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T E Eling

Publications and source records attributed to T E Eling.

At least 55 records · Page 3Linked to original sources
PubMed1995

Analysis and quantitation of splicing variants of the TPA-inducible PGHS-1 mRNA in rat tracheal epithelial cells.

Previously we reported that 12-myristate 13-acetate diester (TPA) and dexamethasone regulate the expression of a putative prostaglandin G/H synthase-1 (PGHS-1) gene in a transformed, immortalized rat tracheal epithelial cell line (EGV-6). Here we report the cloning and sequencing of the cDNA for this gene. Two transcripts of similar size but differing in their 5' ends were detected. One transcript contains the complete 5' coding and noncoding regions, while the other form has an apparently intronic sequence in place of these regions. This aberrantly spliced form lacks codons 1-36. Northern analysis and quantitative PCR indicated that more than 90% of the PGHS-1 mRNA in EGV-6 cells has the aberrantly spliced 5' end. TPA treatment increases the expression of only the complete PGHS-1 mRNA, elevating it to 50% of the total PGHS-1 transcript pool. Levels of the aberrant mRNA are not affected by TPA treatment. A comparison among EGV-6 cells, primary rat tracheal epithelial (RTE) cultures, and rat fibroblasts showed that all three cell lines have similar levels of the aberrantly spliced PGHS-1 mRNA. RTE cells and fibroblasts, unlike EGV-6 cells, also contain properly spliced PGHS-1 mRNA, at levels 100-fold higher than the level of aberrant PGHS-1 mRNA. TPA does not regulate either of the PGHS-1 transcripts in RTE or rat fibroblast cells. These results confirm the induction of functional PGHS-1 mRNA by TPA only in EGV-6 cells.

Amino Acid Sequence↗
PubMed1995

EGF and TPA stimulate de novo synthesis of PGHS-1 and PGHS-2 through different signal transduction pathways.

Epidermal growth factor (EGF) as well as phorbol 12-myristate 13-acetate (TPA) stimulate de novo synthesis of PGHS (prostaglandin H synthase)-1 and PGHS-2 mRNA, resulting in increased production of PGE2 in rat tracheal epithelial cells (RTE, EGV-6 cells). Stimulation of PGE2 production by TPA is more potent than that by EGF. Staurosporine and H-7, protein kinase C (PKC) inhibitors, suppressed the increase of mRNA and PGE2 levels caused by TPA, but not that caused by EGF. On the other hand, methyl 2,5-dihydroxycinnamate, a tyrosine kinase inhibitor (TKI), suppressed the increase of mRNA and PGE2 levels caused by EGF, but not that caused by TPA. These results indicate that EGF stimulates de novo synthesis of PGHS-1 and PGHS-2 mRNA through a signal transduction pathway which is independent from PKC-associated mechanisms but dependent upon the tyrosine kinase activity of the EGF receptor.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Prostaglandin H synthase-catalyzed ring oxygenation of 2-naphthylamine: evidence for two distinct oxidation pathways.

Previous studies showed that prostaglandin H synthase (PHS) cooxidizes 2-naphthylamine (2-NA) to ring-oxygenated products. These metabolites are atypical for a peroxidase-mediated reaction and are completely different from the polymeric nonoxygenated metabolites of 2-NA that are generated with the model peroxidase horseradish peroxidase (HRP). In this study, we investigated possible explanations for the PHS-catalyzed formation of ring-oxygenated 2-NA metabolites. We found that introduction of a peroxyl radical-generated cosubstrate into the HRP/2-NA system resulted in the formation of the same ring-oxygenated products observed in the PHS/2-NA system. 18O2 incorporation studies were utilized to further characterize the source of oxygen in the ring-oxygenated 2-NA metabolites. The data show that, in the case of PHS, ring oxygenation can occur both by peroxyl radical-mediated attack on 2-NA and by direct transfer of peroxide oxygen from PHS to 2-NA.

2-Naphthylamine↗
PubMed1995

Human immunodeficiency virus type 1 envelope protein does not stimulate either prostaglandin formation or the expression of prostaglandin H synthase in THP-1 human monocytes/macrophages.

