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 studied the efficiency of indirect tumor cell purging via enrichment of CD34+ hematopoietic progenitor cells from leukapheresis products (LP) in breast cancer patients based on immunomagnetic selection of CD34+ cells. Detection of tumor cells was made by immunocytochemical staining. In addition, we evaluated the capacity of cytokeratin 19 (CK19)- and a novel epidermal growth factor receptor (EGF-R)-specific reverse transcriptase-polymerase chain reaction (RT-PCR) for monitoring tumor cell depletion. LP from 13 breast cancer patients were analyzed. Twenty-three CD34 selection procedures were performed. A median of 1.4 x 10(10) total nucleated cells ([TNC] range, 0.88 to 3.5 x 10(10)) with a median CD34 purity of 2.5% (range, 0.4% to 6.3%) were entered into the selection procedure. Immunomagnetic CD34 enrichment resulted in a median purity of 83.3% (range, 45% to 95.4%) and a median recovery of 73.2% (range, 22% to 95%). Retransfusion of CD34-selected cells after high-dose chemotherapy resulted in a rapid and sustained hematologic recovery, reaching an absolute neutrophil count of 500/microL at day +10 and platelet count of 20,000/microL at day +11. Tumor cell depletion was quantified by immunocytochemical detection of CK19-positive cells. By this method, a median tumor cell depletion of 1.9 log (range, 0.7 to > 3 log) could be demonstrated. Immunocytochemical detection of tumor cells was more sensitive than RT-PCR, yielding positive results in 81% of LP (17 to 21) versus 58% positive LP (10 of 17). However, EGF-R-based RT-PCR was much more sensitive than CK19-based RT-PCR (10 of 17 v 1 of 17). Despite highly efficient CD34 selection, tumor cells were still detectable after CD34 enrichment using immunocytochemistry and EGF-R-specific RT-PCR. Thus, this novel EGF-R-specific RT-PCR appears to be of value as an additional method to detect contaminating breast cancer cells within LP.
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PMID:Monitoring of tumor cell purging after highly efficient immunomagnetic selection of CD34 cells from leukapheresis products in breast cancer patients: comparison of immunocytochemical tumor cell staining and reverse transcriptase-polymerase chain reaction. 897 10

Detection of the mRNA of selected genes by reverse transcriptase-polymerase chain reaction (RT-PCR) is a sensitive and powerful tool for detecting cancer cells in bone-marrow or peripheral-blood samples. In this study, we determined whether carcinoembryonic antigen (CEA) mRNA is detectable in the peripheral blood of patients with gastrointestinal or breast cancer. In addition, we studied selected patients undergoing surgical procedures to assess whether tumor manipulation during operation enhances cancer-cell dissemination. Peripheral blood from 55 patients with gastrointestinal or breast cancer and from 22 control cases was analysed for CEA mRNA using RT-PCR. For 15 selected cases undergoing curative surgery for cancer, samples were also obtained during and after surgery. The lower limit of detection was 1 to 10 CEA-positive cells diluted among 1 x 10(7) blood mononuclear cells. The test was positive for 20 of the 55 patients with cancer (36%). None of the 22 control samples were positive. An increase in positivity was observed with increasing stage of disease; however, even some patients with early-stage cancer showed positive results. In addition, CEA mRNA could be detected in the peripheral blood during operation in 3 of 13 patients whose pre-operative CEA mRNA in the peripheral blood had been negative. These findings suggest that, (1) RT-PCR amplification of CEA mRNA is an efficient means of detecting circulating solid cancer cells in the peripheral blood, although long-term clinical studies should be done to evaluate its usefulness; (2) not only breast cancer but also gastrointestinal cancer might be better regarded as a systemic disease even in early stages of carcinoma; and (3) surgical manipulation can provoke cancer-cell dissemination.
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PMID:Molecular detection of circulating solid carcinoma cells in the peripheral blood: the concept of early systemic disease. 898 Jan 76

