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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cyclooxygenase 2 (COX-2) overexpression has been described in sporadic colonic neoplasia, but its role in ulcerative colitis (UC) neoplastic progression remains unexplored. Although the specific role of cyclooxygenase in colonic neoplasia is uncertain, its inhibition by nonsteroidal anti-inflammatory drugs decreases the risk of sporadic colonic adenocarcinoma and causes regression of adenomas in familial adenomatous polyposis. To investigate the role of COX-2 in UC-associated neoplasia, we assessed COX-2 protein and mRNA expression throughout the spectrum of UC-associated neoplastic lesions in four total colectomy specimens, using immunocytochemistry and a novel TaqMan reverse transcriptase-polymerase chain reaction assay. The findings were correlated with DNA ploidy and inflammatory activity. We found COX-2 overexpression throughout the neoplastic spectrum in UC (P: < 0.0001, R:(2)=0.53), even in diploid samples that were negative for dysplasia. Overall, neoplastic change explained 53% of the variation in COX-2 expression, whereas inflammatory activity explained only 11%. COX-2 was overexpressed in all aneuploid samples and in 38% of diploid samples (P: = 0.0074). cDNA representational difference analysis was also performed and revealed that COX-2 mRNA was an up-regulated cDNA representational difference analysis difference product. COX-2 overexpression occurs early in UC-associated neoplasia, and the increase cannot be explained by inflammatory activity alone. The data suggest that COX-2-specific inhibitors may have a chemopreventative role in UC but the possibility that they could exacerbate UC inflammatory activity needs to be tested.
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PMID:The role of cyclooxygenase 2 in ulcerative colitis-associated neoplasia. 1098 Jan 13

Ca(2+)-activated K(+) (K(Ca)) channels have been suggested to play a role in the control of endothelial functions such as regulation of vascular tone and cell proliferation. We established a method for single-cell reverse transcriptase-polymerase chain reaction analysis in combination with the patch-clamp technique to characterize K(Ca) channel expression and function in single endothelial cells (ECs) within the endothelial monolayer of intact human mesenteric arteries (MAs) and in disease states. We tested whether endothelial K(Ca) channel expression and function are altered in MAs obtained from patients with colonic adenocarcinoma (CA) compared with those in MAs from non-cancer patients with inactive diverticulitis. Expression of the intermediate-conductance K(Ca) channel (hIK1) was detected in non-cancer and CA patients. In whole-cell patch-clamp measurements, only ECs expressing hIK1 exhibited corresponding K(Ca) currents, whereas respective K(Ca) currents were missing in hIK1-negative ECs. This heterogeneity of hIK1 expression patterns is indicative of a specialized subset of ECs within the endothelial monolayer. In CA patients, compared with non-cancer patients, a 2.5-fold increase in hIK1-expressing ECs per MA was observed (P:<0.05). However, K(Ca) current densities in hIK1-expressing ECs of both groups were similar. In addition to hIK1, expression of the large-conductance K(Ca) channel (hSlo) was detected in single ECs from CA patients. The increased K(Ca) channel expression in CA patients resulted in a 2. 7-fold increase of bradykinin-induced endothelial hyperpolarization compared with controls (P:<0.05). This increased expression and function of K(Ca) channels might indicate an altered functional state of the endothelium in cancer patients and could play a role in tumor angiogenesis.
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PMID:Expression and function of endothelial Ca(2+)-activated K(+) channels in human mesenteric artery: A single-cell reverse transcriptase-polymerase chain reaction and electrophysiological study in situ. 1098 42

