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Biomedical subjects

U Novak

Publications and source records attributed to U Novak.

At least 37 records · Page 2Linked to original sources
PubMed1996

Formation of STAT5-containing DNA binding complexes in response to colony-stimulating factor-1 and platelet-derived growth factor.

Colony-stimulating factor (CSF-1) activates several members belonging to the STAT (signal transducers and activators of transcription) family of transcription factors. We investigated the DNA binding complexes activated by CSF-1 in several cell lines and compared them with complexes activated by platelet-derived growth factor and interleukin 3. Our results indicate that the SIF-A complex activated by CSF-1 and platelet-derived growth factor may contain STAT3/STAT5 heterodimers binding to the high affinity SIF binding site, m67. In addition, both growth factors activate one or several STAT5-containing protein complexes binding to the prolactin-inducible element, PIE. The formation of these complexes was cell type and growth factor specific. Interleukin 3 activated only PIE binding complexes containing STAT5A and STAT5B and did not activate m67 binding complexes. It appears, therefore, that STAT5 cannot bind to m67 as a homodimer, but it can bind if it is dimerized with STAT3, whereas it can bind to the PIE element without being either complexed with STAT3 or any other known STAT protein, possibly as a homodimer or as STAT5A/STAT5B heterodimer. However, in addition, STAT5 may heterodimerize with other proteins and form novel PIE binding complexes.

Amino Acid Sequence↗
PubMed1996

Differential regulation of cell cycle machinery by various antiproliferative agents is linked to macrophage arrest at distinct G1 checkpoints.

There is currently much interest in the mechanisms of action of antiproliferative agents and their effects on cell cycle machinery. In the present study we examined the mechanisms of action of four unrelated agents known to inhibit proliferation of CSF-1-stimulated bone marrow-derived macrophages (BMM). We report that 8-bromo-cAMP (8Br-cAMP) and lipopolysaccharide (LPS) potently reduced CSF-1-stimulated cyclin D1 protein, and cyclin-dependent kinase (cdk) 4 mRNA and protein levels, while the inhibitory effects of the Na+/ H+ antiport inhibitor 5-(N',N'-dimethyl) amiloride (DMA) and interferon gamma (IFN gamma ) were only weak. All agents repressed CSF-1-stimulated retinoblastoma protein phosphorylation. Furthermore, 8Br-cAMP and to a lesser extent IFN gamma, also reduced CSF-1-stimulated levels of E2F DNA binding activity in a macrophage cell line, BAC1.2F5. An explanation for the different effects of the agents is that 8Br-cAMP and LPS were found to arrest BMM in early/mid-G1, while IFN gamma and DMA arrested cells in late G1 or early S phase. These data indicate that (1) different antiproliferative agents can arrest the same cell type at distinct checkpoints in G1 and (2) effects of antiproliferative agents on cell cycle machinery is linked to the position at which they arrest cells in G1.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Aberrant activation of JAK/STAT pathway components in response to G-CSF, interferon-alpha/beta and interferon-gamma in NFS-60 cells.

There is evidence that the cellular responses to cytokines, such as granulocyte colony stimulating factor (G-CSF) and interferons, depend on prior activation of components of the JAK/STAT signalling pathway. We report here that the myeloid cell line NFS-60 shows aberrant JAK/STAT signalling yet elicits expected biological responses to G-CSF and interferons-alpha/beta and gamma. Instead of increased phosphorylation of JAK1 and JAK2 in response to G-CSF and interferon-gamma, and JAK1 and Tyk2 in response to interferon-alpha/beta, we observed only an increase of phosphorylation of Tyk2 in response to all of these cytokines in NFS-60 cells. The subset of STAT proteins being activated in response to these cytokines was unusual as well. G-CSF activated STAT3 and STAT5A, whereas interferons activated, in addition to STAT1 and STAT5 other, as yet unidentified, DNA binding proteins. However, NFS-60 cells show normal biological responses to these cytokines, such as proliferation in response to G-CSF, and reduction of proliferation, induction of an anti-viral response and induction of specific genes in response to interferons.

Animals↗
PubMed1995

Distinct regions of the granulocyte colony-stimulating factor receptor are required for tyrosine phosphorylation of the signaling molecules JAK2, Stat3, and p42, p44MAPK.

