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)

Novel antigen-presenting cells (APCs) were generated using cultured dendritic cells (DCs) and amplified tumor mRNA, and the potential of tumor antigen-reactive T cell induction by the tumor RNA-introduced DCs (DC/tumor RNA) was analyzed in a patient with melanoma antigen-encoding gene (MAGE3)-positive malignant melanoma of the esophagus. DCs were generated from an adherent fraction of peripheral blood mononuclear cells in the presence of granulocyte macrophage colony-stimulating factor and interleukin-4. Tumor mRNA was purified from tumor tissue, amplified in vitro using a T7 RNA polymerase system, and then introduced into DCs by electroporation (150 V/150 microF or 100 V/200 microF). The gene introduction efficiency was 44-55% as measured by enhanced green fluorescent protein reporter gene expression, and the viability of RNA-introduced DCs was approximately 80%. DC/tumor RNA could induce tumor antigen-reactive cytotoxic T lymphocytes (CTLs) in an mRNA-specific manner, but had no effect on the self-antigen-reactive T cells. DC/tumor RNA could induce the polyspecific antigen-reactive CTL responses mediated by both human leukocyte antigen class I and class II molecules, whereas MAGE3 peptide-pulsed DCs induced only the monospecific MAGE3-reactive CTL responses mediated by human leukocyte antigen class I molecules, showing the superiority of the DC/tumor RNA over the DC/peptide. It is suggested that the use of DC/tumor RNA as antigen-presenting cells may be more effective, convenient and practical for the DC-based anti-cancer immunotherapy.
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PMID:Generation of antigen-presenting cells using cultured dendritic cells and amplified autologous tumor mRNA. 1631 11

Minimally invasive intraoperative lymphatic mapping and sentinel node biopsy has become the standard approach for staging the regional lymph nodes for early-stage melanoma. The procedure requires close collaboration of surgeon, pathologist, and nuclear medicine physician. The strength of lymphatic mapping and sentinel node biopsy is its accuracy of detecting occult lymph node metastases. Reverse transcriptase-PCR (RT-PCR) analyses of either fresh-frozen or paraffin-embedded sections of the sentinel lymph nodes have been found to be more sensitive than H&E staining or immunohistochemistry techniques, but lack of specificity and limits in the availability of tissue specimens make this technique impractical for routine use. Three randomized clinical trials are examining the therapeutic value of lymphatic mapping and sentinel node biopsy for melanoma. Preliminary results of the Multicenter Lymphadenectomy Trial I show the high level of accuracy and low morbidity of lymphatic mapping and sentinel node biopsy done through an international working group. The therapeutic value of lymphatic mapping and sentinel node biopsy is still unclear. Multicenter Lymphadenectomy Trial II will test the clinical significance of lymph nodes evaluated by RT-PCR and the value of completion lymph node dissection for patients found to have tumor-positive sentinel lymph nodes by H&E, immunohistochemistry, or RT-PCR. The Sunbelt Melanoma Trial examines the therapeutic value of completion dissection and benefits of Intron A. The ability to detect occult nodal metastases and evaluate the interaction of primary tumor with the regional lymph nodes may provide for better understanding of the metastatic process in patients with melanoma and help to determine the function of the regional lymph nodes as markers of metastases or incubators of tumor cells in the metastatic cascade.
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PMID:Sentinel lymph node biopsy and melanoma biology. 1660 52