The in vitro biosynthesis of metallothionein (MT) has been investigated in RBC precursors from human cord blood in order to support the hypothesis for the nucleated precursor origin of MT in human red blood cells (RBC). Human RBC precursors are obtained by (i) separating glycophorin A(+) (gly A(+)) cells using a magnetic cell sorting (MACS) technique and by (ii) ex vivo expansion of precursors BFU-E (burst forming unit-erythroid) on methylcellulose semi-solid culture media from mononuclear cells of cord blood. Biosynthesis of MT is detected at the protein level, by immuno-histochemical staining using a mouse monoclonal antibody (E9) in ex vivo expanded RBC precursors obtained from BFU-E. Expression of MT is also detected at the mRNA level by MT specific reverse transcriptase polymerase chain reaction (RT-PCR) both in ex vivo expanded precursors from BFU-E and in MACS separated gly A(+) cells. In addition, the expression of the fetal form of MT, MT-0 (also known as MT-1H) at the mRNA level in glycophorin A(+) cells, is also confirmed by cDNA sequencing. With these observations, to our knowledge, MT biosynthesis in human erythroid precursors is reported for the first time. Moreover, the current findings of MT-0 expression at the mRNA level in gly A(+) RBC precursors of hCB has added one more member in the list of cells/organs like fetal liver, human monocytes, non-neoplastic tissues of adenocarcinoma etc., in which the expression of the human fetal form of MT, i.e. MT-0, has also been reported.
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PMID:Metallothionein biosynthesis in human RBC precursors. 1109 34

A coronavirus was isolated from feces of a diarrheic foal and serially propagated in human rectal adenocarcinoma (HRT-18) cells. Antigenic and genomic characterizations of the virus (isolate NC99) were based on serological comparison with other avian and mammalian coronaviruses and sequence analysis of the nucleocapsid (N) protein gene. Indirect fluorescent-antibody assay procedures and virus neutralization assays demonstrated a close antigenic relationship with bovine coronavirus (BCV) and porcine hemagglutinating encephalomyelitis virus (mammalian group 2 coronaviruses). Using previously described BCV primers, the N protein gene of isolate NC99 was amplified by a reverse transcriptase PCR (RT-PCR) procedure. The RT-PCR product was cloned into pUC19 and sequenced; the complete N protein of NC99 (446 amino acids) was then compared with published N protein sequences of other avian and mammalian coronaviruses. A high degree of identity (89.0 to 90.1%) was observed between the N protein sequence of NC99 and published sequences of BCV (Mebus and F15 strains) and human coronavirus (strain OC43); only limited identity (<25%) was observed with group 1 and group 3 coronaviruses. Based on these findings, the virus has been tentatively identified as equine coronavirus (ECV). ECV NC99 was determined to have close antigenic and/or genetic relationships with mammalian group 2 coronaviruses, thus identifying it as a member of this coronavirus antigenic group.
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PMID:Characterization of a coronavirus isolated from a diarrheic foal. 1110 90

This study demonstrates the localization of the prostaglandin (PG)D(2) receptor (DP) within the mucous-secreting globlet cells of the human colon by in situ hybridization, which suggests a role for DP in mucous secretion. Selective high affinity ligands were used, therefore, to evaluate DP regulation of mucous secretion in LS174T human colonic adenocarcinoma cells. The expression of hDP in LS174T cells was confirmed at the mRNA level by reverse transcriptase-polymerase chain reaction, and at the protein level by radioligand binding assays and signal transduction (cyclic AMP accumulation) assays. PGD(2) and the highly selective DP-specific agonist L-644,698 ((4-(3-(3-(3-hydroxyoctyl)-4-oxo-2-thiazolidinyl) propyl) benzoic acid) (racemate)), but not PGE(2) competed for [(3)H]-PGD(2)-specific binding to LS174T cell membranes (K:(i) values of 0.4 nM and 7 nM, respectively). The DP-specific agonists PGD(2), PGJ(2), BW245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropylhydantoin)), and L-644,698 showed similar potencies in stimulating cyclic AMP accumulation (EC(50) values: 45 - 90 nM) and demonstrated the expected rank order of potency. PGE(2) also elicited cyclic AMP production in this cell line (EC(50) value: 162 nM). The activation of cyclic AMP production by PGD(2) and L-644,698, but not PGE(2), was inhibited by the selective DP antagonist BW A868C. Thus, PGD(2) and L-644,698 act through hDP in LS174T cells. PGD(2), L-644,698 and PGE(2) (an established mucin secretagogue) potently stimulated mucin secretion in LS174T cells in a concentration-dependent manner (EC(50)<50 nM). However, BW A868C effectively antagonized only the mucin secretion mediated by PGD(2) and L-644,698 and not PGE(2). These data support a role for the DP receptor in the regulation of mucous secretion.
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PMID:The human prostanoid DP receptor stimulates mucin secretion in LS174T cells. 1113 29