Prostaglandin E2 is observed at elevated levels during human immunodeficiency virus (HIV) infection and thus may contribute to the HIV-dependent immunosuppression. The mechanisms responsible for this increase are not understood. Evidence indicates that the viral envelope proteins perturb membrane signaling mediated by the CD4 receptor, suggesting that the free envelope protein and/or the intact virus may be responsible for the increase in prostaglandin E2 levels. In this study, we have used THP-1 human monocytes and THP-1 cells differentiated by 12-O-tetradecanoylphorbol-13-acetate treatment into macrophages to determine if the HIV envelope protein, gp120, or an anti-CD4 receptor antibody stimulates prostaglandin formation by interacting with the CD4 receptor. Incubation of THP-1 cells with OKT4A antibody greatly stimulated the CD4-p56lck receptor complex as estimated by enhanced p56lck autophosphorylation, while the gp120 gave small but significant responses. Monocytic THP-1 cells poorly metabolized arachidonic acid to prostaglandin E2 and thromboxane B2 as measured by high-pressure liquid chromatography analysis. Western blot (immunoblot) and Northern (RNA) blot analyses revealed that unstimulated monocytes expressed little prostaglandin H synthase 1 and 2 (PGHS-1 and -2). Incubation of the monocytes with lipopolysaccharide, OKT4A, or gp120 did not increase the formation of prostaglandins. The expression of PGHS-1 or PGHS-2 was also not increased. Differentiation of the monocytes to macrophages by 12-O-tetradecanoylphorbol-13-acetate treatment resulted in increased expression of PGHS-1 and increased formation of prostaglandins compared with that for the monocytes. Lipopolysaccharide stimulation of the macrophages increased the formation of prostaglandins and increased the expression of PGHS-2 in the macrophages. However, OKT4A or gp120 preparation, at concentrations that stimulated p56lck autophosphorylation, did not enhance the formation of prostaglandins or the expression of PGHS-1 or PGHS-2. OKT4A and gp120 also did not stimulate the release of arachidonic acid, indicating that phospholipase A2 was not activated by the CD4 receptor in either the THP-1 monocytes or macrophages. These results indicate that activation of the CD4-p56lck receptor signal transduction pathway by the HIV envelope protein does not increase prostaglandin formation.

Animals↗
PubMed1994

Activation of mitogen-activated protein kinases by arachidonic acid and its metabolites in vascular smooth muscle cells.

Previous studies from this laboratory and others suggest that arachidonic acid and its metabolites play important roles in a variety of biological processes such as signal transduction, contraction, chemotaxis, and cell growth and differentiation. Here we studied the effect of arachidonic acid on mitogen-activated protein (MAP) kinases in vascular smooth muscle cells (VSMC). Arachidonic acid activated MAP kinases in VSMC in a time- and dose-dependent manner. Nordihydroguaiaretic acid (NDGA), a potent inhibitor of the lipoxygenase system, significantly blocked the arachidonic acid-induced activation of MAP kinases, whereas indomethacin, an inhibitor of cyclooxygenase, had no effect. In VSMC, arachidonic acid was converted to 15-hydroxyeicosatetraenoic acid (15-HETE); NDGA inhibited the formation of this HETE. Exogenous addition of 15-HETE to VSMC caused stimulation of MAP kinases. Depletion of protein kinase C attenuated both the arachidonic acid- and 15-HETE-induced activation of MAP kinases in VSMC. Together these results suggest that 1) arachidonic acid activates MAP kinases in VSMC; 2) 15-HETE, a 15-lipoxygenase product of arachidonic acid, at least in part, mediates the arachidonic acid effect on MAP kinases; and 3) protein kinase C appears to be important in arachidonic acid activation of MAP kinases. Therefore, MAP kinases may play an important role in arachidonic acid signaling of VSMC growth and function.

Animals↗
PubMed1994

Structure-activity relationship for potentiation of EGF-dependent mitogenesis by oxygenated metabolites of linoleic acid.

