as well as various controls. Immune fluorescent labeling and flow cytometry were used to monitor the reduction in the production of EGFR protein. Quantitative reverse-transcriptase PCR was used to detect the level of EGFR mRNA. Cell count, colony assay, scratch assay, MTT assay in vitro and tumor growth assay in athymic nude mice in vivo were used to assess the functional effects of EGFR silencing on tumor cell growth and proliferation. Our data showed transfection of NSCLC cells with dsRNA resulted in sequence specific silencing of EGFR with 71.31% and 71.78 % decreases in EGFR protein production and 37.04% and 54.92% in mRNA transcription in A549 and SPC-A1 cells respectively. The decrease in EGFR protein production caused significant growth inhibition, i.e.: reducing the total cell numbers by 85.0% and 78.3%, and colony forming numbers by 63.3% and 66.8%. These effects greatly retarded the migration of NSCLC cells by more than 80% both at 24 h and at 48 h, and enhanced chemo-sensitivity to cisplatin by four-fold in A549 cells and seven-fold in SPC-A1. Furthermore, dsRNA specific for EGFR inhibited tumor growth in vivo both in size by 75.06% and in weight by 73.08%. Our data demonstrate a new therapeutic effect of sequence specific suppression of EGFR gene expression by RNAi, enabling inhibition of tumor proliferation and growth. However, in vivo use of dsRNA for gene transfer to tumor cells would be limited because dsRNA would be quickly degraded once delivered in vivo. We thus tested a new bovine lentiviral vector and showed lentivector-mediated RNAi effects were efficient and specific. Combining RNAi with this gene delivery system may enable us to develop RNAi for silencing EGFR into an effective therapy for NSCLC.
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PMID:Silencing the epidermal growth factor receptor gene with RNAi may be developed as a potential therapy for non small cell lung cancer. 1598 32

Vascular endothelial growth factor (VEGF) plays a critical role during normal embryonic angiogenesis and also in the pathological angiogenesis that occurs in a number of diseases, including cancer. K562 human leukemia cells overexpress VEGF, with a shift in isoform production from membrane-bound VEGF189 to the more soluble VEGF165. In the present study, three 19 bp reverse repeated motifs targeting exons 5 and 7 boundary of VEGF165 gene sequence with 9 bp spacer were synthesized and cloned into eukaryotic expression plasmid pGenesil-1 containing U6 shRNA promoter and termination signal of RNA polymerase. The recombinant plasmids pGenesil-VR1, pGenesil-VR2, pGenesil-VR3 and pGenesil-con (plasmid containing random DNA fragment) were transfected into K562 cells, respectively, through lipofectamine reagent. A vector-based small interfering RNA(SiRNA) inhibited VEGF165 mRNA expression by 72% and protein production by 67% in K562 cells. Human microvascular endothelial cell migration induced by conditioned medium from VEGFsi-transfected K562 cells was significantly less than that induced by conditioned medium from K562 cells and control vector-transfected K562 cells. Furthermore, the VEGF shRNA dramatically suppressed tumor angiogenesis and tumor growth in a K562 s.c. xenograft model. Vessel density as assessed by vWF immunohistochemical analysis was also decreased. This strategy provides a novel tool to study the function of various VEGF isoforms and may contribute to VEGF-specific treatment in cancer.
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PMID:Vector-based RNAi approach to isoform-specific downregulation of vascular endothelial growth factor (VEGF)165 expression in human leukemia cells. 1703 51

Monocyte chemotactic protein 3 (MCP-3/CCL7), a CC chemokine able to attract and activate a large panel of leukocytes including natural killer cells and T lymphocytes, could be beneficial in antitumor therapy. Vectors were constructed based on the autonomous parvovirus minute virus of mice (MVMp), carrying the human (MCP-3) cDNA. These vectors were subsequently evaluated in the poorly immunogenic mouse melanoma model B78/H1. The infection of the tumor cells with MCP3-transducing vector at low virus input multiplicities, but not with wild-type virus, strongly inhibited tumor growth after implantation in euthymic mice. In a therapeutic B78/H1 model, repeated intratumoral injections of MCP3-tranducing virus prevented further tumor expansion as long as the treatment was pursued. The antitumor effects of the MCP-3-transducing vector were not restricted to this tumor model since they could also be observed in the K1735 melanoma. The depletion of CD4, CD8, NK cells and of interferon gamma (IFNgamma) in mice implanted with MVMp/MCP3-infected B78/H1 cells abolished the antitumor activity of the vector. The latter data, together with tumor growth in nude mice and reverse-transcriptase (RT)-PCR analyses of MVMp/MCP3-treated tumors, clearly showed that activated CD4, CD8 and NK cells were indispensable for the antineoplastic effect in the B78/H1 tumor. Altogether, our results show that MCP3-transducing parvovirus vectors may be quite potent against poorly or nonimmunogenic tumors, even in conditions where only a fraction of the tumor cell population is efficiently infected with recombinant parvoviruses.
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PMID:MCP-3 (CCL7) delivered by parvovirus MVMp reduces tumorigenicity of mouse melanoma cells through activation of T lymphocytes and NK cells. 1715 74


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