Expression of HER2 and the coamplified genes GRB7 and MLN64 in human breast cancer: quantitative real-time reverse transcription-PCR as a diagnostic alternative to immunohistochemistry and fluorescence in situ hybridization.

Purpose: Accurate testing of HER2 is centrally important for breast cancer therapy and prognosis. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are current standard testing methods. As a potential alternative for assessment of HER2, we explored quantitative real-time reverse transcription-PCR (RT-PCR), a fast and inexpensive method yielding quantitative results insensitive to interobserver variability and amenable to standardized scoring.

Microarray gene expression profiling of B-cell chronic lymphocytic leukemia subgroups defined by genomic aberrations and VH mutation status.

Purpose: Genomic aberrations and mutational status of the immunoglobulin variable heavy chain (VH) gene have been shown to be among the most important predictors for outcome in patients with B-cell chronic lymphocytic leukemia (B-CLL). In this study, we report on differential gene expression patterns that are characteristic for genetically defined B-CLL subtypes.

Materials And Methods: One hundred genetically well-characterized B-CLL samples, together with 11 healthy control samples, were analyzed using oligonucleotide arrays, which test for the expression of some 12,000 human genes.

Tissue-wide expression profiling using cDNA subtraction and microarrays to identify tumor-specific genes.

With the objective of discovering novel putative intervention sites for anticancer therapy, we compared transcriptional profiles of breast cancer, lung squamous cell cancer (LSCC), lung adenocarcinoma (LAC), and renal cell cancer (RCC). Each of these tumor types still needs improvement in medical treatment. Our intention was to search for genes not only highly expressed in the majority of patient samples but which also exhibit very low or even absence of expression in a comprehensive panel of 16 critical (vital) normal tissues.

CDNA microarray gene expression analysis of B-cell chronic lymphocytic leukemia proposes potential new prognostic markers involved in lymphocyte trafficking.

Human cancer is characterized by complex molecular perturbations leading to variable clinical behavior, often even in single-disease entities. We performed a feasibility study systematically comparing large-scale gene expression profiles with clinical features in human B-cell chronic lymphocytic leukemia (B-CLL). cDNA microarrays were employed to determine the expression levels of 1,024 selected genes in 54 peripheral blood lymphocyte samples obtained from patients with B-CLL.

A comparative cell-based high throughput screening strategy for the discovery of selective tyrosine kinase inhibitors with anticancer activity.

Growth factor receptor tyrosine kinases (RTK) have been implicated in tumor growth, metastasis and angiogenesis, and are thus considered promising targets for therapeutic intervention in malignant diseases. We present a novel drug discovery strategy to find inhibitors of RTKs based on comparative screening of compound libraries employing functional cellular assays. Cell lines stably expressing HER2 and the receptors for hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF) have been established.

Activation of intercellular adhesion molecule 1 expression by Helicobacter pylori is regulated by NF-kappaB in gastric epithelial cancer cells.

Interactions between leukocytes and epithelial cells may play a key role in Helicobacter pylori-associated gastric mucosal inflammation. This process is mediated by various cell adhesion molecules. The present study examined the molecular mechanisms leading to H.

Gene expression profiling in drug discovery and development.

Recent advances in technology, especially the merger between molecular biology, automation technology and computer science, are rapidly changing the manner in which pharmaceutical companies will discover new drug targets and develop new therapeutic drugs. One example of such a merger between different disciplines has been the development of technology platforms, such as cDNA microarrays and oligonucleotide arrays, allowing the generation of comprehensive gene expression profiles in a previously insurmountable throughput. Hereby, the major limitation is currently shifting from the efficient generation of biological information to the extraction of biological knowledge.

Regulation of intercellular adhesion molecule-1 gene by tumor necrosis factor-alpha is mediated by the nuclear factor-kappaB heterodimers p65/p65 and p65/c-Rel in the absence of p50.