Epidermal growth factor induces the oxygenation of linoleic acid in Syrian hamster embryo fibroblasts, and the lipoxygenase-derived products potentiate the mitogenic signal. We have further characterized the linoleate metabolites of growth factor-activated cells by chiral phase HPLC analysis. The primary product was identified as the pure (S) enantiomer of 13-hydroxyoctadecadienoic acid (HODE). In comparison to 13(R)-HODE isomer, only the biologically derived 13(S)-HODE was active in augmenting DNA synthesis as assessed by [3H]thymidine incorporation. To extend these investigations, we defined the structural requirements of analogous lipid compounds necessary for stimulation of mitogenesis in these cells. Carbon-chain length, degree of unsaturation, type of oxidized functionality, position of oxygenated moiety, double-bond geometry, and chirality were all identified as factors that modulate the mitogenic activity of related compounds. The results demonstrate a high degree of specificity for (S)-isomer hydro(pero)xylinoleic acid metabolites in stimulating DNA synthesis and further define the relationship between linoleic acid metabolism and growth-factor-dependent cell growth.

Animals↗
PubMed1994

Cellular proliferation and lipid metabolism: importance of lipoxygenases in modulating epidermal growth factor-dependent mitogenesis.

In this article we have reviewed and discussed the results of our investigation of lipid metabolites as modulators of epidermal growth factor (EGF) signaling pathways. We have studied epidermal growth factor-dependent mitogenesis in BALB/c 3T3 and Syrian hamster embryo (SHE) cells in culture. We observed that EGF stimulates the formation of prostaglandins in BALB/c 3T3 cells and their formation appears to be necessary for EGF dependent mitogenesis. EGF did not stimulate PGE2 formation in SHE cells and in fact, exogenously added PGE2 inhibited mitogenesis. In both cell lines, EGF stimulated the formation of lipoxygenase-derived 13(S)-hydroxyoctadecadienoic acid (13-HODE) and inhibition of 13-HODE formation attenuated mitogenesis. The addition of 13-(S)-HODE enhanced EGF-dependent mitogenesis but when added alone, the compound was not mitogenic. Other metabolites, including lipoxygenase metabolites of arachidonic acid, were either weak simulators of EGF-dependent mitogenesis or essentially inactive. The 13(S)-HODE appears to be formed by an apparently unique lipoxygenase that is regulated by the tyrosine kinase activity of the EGF receptor. The mechanisms by which lipids, particularly the lipoxygenase-derived linoleic acid metabolites, modulate the EGF signaling pathways leading to cell proliferation is discussed. The possible significance of lipoxygenase and prostaglandin H synthase-dependent metabolism of unsaturated fatty acids in breast and colon cancer is also discussed.

3T3 Cells↗
PubMed1993

Effects of prostaglandins and hydroxyoctadecadienoic acid on epidermal growth factor-dependent DNA synthesis and c-myc proto-oncogene expression in Syrian hamster embryo cells.

Epidermal growth factor (EGF) stimulates DNA synthesis in quiescent Syrian hamster embryo (SHE) cells. Work in the present authors' laboratory has shown that the formation of 9- and 13-hydroxyoctadecadienoic acid (HODEs), 15-lipoxygenase-derived metabolites of linoleic acid, are involved in the mitogenic response to EGF in these cells (Glasgow et al. (1992) J. Biol. Chem. 267, 10771-10779). SHE cells also produce prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha). We now report the effects of HODEs and prostaglandins on EGF-dependent expression of the growth-related proto-oncogene c-myc in SHE cells. Treatment of cells with eicosatetraynoic acid (ETYA), which blocks EGF-dependent HODE formation, inhibited the mitogenic response to EGF, while exogenous 13-HODE potentiated EGF-dependent DNA synthesis. However, neither ETYA or 13-HODE altered the accumulation of c-myc mRNA in response to EGF. In contrast, PGE2 inhibited EGF-induced DNA synthesis and down-regulated EGF-stimulated c-myc mRNA accumulation in a dose-dependent manner, whereas PGF2 alpha had no effect on these responses. PGE2, but not PGF2 alpha, induced a rapid increase in cAMP formation, and both forskolin and 8-(4-chlorophenylthio)-cAMP mimicked the inhibitory effects of PGE2 on EGF-dependent DNA synthesis and c-myc mRNA accumulation, suggesting that the involvement of cAMP. The results indicate that the modulation of EGF-dependent DNA synthesis by PGE2, but not by HODEs, is associated with altered expression of the proto-oncogene c-myc in SHE cells.