Formalin-fixed, paraffin-embedded tissue is the most widely available material for retrospective clinical studies. In combination with the potential of genomics, these tissues represent an invaluable resource for the elucidation of disease mechanisms and validation of differentially expressed genes as novel therapeutic targets or prognostic indicators. We describe here an approach that, in combination with laser-assisted microdissection allows quantitative gene expression analysis in formalin-fixed, paraffin-embedded archival tissue. Using an optimized RNA microscale extraction procedure in conjunction with real-time quantitative reverse transcriptase-polymerase chain reaction based on fluorogenic TaqMan methodology, we analyzed the expression of a panel of cancer-relevant genes, EGF-R, HER-2/neu, FGF-R4, p21/WAF1/Cip1, MDM2, and HPRT and PGK as controls. We demonstrate that expression level determinations from formalin-fixed, paraffin-embedded tissues are accurate and reproducible. Measurements were comparable to those obtained with matching fresh-frozen tissue and neither fixation grade nor time significantly affected the results. Laser microdissection studies with 5-microm thick sections and defined numbers of tumor cells demonstrated that reproducible quantitation of specific mRNAs can be achieved with only 50 cells. We applied our approach to HER-2/neu quantitative gene expression analysis in 54 microdissected tumor and nonneoplastic archival samples from patients with Barrett's esophageal adenocarcinoma and showed that the results matched those obtained in parallel by fluorescence in situ hybridization and immunohistochemistry. Thus, the combination of laser-assisted microdissection and real-time TaqMan reverse transcriptase-polymerase chain reaction opens new avenues for the investigation and clinical validation of gene expression changes in archival tissue specimens.
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PMID:Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue. 1115 80

The matrix metalloproteinase matrilysin has been implicated in the progression of gastrointestinal and other cancers. The aim of this study was to examine matrilysin mRNA expression and determine whether it is correlated with K-ras mutations and/or progression of pancreatic carcinoma. Using the semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we analyzed 11 pancreatic cancer cell lines and 70 pancreatic adenocarcinoma tissues for matrilysin mRNA expression. The results were correlated with clinicopathological characteristics and K-ras mutations. Significant amounts of matrilysin mRNA were detected in six of the eight cell lines with K-ras mutations but not in the three cell lines with wild-type K-ras. Matrilysin mRNA was detected in 57 (81.4% ) of the 70 tumor tissues and in all of the eight liver metastases, but not in any of the adjacent non-tumorous tissues. Matrilysin expression was significantly correlated with the size of tumor, tumor spreading, lymph node metastasis, advanced pathologic tumor-node- metastasis stage and K-ras mutations. The relative amounts of matrilysin mRNA in tumor tissues increased with increase in tumor stage and were highest in liver metastatic tumor tissues. Our results suggest that matrilysin, the expression of which is correlated with K-ras mutations, plays a key role in tumor growth and progression of pancreatic carcinoma.
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PMID:Association of matrilysin mRNA expression with K-ras mutations and progression in pancreatic ductal adenocarcinomas. 1140 48

