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Biomedical subjects

R A Heller

Publications and source records attributed to R A Heller.

At least 19 recordsLinked to original sources
PubMed2000

Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis.

The molecular mechanisms of pulmonary fibrosis are poorly understood. We have used oligonucleotide arrays to analyze the gene expression programs that underlie pulmonary fibrosis in response to bleomycin, a drug that causes lung inflammation and fibrosis, in two strains of susceptible mice (129 and C57BL/6). We then compared the gene expression patterns in these mice with 129 mice carrying a null mutation in the epithelial-restricted integrin beta6 subunit (beta6(-/-)), which develop inflammation but are protected from pulmonary fibrosis. Cluster analysis identified two distinct groups of genes involved in the inflammatory and fibrotic responses. Analysis of gene expression at multiple time points after bleomycin administration revealed sequential induction of subsets of genes that characterize each response. The availability of this comprehensive data set should accelerate the development of more effective strategies for intervention at the various stages in the development of fibrotic diseases of the lungs and other organs.

Animals↗
PubMed2000

Use of oligonucleotide arrays to analyze drug toxicity.

The advent of oligonucleotide arrays allows the simultaneous analysis of the expression of thousands of genes. This powerful technology, highly dependent on advanced analysis tools, can transform the level of information currently available on the mechanisms underlying drug-related toxicity. It is now possible to analyze the global transcriptional response to a drug and determine the global pathways associated with the effects of this agent. This analysis can be performed on samples from patients that developed a toxic effect, on cells exposed to the toxic agent, and in animal models of toxicity. Especially useful is the comparison of transcriptional responses in animals susceptible to drug-induced disease with those of genetically modified animals that are resistant to this effect. This analytic strategy allows the delineation of specific mechanisms relevant and specific to drug-induced toxicity and thus might lead to novel therapeutic interventions in these toxic reactions.

Animals↗
PubMed1998

Microarrays: biotechnology's discovery platform for functional genomics.

Advances in microarray technology enable massive parallel mining of biological data, with biological chips providing hybridization-based expression monitoring, polymorphism detection and genotyping on a genomic scale. Microarrays containing sequences representative of all human genes may soon permit the expression analysis of the entire human genome in a single reaction. These 'genome chips' will provide unprecedented access to key areas of human health, including disease prognosis and diagnosis, drug discovery, toxicology, aging, and mental illness. Microarray technology is rapidly becoming a central platform for functional genomics.

Biotechnology↗
PubMed1997

Discovery and analysis of inflammatory disease-related genes using cDNA microarrays.

cDNA microarray technology is used to profile complex diseases and discover novel disease-related genes. In inflammatory disease such as rheumatoid arthritis, expression patterns of diverse cell types contribute to the pathology. We have monitored gene expression in this disease state with a microarray of selected human genes of probable significance in inflammation as well as with genes expressed in peripheral human blood cells. Messenger RNA from cultured macrophages, chondrocyte cell lines, primary chondrocytes, and synoviocytes provided expression profiles for the selected cytokines, chemokines, DNA binding proteins, and matrix-degrading metalloproteinases. Comparisons between tissue samples of rheumatoid arthritis and inflammatory bowel disease verified the involvement of many genes and revealed novel participation of the cytokine interleukin 3, chemokine Gro alpha and the metalloproteinase matrix metallo-elastase in both diseases. From the peripheral blood library, tissue inhibitor of metalloproteinase 1, ferritin light chain, and manganese superoxide dismutase genes were identified as expressed differentially in rheumatoid arthritis compared with inflammatory bowel disease. These results successfully demonstrate the use of the cDNA microarray system as a general approach for dissecting human diseases.

Arthritis, Rheumatoid↗
PubMed1997

Transcriptional control of matrix metalloproteinases and the tissue inhibitors of matrix metalloproteinases.

Matrix metalloproteinases (MMPs) are enzymes with important roles in a variety of normal physiological processes; these same enzymes are also operative in a range of pathologies. The proteins known as the tissue inhibitors of matrix metalloproteinases (TIMPs) act to limit the enzymatic function of the MMPs. MMPs and TIMPs can be divided into two groups with respect to gene expression: the majority exhibit inducible expression and a small number are produced constitutively or are expressed at very low levels and are not inducible. Among the agents that induce MMP and TIMP production are the inflammatory cytokines TNF alpha and IL1 beta. A marked cell type specificity is a hallmark of both MMP and TIMP gene expression (i.e., a limited number of cell types can be induced to make these proteins). An analysis of the control elements in the promoter regions of these proteins reveals a correlation between the presence of both AP-1 and Ets binding sites and inducible expression. The chromosomal locations of most of the MMPs and TIMPs have been verified; these data will provide the basis for investigations into possible correlations between mutations in these genes and disease states.

