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T Emrich

Publications and source records attributed to T Emrich.

10 recordsLinked to original sources
PubMed2001

Ribozyme cleavage of telomerase mRNA sensitizes breast epithelial cells to inhibitors of topoisomerase.

Telomerase activity is necessary and sufficient for immortality in many cells and hence represents a prime target for antitumor strategies. Here, we show that a hammerhead ribozyme cleaves human telomerase (hTERT) mRNA in vitro. Stable transfection in clones of the human breast tumor line MCF-7 and the immortal breast cell line HBL-100 results in expression of the ribozyme, diminishes the abundance of hTERT mRNA, and inhibits telomerase activity. This led to shortened telomeres, inhibition of net growth, and induction of apoptosis. In HBL-100 mass cultures infected with a ribozyme-expressing adenovirus diminution of hTERT mRNA, attenuation of telomerase activity, inhibition of net growth, and induction of apoptosis was found as well. Attenuation of telomerase activity increased the sensitivity of HBL-100 and MCF-7 clones specifically to inhibitors of topoisomerase. Likewise, expression of exogenous telomerase in originally telomerase-negative human fibroblasts decreased their sensitivity to topoisomerase poisons but not to a number of other cytotoxic drugs. The data validate a ribozyme approach for telomerase inhibition therapy in cancer and suggest that it might be combined advantageously with topoisomerase-directed chemotherapy.

Adenocarcinoma↗
PubMed2001

Analysis of human telomerase reverse transcriptase mRNA (hTERT) expression in myxoid liposarcomas using LightCycler real-time quantitative reverse transcriptase-polymerase chain reaction.

We describe a convenient, nonradioactive reverse transcription--polymerase chain reaktion (RT-PCR) method for the rapid and accurate quantitative detection of the human telomerase catalytic subunit human telomerase reverse transcriptase (hTERT) mRNA. The LightCycler TeloTAGGG hTERT Quantification Kit (Roche Molecular Biochemicals) was designed to be used for the highly sensitive and quantitative detection of hTERT mRNA relative to the house-keeping gene porphobilinogen deaminase (PBGD). As a tumor progression model, we investigated 26 myxoid liposarcomas (11 pure myxoid grade I, 15 myxoid/round cell grade II/III) for the hTERT expression level and compared the results of the new method with former measurements performed in silver-stained polyacrylamide gels. Both methods revealed similar results, with real-time RT-PCR being the more accurate quantification technique, which also saves time and material. Elevated hTERT expression (cut-off ratio x 100 at 1.3) was an indicator of round cell components and hence for tumor progression in myxoid liposarcoma. The new method is capable of differentiating between pure myxoid and myxoid/round cell liposarcomas for hTERT-expression more accurately.

Computer Systems↗
PubMed2001

Real-time quantitative PCR of telomerase mRNA is useful for the differentiation of benign and malignant pancreatic disorders.

The presence of telomerase activity has been proposed as a specific and sensitive marker for malignant tissue, and positivity rates of up to 95% have been reported in pancreatic cancer. In the present study telomerase activity analysis was reevaluated in 29 pancreatic cancer tissues compared with 36 chronic pancreatitis tissues and 21 normal controls, and a study was made of whether malignant and benign pancreatic disorders can be better differentiated using a novel technique real-time quantitative PCR analysis-analyzing telomerase mRNA expression. Telomerase activity was present in 35% (10 of 29) of pancreatic cancer samples, 3% (one of 36) of chronic pancreatitis samples, and none of the normal pancreatic tissue samples in the TRAP assay. Real-time quantitative PCR analysis revealed the presence of telomerase mRNA expression in 50% (10 of 20) of normal, 86% (31 of 36) of chronic pancreatitis, and 90% (26 of 29) of pancreatic cancer samples. However, quantification of the expression data revealed that the relative increase above normal was 5.5 (range, 3.5-8.6) for chronic pancreatitis and 23.9 (range, 18.6-30.7) for pancreatic cancer samples (p < 0.01). No relationship was found between telomerase activity and the fold increase of telomerase mRNA above normal and gender, patient age, tumor stage, or tumor grade. These data indicate that detection of telomerase activity using the TRAP assay has limitations in differentiating benign and malignant pancreatic disorders. However, telomerase mRNA analysis by real-time quantitative PCR analysis allows a highly sensitive detection and differentiation of pancreatic cancer from normal pancreas and chronic pancreatitis and thereby may serve as a new reliable, easy, and effective diagnostic tool for cancer diagnosis.