This study was aimed at identifying the molecular mechanisms by which ceramide inhibits telomerase activity in the A549 human lung adenocarcinoma cell line. C(6)-ceramide (20 microm) caused a significant reduction of telomerase activity at 24 h as detected using the telomeric repeat amplification protocol, and this inhibition correlated with decreased telomerase reverse transcriptase (hTERT) protein. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analyses showed that C(6)-ceramide significantly decreased hTERT mRNA in a time-dependent manner. Electrophoretic mobility shift and supershift assays demonstrated that the binding activity of c-Myc transcription factor to the E-box sequence on the hTERT promoter was inhibited in response to C(6)-ceramide at 24 h. These results were also confirmed by transient transfections of A549 cells with pGL3-Basic plasmid constructs containing the functional hTERT promoter and its E-box deleted sequences cloned upstream of a luciferase reporter gene. Further analysis using RT-PCR and Western blotting showed that c-Myc protein but not its mRNA levels were decreased in response to C(6)-ceramide at 24 h. The effects of ceramide on the c-Myc protein were shown to be due to a reduction in half-life via increased ubiquitination. Similar results were obtained by increased endogenous ceramide levels in response to nontoxic concentrations of daunorubicin, resulting in the inhibition of telomerase and c-Myc activities. Furthermore, the elevation of endogenous ceramide by overexpression of bacterial sphingomyelinase after transient transfections also induced the inhibition of telomerase activity with concomitant decreased hTERT and c-Myc protein levels. Taken together, these results show for the first time that both exogenous and endogenous ceramides mediate the modulation of telomerase activity via decreased hTERT promoter activity caused by rapid proteolysis of the ubiquitin-conjugated c-Myc transcription factor.
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PMID:Molecular mechanisms of ceramide-mediated telomerase inhibition in the A549 human lung adenocarcinoma cell line. 1144 Oct 1

Urotensin II is the most potent vasoconstrictor peptide identified so far. Expression of urotensin II and urotensin II receptor mRNAs was studied in various human tumor cell lines by reverse transcriptase polymerase chain reaction (PCR) method. Secretion of urotensin II by these tumor cells was studied by radioimmunoassay. The tumor cell lines studied were T98G glioblastoma cells, IMR-32 neuroblastoma cells, NB69 neuroblastoma cells, BeWo choriocarcinoma cells, SW-13 adrenocortical carcinoma cells, DLD-1 colorectal adenocarcinoma cells and HeLa cervical cancer cells. Urotensin II mRNA was expressed in 6 tumor cell lines except for NB69 neuroblastoma cells. Urotensin II receptor mRNA was expressed in all 7 tumor cell lines. A significant amount of urotensin II-like immunoreactivity was detected only in the culture medium of SW-13 adrenocortical carcinoma cells by radioimmunoassay. Sephadex G-50 column chromatography showed that the urotensin II-like immunoreactivity in the culture medium extract was eluted earlier than synthetic human urotensin II, suggesting that SW-13 cells secreted higher molecular weight materials, perhaps partially processed forms of the urotensin II precursor. Reverse phase high-performance liquid chromatography (HPLC) showed three immunoreactive peaks, one of which was eluted in the position of urotensin II. The present study has shown for the first time expression of urotensin II and urotensin II receptor mRNAs in various tumor cell lines and the secretion of urotensin II-like immunoreactivity by SW-13 adrenocortical carcinoma cells.
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PMID:Expression of urotensin II and urotensin II receptor mRNAs in various human tumor cell lines and secretion of urotensin II-like immunoreactivity by SW-13 adrenocortical carcinoma cells. 1144 48

Application of recombinant adenoviral vectors for cancer gene therapy is currently limited due to lack of specificity for tumor cells. For gastric and esophageal adenocarcinoma, we present here that the relative abundant expression of the primary adenovirus receptor, coxsackie/adenovirus receptor (CAR), on normal epithelium compared to carcinoma favors the transduction of the epithelium. As such, to achieve specific transduction of cancer cells, targeting approaches are required that ablate the binding of the virus to CAR and redirect the virus to tumor-specific receptors. By immunohistochemistry and reverse transcriptase polymerase chain reaction assays, we demonstrate a marked difference in expression of the human epithelial cell adhesion molecule (EpCAM) between normal and (pre)malignant lesions of the stomach and esophagus. Based on this, we explored the feasibility of using EpCAM to achieve gastric and esophageal adenocarcinoma selective gene transfer. Adenoviral vectors redirected to EpCAM using bispecific antibodies against the adenovirus fiber-knob protein and EpCAM specifically infected gastric and esophageal cancer cell lines. Using primary human cells, an improved ratio of tumor transduction over normal epithelium transduction was accomplished by the EpCAM-targeted vectors. This study thus indicates that EpCAM-targeted adenoviral vectors may be useful for gastric and esophageal cancer-specific gene therapy in patients.
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PMID:Selective gene delivery toward gastric and esophageal adenocarcinoma cells via EpCAM-targeted adenoviral vectors. 1147 54


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