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)

The most frequent site of breast cancer metastasis is bone suggesting that some breast cancers express proteins that facilitate this process. We evaluated whether a highly metastatic breast cancer cell line, MDA-MB-231, and a less metastatic breast cancer cell line, MCF-7, contain bone morphogenetic proteins (BMP). Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that MDA and MCF-7 cells contain mRNAs for BMP receptors IA, IB and II. RT-PCR indicated the presence of mRNAs for BMPs 2 and 3 but not 4 and 7 in breast cells. Using a RT-PCR strategy with molecular beacons, we found that the mRNA for BMP2 in MDA cells was decreased by 75% after a sublethal dose of radiation. An ELISA using an antibody specific for BMP2 demonstrated that BMP2 protein was reduced after radiation of MDA cells. The mRNA for BMP2 was expressed to a lesser extent in MCF-7 cells than MDA cells and was not altered after radiation treatment of MCF-7 cells as demonstrated by molecular beacon RT-PCR. Recombinant human BMP2 decreased the proliferation of MDA cells to a greater extent than MCF-7 cells. These results expand the number of tissues that contain BMPs and demonstrate the effect of this signalling pathway of the growth state of these tissues.
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PMID:Identification of bone morphogenetic proteins and their receptors in human breast cancer cell lines: importance of BMP2. 1062 28

We have developed a new assay, ISET (isolation by size of epithelial tumor cells), which allows the counting and the immunomorphological and molecular characterization of circulating tumor cells in patients with carcinoma, using peripheral blood sample volumes as small as 1 ml. Using this assay, epithelial tumor cells can be isolated individually by filtration because of their larger size when compared to peripheral blood leukocytes. ISET parameters were defined using peripheral blood spiked with tumor cell lines (HepG2, Hep3B, MCF-7, HeLa, and LNCaP). ISET can detect a single, micropipetted tumor cell, added to 1 ml of blood. We also demonstrate that fluorescence in situ hybridization can be used to perform chromosomal analyses on tumor cells collected using ISET. Polymerase chain reaction-based genetic analyses can be applied to ISET-isolated cells, and, as an example, we demonstrate homozygous p53 deletion in single Hep3B cells after filtration and laser microdissection. Finally, we provide evidence for the in vivo feasibility of ISET in patients with hepatocellular carcinoma undergoing tumor resection. ISET, but not reverse transcriptase-polymerase chain reaction, allowed analysis of cell morphology, counting of tumor cells, and demonstration of tumor microemboli spread into peripheral blood during surgery. Overall, ISET constitutes a novel approach that should open new perpectives in molecular medicine.
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PMID:Isolation by size of epithelial tumor cells : a new method for the immunomorphological and molecular characterization of circulatingtumor cells. 1062 54

The presence of an exon 1' sequence in the estrogen receptor alpha (ERalpha) mRNA was detected in different stocks of ER-positive MCF-7 human breast cancer cells by reverse transcriptase polymerase chain reaction (RT-PCR) and ribonuclease protection analysis (RPA), but not by Northern blot analysis. This mRNA, however, was not detectable in ERalpha-positive ZR-75-1 or ERalpha-negative MDA-MB-231 breast cancer cells, suggesting that exon 1' ER mRNA is differentially expressed in some but not all ER-positive cell lines, and then, only at very low levels.
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PMID:Differential expression of an estrogen receptor messenger RNA containing exon 1' sequences in MCF-7 breast cancer cell line stocks. 1068 May 97

Angiopoietin-1 (Ang1) has been shown to act as an angiogenic promoter in embryonic angiogenesis by promoting vascular branching, pericyte recruitment and endothelial survival. We have investigated the role of Ang1 in tumour neovascularization under clinical conditions and in animal models. The expression of Ang1 in clinical breast cancer specimens was analysed by using laser-capture microdissection and reverse transcriptase-linked polymerase chain reaction (RT-PCR) on RNA isolated from the samples. Despite the expression of Ang1 in many human breast cancer cell lines, the gene was expressed in only three of 21 breast cancer clinical specimens, even though its receptor, Tie2, is abundant in the vasculature of all of these tumours. Ang1 was then overexpressed in a human breast cancer cell line (MCF-7) on its own and in conjunction with FGF1, an angiogenic factor shown to be able to increase the tumorigenicity of MCF-7 cells. High concentrations of Ang1 were produced in the conditioned media of the transfected cells (range 156-820 ng ml(-1)). However, in contrast to its physiological role as promoter of angiogenesis, overexpression of Ang1 did not enhance tumour growth, but instead caused up to a 3-fold retardation of tumour growth (P = 0.003).
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PMID:Expression and function of angiopoietin-1 in breast cancer. 1102 28