Human intercellular adhesion molecule-1 (ICAM-1) plays an important role in immune responses as the major specific ligand for the beta2-integrins LFA-1 and Mac-1. During the inflammatory process, ICAM-1 expression is stimulated by various proinflammatory cytokines. We have examined the mechanisms of transcriptional control involved in the stimulation of ICAM-1 gene expression by tumor necrosis factor-alpha (TNF-alpha) and by the nuclear factor-kappaB (NF-kappaB) family of transcription factors in the Ad5-transformed human embryonal kidney cell line 293.

Transcriptional regulation of the human intercellular adhesion molecule-1 gene: a short overview.

Human intercellular adhesion molecule-1 (ICAM-1), a transmembrane glycoprotein that functions as a ligand for lymphocyte function-associated antigen-1, plays an important role in mediating cell-cell interactions in inflammatory reactions. It is induced by proinflammatory cytokines such as interleukin-1, tumor necrosis factor-alpha or interferon-gamma, as well as by phorbol esters, retinoic acid and lipopolysaccharide. Furthermore, ICAM-1 is upregulated by interleukin-6, which suggests that it belongs to the family of acute phase response genes.

Heterodimeric retinoic acid receptor-beta and retinoid X receptor-alpha complexes stimulate expression of the intercellular adhesion molecule-1 gene.

Human intercellular adhesion molecule-1 (ICAM-1), a specific ligand for the leukocyte-function associated antigen-1 and for Mac-1, plays an important role in immune responses. ICAM-1 expression is regulated by various proinflammatory cytokines, phorbol myristate acetate, and retinoic acid. In this study, we investigated the mechanisms of transcriptional control involved in the stimulation of ICAM-1 gene expression by retinoic acid in Cos-1 cells.

Functional characterization of the human neurokinin receptors NK1, NK2, and NK3 based on a cellular assay system.

The neurokinin receptor family is known to modulate phospholipase C activity. In order to find new compounds modulating the activity of these receptors we have developed a cellular screening system that measures the biological activity of receptors coupled to the IP3/DAG signal transduction pathway via the transcriptional activation of a reporter gene. For the establishment of neurokinin test cell lines the reporter cell line A20, stably transformed with the luciferase gene under the control of a promoter containing TPA response elements (TRE), which did not respond to neurokinin agonists, was used.

Regulation of intercellular adhesion molecule-1 expression by retinoic acid: analysis of the 5' regulatory region of the gene.

Human intercellular adhesion molecule-1 (ICAM-1), a specific ligand for the lymphocyte-function-associated antigen-1, plays an important role in immune responses. ICAM-1 expression is regulated by various proinflammatory cytokines, by PMA, and by retinoic acid. In this study, we have investigated the mechanisms of transcriptional control involved in the stimulation of ICAM-1 gene expression by retinoic acid in SK-N-SH cells.

Saccharomyces cerevisiae cdc15 mutants arrested at a late stage in anaphase are rescued by Xenopus cDNAs encoding N-ras or a protein with beta-transducin repeats.

We have constructed a Xenopus oocyte cDNA library in a Saccharomyces cerevisiae expression vector and used this library to isolate genes that can function in yeast cells to suppress the temperature sensitive [corrected] defect of the cdc15 mutation. Two maternally expressed Xenopus cDNAs which fulfill these conditions have been isolated. One of these clones encodes Xenopus N-ras.

Functional testing of human dopamine D1 and D5 receptors expressed in stable cAMP-responsive luciferase reporter cell lines.

A large number of G-protein coupled receptors are known to modulate adenylyl cyclase activity. In order to find new compounds modulating the activity of specific receptor subtypes we developed a cellular screening system that measures the biological activity of drugs acting on receptors rather than merely their binding characteristics. The activity of the receptor coupling to the cAMP signal transduction pathway was measured via transcriptional activation of a reporter gene.

Establishment of a cellular assay system for G protein-linked receptors: coupling of human NK2 and 5-HT2 receptors to phospholipase C activates a luciferase reporter gene.

A functional cellular assay system was developed for the detection of substances modulating the activity of G protein-coupled receptors, linked to the phospholipase C second messenger system. The human adenocarcinoma cell line A549 was transformed with the Photinus pyralis luciferase gene under the control of the ICAM-1 gene 5'regulatory region and, subsequently, stably transfected with the human neurokinin 2 (NK2) receptor gene. The ICAM-1 promoter is known to be inducible via the phospholipase C signal transduction pathway.

