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)

We examined the expression of ornithine decarboxylase (ODC) mRNA in 53 female cases of breast cancer by a reverse transcriptase-polymerase chain reaction (RT-PCR) assay to determine the clinicopathologic significance of its expression. A significantly higher expression of ODC mRNA was, observed in younger patients than in older patients. The patients with a larger sized tumour possessed a significantly higher expression of ODC mRNA. In addition, the cases with a poor prognosis showed significantly higher expression of ODC. Previous studies have reported in vivo and in vitro correlation between the expression of ODC and c-myc genes in human carcinomas. We disclosed a significant correlation between these genes in primary breast carcinomas. We conclude that the expression of ODC may potentially be a new biological marker for breast carcinoma.
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PMID:Expression of ornithine decarboxylase mRNA and c-myc mRNA in breast tumours. 947 98

Telomerase, a cellular reverse transcriptase, has been detected in the majority of human malignant tumors, where it provides an escape mechanism from proliferative limitations due to progressive telomere erosion with each cell division. In this study, we used a non-radioactive telomeric repeat amplification protocol (TRAP) with an internal telomerase assay standard for the detection and semiquantitative analysis of 98 single frozen sections of normal breast tissue and benign and malignant breast lesions on an automated laser-fluorescence sequencer. Telomerase activity was detected in 36 of 40 (90%) infiltrating breast carcinomas, whereas no activity was found in nonmalignant breast tissues including blunt duct adenosis, papilloma, ductal hyperplasia and atypical ductal hyperplasia. However, telomerase activity was detected in 59% of ductal in situ carcinomas, suggesting that telomerase reactivation is an early event in breast carcinogenesis. We found a positive correlation between telomerase activity levels and cell proliferation determined by MIB1 immunostaining. No correlation, however, could be demonstrated between telomerase activity and other known breast cancer prognostic indicators. Telomerase activity was also detected in 60% of fibroadenomas indicating that careful interpretation of analysis of telomerase activity in fine needle aspirates is required, since low telomerase activity may not necessarily be an indicator of malignancy in breast tissue.
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PMID:Telomerase activity in human proliferative breast lesions. 947 5

Although peripheral blood and bone marrow are usually readily available from patients, present techniques of RNA extraction are tedious, require millilitres of starting material and removal of red blood cells before RNA purification. Further, successful reverse transcriptase polymerase chain reaction (RT-PCR) amplification requires the removal of haemoglobin derivatives which interfere with the PCR process. Recently, one step rapid use reagents have become available, claiming to be useful for obtaining high quality RNA from microlitre quantities of whole blood drawn directly from the patient. Their use to date in clinical samples appears limited with little information in the literature documented. In an attempt to overcome this, we tested the Trizol-LS, RNA-STAT-50 and Ultraspec-3 reagents upon a statistically significant number of clinical isolates of fresh and cryopreserved peripheral blood, bone marrow, blood apheresis products and a breast cancer cell line (MCF7) in order to evaluate whether these methods could be applied to routine laboratory use in an RT-PCR method capable of detecting rare gene expression. Our findings showed that there was some variation in the quality of RNA extracted which was indicated by absorbance spectrophotometry at 260 and 280 nm. 1% agarose gel electrophoresis showed that each of these methods could yield total RNA capable of generating the signature 18S and 28S rRNA bands. Using the Kruskal-Wallis non-parametric anova test combined with Dunn's multiple comparison test, the only statistically significant difference (p<0.05) indicated that Trizol-LS was more reliable than RNA-STAT-50-LS and Ultraspec-3 at extracting RNA from fresh peripheral blood. RNA extracted with the Trizol-LS and RNA STAT-50 reagents was successfully amplified in a multiplex RT-PCR reaction for detection of the multi-drug resistance related genes MDR1, the multi-drug resistance related protein (MRP) and topoisomerase IIalpha. Low level MDR1 gene expression could be detected in frozen whole blood. However, PCR products were only seen when the anti-coagulant heparin was removed from all samples prior to cDNA production. RT-PCR amplification was not 100% successful with RNA extracted with Ultraspec-3 reagent. In conclusion, we found that the RNA extracted from whole blood with the Trizol-LS and the RNA-STAT-50 are suitable for use in clinically relevant molecular biology protocols that analyze rare event genes without further purification. Our results indicated that the Trizol-LS reagent was generally more consistent in obtaining a pure and sufficient quantity of RNA from patient material as shown by the mean result of purity and quantity in comparison to either Ultraspec-3 or RNA-STAT-50-LS reagents. Ultraspec-3 is not easily suited for direct use with whole blood products.
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PMID:Evaluation of three rapid RNA extraction reagents: relevance for use in RT-PCR's and measurement of low level gene expression in clinical samples. 948 49