Animals↗
PubMed1996

Cytokine control of interstitial collagenase and collagenase-3 gene expression in human chondrocytes.

Human collagenase-3 expression, previously seen only in a breast tumor tissue, is here shown to be expressed in primary human chondrocytes derived from the joint tissue and in transformed human chondrocytes. Its mRNA is inducible by the inflammatory cytokines interleukin-1beta plus tumor necrosis factor-alpha, but not by phorbol 12-myristate 13-acetate and only slightly by the growth factors platelet-derived growth factor and epidermal growth factor. Human synovial fibroblasts, another prominent cell type in the joint tissue, do not produce collagenase-3 message. Expression of the murine collagenase, which is possibly the counterpart of human collagenase-3, is induced by interleukin-1beta plus tumor necrosis factor-alpha, and its full induction requires the presence of the transcription factor, c-FOS. This family of transcription factors also plays a role in induction of human collagenase-3, since it binds to the AP-1 site of this matrix metalloproteinase.

Animals↗
PubMed1994

The human stromelysin promoter contains a previously unreported 1.0-kb sequence.

Cloning and characterization of the promoter region controlling the gene encoding human stromelysin (Str) has been previously reported [Quinones et al., J. Biol. Chem. 264 (1989) 8339-8344]. We have characterized independently isolated genomic clones of the STR promoter, designated pSKStrB and 682, that are considerably different from the published sequence. Although the sequences up to an XbaI site at -480 of the 5' regions are identical, a novel 1.0-kb segment exists upstream from -480. This sequence is absent from the published clone, but its presence in the genomic DNA from twelve individuals has been confirmed by both PCR analysis and restriction mapping. Upstream of the novel 1-kb segment, the sequence of the published clone reappears, but in pSKStrB exists in inverse orientation.

Base Sequence↗
PubMed1994

Mechanisms of tumor necrosis factor cytotoxicity and the cytotoxic signals transduced by the p75-tumor necrosis factor receptor.

We have here provided evidence that TNF mediated cytotoxicity is genetically, pharmacologically, and temporally distinct from the cytotoxicity mediated by TNF/CHX. Most studies on TNF cytotoxicity have been done by the combined use of TNF/CHX. The relevance of this approach to the physiological mechanisms underlying cytotoxicity by TNF alone is at present unclear. We have described a system in which overexpression of the p75-TNFR causes TNF-resistant cells to become TNF-sensitive. These cells are killed by TNF alone in a very short period of time and they are a useful system to study the mechanism of TNF-cytotoxicity.

Animals↗
PubMed1993

Chimeric tumor necrosis factor-TrkA receptors reveal that ligand-dependent activation of the TrkA tyrosine kinase is sufficient for differentiation and survival of PC12 cells.

To elucidate the function of the two nerve growth factor (NGF) receptors, p75NGFR and p140trk, chimeric molecules were constructed of tumor necrosis factor (TNF) and NGF receptors. Rat PC12 pheochromocytoma cells transiently transfected with TNF-p140trk chimeras, which contain the extracellular domain of TNF receptor and the transmembrane and cytoplasmic domains of p140trk, showed TNF-dependent neuronal differentiation and cell survival. The activity of TNF-p140trk chimeras was completely blocked by the tyrosine kinase inhibitor K252a, and TNF was unable to induce neurite elongation in PC12 cells transfected with a tyrosine kinase-defective chimeric receptor. The TNF-p75NGFR chimeras, which contain the cytoplasmic domain of p75NGFR, were nonfunctional. Our results suggest that p140trk may function as ligand-activated homodimers and that ligand-mediated activation of the cytoplasmic domain of p140trk alone is sufficient for inducing a neuronal phenotype.

Animals↗
PubMed1993

Cytoplasmic phospholipase A2 activity and gene expression are stimulated by tumor necrosis factor: dexamethasone blocks the induced synthesis.

The interaction of tumor necrosis factor alpha (TNF) with its two membrane-bound receptors initiates intracellular events in which arachidonic acid and its derivatives are involved. In HeLa cells, TNF treatment induces an arachidonic acid-selective, Ca(2+)-dependent cellular phospholipase A2 (cPLA2). By itself, TNF causes a modest increase in cPLA2 activity, but with the Ca2+ ionophore A23187 it provides a strong synergistic action. Within minutes in response to TNF, cPLA2 becomes phosphorylated and in the presence of Ca2+ produces a 3- to 4-fold increase in activity. TNF also increases cPLA2 mRNA and protein expression, an estimated 5-fold increase in an 8-hr period. This increase in cPLA2 activity occurs, therefore, in a biphasic time-dependent manner. Dexamethasone, known to antagonize the action of TNF, is here shown to inhibit TNF-induced gene expression and to prevent the second phase of increase in cPLA2 activation. Our results suggest that the cPLA2 activation may provide a regulatory function and may explain the proinflammatory action of TNF.