The protein tyrosine kinases JAK1 and JAK2 are phosphorylated tyrosine after the interaction of granulocyte colony-stimulating factor (G-CSF) with its transmembrane receptor. So too is Stat3, a member of the STAT family of transcriptional activators thought to be activated by the JAK kinases. Truncated G-CSF receptor (G-CSF-R) mutants were used to determine the different regions of the cytoplasmic domain necessary for tyrosine phosphorylation of the signaling molecules JAK2, Stat3, and p42, p44MAPK. We have shown that G-CSF-induced tyrosine phosphorylation and kinase activation of JAK2 requires the membrane proximal 57 amino acids of the cytoplasmic domain. In contrast, maximal Stat3 tyrosine phosphorylation required amino acids 96 to 183 of the G-CSF-R cytoplasmic domain, Stat3 DNA binding could occur with a receptor truncated 96 amino acids from the transmembrane domain and containing a single tyrosine residue, but was reduced in comparison with the full-length receptor. Together with the tyrosine phosphorylation of Stat3, this finding suggests that additional Stat3 does not appear to be required for proliferation. MAP kinase tyrosine phosphorylation correlated with both the proliferative response and JAK2 activation.

Amino Acid Sequence↗
PubMed1995

Colony-stimulating factor 1-induced STAT1 and STAT3 activation is accompanied by phosphorylation of Tyk2 in macrophages and Tyk2 and JAK1 in fibroblasts.

Colony-stimulating factor 1 (CSF-1) causes the activation of STAT1 and STAT3 transcription factors in bone marrow macrophages (BMM), in the macrophage cell line BAC1.2F5, and in fibroblasts that express the wild-type receptor for CSF-1. Fibroblasts expressing a mutant receptor in which the tyrosine 809 is replaced with phenylalanine do not activate STAT proteins in response to CSF-1. The activation of the STAT proteins in BMM is accompanied by tyrosine phosphorylation of Tyk2. In fibroblasts, the activation of the STAT proteins is accompanied by tyrosine phosphorylation of Tyk2 and JAK1. We propose that these JAK kinases are subjected to very rapid phosphorylation in response to CSF-1, followed by rapid dephosphorylation. Furthermore, we propose that kinases other than JAK kinase may be involved in the phosphorylation of the STAT proteins in response to CSF-1.

3T3 Cells↗
PubMed1994

Cytokine modulation of plasminogen activator inhibitor-1 (PAI-1) production by human articular cartilage and chondrocytes. Down-regulation by tumor necrosis factor alpha and up-regulation by transforming growth factor-B basic fibroblast growth factor.

Recombinant human cytokines were examined for their effects on plasminogen activator inhibitor-1 (PAI-1) production by human articular cartilage and chondrocyte monolayer cultures. Cartilage and chondrocytes were cultured with and without added cytokines and the conditioned media assayed for PAI-1 by a specific enzyme-linked immunosorbent assay, and mRNA levels determined by Northern blot analysis. Tumor necrosis factor alpha (TNF alpha) reduced, and transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (bFGF) increased, the levels of PAI-1 antigen and mRNA in the culture fluids and cell extracts, respectively. The effects of TNF alpha and TGF-beta on PAI-1 antigen levels were both time- and concentration-dependent; optimum doses being 10-100 pM TNF alpha and 0.4-0.8 nM TGF-beta, with each cytokine exerting its effect on PAI-1 antigen levels within 8 h of addition to culture. TNF alpha (and interleukin-1 alpha) also countered the effects of TGF-beta and bFGF. The anti-inflammatory drugs, indomethacin and dexamethasone, did not appear to modulate PAI-1 levels in cultures of cartilage tissue. The inhibition of PAI-1 levels by cytokines and reagents which stimulate cartilage resorption (i.e., TNF alpha, interleukin-1 alpha, retinoic acid) and enhancement by cytokines which counter it (i.e., TGF-beta, bFGF) further implicate plasminogen activator in the mechanism(s) of cartilage degradation in diseases such as arthritis.

Aged↗
PubMed1994

Regulation of the urokinase gene by the retinoblastoma protein.

The promoter of the human urokinase plasminogen activator (uPA) gene contains a sequence identical with the retinoblastoma control element (RCE) of the murine c-fos gene, as well as several Sp1 binding sites. In a number of cell lines, the uPA promoter is activated during enforced expression of the retinoblastoma protein, pRB. Electrophoretic mobility-shift assays revealed that the RCE sequence of the uPA gene forms only one specific DNA-protein complex that does not contain pRB. The formation of the RCE-protein complex can be inhibited by 20 molar excess of the unlabeled RCE sequences and by 5 molar excess of the unlabeled E2F binding site. The RCE of the human uPA gene interacts specifically with a protein, which appears to be distinct from members of the E2F family of proteins, Sp1, ATF2, and Elf-1, which are all transcription factors shown to be regulated by pRB.