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

Transformed cells express high levels of non-telomeric reverse-transcriptase (RT) activity of retrotransposon and endogenous retrovirus origin. We previously reported that RT inhibition, either pharmacological or through transient silencing of RT-encoding LINE-1 (L1) elements by RNA interference (RNAi), reduced proliferation, induced differentiation and reprogrammed gene expression in human tumorigenic cell lines. Moreover, the antiretroviral drug efavirenz antagonized tumor progression in animal models in vivo. To get insight into the role of retroelements in tumorigenesis, we have now produced two cell lines derived from A-375 melanoma, in which the expression of either L1 retrotransposon, or HERV-K endogenous retrovirus, was stably suppressed by RNAi. Compared to the parental A-375 cell line, cells with stably interfered L1 expression show a lower proliferation rate, a differentiated morphology and lower tumorigenicity when inoculated in nude mice. L1 silencing modulates expression of several genes and, unexpectedly, also downregulates HERV-K expression. In HERV-K interfered cells, instead, L1 expression was unaffected, and cell proliferation and differentiation remained unchanged compared to parental A-375 cells. In vivo, however, their tumorigenic potential was found to be reduced after inoculation in nude mice. These results suggest that L1 and HERV-K play specific and distinct roles in cell transformation and tumor progression.
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PMID:Distinct roles for LINE-1 and HERV-K retroelements in cell proliferation, differentiation and tumor progression. 1723 20

Melanoma antigen recognized by T cells 1 (MART-1) and tyrosinase-related protein-2 (TRP-2) are two useful markers for immunohistochemical detection of melanocytic tumors. However, these markers may be passively acquired (phagocytosed) rather than actively synthesized. Reverse transcriptase in situ polymerase chain reaction (RT in situ PCR) can amplify even small amounts of specific mRNA in cells and therefore confirm the cellular source of a marker. We developed a one-step RT in situ PCR procedure in which Thermus thermophilus DNA polymerase synthesizes and amplifies cDNA from mRNA in a single reaction mixture. To examine its practicability and feasibility with formalin-fixed, paraffin-embedded (FFPE) tissue, we compared the results of one-step RT in situ PCR with those of immunohistochemistry (IHC). MART-1 mRNA was identified in the cytoplasm of lesional cells from 23/26 primary melanomas (92%), 9/9 metastatic melanomas (100%) and 5/6 nevi (83%). MART-1 epitope was detected by IHC in 23/24 primary melanomas (96%), 9/9 metastatic melanomas (100%) and 5/6 nevi (83%). TRP-2 mRNA was identified in the cytoplasm of lesional cells from 17/26 primary melanomas (65%), 6/9 metastatic melanomas (67%) and 4/6 nevi (67%). TRP-2 epitope was detected by IHC in 20/24 primary melanomas (83%), 9/9 metastatic melanomas (100%) and 4/6 nevi (67%). Both techniques detected MART-1 and TRP-2 in FFPE melanoma cell lines. Neither marker was detected in squamous cell carcinomas or basal cell carcinomas by RT in situ PCR or IHC. We conclude that the RT in situ PCR technique can be successfully applied to FFPE tissue to determine the cellular sources of gene expression observed by conventional PCR approaches.
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PMID:RT in situ PCR detection of MART-1 and TRP-2 mRNA in formalin-fixed, paraffin-embedded tissues of melanoma and nevi. 1820 35

Reverse transcriptase (RT) inhibitors are emerging as a novel class of anticancer differentiating agents, active in several human tumor cell models, such as melanoma and prostate, thyroid and colon carcinoma. Indeed, much evidence suggests that they may act by inhibiting endogenous RT, a gene highly expressed in undifferentiated and transformed cells. We therefore evaluated whether endogenous RT may represent a new molecular target in the treatment of human renal clear-cell carcinoma, a neoplasm with very low sensitivity to standard pharmacological therapies. Efavirenz and nevirapine, 2 non-nucleosidic RT inhibitors commonly used in HIV patients, either induced a reversible downregulation of cell proliferation or enhanced cell differentiation in primary cultures of human renal carcinoma cells characterized by high levels of endogenous RT activity. Both agents upregulated the expression of the vitamin D receptor and calbindin 28k genes, which are constitutively expressed in renal tubular cells, and induced vitamin D signaling by enhancing the ability of tumor cells to upregulate the vitamin D-dependent gene, CYP24. Furthermore, efavirenz- and nevirapine-differentiated tumor cells exhibited an immunogenic phenotype with an increased expression of HLA-I and CD40 antigens and an enhanced ability to elicit a specific T-cell response in mixed lymphocyte/tumor-cell cultures. Indeed, renal carcinoma cells exposed to efavirenz induced a CD8(+)CCR7-CD45RA(-) effector memory T-cell phenotype, whereas untreated RCC cells induced a CD8(+)CCR7(+)CD45RA(-) central memory T-cell phenotype. These data suggest that RT inhibitors may be a novel tool in the treatment of human renal clear-cell carcinoma, potentially able to enhance the immunogenic potential of tumor cell.
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PMID:Reverse transcriptase inhibitors induce cell differentiation and enhance the immunogenic phenotype in human renal clear-cell carcinoma. 1835 78

