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Biomedical subjects

T E Eling

Publications and source records attributed to T E Eling.

At least 37 records · Page 2Linked to original sources
PubMed1999

The linoleic acid metabolite, 13-HpODE augments the phosphorylation of EGF receptor and SHP-2 leading to their increased association.

In previous studies with Syrian hamster embryo fibroblasts, we found that a specific lipoxygenase metabolite of linoleic acid, 13(S)-hydroperoxyoctadecadienoic acid (HpODE), enhanced epidermal growth factor (EGF) signal transduction in a tumor suppressor gene plus phenotype (supB+); with a diminished response to 13(S)-HpODE in a tumor suppressor gene minus phenotype (supB-). This differential response was attributed to differences in the rate of EGF receptor (EGFR) dephosphorylation. To further define the molecular basis for these observations, in this report we examine the interaction of phosphorylated EGFR with the SH2 domain-containing protein tyrosine phosphatase, SHP-2, a positive modulator of EGF dependent cell growth. SHP-2 associated with phosphorylated EGFR to a greater extent in supB+ cells when compared to supB-. This differential association could not be accounted for by differences between suppressor gene phenotypes in SHP-2 protein level or mutations in the molecular sequence. The addition of 13(S)-HpODE stimulated a concentration-dependent increase in EGF-dependent phosphorylation of SHP-2 and its association with EGFR. A more dramatic response was observed in the supB+ cells. Differences in SHP-2 interaction with EGFR may account, in part, for phenotypic differences in the growth rates and responsiveness to EGF between the supB+ and supB- cells. EGFR-SHP-2 association appears to play an important role in the regulation of EGFR signal transduction.

Animals↗
PubMed1999

Introduction of full-length APC modulates cyclooxygenase-2 expression in HT-29 human colorectal carcinoma cells at the translational level.

Mutation of the adenomatous polyposis coli (APC) gene is associated with the earliest stages of colorectal tumorigenesis and appears to be responsible for the hereditary condition familial adenomatous polyposis (FAP). Evidence indicates that cyclooxygenase-2 (COX-2) is induced and at elevated levels in human colorectal cancers and in the polyps of mouse FAP models. We have used HT-29 cells, a human colorectal carcinoma cell line with a mutant carboxy-truncated APC gene, in which intact APC gene has been introduced under the control of an inducible promoter. These HT-29-APC cells provide a suitable model system to examine how COX-2 expression becomes dysregulated after loss of APC function. Induction of full-length APC causes the HT-29-APC cells to undergo apoptosis. However, differentiation, as measured by alkaline phosphatase activity, is not induced upon expression of full-length APC. Full-length APC protein has been shown to bind the intracellular protein beta-catenin and, as a result, the Lef/Tcf transcription factors are down-regulated. Analysis of APC immunoprecipitates demonstrate a time-dependent increase of beta-catenin interacting with full-length APC. Thus, the Lef/Tcf signaling pathway is intact at this point in these cells. Furthermore, upon expression of full-length APC, COX-2 protein expression is down-regulated while COX-2 mRNA levels remain the same. These data indicate that APC plays a role, either directly or indirectly, in the translational regulation of COX-2. Treatment of the HT-29-APC cells with sodium butyrate, an inducer of apoptosis, does not alter COX-2 protein expression. Thus, COX-2 down-regulation appears to be APC specific and not just due to apoptotic induction. APC appears to uniquely regulate COX-2 expression. The mechanism by which COX-2 protein expression is down-regulated in the HT-29-APC cells is under investigation.

Adenomatous Polyposis Coli Protein↗
PubMed1998

Nitric oxide trapping of tyrosyl radicals generated during prostaglandin endoperoxide synthase turnover. Detection of the radical derivative of tyrosine 385.

Tyrosyl radicals have been detected during turnover of prostaglandin endoperoxide H synthase (PGHS), and they are speculated to participate in cyclooxygenase catalysis. Spectroscopic approaches to elucidate the identity of the radicals have not been definitive, so we have attempted to trap the radical(s) with nitric oxide (NO). NO quenched the EPR signal generated by reaction of purified ram seminal vesicle PGHS with arachidonic acid, suggesting that NO coupled with a tyrosyl radical to form inter alia nitrosocyclohexadienone. Subsequent formation of nitrotyrosine was detected by Western blotting of PGHS incubated with NO and arachidonic acid or organic hydroperoxides using an antibody against nitrotyrosine. Both arachidonic acid and NO were required to form nitrotyrosine, and tyrosine nitration was blocked by the PGHS inhibitor indomethacin. The presence of superoxide dismutase had no effect on nitration, indicating that peroxynitrite was not the nitrating agent. To identify which tyrosines were nitrated, PGHS was digested with trypsin, and the resulting peptides were separated by high pressure liquid chromatography and monitored with a diode array detector. A single peptide was detected that exhibited a spectrum consistent with the presence of nitrotyrosine. Consistent with Western blotting results, both NO and arachidonic acid were required to observe nitration of this peptide, and its formation was blocked by the PGHS inhibitor indomethacin. Peptide sequencing indicated that the modified residue was tyrosine 385, the source of the putative catalytically active tyrosyl radical.

Amino Acid Sequence↗
PubMed1998

Inhibition of EGF-dependent mitogenesis by prostaglandin E2 in Syrian hamster embryo fibroblasts.