Animals↗
PubMed1993

Production of leukemia inhibitory factor by human articular chondrocytes and cartilage in response to interleukin-1 and tumor necrosis factor alpha.

OBJECTIVE: To investigate the production of leukemia inhibitory factor (LIF) by human articular chondrocytes and cartilage. METHODS: Chondrocytes and cartilage were cultured with and without added cytokines, and the conditioned media assayed for LIF by a specific radioreceptor competition assay. RESULTS: Interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) stimulated LIF production by chondrocyte monolayers and cartilage. LIF was generally not detected in unstimulated cultures. Northern blot analysis showed increased amounts of LIF messenger RNA in IL-1- and TNF alpha-treated chondrocyte cultures. CONCLUSION: Chondrocytes, stimulated by IL-1 and/or TNF alpha, are potential contributors to the elevated levels of LIF observed in the synovial fluids of patients with rheumatoid arthritis and other inflammatory arthritides.

Aged↗
PubMed1991

Human articular cartilage and chondrocytes produce hemopoietic colony-stimulating factors in culture in response to IL-1.

The hemopoietic CSF, granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF), are cytokines that mediate the clonal proliferation and differentiation of progenitor cells into mature macrophages and/or granulocytes. We have employed an all-human cell culture system, specific ELISA for GM-CSF and G-CSF, and Northern analysis to investigate whether chondrocytes are a potential source of CSF in rheumatoid disease. We report that human rIL-1 stimulated in a dose-dependent manner the production of GM-CSF and G-CSF by human articular cartilage and chondrocyte monolayers in organ and cell culture, respectively. Increased levels of the CSF Ag were detected after 2 to 8 h stimulation with IL-1, and the optimum dose of IL-1 was 10 to 100 U/ml (0.06 to 0.6 nM IL-1 alpha; 0.02 to 0.2 nM IL-1 beta); neither CSF was detectable in nonstimulated cultures nor in IL-1-stimulated cultures treated with actinomycin D or cycloheximide, indicating the requirement for de novo RNA and protein synthesis. The IL-1-mediated increase in GM-CSF could also be inhibited by the corticosteroid, dexamethasone, but not by the cyclo-oxygenase inhibitor, indomethacin. Although having little effect when tested alone, TNF-alpha and lymphotoxin (TNF-beta) could synergize with IL-1 for the production of GM-CSF. Basic fibroblast growth factor, platelet-derived growth factor, and IFN-alpha and IFN-gamma each had no effect on GM-CSF levels. Results obtained by Northern analysis of chondrocyte total RNA reflected those found for the CSF Ag, namely that CSF mRNA levels were elevated in response to IL-1, but not TNF, and that there was synergy between these two cytokines. We propose that chondrocyte CSF production in response to IL-1, and the concurrent destruction of cartilage by IL-1, could provide a mechanism for the chronic nature of rheumatoid disease.

Cartilage↗
PubMed1991

A labile repressor acts through the NFkB-like binding sites of the human urokinase gene.

Transcription of the human urokinase type plasminogen activator (uPA) gene in HeLa cells is induced by phorbol myristate acetate (PMA), interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha). The response to these factors is rapid, independent of new protein synthesis and amplified in the presence of an inhibitor of protein synthesis, indicating the presence of a labile repressor. A DNA element, similar to the binding site for the transcription factor NFkB, is located around--1865 with respect to the start site of transcription in the uPA promoter and confers superinducibility by these agents in the presence of cycloheximide (CHX). A synthetic copy of this element confers superinducibility on a minimal uPA gene promoter and on the thymidine kinase (TK) gene promoter linked to the chloramphenicol acetyl transferase (CAT) gene. CHX alone does not increase transcription from these constructs in HeLa cells, although it superinduces the effects of PMA, IL-1 and TNF alpha. A second NFkB-like binding site located at around--1835 is not capable of conferring transcriptional activation under the same conditions. Our results suggest that maximal transcriptional activation of the uPA gene by PMA, IL-1 and TNF alpha requires the induction of NFkB activity and the decay of a short lived repressor protein, possibly IkB.

Base Sequence↗
PubMed1991

Recombinant human interleukin-1 inhibits plasminogen activator inhibitor-1 (PAI-1) production by human articular cartilage and chondrocytes.