A modified reverse transcriptase-polymerase chain reaction (RT-PCR) technique was established with the aim of monitoring the tumor cell contamination in peripheral blood stem cells harvested from breast cancer patients. In an experimental approach, single cell suspensions of different breast cancer cell lines were mixed to normal peripheral blood mononuclear cells in order to 1) determine the sensitivity of tumor cell detection within PBMC and 2) compare polymerase chain reaction in its capacity of monitoring the efficiency of immunomagnetic purging using the magnetic cell separation (MACS) system to immunocytochemical staining. Several target sequences were assessed for their indicative potential and specificity allowing the detection of breast cancer cells by RT-PCR. Among the sequences evaluated, epithelial growth factor receptor (EGF-R) mRNA and Cytokeratin 19 mRNA were shown to be highly specific and sensitive markers for the detection of breast cancer cells within normal peripheral blood mononuclear cells and for the evaluation of the efficiency in immunomagnetic purging. In addition, we were able to show that the MACS is a potent and efficient tool for the selection of tumor cells from peripheral blood mononuclear cells, thus establishing its value for clinical scale immunomagnetic purging.
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PMID:Reverse transcriptase-polymerase chain reaction (RT-PCR)-controlled immunomagnetic purging of breast cancer cells using the magnetic cell separation (MACS) system: a sensitive method for monitoring purging efficiency. 898 8

This paper examines the expression of fibroblast growth factor 2 (FGF-2) in the malignant human breast. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess the level of expression of FGF-2 in a series of 51 patients clinically followed up for a median of 84 months (Luqmani et al, 1992). Immunohistochemistry and Western blotting were used to show that the level of FGF-2 in breast tissues correlated with the amount of FGF-2 mRNA. FGF-2 was present in both malignant and non-malignant breast, although less was expressed in malignant tissues as determined by all three methods. Immunohistochemistry on frozen sections of breast tissue showed expression of FGF-2 in myoepithelial and epithelial cells in non-malignant samples and generally lower or undetectable levels of staining in malignant epithelial cells. The results obtained by immunohistochemistry correlated well with RT-PCR data showing similar levels of FGF-2 and FGF-2 mRNA expression in samples. No correlation was found between FGF-2 mRNA expression and T stage, nodal status or oestrogen receptor status. However, Kaplan-Meier survival plots show that higher levels of FGF-2 are associated with improved overall and disease-free survival. We suggest that FGF-2 expression may have value as a prognostic indicator in breast cancer.
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PMID:Fibroblast growth factor 2 in breast cancer: occurrence and prognostic significance. 900 May 94

Thrombospondin-1 (TSP1) is a Mr 450,000 extracellular matrix glycoprotein that modulates tumor growth, angiogenesis, and metastasis. Of the five structurally different TSPs described to date, only TSP2 is similar to TSP1 in terms of its molecular architecture, and TSP2 also modulates angiogenesis. Angiogenesis plays a relevant role in the biological aggressiveness of breast cancer, and TSP1 is present in the tumor stroma (termed desmoplasia) of invasive human breast ductal carcinoma not otherwise specified (NOS). The present study was designed to identify and quantify TSP1 and TSP2 mRNAs in normal, benign, and neoplastic human breast tissues using the reverse transcriptase PCR technique. We found that TSP2, like TSP1, was expressed in human breast tissues, and that TSP1 and TSP2 mRNA expression in invasive breast carcinoma NOS was significantly increased compared to that observed in normal and benign tissues. The expression of TSP1 and TSP2 in invasive breast ductal carcinoma NOS did not significantly correlate with any of the prognostic factors studied (tumor size, lymph node status, morphology, and hormone receptor status). However, when our study population was divided according to the quantity of tumor stroma, TSP1 (and possibly TSP2) mRNA expression and microvessel counts in desmoplastic-rich stroma of breast carcinoma NOS were significantly increased compared to those observed in desmoplastic-poor stromata.
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PMID:Thrombospondin-1 and -2 messenger RNA expression in normal, benign, and neoplastic human breast tissues: correlation with prognostic factors, tumor angiogenesis, and fibroblastic desmoplasia. 901 63