Adult↗
PubMed1997

Abnormal expression of the B-cell homing chemokine receptor BLR1 during the progression of acquired immunodeficiency syndrome.

The putative chemokine receptor BLR1 has been identified as the first G-protein-coupled receptor involved in B-cell migration and in microenvironmental homing to B-cell follicles and to germinal centers. In healthy individuals, expression of BLR1 is restricted to all mature recirculating B cells and to a subpopulation of T-helper memory cells. In the present study, we analyzed the distribution of BLR1 on defined lymphocyte subsets during the progression of acquired immunodeficiency syndrome. It is shown that the proportion of T-helper memory cells coexpressing BLR1 continuously decreases during the infection, whereas a high proportion of gamma/delta T cells expressing BLR1 can be found in peripheral blood. The latter subpopulation is restricted to lymphoid tissues in healthy individuals. Most interestingly, in 75% of all human immunodeficiency virus (HIV)+ individuals, peripheral blood B cells were identified as not expressing BLR1 and phenotypically resembling germinal center cells of lymphoid tissue. Using BLR1 as a marker molecule, this study identifies peripheral blood lymphocytes in HIV+ individuals that are usually restricted to lymphoid tissue in healthy individuals. Because HIV infection is active in lymphoid tissue even at the clinically latent stage, aberrant expression of the B-cell homing chemokine receptor BLR1 might be an early indicator for the onset of destruction of lymphoid tissue.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Expression of the G-protein--coupled receptor BLR1 defines mature, recirculating B cells and a subset of T-helper memory cells.

The G-protein-coupled receptor BLR1 related to receptors for chemokines and neuropeptides has been identified as the first lymphocyte-specific member of the gene family characterized by seven transmembrane-spanning regions. Using a high-affinity anti-BLR1 monoclonal antibody (MoAb) and three-color flow cytometry it is shown that BLR1 expression on peripheral blood cells is limited to B cells and to a subset of CD4+ (14%) and CD8+ (2%) lymphocytes. T cells expressing BLR1 were positive for CD45R0, were negative for interleukin-2 receptors, show high levels of CD44, and show low levels of L-selectin. The majority of CD4+ cells originating from secondary lymphatic tissue, but none of cord blood-derived T cells, express BLR1. These observations suggest that BLR1 is a marker for memory T cells. Furthermore, BLR1 expression was detected on all CD19+ peripheral or tonsillar B lymphocytes, but only on a fraction of cord blood cells and bone marrow cells expressing CD19, sIgM, or sIgD. Interestingly, activation of both mature B and T cells by CD40 MoAb and CD3 MoAb, respectively, led to complete downregulation of BLR1. These data suggest that the G-protein-coupled receptor BLR1 is involved in functional control of mature recirculating B cells and T-helper memory cells participating in cell migration and cell activation.

Adult↗
PubMed1994

Transmembrane topology of the lymphocyte-specific G-protein-coupled receptor BLR1: analysis by flow cytometry and immunocytochemistry.

The blr1 gene encodes the first member of the superfamily of G-protein-coupled receptors showing a lymphocyte- and differentiation-specific expression pattern. To study the synthesis of the BLR1-protein and to characterize the receptor we fused its coding region to a sequence derived from human c-MYC allowing the detection of the corresponding fusion protein by an anti-myc-antibody. Expression of the epitope-tagged BLR1 in human embryonic kidney 293 cells to high levels showed an apparent molecular mass for BLR1 of 50 kDa. This is reduced to 40 kDa following treatment of the cells with tunicamycin indicating the presence of N-linked glycosylation. Furthermore expression of amino- and carboxyl-terminally tagged BLR1 demonstrated that BLR1 is an integral protein of the plasma membrane inserted therein in the predicted orientation. Using this efficient expression system we generated a monoclonal antibody (mAb 8B2) against human BLR1. Flow cytometric analysis of peripheral blood lymphocytes confirms the lymphocyte-specific expression of BLR1. The highly efficient expression of BLR1 in 293 cells and the generated mAb offers a powerful tool for further characterization of this receptor and analysis of its function on lymphocytes and during B-cell development.

