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)

Mucins, including MUC-1, are generally considered to be products of epithelial tissues and of their tumors. To examine the possible expression of MUC-1 in other cell types, a panel of human epithelial and non-epithelial tumor cell lines was studied by reverse transcriptase polymerase chain reaction (RT-PCR), Northern blot analysis, immunocytology and radioimmunoprecipitation. Using the highly sensitive RT-PCR method, products corresponding to the non-repetitive 5' and 3' MUC-1 sequences were detected in all the cell lines examined. Amplified products lacking the tandem repeat region of MUC-1, including a new short form (designated MUC-1/Z) different from the previously reported MUC-1/Y protein, were also detected in most cell lines tested. Northern blot analysis, using a probe to the variable number tandem repeat (VNTR) region, confirmed the presence of MUC-1 mRNA in the astrocytoma, melanoma and neuroblastoma cell lines studied. MUC-1 protein was detected by immunocytology in these cell lines using monoclonal antibody (MAb) 139H2. Immunoprecipitation analysis with [3H]-glucosamine-labeled cell lysates and MAb 139H2 or an antibody to the cytoplasmic domain, CT-1, detected MUC-1 protein in 2 epithelial cell lines, an astrocytoma cell line (SK-MG-4) but not in the melanoma and neuroblastoma cell lines studied. Northern blot analysis using a probe to the 3' end of MUC-1 mRNA, confirmed the presence of MUC-1 mucin and also identified short products corresponding to the size of the short variant forms. Protein products corresponding to the MUC-1/Y and MUC-1/Z variant forms were not observed using either [3H]-glucosamine-labeled OVCAR-3 cells or [3H]-amino acid-labeled MCF-7 cells and either CT-1 antibody or MAb 232A1, detecting an epitope to the C-terminal region. Thus, depending on the sensitivity of the assay used, varying amounts of MUC-1 mRNA and protein could be detected in non-epithelial tumor cell lines. Although the amounts of MUC-1 in these cell lines are much lower than in carcinomas, it is possible that MUC-1 mucin serves a similar function in non-epithelial as in epithelial cells. The possible role of MUC-1/Y and MUC-1/Z variant forms in these cell lines is not understood.
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PMID:Comparison of MUC-1 mucin expression in epithelial and non-epithelial cancer cell lines and demonstration of a new short variant form (MUC-1/Z). 921 28

The presence of tumour cells in the circulation may predict disease recurrence and metastasis. To improve on existing methods of cytological or immunocytological detection, we have developed a sensitive and quantitative technique for the detection of carcinoma cells in blood, using the reverse transcriptase polymerase chain reaction (RT-PCR) identifying transcripts of the pancarcinoma-associated tumour marker EGP-2 (KSA or 17-1A antigen). The amount of EGP2 mRNA was quantified using an internal recombinant competitor RNA standard with known concentration and which is both reversely transcribed and co-amplified in the same reaction, allowing for a reliable assessment of the initial amount of EGP2 mRNA in the sample. Calibration studies, seeding blood with MCF-7 breast carcinoma cells, showed that the assay can detect ten tumour cells among 1.0 x 10(6) leucocytes. The PCR assay revealed that normal bone marrow expresses low levels of EGP2 mRNA, although immunocytochemistry with the anti-EGP2 MAb MOC31 could not identify any positively stained cell. Analyses using this RT-PCR assay may prove to have applications to the assessment of circulating tumour cells in clinical samples.
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PMID:A quantitative reverse transcriptase polymerase chain reaction-based assay to detect carcinoma cells in peripheral blood. 921 28

Differential display was used to identify genes which were differentially expressed when HT-29 human colon adenocarcinoma cells were grown as monolayers attached to plastic or as colonies in soft agarose. One of the gel bands differentially displayed corresponded to a 171 bp fragment that showed 99% identity with a sequence of mRNA for human calnexin. The decrease in calnexin gene expression by HT-29 cells growing as colonies in soft agarose was confirmed by reverse transcriptase-PCR (RT-PCR) using calnexin-specific primers. We also used RT-PCR to show that the expression of calnexin was decreased in HT-29 cells and MCF-7 human breast adenocarcinoma cells growing in suspension in poly(hydroxyethyl methacrylate)-coated wells compared to cells growing as monolayers. The results suggest that there is a signal for the up-regulation of calnexin expression when cells contact a substrate which allows cell adhesion.
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PMID:The expression of the molecular chaperone calnexin is decreased in cancer cells grown as colonies compared to monolayer. 929 53