We used a reverse transcriptase-polymerase chain reaction method for squamous-cell carcinoma (SCC) antigen mRNA to detect circulating tumour cells in patients with carcinoma of the uterine cervix. The sensitivity of the method, as determined by cell spiking experiments, was 10 cultured A431 cells among 10(6) white blood cells. Circulating tumour cells were detected in 6 of 15 patients. In our control group of 24 women, SCC antigen mRNA was detected in 2 pregnant women at term. We followed up the patients for 24 months after sampling and evaluated the outcome. Three out of 6 patients positive for SCC antigen mRNA have relapsed. Additionally, 1 patient has developed breast cancer. In the group of 9 patients negative for SCC antigen mRNA there has been 1 relapse and 1 case of progression of disease. These results suggest that detection of SCC antigen mRNA in peripheral blood by RT-PCR could be useful for staging and evaluation of prognosis in epidermoid carcinoma of the uterine cervix.
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PMID:Detection of squamous-cell carcinoma antigen-expressing tumour cells in blood by reverse transcriptase-polymerase chain reaction in cancer of the uterine cervix. 903 73

The expression of midkine (MK), a growth/differentiation factor, was assessed in 34 surgically resected specimens of primary breast cancer or mastopathy. Using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, all of the non-cancerous and cancerous tissues were found to express MK except for one breast cancer specimen. Northern blot analysis revealed that MK mRNA was also expressed in the normal breast tissues examined. Immunohistochemical analysis of the MK protein was performed on a limited number of the specimens, showing that some cancerous tissues were immunoreactive with anti-MK antibodies. Furthermore, using RT-PCR analysis, expression of not only the wild-type but also a truncated form of MK, which was recently found in various human tumor cell lines, was detected in 6 of 26 cancerous tissues but not in non-cancerous tissues.
Breast Cancer Res Treat 1997 Mar
PMID:Midkine expression in human breast cancers: expression of truncated form. 906 93

A variant form of the human oestrogen receptor (ER) mRNA lacking sequences encoded within exon 5 has been described (Fuqua SAW, Fitzgerald SD, Chamness GC, Tandon AK, McDonnell DP, Nawaz Z, O'Malloy BW, McGuire WL 1991, Cancer Res 51: 105-109). We have examined the expression of the exon 5-deleted ER (HE delta5) mRNA variant in breast biopsies using reverse transcriptase polymerase chain reaction (RT - PCR). HE delta5 mRNA was present in only 13% of non-malignant breast tissues compared with 32% of carcinomas (95% CI, P=0.05). Presence of the HE delta5 mRNA was associated with the presence of immunohistochemically detected ER (P=0.015) and progesterone receptor (PR) (P=0.02). There was a positive correlation between the presence of HE delta5 and disease-free survival (P=0.05), suggesting that the presence of HE delta5 may be an indicator of better prognosis. We have raised a monoclonal antibody specific to the C-terminal amino acids of HE delta5. This antibody recognized the variant but not the wild-type ER protein. We show that HE delta5 protein is present in breast cancer using immunohistochemical techniques. We also analysed trans-activation by HE delta5 in mammalian cells and showed that, in MCF-7 cells, HE delta5 competes with wild-type ER to inhibit ERE-dependent trans-activation. Our results indicate that this variant is unlikely to be responsible for endocrine resistance of breast cancer, but its presence at both the mRNA and protein level suggest that it may, nevertheless, be involved in regulating the expression of oestrogen-responsive genes in breast cancer.
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PMID:Presence of exon 5-deleted oestrogen receptor in human breast cancer: functional analysis and clinical significance. 909 67