The plasma membrane Ca(2+) pump is a key regulator of cytosolic free Ca(2+). Recent studies have demonstrated the dynamic expression of the plasma membrane Ca(2+) pump in a variety of cell types. Furthermore, alterations in plasma membrane calcium pump activity have now been implicated in human disease. In this study, the development of a technique to quantitatively assess mRNA expression of the human plasma membrane Ca(2+) ATPase (PMCA1) isoform of the plasma membrane Ca(2+) pump, using a real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) assay in a human breast epithelial cell line (MCF-7) is described. The sequences of the PMCA1 primers and probe for real-time RT-PCR are presented. The results also indicate that PMCA1 mRNA can be normalized to both 18S ribosomal RNA (18S rRNA) and human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) in MCF-7 cells. Real-time RT-PCR will be most useful in assessing PMCA1 mRNA expression in cases where only low amounts of RNA are available and/or when numerous samples must be assessed simultaneously.
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PMID:Development of a real-time RT-PCR assay for plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA levels in a human breast epithelial cell line. 1139 29

The presence of estrogen receptors (ERs) in breast carcinomas is important for clinical response to endocrine therapy. However, the cellular mechanisms following ER activation are not fully understood. It has been indicated that expression of the ER is associated with the expression of c-erbB-4. To address this question, 103 breast carcinoma samples were studied using reverse transcriptase polymerase chain reaction (RT-PCR) analysis after application of a microselection method for RNA isolation. Total RNA for RT-PCR was isolated from 20-microm-thick frozen sections, which were made from microselected areas. Paraffin blocks from 98 of these 103 tumors were also immunohistochemically examined. Significant associations between ER-alpha and c-erbB-4 mRNA and protein expressions were found in the present study with both methods. One-fourth of the tumors did not express ER-alpha (22%, 24%, and 26% with chemical binding, immunohistochemistry, and RT-PCR, respectively). About one-half of the ER-alpha negative tumors did not express c-erbB-4 on both mRNA and protein levels (48% with RT-PCR and 46% with immunohistochemistry, P=0.001 for both methods). The endocrine therapy responsive breast cancer cell lines MCF-7 and T47-D were positive for both ER-alpha and c-erbB-4 expression, while the endocrine therapy nonresponsive breast cancer cell lines MDA-MD-231 and SK-BR-3 were not. Thus, we confirm the association between the expression of ER-alpha and c-erbB-4 mRNA and protein in breast carcinomas, indicating a role for c-erbB-4 in estrogen signal transduction.
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PMID:Estrogen receptor-alpha and C-ERBB-4 expression in breast carcinomas. 1149 42

Differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) was used to identify a novel retrovirus, designated SC1, that is expressed at high levels in rat granulosa cells and prepubertal Sertoli cells. The initial DDRT-PCR screen was performed using RNA from cultured prepubertal rat Sertoli cell, liver, and brain samples. SC1 was detected in the prepubertal rat Sertoli cell samples but not in those from liver and brain. SC1 cDNA was 6 kilobases in length and contained regions encoding for the gag, pol, and env retroviral proteins. Northern blot analysis failed to detect expression of the SC1 gene in total RNA isolated from adult brain, heart, spleen, lung, liver, skeletal muscle, kidney, prostate, and epididymis. Similarly, Northern blot analysis of testes from rats at various ages of development showed that high-level expression of the SC1 gene was limited to prepubertal testis samples. In situ hybridization analysis localized the SC1 mRNA to the seminiferous tubules of prepubertal testes and at a much lower level in Sertoli cells of adult testes. Northern blot analysis of total RNA isolated from Sertoli cells from 20-, 27-, and 35-day-old rat Sertoli cells and type A spermatogonia, pachytene spermatocytes, and round spermatids showed expression of the SC1 gene to be restricted to 20- and 27-day-old Sertoli cells, with no expression detected in germ cells. Furthermore, Northern blot analysis also showed expression of the SC1 gene in rat ovaries, and the level of expression was affected during eCG/hCG-induced ovulation. Expression of SC1 mRNA was localized by in situ hybridization of eCG-treated ovaries to the granulosa cell layer in developing follicles. Southern blot analysis showed SC1 to be endogenous in the rat and absent in mouse and human cell genomes. Transient transfection assays using the SC1 promoter region showed high promoter activity in MSC-1 and cultured prepubertal rat Sertoli cells, and no activity in 3T3 or MCF-7 cell lines.
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PMID:Identification of a novel retrovirus expressed in rat Sertoli cells and granulosa cells. 1156 55