Circulating prostate cells can be detected in cancer patients by using reverse transcriptase-PCR (RT-PCR) assay for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) mRNA. A quality-control study involving a conventional RT-PCR assay was performed and, surprisingly, detected both transcripts in many negative control cell lines and in normal blood samples. The existence of an illegitimate transcription of the PSA and PSM genes was evidenced by sequence analysis of several PSM and PSA-PCR products. Sequencing indeed demonstrated the presence of a PSA or PSM polymorphism in some but not all the cell lines and patient samples, as well as a heterozygous mutation (G to A; Asp to Asn) in the Jurkat cell line. Moreover, the amount of PSA transcript in MCF-7, a PSA-negative breast line, increased after incubation with cycloheximide. Interestingly, the frequency of positivity was as high as 12% in male samples if only tested once, but dropped to 3% upon multiple testing of the same cDNA. This highlights the stochastic effects in RT-PCR results at high sensitivity, hence the importance of repetitive testing in clinical samples. Decreasing the number of cycles avoided the amplification of illegitimate transcripts but also affected the limit of detection, as evidenced with PSA and PSM cDNA containing plasmids, mixing of LNCap with normal blood samples, and the PSA-PSM-negative K562 cell line. The current data raise the need for a multicentric standardization of the RT-PCR methodology used to amplify PSA and PSM transcripts.
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PMID:Expression of prostate-specific antigen and prostate-specific membrane antigen transcripts in blood cells: implications for the detection of hematogenous prostate cells and standardization. 951 Aug 50

In many cases of human cancer, the appearance of hypersialylated glycan structures is related to a precise stage of the disease; this may depend on altered regulation of one or more sialyltransferases genes. Since several distinct sialyltransferase enzymes arising from different unique genes transfer sialic acid residues in the same linkage onto the same acceptor, it is impossible to precisely determine which enzyme is involved in the observed phenotype based on enzymatic assays. We have developed a very sensitive and highly reproducible multiplex reverse transcriptase-polymerase chain reaction technique in order to monitor the expression of four human sialyltransferases genes ST6Gal I, ST3Gal I, ST3Gal III and ST3Gal IV in small cell samples. Multiplex PCR amplification using specific primers for each sialyltransferase and detection of amplification products by polyacrylamide gel electrophoresis is a method that is fast and easy to handle and has proven to be useful for establishing sialyltransferase patterns of expression in breast immortalized cell line HBL100 as well as in breast cancer cell lines MCF-7/6, MCF-7/AZ and MDA.
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PMID:Multiplex RT-PCR method for the analysis of the expression of human sialyltransferases: application to breast cancer cells. 953 Sep 53

The aim of this study was to investigate certain genes for their suitability as molecular markers for detection of breast carcinoma cells using the reverse transcriptase-polymerase chain reaction (RT-PCR). RNA was prepared from MCF-7 breast carcinoma cells and peripheral blood leucocytes of healthy female volunteers. This RNA was screened for mRNA of MUC1, cytokeratin 19 (CK19) and CD44 (exons 8-11) by RT-PCR and the results validated by Southern blots. Variable degrees of expression of MUC1 and CD44 (exons 8-11) were detected in normal peripheral blood, rendering these genes non-specific for epithelial cells and therefore unsuitable for use as markers to detect breast carcinoma cells. Although CK19 mRNA was apparently specific, it was deemed unsuitable for use as a marker of breast cancer cells in light of its limited sensitivity. Furthermore, an attempt at using nested primers to increase sensitivity resulted in CK19 mRNA being detected after two amplification rounds in blood from healthy volunteers.
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PMID:Putative markers for the detection of breast carcinoma cells in blood. 957 23