Arachidonic Acid↗
PubMed1992

Cell killing and induction of manganous superoxide dismutase by tumor necrosis factor-alpha is mediated by lipoxygenase metabolites of arachidonic acid.

The signalling pathways utilized by tumor necrosis factor-a (TNF) to elicit its actions have been examined in TA1 adipogenic cells. A role for lipoxygenase metabolites of arachidonic acid as mediators of TNF action in the induction of c-fos has been described. In this paper we report that acute cytotoxicity elicited by TNF, in the presence of cycloheximide (CHX), also utilizes this pathway since inhibitors of lipoxygenase action fully prevent TNF/CHX killing of several cell lines. Our data reveal that TNF induction of manganous superoxide dismutase (MnSOD) is also dependent upon lipoxygenase activity. Radical scavengers such as NAC and PDTC prevent TNF/CHX-induced cell killing and reduce MnSOD induction by TNF. Therefore, cell death by TNF/CHX treatment occurs via a pathway in which lipoxygenase products directly or indirectly operate via the generation of superoxide anions.

3T3 Cells↗
PubMed1992

The p70 tumor necrosis factor receptor mediates cytotoxicity.

Tumor necrosis factor alpha (TNF) selectively kills tumor cells, but this specificity is not clearly understood. Two distinctly different cell surface receptors (TNFRs), proteins of 55 kd (p55) and 70-80 kd (p70), mediate TNF action. Mouse TA1 cells are not killed by human (h) TNF, but are killed by mouse (m) TNF alone. Since the mouse p70 TNFR is recognized only by mTNF, these results implicate p70 receptor action in TA1 cell killing. Human HeLa cells have mainly the p55 receptor and are not killed by hTNF alone. When transfected with the human p70 TNFR, HeLa p70 die within 24 hr. HeLa p70 cells also show reduced c-fos and manganous superoxide dismutase induction by TNF. NIH 3T3 mouse fibroblasts are sensitive to only mTNF, but overexpression of the human p70 receptor causes cell death by hTNF and increased sensitivity to mTNF. These results provide a direct function for the p70 TNFR in TNF-induced cytotoxicity.

3T3 Cells↗
PubMed1992

CCAAT/enhancer binding protein expression is rapidly extinguished in TA1 adipocyte cells treated with tumor necrosis factor.

Tumor necrosis factor (TNF) has been shown to have diverse effects on a wide variety of cell types. In the mouse adipogenic TA1 cell line, TNF completely abolishes differentiation and reverts fully differentiated fat cells into fibroblasts. This block in differentiation and its reversal is due to the rapid reduction in the expression of adipose-specific genes. This study reports that the transcription factor, CCAAT/enhancer binding protein (C/EBP), previously reported to promote the differentiation of 3T3-L1 adipocytes, is expressed in TA1 cells. During their growth in culture, the levels of C/EBP, as evidenced by its cellular levels of specific mRNA, protein, and DNA binding activity, increase dramatically when cells reach confluence and proceed to differentiate. Addition of TNF to cultured preadipocytes or fully differentiated adipocytes rapidly reduces C/EBP levels and is accompanied by the decrease in expression of adipose-specific genes. C/EBP binding sites occur in several adipose-specific genes, and here it is demonstrated that its presence in a novel adipose-specific gene, Clone 47, also referred to as FSP27, may be responsible for the strong down-regulation of the expression of the Clone 47 (FSP27) promoter-linked chloramphenicol acetyl transferase gene by TNF. This study proposes that the loss of C/EBP in response to TNF treatment may in part explain the loss of the adipocyte differentiated state.

Adipose Tissue↗
PubMed1992

Rapid screening of libraries with radiolabeled DNA sequences generated by PCR using highly degenerate oligonucleotide mixtures.

The PCR technique can use protein-derived oligonucleotide sequences as primers to develop probes for screening recombinant libraries. Here we report a method with highly degenerate mixtures of oligonucleotides as primers for the PCR that eliminates the need to identify or isolate the DNA sequences derived by PCR. The method uses the pool of PCR-generated DNA sequences radiolabeled during the extension reaction as a probe, combined with highly stringent hybridization and wash conditions that permit only homologous sequences to hybridize and therefore target desired clones. This technique was used successfully to clone the receptor for tumor necrosis factor.