Disseminated epithelial tumor cells have been detected in the bone marrow and blood of cancer patients by means of immunocytochemical or immunofluorescent staining of cytocentrifuge slides, multiparameter flow cytometry, and reverse transcriptase-polymerase chain reaction. However, it is hardly possible using such methods to detect tumor cells at a frequency below 10(-6). To increase the sensitivity of these detection techniques we have developed a new technology for the enrichment of disseminated epithelial tumor cells from hematopoietic cell samples by high-gradient magnetic cell sorting (MACS). Cells are permeabilized and fixed and carcinoma cells are magnetically labeled specifically with an anti-cytokeratin 8 monoclonal antibody (mAb) directly conjugated to superparamagnetic microbeads. Magnetically labeled cells are enriched on high-gradient magnetic columns. Tumor cells are detected in the enriched cell fraction by flow cytometry, fluorescence microscopy, or immunocytochemisty. In this study we demonstrated the method using a model system in which five to 5,000 cells from a breast cancer cell line were seeded into blood cell samples from a healthy donor containing 1.2 x 10(8) leukocytes. Tumor cells were 10,477+/-4242 (n=25)-fold magnetically enriched, and 57.7%+/-16.9% (n=33) of the initially seeded tumor cells were recovered. Applying the method to 20-40 mL blood samples from patients with advanced carcinomas of the breast, prostate, colon, rectum, or lung, we were able to detect between one and 6.8 x 10(4) cytokeratin-expressing tumor cells in 21 of 34 patients. This corresponds to frequencies of tumor cells between 6.8 x 10(-9) and 1.1 x 10(-3) among nucleated cells in the original sample. Enriched tumor cells were further analyzed for expression of tissue-specific and prognostic markers such as breast mucin glycoproteins, erbB2, and CD44v6 for additional characterization and to confirm their tumor origin. The technique described could become a valuable tool for the quantification and molecular characterization of metastatic carcinoma cells in hematopoietic tissue, and may ultimately prove useful in the diagnosis, prognosis, and monitoring of patients with carcinoma.
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PMID:Immunomagnetic enrichment of disseminated epithelial tumor cells from peripheral blood by MACS. 950 22

Identification and quantitative evaluation of drug resistance markers are essential to assess the impact of multidrug resistance (MDR) in clinical oncology. The MDR1 gene confers pleiotropic drug resistance in tumour cells, but other molecular mechanisms are also involved in drug resistance. In particular, the clinical pattern of expression of the other MDR-related genes is unclear and their interrelationships are still unknown. Here, we report standardization of the procedures used to determine a reliable method of semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) using a standard series of drug-sensitive and increasingly resistant cell lines to evaluate the expression of three MDR-related genes, i.e. MDR1 (multidrug resistance gene 1), MRP (multidrug resistance related protein) and GSTp (glutathione-S-transferase p), reported to be endogenous standard genes for normalization of mRNAs. A total of 74 breast cancer surgical biopsies, obtained before any treatment, were evaluated by this method. When compared with classical clinical and laboratory findings, GSTp mRNA level was higher in diploid tumours. However, the main finding of our study suggests a clear relationship between two of these MDR-related gene expressions, namely GSTp and MRP. This finding provides new insight into human breast tumours, which may possibly be linked to the glutathione conjugate carrier function of MRP. Well defined semiquantitative RT-PCR procedures can therefore constitute a powerful tool to investigate MDR phenotype at mRNA levels of different related genes in small and precious tumour biopsy specimens.
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PMID:Comparative evaluation by semiquantitative reverse transcriptase polymerase chain reaction of MDR1, MRP and GSTp gene expression in breast carcinomas. 951 46