Using reverse transcriptase-polymerase chain reaction amplification it was possible to detect the presence of type 1 human estrogen sulfotransferase (hEST1) mRNA in the hormone-dependent: MCF-7 and T-47D, and hormone-independent: MDA-MB-231 and MDA-MB-468, human breast cancer cells. The expression of this mRNA is significantly higher in the MDA-MB-468 cells and a correlation of this mRNA expression with the enzymatic activity was observed. The progestin promegestone (R-5020) at a low concentration (5 x 10(-7) M) can significantly increase the estrogen sulfotransferase activity and its mRNA in the hormone-dependent MCF-7 and T-47D cells. As estrogen sulfates are biologically inactive, the stimulatory effect on sulfotransferase by promegestone may open attractive possibilities in the control of estradiol in human breast cancer.
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PMID:Human estrogen sulfotransferase (hEST1) activities and its mRNA in various breast cancer cell lines. Effect of the progestin, promegestone (R-5020). 974 35

The dietary phytoestrogen, daidzein, produced a biphasic response in cell proliferation of cultured, estrogen-responsive human breast carcinoma MCF-7 cells. Cell growth was stimulated at a daidzein concentration of 0.25 microg/ml whereas the addition of daidzein at concentrations >25 microg/ml significantly inhibited cell growth in a dose-dependent fashion, resulting in an IC50 value of 50 microg/ml. Upon exposure to 50 microg/ml of daidzein, cell morphology was severely altered, cell volume decreased, and condensation of the chromosomes was clearly noticeable. To identify genes whose expression were inhibited by daidzein, a differential display reverse transcriptase polymerase chain reaction assay (DD-RT-PCR) was performed and the cDNA fragments of several daidzein-regulated genes were visualized. The sequence of one of the cloned cDNA fragments that showed differential mRNA expression level in response to daidzein at a concentration of 50 microg/ml had a high homology with a cDNA expressed in fetal human brain, EST 06411.
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PMID:Differential display screening for specific gene expression induced by dietary nonsteroidal estrogen. 989 Jul 44

Six endometrial cancer cell lines (Ishikawa, EIIL, HEC1A, 6, 50 and 59), one breast cancer cell line (MCF-7) and two ovarian cancer cell lines (OVHS-1, HRA) were treated for 24 or 168 h with a gonadotropin-releasing hormone (GnRH) analogue, Buserelin acetate, and the cellular growth profile was studied. All these cell lines except for the HRA line had positive GnRH receptor mRNA expression detected by reverse transcriptase polymerase chain reaction. GnRHa suppressed cell growth after 168 h of exposure, but not after 24 h. Suppression of cell growth by the exposure to cis-platinum (CDDP, 10 nM for 24 h) was significantly increased in the presence of GnRHa for 168 h. The mechanism of this growth inhibition was tested by examining both RNA components of human telomerase (hTR) expression and telomerase activity. The results showed that GnRHa inhibits telomerase activity without altering the RNA component of telomerase expression. The present data suggest that GnRH analogue may modulate endometrial, breast and ovarian cancer cell growth through modifying the telomerase activity. Since GnRHa increased the cytotoxic effects of CDDP and GnRHa is a compound of high patient compliance, the value of GnRHa as a tumor sensitizer to CDDP should be further tested in clinical trials.
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PMID:In vitro effects of gonadotropin-releasing hormone (GnRH) analogue on cancer cell sensitivity to cis-platinum. 1038 Nov 37

Nitric oxide can both stimulate and suppress apoptosis. By reverse transcriptase-polymerase chain reaction and sequencing we show that human breast cancer (MCF-7) cells express endothelial cell nitric-oxide synthase (ecNOS), but not other nitric-oxide synthase isoforms. Inhibition of ecNOS activity in MCF-7 cells increased tumor cell apoptosis, and this effect was also seen following treatment with an NO scavenger. In addition, low concentrations of the NO donor sodium nitroprusside inhibited, whereas high concentrations stimulated MCF-7 cell apoptosis. The ecNOS promoter was found to contain a specific binding site for the apoptosis-regulating protein p53. In co-transfection studies wild-type, but not mutant, p53 down-regulated transcription of an ecNOS promoter-luciferase reporter gene construct. In addition, NO donors up-regulated p53 protein levels in MCF-7 cells. These data point to a previously unrecognized p53-dependent regulation of ecNOS expression that may be important both for regulating apoptosis and for avoiding the generation of genotoxic quantities of NO.
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PMID:Endogenous endothelial cell nitric-oxide synthase modulates apoptosis in cultured breast cancer cells and is transcriptionally regulated by p53. 1060 25


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