Amino Acid Sequence↗
PubMed1993

Topological characterization of the lymphoid-specific seven transmembrane receptor BLR1 by epitope-tagging and high level expression.

We have tagged the human lymphocyte-specific G-protein-coupled receptor BLR1 either with an amino-terminal or a carboxyl-terminal epitope-tag recognized by an anti-MYC monoclonal antibody. Flow cytometry was used to determine the efficiency of transient transfections and to establish human embryonic kidney 293 cell clones showing stable high level expression of BLR1. Analysis of permeabilized versus non-permeabilized transfected 293 cells demonstrated that BLR1 is an integral plasma membrane protein, topologically oriented therein as predicted for other members of this class of seven pass membrane receptors. In addition, BLR1 was expressed in 293 cells to high levels as a glycosylated membrane protein. The easily detectable and assayable expression of tagged G-protein-coupled receptors, as exemplified for BLR1 in 293 cells, provides a suitable system for further functional studies and offers an efficient screening tool for the identification of receptor-specific antibodies, ligands, or receptor-associated proteins.

Amino Acid Sequence↗
PubMed1993

A general method for screening mAbs specific for G-protein coupled receptors as exemplified by using epitope tagged BLR1-transfected 293 cells and solid-phase cell ELISA.

Monoclonal antibodies (mAb) against G-protein coupled receptors are rare. In this study we describe a cell ELISA-based screening system for monoclonal antibodies specific for the G-protein coupled receptor BLR1 (Eur. J. Immunol. 1992. 22:2795) using human embryonic kidney 293 cells transfected with a modified human BLR1 cDNA directing the synthesis of an epitope tagged BLR1 protein. Lou/C rats were immunized with BLR1 transfected, tagged 293 cells and after fusion of spleen cells with X63 Ag8.653 myeloma cells supernatants were tested for BLR1 specific antibodies by comparing the binding to BLR1 transfected 293 cells and to untransfected control cells immobilised on poly-L-lysine coated microtiter plates. Cells were fixed with 2% paraformaldehyde and permeabilized using digitonin in order to allow binding of mAb directed against intracellular epitopes. This mild fixation retained excellent morphology of 293 cells and allowed reliable binding to the trays. Screening of approximately 2500 supernatants identified 19 antibodies binding to BLR1 transfected 293 cells but not to control 293 cells. One of these mAb specifically bound to the G-protein coupled receptor BLR1.

Animals↗
PubMed1992

Differentiation-specific expression of a novel G protein-coupled receptor from Burkitt's lymphoma.

Deregulation of the proto-oncogene MYC by specific chromosomal translocations has been shown to be essential but not sufficient for the development of Burkitt's lymphoma (BL). To identify other genes which either mark important steps in tumorigenesis or which reflect the cellular differentiation state of BL cells we have compared tumor cells to immortalized lymphoblastoid B cells by subtractive hybridization. We have identified a complementary DNA clone which encodes a novel member of the superfamily of GTP-binding (G) protein-coupled receptors, designated BLR1. The corresponding mRNA is expressed in BL and lymphatic tissues but not in other cell lines either of the B cell lineage or of other hematopoietic or non-hematopoietic origin. This exclusive expression of BLR1 and the oncogenic potential of this receptor class supports the hypothesis that BLR1 exerts a regulatory function in BL lymphomagenesis and/or B cell differentiation. Moreover, the protein sequence is highly related to that of receptors for the cytokine interleukin (IL)-8 and other neutrophil chemoattractants. We conclude that BLR1 may represent a potential candidate involved in the process of physiologic trafficking, cell-cell interactions, and activation of mature B lymphocytes in lymphatic tissues.

Amino Acid Sequence↗