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

A method based on the reverse transcriptase-polymerase chain reaction (RT-PCR) was developed that allows the determination of relative mRNA expression levels in fine-needle aspirates from human tumors. The method was developed for the c-erbB-2 gene, using the porphobilinogen deaminase (PBGD) gene as an internal standard. It was validated for mRNA isolated from cell lines and for material obtained by fine-needle aspiration from human breast cancer. Gene expression levels were determined by measuring the activity of radiolabeled RT-PCR-amplified gene-specific bands with a phosphor imager. At least four points are measured on the log-linear part of the amplification cycle versus signal intensity curves, and subsequently the distance between the curves of the gene of interest and that of an internal standard gene is used to calculate the relative expression levels. The method worked equally well with the BRCA1 gene, illustrating that it can be generalized to other genes. The method is suitable to measure or monitor semiquantitively gene expression levels in accessible human tumors in situ.
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PMID:A method to monitor mRNA levels in human breast tumor cells obtained by fine-needle aspiration. 955 96

The participation of viruses in mammary carcinogenesis has been largely studied in animals. A model similar to the mouse mammary tumor virus (MMTV) was previously proposed. Several lines of research supported the participation of MMTV in human breast cancer, but these evidences were contradicted when further research was performed. One major issue was the presence of human endogenous retroviral sequences that confounded results reporting MMTV-like sequences in human breast cancer. To overcome this problem we selected a 660 bp sequence of the MMTV env gene with low homology to endogenous sequences and search for a sequence to it using the polymerase chain reaction (PCR). The sequence was found in 38% of the human breast cancers and in 2% of the normal breasts studied. The sequence was not present in tumors from other organs. It was 90-98% homologous to MMTV and only 18% to human endogenous retrovirus (HERV) K-10. It was also detected in some of the positive tumors by Southern blot hybridization using one of the cloned 660 bp as a probe. Using reverse transcriptase PCR, it was possible to demonstrate that the 660 bp sequence is expressed in the majority of the tumors. Also, preliminary experiments revealed that sequences related to the LTR and gag genes of MMTV were present in the DNA of breast tumors. The origin of the MMTV-like sequences in tumor DNA could be the result of integrated MMTV-like sequences derived from a human mammary virus or may represent unknown endogenous sequences that can only be detected in breast tumors.
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PMID:[Searching for retroviral sequences related to human breast cancer]. 956 45

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

In the present study, we have employed a unique breast cancer cell line (Met-1, which was derived from a high metastatic potential tumor in transgenic mice expressing polyomavirus middle T oncogene) to study the role of CD44 variant isoform(s) in the regulation of metastatic breast tumor cell behavior. The results of reverse transcriptase-polymerase chain reaction, Southern blot, nucleotide sequencing, immunoprecipitation, and immunoblot analyses indicated that these cells express a major CD44 isoform (molecular weight approximately 260 kDa) containing a v3,8-10 exon insertion (designated as CD44v3,8-10). In addition, we have determined that CD44v3,8-10 binds specifically to the cytoskeletal proteins such as ankyrin. Biochemical analyses, using competition binding assays and a synthetic peptide identical to NGGNGTVEDRKPSEL (a sequence located between aa480 and aa494 of CD44v3,8-10) indicate that this 15-amino acid peptide binds specifically to the cytoskeletal protein ankyrin (but not to fodrin or spectrin). This peptide competes effectively for ankyrin binding to CD44v3,8-10. Therefore, we believe that the sequence 480NGGNGTVEDRKPSE494L, located at the cytoplasmic domain of CD44v3,8-10, is required for the ankyrin binding. We have also detected that CD44v3,8-10-containing Met-1 cells are capable of forming membrane spikes or "invadopodia" structures and undergo active migration processes. Treatments of Met-1 cells with certain agents including anti-CD44v3 antibody, cytochalasin D (a microfilament inhibitor), and W-7 (a calmodulin antagonist), but not colchicine (a microtubule disrupting agent) effectively inhibit "invadopodia" formation and subsequent tumor cell migration. Further analyses using zymography assays and double immunofluorescence staining indicated that CD44v3,8-10 is closely associated with the active form of matrix metalloproteinase, MMP-9, in a complex within "invadopodia" structures. These findings suggest that CD44v3,8-10 plays an important role in linking ankyrin to the membrane-associated actomyosin contractile system required for "invadopodia" formation (coupled with matrix degradation activities) and tumor cell migration during breast cancer progression.
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PMID:CD44v(3,8-10) is involved in cytoskeleton-mediated tumor cell migration and matrix metalloproteinase (MMP-9) association in metastatic breast cancer cells. 961 60


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