N6-Benzoyladenine-cyanoborane (2), and 6-triphenylphosphonylpurine-cyanoborane (3) were selected for investigation of cytotoxicity in murine and human tumor cell lines, effects on human HL-60 leukemic metabolism and DNA strand scission to determine the feasibility of these compounds as clinical antineoplastic agents. Compounds 2 and 3 both showed effective cytotoxicity based on ED(50) values less than 4 mug/ml for L1210, P388, HL-60, Tmolt(3), HUT-78, HeLa-S(3) uterine, ileum HCT-8, and liver Hepe-2. Compound 2 had activity against ovary 1-A9, while compound 3 was only active against prostate PL and glioma UM. Neither compound was active against the growth of lung 549, breast MCF-7, osteosarcoma HSO, melanoma SK2, KB nasopharynx, and THP-1 acute monocytic leukemia. In mode of action studies in human leukemia HL-60 cells, both compounds demonstrated inhibition of DNA and protein syntheses after 60 min at 100 muM. These compounds inhibited RNA synthesis to a lesser extent. The utilization of the DNA template was suppressed by the compounds as determined by inhibition of the activities of DNA polymerase alpha, m-RNA polymerase, r-RNA polymerase and t-RNA polymerase, which would cause adequate inhibition of the synthesis of both DNA and RNA. Both compounds markedly inhibited dihydrofolate reductase activity, especially in compound 2. The compounds appeared to have caused cross-linking of the DNA strands after 24 hr at 100 muM in HL-60 cells, which was consistent with the observed increased in ct-DNA viscosity after 24 hr at 100 muM. The compounds had no inhibitory effects on DNA topoisomerase I and II activities or DNA-protein linked breaks. Neither compound interacted with the DNA molecule itself through alkylation of the nucleotide bases nor caused DNA interculation between base pairs. Overall, these antineoplastic agents caused reduction of DNA and protein replication, which would lead to killing of cancer cells.
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PMID:Synthesis and cytotoxicity of cyanoborane adducts of n6-benzoyladenine and 6-triphenylphosphonylpurine. 1847 22

Reverse transcriptase-polymerase chain reaction (RT-PCR)-mediated detection of melanoma cells may be a prognostic factor for disease outcome. We investigated the presence of melanoma cells in lymphatic drainage and blood in melanoma patients after lymph node dissection (LND) via the highly sensitive multimarker (MM) RT-PCR assay. We collected 24-h lymph fluid (LY) and peripheral blood (BL) from 107 stage III melanoma patients after radical LND (59 axillary and 48 ilioinguinal LND). Tyrosinase, MART1 and uMAGE mRNA levels were determined by RT-PCR to detect melanoma cells, and the presence of at least one marker signified a positive result. All patients underwent follow-up (median for survivors, 21 months, range: 4-37 months). Forty patients (37.4%) were positive for LY MM RT-PCR and 28 (26.2%) were positive based on BL MM RT-PCR. No differences for disease-free survival (DFS) curves according to BL MM RT-PCR were observed, but we found significant differences in the estimated 24-month DFS rate for patients with at least one marker and those without any marker in lymph fluid [18.9% (95% confidence interval: 1.4-37.5%) and 42.1% (95% confidence interval: 29.7-54.5%), median: 9.9 and 15.3 months, respectively] (P=0.04). Detection of multiple markers in lymph fluid correlated with shorter DFS. Approximately 37% of lymph fluid after radical LND were positive by MM RT-PCR, which correlated significantly with early melanoma recurrences and shorter survival. The LY MM RT-PCR seems to be an effective prognostic tool for stage III melanoma patients. The MM RT-PCR analysis of single peripheral blood sample in these patients did not have additional prognostic value.
Melanoma Res 2008 Aug
PMID:Molecular staging by multimarker reverse transcriptase-polymerase chain reaction assay of lymphatic drainage and blood from melanoma patients after lymph node dissection. 1862 8