Animals↗
PubMed1993

ESR studies on reactivity of protein-derived tyrosyl radicals formed by prostaglandin H synthase and ribonucleotide reductase.

Using ESR spectroscopy, the ability of enzyme inhibitors to quench protein-derived tyrosyl radicals was studied in two different enzymes, prostaglandin H synthase and ribonucleotide reductase. The prostaglandin H synthase inhibitors indomethacin, eugenol, and MK-410 effectively prevent the formation of tyrosyl radicals during the oxidation of arachidonic acid by prostaglandin H synthase from ram seminal vesicles. A direct reaction with preformed tyrosyl radicals was observed only with eugenol. The other prostaglandin H synthase inhibitors were ineffective. The ribonucleotide reductase inhibitors hydroxyurea and 4-hydroxyanisole, which effectively inactivate the tyrosyl radical in the active site of ribonucleotide reductase present in tumor cells, exhibit a different reactivity with tyrosyl radicals formed by prostaglandin H synthase. Hydroxyurea quenches preformed tyrosyl radicals in prostaglandin H synthase weakly, whereas 4-hydroxyanisole does not quench tyrosyl radicals in prostaglandin H synthase at all. Eugenol, which quenches preformed prostaglandin H synthase-derived tyrosyl radicals, also quenches the tyrosyl radical in ribonucleotide reductase. The results suggest that the reactivity of protein-linked tyrosyl radicals in ribonucleotide reductase and those formed during prostaglandin H synthase catalysis are very different and have unrelated roles in enzyme catalysis.

Animals↗
PubMed1993

Phorbol ester and epidermal growth factor enhance the expression of two inducible prostaglandin H synthase genes in rat tracheal epithelial cells.

Previous studies from our laboratory suggested that phorbol 12-myristate 13-acetate (TPA) stimulates prostaglandin E2 (PGE2) production by inducing de novo synthesis of prostaglandin H synthase (PHS) in a rat tracheal cell line. We report here an extension of this work to further elucidate the mechanisms by which TPA (and epidermal growth factor) stimulates PGE2 production. We used the rat tracheal cell line EGV6, which has a lower basal level of PGE2 production and responds to TPA and EGF stimulation with a much greater increase in PGE2 synthesis than the previously used cell line, Incubation of EGV6 cultures with TPA or EGF resulted in a time- and dose-dependent increase in PGE2 synthesis up to 40-fold and 6-fold, respectively. Serum also stimulated PGE2 synthesis, while bombesin, retinoic acid, and bacterial lipopolysaccharide did not. PHS protein levels in microsomal preparations from the cells were estimated by Western analysis. Antibodies raised against murine PHS-2 cross reacted with the EGV-6 PHS while several antibody preparations that react with PHS-1 from ram or mouse reacted poorly with the cellular preparation. TPA treatment increased the de novo synthesis of PHS-2 while dexamethasone treatment reduced the response to TPA. Northern blot analysis of mRNA from EGV6 cultures using a ram PHS cDNA revealed a 2.8- and a 4.5- to 4.9-kb (designated 4.9 kb) transcript. Treatment with TPA or EGF increased the expression of both transcripts and this effect was further enhanced by cyclohexamide. To further define the PHS mRNA species of EGV6 cells, two well-characterized murine PHS cDNA probes were used. The constitutive murine PHS cDNA probe hybridized only with the 2.8-kb transcript, and the inducible murine PHS cDNA hybridized only with the 4.9-kb transcript. The rates of induction as well as degradation of the 4.9-kb PHS mRNA were much more rapid than those of the 2.8-kb mRNA species. Dexamethasone partially inhibited the induction of both PHS transcripts by TPA. Southern analysis of genomic EGV6 DNA indicated the presence of two distinct PHS genes in these cells. Taken together these findings indicate that two PHS genes are expressed in rat tracheal epithelial cells. In contrast to the PHS genes expressed in murine (and chicken) fibroblasts in which only the gene coding for the larger mRNA species is transcriptionally regulated, in the rat tracheal cells both genes are positively regulated by TPA and EGF and downregulated by glucocorticoids.