Cloning of the human gene for intercellular adhesion molecule 1 and analysis of its 5'-regulatory region. Induction by cytokines and phorbol ester.

Human intercellular adhesion molecule-1 (ICAM-1), a specific ligand for the lymphocyte function-associated Ag-1 (LFA-1), plays an important role in leukocyte-endothelial cell interactions. It is induced by proinflammatory cytokines such as IL-1, TNF-alpha, or IFN-gamma. However, little is known concerning the intracellular regulatory mechanisms which trigger ICAM-1 up-regulation.

Cloning and nucleotide sequence of cDNA encoding human erythrocyte band 7 integral membrane protein.

cDNA clones encoding the human erythrocyte band 7 membrane protein were isolated by immunoscreening from bone marrow and HeLa cell lambda gt 11 cDNA libraries, and their nucleotide sequences were determined. HeLa- and bone marrow cell-derived sequences were identical, except for one nucleotide; the deduced sequence of 287 amino acids was confirmed by sequence identity with peptides of the erythroid protein. Structure analysis assigned band 7 protein to the type Ib transmembrane proteins.

Cloning and sequencing of rat plectin indicates a 466-kD polypeptide chain with a three-domain structure based on a central alpha-helical coiled coil.

We have determined the complete cDNA sequence of rat plectin from a number of well-characterized overlapping lambda gt11 clones. The 4,140-residue predicted amino acid sequence (466,481 D) is consistent with a three-domain structural model in which a long central rod domain, having mainly an alpha-helical coiled coil conformation, is flanked by globular NH2- and COOH-terminal domains. The plectin sequence has a number of repeating motifs.

Molecular cloning and expression of human and rat tumor necrosis factor receptor chain (p60) and its soluble derivative, tumor necrosis factor-binding protein.

Tumor necrosis factor-alpha (TNF-alpha), a protein released by activated macrophages, is involved in a wide variety of human diseases including septic shock, cachexia, and chronic inflammation. TNF binding protein (TNF-BP), a glycoprotein with high affinity to TNF-alpha isolated from urine, acts as an inhibitor of TNF-alpha by competing with the cell-surface TNF receptor. We report here the partial amino acid sequencing of human TNF-BP as well as the isolation, sequence, and expression of cDNA clones encoding a human and rat TNF receptor.

A soluble form of intercellular adhesion molecule-1 inhibits rhinovirus infection.

Rhinoviruses belong to the picornavirus family and cause about 50% of common colds. Most rhinoviruses and some coxsackie viruses share a common receptor on human cells. The glycoprotein intercellular adhesion molecule-1 (ICAM-1) has recently been identified as the cellular receptor for the subgroup of rhinoviruses known as the major groups.

Cloning and expression of cDNA for human vascular anticoagulant, a Ca2+-dependent phospholipid-binding protein.

Based on sequence information from tryptic peptides an almost full-size cDNA coding for the human vascular anticoagulant was isolated from a placental cDNA library and sequenced. The coding region was cloned into an Escherichia coli expression vector and the protein expressed at high levels. The recombinant protein was purified and found to be indistinguishable from its natural counterpart in several biological assays.

Primary structure of ICAM-1 demonstrates interaction between members of the immunoglobulin and integrin supergene families.

Intercellular adhesion molecule 1 (ICAM-1) is a 90 kd inducible surface glycoprotein that promotes adhesion in immunological and inflammatory reactions. ICAM-1 is a ligand of lymphocyte function-associated antigen-1 (LFA-1), an alpha beta complex that is a member of the integrin family of cell-cell and cell-matrix receptors. ICAM-1 is encoded by an inducible 3.

Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning.

We have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library.

Selective regulation of trypsin gene expression by calcium and by glucose starvation in a rat exocrine pancreas cell line.

Treatment of the rat pancreatic acinar cell line AR4-2J with the calcium ionophore A23187 selectively increases, within a few hours, the steady-state level of trypsin mRNA. Addition of the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate potentiates the calcium-induced increase. The mRNA level of the other tested exocrine pancreatic genes decreases.