Osteoblasts are established targets of estrogen action in bone. We screened 66 conditionally immortalized clonal human osteoblast cell lines for estrogen receptors (ERs) using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for ER alpha mRNA and transactivation of adenovirus-estrogen response element (ERE)-tk-luciferase by 17 beta-estradiol (17 beta-E2) for functional ER protein. One of these cell lines, termed HOB-03-CE6, was chosen for further characterization. The cells, which were conditionally immortalized with a temperature-sensitive SV40 large T antigen, proliferated at the permissive temperature (34 degrees C) but stopped dividing at the nonpermissive temperature (> or = 39 degrees C). Alkaline phosphatase activity and osteocalcin secretion were upregulated by 1 alpha, 25-dihydroxyvitamin D3 in a dose-dependent manner. The cells also expressed type I collagen and other bone matrix proteins, secreted a variety of growth factors and cytokines, formed mineralized nodules based on alizarin red-S and von Kossa histochemical staining, and responded to dexamethasone, all-trans retinoic acid, and transforming growth factor-beta 1. This cell line expressed 42-fold less ER message than MCF-7 human breast cancer cells, as determined by quantitative RT-PCR. However, adenovirus-ERE-tk-luciferase activity was upregulated three- to fivefold in these cells by 17 beta-E2 with an EC50 of 64 pM. Furthermore, this upregulation was suppressed by co-treatment with the anti-estrogen ICI-182, 780. Cytosolic extracts of these cells specifically bound [125I]-17 beta-E2 in a concentration-dependent manner with a Bmax of 2.7 fmoles/mg protein (approximately 1,200 ERs/cell) and a Kd of 0.2 nM. DNA gel-shift analysis using a [32P]-ERE demonstrated the presence of ERs in nuclear extracts of these cells. Moreover, binding of the extracts to this ERE was blocked by a monoclonal antibody to the human ER DNA-binding domain. We evaluated these cells for 14 of 20 reported endogenous responses to 17 beta-E2 in osteoblasts. Although most of these responses appeared to be unaffected by the steroid, 17 beta-E2 suppressed parathyroid hormone-induced cAMP production, as well as basal interleukin-6 mRNA expression; conversely, the steroid upregulated the steady-state expression of alkaline phosphatase message in these cells. In summary, we have identified a clonal, conditionally phenotypic, human osteoblast cell line that expresses functional ERs and exhibits endogenous responses to 17 beta-E2. This cell line will be a valuable in vitro model for exploring some of the molecular mechanisms of estrogen action in bone.
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PMID:Functional properties of a conditionally phenotypic, estrogen-responsive, human osteoblast cell line. 913 93

Previous studies related mouse mammary tumor virus (MMTV) to human breast cancer. However, the presence of human endogenous retroviruses (HERs) confounded these results. We selected a 660-bp sequence of the MMTV env gene with low homology to HER (or any other known gene) and searched for a sequence homologous to it, using the polymerase chain reaction (PCR). The 660-bp sequence was detected in 131 (39%) of 335 unselected breast cancers, in 2 (6.9%) of 29 fibroadenomas, and in 2 (1.65%) of 121 normal breast specimens. The sequence was not present in normal tissues, or in other human cancers or cell lines. Cloning and sequencing of the 660-bp sequence revealed that it is 95-98% homologous to MMTV env gene, but not the known HERs or other viral or human gene. Southern blot hybridization using labeled cloned sequences demonstrated that the 660-bp sequence was present in very low copy number as a 6-8 kb EcoRI fragment only in breast cancer samples and in some of the human breast cancer cell lines that were positive by PCR. Preliminary experiments using reverse transcriptase (RT)-PCR indicated that expression of the 660-bp sequence can be detected in 65% of the positive tumors. We were also able to identify in breast cancer DNA a segment of 1.6 kb comprising LTR and env gene sequences, which are homologous to MMTV, but not to the HERs. 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.
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PMID:Possibilities of a viral etiology for human breast cancer. A review. 915 17


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