Animals↗
PubMed1993

Prostaglandin H synthase-dependent formation of the direct-acting mutagen 2-nitro-3-methylimidazo[4,5-f]quinoline (nitro-IQ) from IQ.

The mutagenic effects of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) following activation by ram seminal vesicle microsomes (RSVM, a source of prostaglandin H synthase, PHS) were studied in Salmonella typhimurium tester strains possessing elevated levels of acetyl-CoA: arylamine N-acetyltransferase (NAT). The metabolites formed by RSVM were extracted and fractionated by high pressure liquid chromatography (HPLC). One isolable product accounted for most of the direct-acting mutagenicity observed in the extracts. The metabolite was identified as 2-nitro-3-methylimidazo[4,5-f]quinoline (nitro-IQ). Since nitro-IQ is a potent direct-acting mutagen, its role in IQ genotoxicity warrants further study.

Animals↗
PubMed1992

Modulation of c-jun and jun-B messenger RNA and inhibition of DNA synthesis by prostaglandin E2 in Syrian hamster embryo cells.

Fatty acid metabolites such as prostaglandins are important regulators of DNA synthesis and cell proliferation. However, the mechanisms involved in this regulation are unclear. We have examined the effects of several fatty acid metabolites on the expression of the growth-related genes c-jun and jun-B in Syrian hamster embryo cells. Treatment of cells with prostaglandin E2 (PGE2) resulted in the inhibition of epidermal growth factor (EGF)-induced DNA synthesis and c-jun mRNA accumulation, whereas PGE2 augmented EGF-stimulated jun-B mRNA and markedly stimulated jun-B accumulation in the absence of EGF. Treatment of cells with PGE2 resulted in rapid accumulation of cyclic AMP (cAMP), whereas prostaglandin F2 alpha did not stimulate cAMP formation and did not alter EGF-stimulated DNA synthesis or accumulation of c-jun or jun-B mRNA. Forskolin and 8-(4-chlorophenylthio)-cAMP mimicked the effects of PGE2 on DNA synthesis and on the expression of c-jun and jun-B, suggesting the involvement of cAMP. We have shown that EGF-induced DNA synthesis requires the formation of hydroxyoctadecadienoic acids, formed from linoleic acid by a 15-lipoxygenase, in Syrian hamster embryo cells (Glasgow et al., J. Biol. Chem., 267: 10771-10779). Inhibition of this 15-lipoxygenase blocked EGF-dependent hydroxyoctadecadienoic acid formation and mitogenesis but did not affect EGF-stimulated c-jun or jun-B mRNA accumulation. The data suggest that the modulation of EGF-dependent DNA synthesis by PGE2 is associated with altered expression of c-jun and jun-B in Syrian hamster embryo cells. In contrast, hydroxyoctadecadienoic acids appear to act downstream or divergent from c-jun and jun-B expression in the regulation of EGF-dependent DNA synthesis.

Animals↗
PubMed1992

Spectral analysis of the protein-derived tyrosyl radicals from prostaglandin H synthase.

We have analyzed the low temperature EPR spectra of the protein-derived tyrosyl radicals detected upon addition of arachidonic acid or 5-phenyl-4-pentenyl-1-hydroperoxide (PPHP) to prostaglandin H synthase. With either arachidonic acid or PPHP the initial radical detected is a doublet (peak-to-trough = 35 Gauss) that disappears rapidly and is replaced by a broad singlet (peak-to-trough = 30 Gauss) followed by a narrow singlet (peak-to-trough = 26.5 Gauss). The relative amounts of these signals vary with time and concentration of arachidonic acid. The three tyrosyl radical signals were subjected to computer simulation and power saturation analysis. The data establish that there are only two distinct tyrosyl radical species, the doublet and the narrow singlet. The broad singlet seen at intermediate times and at low arachidonic acid concentrations is a composite of the doublet and the narrow singlet. The composition of the broad singlet in incubations of prostaglandin H synthase with 0.5 mM arachidonic acid is approximately 40% doublet and 60% singlet. The broad singlet signal does not represent a distinct tyrosyl radical species.