Caspase-8 (Fas-associating protein with death domain-like interleukin-1beta- converting enzyme [FLICE]/MACH/Mch5) belongs to a family of cysteine proteases presumed to be the apex of the apoptotic signaling pathways. We recently reported the presence of a novel isoform of caspase-8, named caspase-8L, generated by the alternative splicing of human caspase-8 gene, from human peripheral blood lymphocytes by reverse transcriptase-polymerase chain reaction. We herein report a functional analysis of caspase-8L in the Fas-mediated apoptotic pathway. Caspase-8L is missing the catalytic site of caspase-8 but retains 2 N-terminal repeats of the death-effector domain. The caspase-8L messenger RNA was detected in various tissues but not in any cell lines examined. In human peripheral blood lymphocytes, caspase-8L was strongly suggested to be expressed at the protein level. In MCF-7 cells, caspase-8L transfection itself did not affect cell viability but instead inhibited the apoptosis induced by the cotransfection of caspase-8 in a dominant negative manner. Moreover, Fas-mediated apoptosis was inhibited in caspase-8L-transfected Jurkat cells, which were associated with a reduction in the caspase-8 catalytic activity. In vitro binding assays demonstrated that caspase-8L bound to FADD (Fas-associating protein with death domain) and caspase-8a and blocked the binding of caspase-8 to FADD. In in vivo binding assays, transfected caspase-8L bound to endogenous FADD. Thus, caspase-8L acts as an inhibitor of caspase-8 by interfering with the binding of caspase-8 to FADD and is involved in the regulation of Fas-mediated apoptosis.
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PMID:Characterization of caspase-8L: a novel isoform of caspase-8 that behaves as an inhibitor of the caspase cascade. 1201 Aug 9

The effects of estradiol (E2) and progesterone on the oxytocin receptor (OTR) were investigated in MCF-7 and Hs 578T human breast cancer cell lines. OTR messenger RNA and protein were identified by reverse transcriptase polymerase chain reaction (PCR) and solution-phase hybridization-RNase protection assay, and Western blot analysis, respectively, in cell lines and in cancerous breast tissue removed from women at mastectomy. Cells were exposed to E2, progesterone, or vehicle (each steroid, 10(-10)-10(-6) M) for 24 h and harvested for extraction of RNA. The OTR PCR product was increased by E2 (10(-7) M, p < 0.05, or 10(-6) M, p < 0.01 vs control) and decreased by progesterone (control vs 10(-7) or 10(-6) M, each p < 0.005). Hs578T cells were cultured in the presence or absence of E2 (10(-6) M) or progesterone (10(-6) M) for 24 h and binding was measured. For the E2-exposed cells, the Kd (p < 0.05), and Bmax (p < 0.01) were higher whereas for the progesterone-treated cells the Kd (p < 0.05) and Bax were lower than control cells. E2 and progesterone not only regulate OTR expression and binding in normal mammary myoepithelium but also in malignant mammary cell lines.
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PMID:Estradiol and progesterone regulate oxytocin receptor binding and expression in human breast cancer cell lines. 1216 28

Minimal residual disease (MRD) evaluation in breast cancer patients is a promising tool to improve current staging procedures. In a previous work employing a CK-19-based reverse transcriptase-polymerase chain reaction (RT-PCR) technique for MRD detection, we identified a group of women who exhibited persistent negativity for this assay and for whom this technique was considered noninformative. In order to improve the yield of MRD detection in these patients, we evaluated the usefulness of RT-PCR detection of c-erbB-2 expression. We were able to detect up to 1 MCF-7 cell (positive for c-erbB-2 expression) in a mixture of 1,000,000 CCRF-CEM cells (negative for c-erbB-2 expression). We evaluated the specificity of this technique in the peripheral-blood mononuclear cells (PBMCs) of 20 healthy women and found that 2 of these women were positive for c-erbB-2 expression. In the PBMCs of a group of 16 women with breast cancer, 25% of the samples were positive for c-erbB-2 expression before chemotherapy. Except for race (P = 0.017), no other significant correlations were found, including c-erbB-2 expression in the primary tumor by immunoperoxidase. Interestingly, in the subgroup of 6 patients for whom this technique was informative, we found that 80% of the samples obtained while on chemotherapy were negative compared to only 10% obtained off treatment (P = 0.017). Additionally, 2 patients for whom CK-19 expression was noninformative had at least 1 c-erbB-2-positive sample. We conclude that this technique might be useful for MRD detection in breast cancer patients, but further studies are necessary to confirm our findings.
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PMID:Peripheral blood c-erbB-2 expression by reverse transcriptase-polymerase chain reaction in breast cancer patients receiving chemotherapy. 1219 78


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