Human articular cartilage and chondrocyte monolayers in culture constitutively produced plasminogen activator inhibitor-1 (PAI-1) protein and mRNA, as assessed by a specific enzyme-linked immunosorbent assay and Northern blotting analysis, respectively. Recombinant human interleukin-1 (IL-1) invoked a dose-dependent inhibition of PAI-1 production in both cartilage and chondrocyte cultures. The inhibitory effect of IL-1 was observed between 2-8h after addition of the cytokine, while the optimal dose was between 10-100U/ml IL-1 alpha (57-570pM IL-1 alpha). Results obtained by Northern analysis of chondrocyte total RNA reflected those found for the PAI-1 antigen, namely, that nontreated chondrocytes showed PAI-1 mRNA which was reduced by IL-1 treatment. To our knowledge, this is the first report where IL-1 has been found to inhibit PAI-1 expression. Since IL-1 has been shown before to cause human cartilage destruction and a correlated change in plasminogen activator activity, it could be that a concomitant reduction in PAI-1 levels by IL-1 may be significant in the control of these changes in cartilage.

Antibodies, Monoclonal↗
PubMed1990

High-level beta-globin expression after retroviral transfer of locus activation region-containing human beta-globin gene derivatives into murine erythroleukemia cells.

The locus activation region (LAR) of the human beta-globin-like gene cluster is characterized by a group of four DNase I hypersensitive sites, which arise specifically in erythroid tissues and are required for a normal pattern of beta-globin-like gene expression. The hypersensitive sites are found at positions 6.1, 10.9, 14.7, and 18 kilobase pairs (kbp) 5' of the epsilon-globin gene. Recently functional assays of the LAR that tested determinants for all four hypersensitive sites showed that expression of the human beta-globin gene was increased to normal or near-normal levels in both transgenic mice and erythroid cells. We constructed retroviral vectors with a human beta-globin gene and the determinant for a single hypersensitive site and measured beta-globin gene expression after retroviral infection of murine erythroleukemia cells. Fragments for the hypersensitive sites at -18 or -10.9 kbp increased human beta-globin RNA levels respectively to 35% or 132% of the endogenous mouse beta maj-globin RNA level. In addition, greater expression was also observed for the neomycin phosphotransferase RNA, which was transcribed from the retroviral LTR, showing that the LAR fragments activated expression from a heterologous promoter. In the context of gene-transfer experiments ultimately aimed at gene therapy, our results show that LAR determinants lead to an increased level of human beta-globin RNA expression after retroviral transfer into erythroid cells. But inclusion of LAR determinants in retroviral vectors also entails the potential risk of activating the expression of nonglobin genes in erythroid cells.

Animals↗
PubMed1990

The block to transcription elongation is promoter dependent in normal and Burkitt's lymphoma c-myc alleles.

Aberrant c-myc expression patterns occur in human Burkitt's lymphoma cells, which consistently exhibit c-myc chromosomal translocations, mutations within and flanking the translocated allele, a loss of the block to transcription elongation in exon 1, and a promoter shift to use of the upstream P1 promoter. To define the mechanism responsible for the loss of transcription elongation blockage and resulting c-myc deregulation in Burkitt's lymphoma, we analyzed transcription patterns after transfer of normal and Burkitt's lymphoma c-myc alleles into murine cells and Xenopus oocyte germinal vesicles. We have determined that although the mutations within and surrounding several Burkitt's lymphoma c-myc alleles are not sufficient, in themselves, to abrogate the transcription elongation block, transcription initiation from the P2 promoter may be necessary to obtain the block to transcription elongation. To test directly the role of c-myc promoters in programming transcription elongation blockage, we analyzed transcription patterns from in vitro mutagenized c-myc genes containing deletions of either the P1 or P2 promoter. These data confirm that P1-initiated c-myc transcripts do not terminate at discrete sites near the 3' end of exon 1, whereas P2-initiated transcripts either terminate or read through the transcription block signals. Therefore, overexpression and/or constitutive expression from the c-myc P1 promoter may contribute to increased readthrough transcription in Burkitt's lymphoma cells and, hence, to aberrant expression patterns or levels of c-myc steady-state transcripts. In addition, the ability of normal cells to modulate c-myc P2-initiated transcription to either read through or to block elongation provides a fine control mechanism over c-myc steady-state RNA levels.

Alleles↗
PubMed1990

Retroviral vectors for the beta-globin gene that demonstrate improved titer and expression.