Alkenes↗
PubMed1992

Modulation of the epidermal growth factor mitogenic response by metabolites of linoleic and arachidonic acid in Syrian hamster embryo fibroblasts. Differential effects in tumor suppressor gene (+) and (-) phenotypes.

Specific metabolites of arachidonic and linoleic acid have been proposed as serving a regulatory function in growth factor signal transduction in fibroblasts. In studies with Syrian hamster embryo (SHE) fibroblasts, we found lipoxygenase inhibitors to be potent blockers of epidermal growth factor (EGF)-dependent mitogenesis. Analytical chemical characterization of arachidonic and linoleic acid metabolism in SHE cells demonstrated that the major lipoxygenase product was 13-hydroxyoctadecadienoic acid (HODE). EGF stimulation of quiescent SHE cells resulted in an enhancement of HODE biosynthesis. The primary arachidonate products were prostaglandin E2 and F2 alpha formed via the cyclooxygenase pathway. Inhibition of cyclooxygenase activity did not alter the EGF-mitogenic response in SHE cells. Addition of lipoxygenase-derived linoleate metabolites (10(-10)-10(-6) M) produced a 2-4-fold potentiation of EGF-stimulated [3H]thymidine incorporation in SHE cells. Interestingly, the linoleate products did not enhance the EGF mitogenic effect in variant SHE cells that had lost tumor suppressor gene function. These results were confirmed by autoradiographic studies of DNA synthesis and suggest that loss of tumor suppressor phenotype correlates with a lack of responsiveness to linoleate products in signal transduction. In studies on the mechanism of EGF regulation of linoleic acid metabolism, inhibitors of EGF receptor tyrosine kinase activity were observed to block EGF-stimulated HODE biosynthesis. In addition, both cyclohexamide and actinomycin D attenuated the ability of EGF to increase linoleic acid metabolism in SHE cells. EGF induction of the linoleate pathway appears to be linked to activation of the EGF receptor and may be modulated at transcriptional or translational levels.

Animals↗
PubMed1992

Enhanced mutagenicity of anisidine isomers in bacterial strains containing elevated N-acetyltransferase activity.

In previous studies on the mutagenicity of anisidine isomers, the ortho isomer was considered to be mutagenic towards standard Ames tester strains, while the para isomer gave equivocal results. In the present study we show that both para- and ortho-anisidine isomers are mutagenic in a Salmonella typhimurium tester strain containing elevated levels of N-acetyltransferase (YG1029). p-Anisidine gave a positive mutagenic response using either hamster S9 or ram seminal vesicle microsomes (RSVM) as an activating system, while o-anisidine gave a positive response only with the hamster S9 fraction. The mutagenic response from p-anisidine was greater than with o-anisidine in each case. In tests with p-anisidine and RSVM, the addition of arachidonic acid was not necessary to observe a mutagenic response. Catalase produced a dose-dependent decrease in the mutagenic response with p-anisidine and RSVM; this indicates that endogenous hydrogen peroxide from the bacteria acts as a substrate for the peroxidase activity of RSVM prostaglandin H synthase. These results demonstrate that both anisidine isomers are mutagenic and that N-acetyltransferase enzymes play an important role in their metabolism to mutagenic species.

Arylamine N-Acetyltransferase↗
PubMed1992

Xenobiotic metabolism by prostaglandin H synthase.

During the metabolism of arachidonic acid by prostaglandin H synthase many chemicals including carcinogens are metabolized. These chemicals are metabolized by either the peroxidase activity of prostaglandin H synthase, the peroxyl radicals generated during arachidonic acid oxygenation, or a combination of these two mechanisms. In many cases, the chemical metabolism results in the formation of reactive metabolites that have mutagenic activity and potential carcinogenic activity. In other cases, the chemicals are detoxified. Chemical metabolism that occurs during arachidonic acid oxygenation may be an important determinate of chemical toxicity in extra-hepatic tissues.

Animals↗