Amino Acid Sequence↗
PubMed1991

Tumor necrosis factor receptor genes, TNFR1 and TNFR2, on human chromosomes 12 and 1.

Tumor necrosis factor, TNF, is a 17-kDa protein secreted by macrophages and classified as a cytokine. TNF binds to high-affinity receptors on the cell surface and is involved in a wide variety of biological responses. There are at least two types of receptors, tumor necrosis factor receptors 1 and 2 (TNFR1 and TNFR2). The genes for TNFR1 a 55-kDa protein, and TNFR2, a 70-kDa protein, have been mapped to human chromosomes 1 12 (12pter-cen) and (1pter-p32), respectively, by Southern blot analysis of human x Chinese hamster somatic cell hybrid panels. Recently, the corresponding genes in the mouse have been mapped to chromosomes 4 and 6 in regions that are conserved on human chromosomes 1 and 12.

Animals↗
PubMed1990

Amplified expression of tumor necrosis factor receptor in cells transfected with Epstein-Barr virus shuttle vector cDNA libraries.

As an approach to isolate the cell-surface receptor for tumor necrosis factor (TNF), we have developed transfectants of human B-lymphoblastoid cells (UC cells) that overexpress the TNF receptor. These transfectants were isolated from UC cells transfected with cDNA libraries of HeLa or NG108 cells constructed in the mammalian expression vector EBO-pcD. This vector contains the Epstein-Barr virus origin of replication (ori-P) plus the EBNA-1 gene conferring replication function to ori-P and, therefore, the ability to replicate autonomously within the transfected cell (Margolskee, R.F., Kavathas, P., and Berg, P. (1988) Mol. Cell. Biol. 8, 2837-2947). Cells overexpressing the TNF receptor were identified and separated by the binding of fluoresceinated TNF and flow cytometric selection. Scatchard analysis of 125I-TNF binding data revealed a single class of high affinity receptors with a dissociation constant (Kd) of 0.2 to 2 nM and a receptor density of about 150,000 per cell, an increase of approximately 150-fold over UC cells. Cross-linking of receptor-ligand with bis-sulfosuccinimidyl suberate followed by polyacrylamide gel electrophoresis gave estimates of 87 and 104 kDa for the size of the complex. Based on its ability to bind TNF, a 68-kDa receptor protein was identified in cell extracts enriched for the receptor by using immobilized wheat germ agglutinin and TNF affinity chromatography. The difference in the estimated size of the receptor and the receptor-ligand complexes demonstrates that TNF binds to the receptor as a monomer or a dimer. Analysis of cDNA sequences conferring receptor amplification in transfectants revealed that plasmid DNA was present at 30 or more copies per cell, most likely integrated into the genomic DNA or organized into high molecular weight catenanes, and autonomously replicating units could not be recovered. Therefore, while this vector was useful in generating stable receptor-amplified cells, it was not maintained as a recoverable episome.

Animals↗
PubMed1990

Complementary DNA cloning of a receptor for tumor necrosis factor and demonstration of a shed form of the receptor.

Tumor necrosis factor (TNF) receptor (TNFR) was isolated as a 68-kDa glycoprotein from UC/HeLa 2-5 cells developed from a parental B-cell line (UC cells) to overexpress the receptor. Tryptic digests of two separate TNFR preparations provided amino acid sequences of four different peptides. Amino-terminal analysis indicated the presence of the amino-acid sequence Val-Ala-Phe-Thr-Pro, reported to be the amino-terminal sequence of a 30-kDa urinary TNF-binding protein II. Examination of the cultured medium of UC/HeLa 2-5 cells showed an abundance of a 40-kDa TNF-binding protein, indicating that the previously cited 30-kDa TNF-binding protein II is likely to be a shed form of the TNFR. Based on the peptide sequences, oligonucleotides were synthesized, and two of these were used as primers in the polymerase chain reaction to amplify cDNA sequences from poly(A)+ RNA of UC/HeLa 2-5 cells. These PCR fragments were radiolabeled and used to screen a cDNA library made from UC/HeLa 2-5 mRNA. Further analysis identified cDNA sequences that encoded the amino acid sequences of all four TNFR peptides. RNA blot-hybridization analysis of UC/HeLa 2-5 mRNA revealed a 3.8-kilobase transcript of the same size as the mRNA in the parental UC cells. Genomic Southern blots indicated the presence of a single gene in parental cells and a second, amplified gene in TNFR-overexpressing cells, suggesting amplification of the transfected gene as a possible mechanism for the increase in TNFR numbers in UC/HeLa 2-5 cells.

Amino Acid Sequence↗