To study the feasibility of a therapy for thalassemia based on addition of a correctly functioning globin gene to bone marrow stem cells, we have developed retroviral vectors that can transfer the human beta-globin gene into pluripotent hematopoietic stem cells of the mouse. Mice reconstituted with virus-infected bone marrow cells showed long-term tissue-specific expression of human beta-globin RNA and protein. Recently, we have redesigned the retroviral vector to improve the efficiency of stem cell infection and to raise the level of globin expression obtained from the virally transduced gene. Removal of a portion of the second intron of the beta-globin gene resulted in the accumulation of a higher level of full-length viral RNA in retrovirus packaging cell lines, and these cell lines produced beta-globin virus particles at substantially higher titers. Addition of fragments from the locus activation region (LAR) of the beta-like globin gene cluster to the retroviral vectors increased beta-globin expression in infected murine erythroleukemia (MEL) cells. Fragments from the -18 and -10.9 kbp DNase I-hypersensitive sites of the LAR increased human beta-globin RNA levels to 35% and 132% of the endogenous mouse beta maj-globin RNA level, respectively. Increased expression was also found for neomycin phosphotransferase RNA, which was transcribed from the retroviral long terminal repeat (LTR), showing that the LAR fragments also activated expression from a nearby heterologous promoter. These results are discussed in the context of the efficacy and safety of gene therapy for chronic anemia in humans.

Animals↗
PubMed1989

A new gastric carcinoma cell line (LIM1839) derived from a young Caucasian male.

We describe a new and unique gastric carcinoma cell line (LIM1839) derived from a young Caucasian male with rapidly progressing disease. The cell line grows with a pleomorphic morphology and has been in continuous culture for more than 3 years. The cells cannot be cloned in semi-solid agar or grown in nude mice despite numerous attempts. The karyotype of the cultured cells is highly abnormal with a large number of structural and numerical changes. Some chromosomes are dicentric and this feature has persisted in this culture. The cells express one of the small-intestinal dipeptidases, aminopeptidase N, but do not express dipeptidyl peptidase IV or the disaccharidases, sucrase isomaltase or maltase glucoamylase. The cells express high levels of EGF receptors and of messenger RNA for insulin-like growth factor II.

Adenocarcinoma↗
PubMed1989

Expression of transforming growth factor alpha messenger RNA in the normal and neoplastic gastro-intestinal tract.

The presence of TGF alpha mRNA has been reported previously to occur in primary colon cancers. We report the expression of the normal 4.5 kb TGF alpha transcript in the mucosa of the normal human gastro-intestinal tract from oesophagus through to colon. The highest levels of human TGF alpha mRNA occurred in the duodenum but significant levels were present in all of the mucosa. Similarly, in the rat gastro-intestinal tract, TGF alpha transcripts were detected in the lower gastro-intestinal tract mucosa. The relative abundance of the TGF alpha mRNA appeared to decrease in distal regions of the gastro-intestinal tract. The level of the TGF alpha mRNA was similar in both the normal and the neoplastic colon tissue. Similarly, in 2 patients with carcinomas, the TGF alpha mRNA was expressed at similar levels in the tumour and in adjacent mucosa. Although TGF alpha mRNA is associated with transformed cells from the gastro-intestinal tract, the presence of this mRNA at equivalent concentrations in normal mucosa suggests that over-production of TGF alpha is not an essential feature of carcinomas in the gastro-intestinal tract.

Animals↗
PubMed1989

Long-term expression of the human beta-globin gene after retroviral transfer into pluripotent hematopoietic stem cells of the mouse.

We have studied the regulation of the human beta-globin gene after retroviral transfer into a variety of transformed and normal hematopoietic cells. After transfer into murine erythroleukemia cells (MEL) expression from the human beta-globin gene responds to inducers of erythroid maturation in parallel to the endogenous murine globin genes. After infection of human BFU-E, RNA expression from the virally-transferred beta-globin gene was measured at 2.5%-5% of the endogenous beta-globin level. The most improved globin vectors can transfer the human beta-globin gene into pluripotent hematopoietic stem cells in mouse bone marrow. Mice reconstituted with infected marrow show human beta-globin RNA and protein expression in peripheral blood cells for over 4 months. In these animals, both myeloid and lymphoid cells carry the integrated provirus at a level of about 1 copy per cell. In serial transplantation experiments, bone marrow from these animals is capable of repopulating secondary and tertiary recipient animals which go on to show long-term human beta-globin expression. Retroviral vectors thus provide a practical way to refine models of globin gene regulation through in vivo tests and to evaluate the feasibility of protocols for gene addition therapy.

Animals↗