Although microRNAs (miRNAs) are key regulators of gene expression in normal human physiology and disease, transcriptional regulation of miRNAs is poorly understood, because most miRNA promoters have not yet been characterized. We identified the proximal promoters of 175 human miRNAs by combining nucleosome mapping with chromatin signatures for promoters. We observe that one-third of intronic miRNAs have transcription initiation regions independent from their host promoters and present a list of RNA polymerase II- and III-occupied miRNAs. Nucleosome mapping and linker sequence analyses in miRNA promoters permitted accurate prediction of transcription factors regulating miRNA expression, thus identifying nine miRNAs regulated by the MITF transcription factor/oncoprotein in melanoma cells. Furthermore, DNA sequences encoding mature miRNAs were found to be preferentially occupied by positioned-nucleosomes, and the 3' end sites of known genes exhibited nucleosome depletion. The high-throughput identification of miRNA promoter and enhancer regulatory elements sheds light on evolution of miRNA transcription and permits rapid identification of transcriptional networks of miRNAs.
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PMID:Chromatin structure analyses identify miRNA promoters. 1905 95

Primary poorly differentiated (small round and non-small) sinonasal neoplasms comprise histogenetically and biologically diverse entities with overlapping morphologic features. Because of the limited initial biopsy tissue materials, differential diagnostic difficulties may arise and complicate timely management of some cases. We used immunohistochemical and molecular marker analyses in a large cohort of these tumors to optimize their early diagnosis and classification. Fifty-two tumors of the skull base and sinonasal regions and, for comparison, 19 poorly differentiated neoplasms of other head and neck sites were analyzed by a panel of immunohistochemical markers including those of epithelial, mesenchymal, melanocytic, and neuroectodermal origin using tissue microarray. Reverse transcriptase-polymerase chain reaction analysis of messenger RNA for EWS-FLI1 and PAX-FKHR fusion transcripts and the human achaete-scute homolog-1 gene was performed on 24 of the 52 sinonasal tumors and the 19 tumors of other sites for comparison. The immunohistochemical results substantiated the phenotypic assessment and the initial diagnosis in 49 of the 52 tumors. In 4 instances the integrated markers and phenotypic analyses led to reclassification of 3 tumors and confirmed the histogenesis of a mesenchymal tumor with aberrant cytokeratin expression. Molecular analysis of the EWS-FLI1 fusion gene transcript revealed 4 (9.3%) of the 43 tumors to be positive; all were Ewing sarcomas. The human achaete-scute homolog-1 gene transcript was identified in 10 (23.8%) of 42 tumors: 3 of 6 neuroblastomas, all 4 neuroendocrine carcinomas, and 1 each in sinonasal undifferentiated carcinoma, rhabdomyosarcoma, and melanoma. The PAX-FKHR fusion transcript was not detected in any tumors. We conclude that (1) an integrated morphologic and biomarker algorithm may better optimize the early diagnosis of poorly differentiated sinonasal and skull-base tumors; (2) molecular analysis may assist in future biological stratification of certain classes of these tumors; and (3) the human achaete-scute homolog-1 gene transcript is a nonspecific marker for the diagnosis of neuroblastoma.
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PMID:Molecular and phenotypic analysis of poorly differentiated sinonasal neoplasms: an integrated approach for early diagnosis and classification. 1915 Jan 7


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