Lipid metabolism can play an important role in the development and progression of human cancers. We have used Syrian hamster embryo (SHE) fibroblasts as a model system to study how lipid metabolites can alter cell proliferation and apoptosis. For example, the linoleic acid metabolite 13(S)-HpODE enhances EGF-dependent growth by inhibiting de-phosphorylation of the EGFR which leads to activation of the MAP kinase pathway. In contrast, the arachidonic acid metabolite, PGE2, inhibits EGF-dependent mitogenesis and the expression of the proto-oncogenes c-myc, c-jun, and jun-B. In this study, we have investigated the mechanism by which PGE2 attenuates these responses by studying the EGF signaling cascade in SHE cells. PGE2 pretreatment caused a concentration-dependent decrease in EGF-dependent phosphorylation of MAP kinase and a corresponding inhibition of EGF-stimulated MAP kinase activity. Pretreatment of the SHE cells with PGE2 had little effect on the magnitude of EGF-dependent receptor auto-phosphorylation and the phosphorylation of GAP suggesting a down-stream target. Treatment of cells with forskolin and EGF causes similar inhibition of MAP kinase phosphorylation as observed with PGE2 and EGF. Since PGE2 elevates cAMP in these cells, it may act by altering cAMP accumulation. Raf-1 activity can be inhibited by a cAMP-dependent process. Raf-1 activity, measured by phosphorylation of Mek-1, was attenuated by the addition of PGE2. To determine if inhibition of Raf-1 activity causes inhibition of the MAP kinase pathway, cells were concomitantly incubated with PGE2 and EGF. Inhibition of MAP kinase phosphorylation was observed. From these data, we propose that in SHE cells PGE2 increases cAMP levels, which in turn causes inhibition of Raf-1 activity. The MAP kinase pathway is thus downregulated which decreases mitogenesis and proto-oncogene expression. This study demonstrates that an arachidonic acid metabolite can modulate phosphorylation and activity of key signal transduction proteins in a growth factor mitogenic pathway.

Animals↗
PubMed1997

The linoleic acid metabolite, (13S)-hydroperoxyoctadecadienoic acid, augments the epidermal growth factor receptor signaling pathway by attenuation of receptor dephosphorylation. Differential response in Syrian hamster embryo tumor suppressor phenotypes.

In Syrian hamster embryo (SHE) fibroblasts, epidermal growth factor receptor (EGFR) tyrosine kinase activity regulates the metabolism of endogenous linoleic acid to (13S)-hydroperoxyoctadecadienoic acid (13S)-HPODE). (13S)-HPODE stimulates EGF-dependent mitogenesis in a SHE cell phenotype, which expresses tumor suppressor genes (supB+), but was not effective in a variant that does not express these suppressor genes (supB-). In the present study, we have investigated the potential effects of this lipid metabolite on the EGFR signaling pathways in these two SHE cell lines. Treatment of quiescent SHE cells with EGF produced a rapid, transient increase in the tyrosine phosphorylation of EGFR. Dependence on EGF concentration for EGFR tyrosine phosphorylation was similar in both SHE cell lines, but a more prolonged phosphorylation was detected in the supB- variant. Incubation of supB+ cells with (13S)-HPODE and EGF increased EGFR autophosphorylation and tyrosine phosphorylation on several signaling proteins with Src homology-2 domains including GTPase-activating protein. The lipid metabolite did not significantly alter EGF-dependent tyrosine phosphorylation in the supB- variant. Tyrosine phosphorylation of mitogen-activated protein (MAP) kinase was also measured. The addition of (13S)-HPODE increased the extent and duration of MAP kinase tyrosine phosphorylation in supB+ cells but not in the supB- variant. MAP kinase activity in supB+ cells, as measured in immunoprecipitates from cells after the addition of EGF, was increased by the presence of (13S)-HPODE. The addition of (13S)-HPODE did not directly alter EGFR kinase activity or the internalization of the EGFR. However, the addition of (13S)-HPODE to supB+ cells extended the tyrosine phosphorylation of the EGFR in response to EGF. The dephosphorylation of the EGFR was measured directly, and a slower rate was observed in the supB- compared with the supB+ cells. Incubation of the supB+ cells with (13S)-HPODE attenuated the dephosphorylation of the EGFR. Thus, (13S)-HPODE stimulates EGF-dependent mitogenesis and up-regulation of EGF-dependent tyrosine phosphorylation by inhibiting the dephosphorylation of the EGFR. This study shows that a metabolite of an essential dietary fatty acid, linoleic acid, can modulate tyrosine phosphorylation and activity of key signal transduction proteins in a growth factor mitogenic pathway.

Animals↗
PubMed1997

Nitric oxide trapping of the tyrosyl radical of prostaglandin H synthase-2 leads to tyrosine iminoxyl radical and nitrotyrosine formation.

The determination of protein nitrotyrosine content has become a frequently used technique for the detection of oxidative tissue damage. Protein nitration has been suggested to be a final product of the production of highly reactive nitrogen oxide intermediates (e. g. peroxynitrite) formed in reactions between nitric oxide (NO.) and oxygen-derived species such as superoxide. The enzyme prostaglandin H synthase-2 (PHS-2) forms one or more tyrosyl radicals during its enzymatic catalysis of prostaglandin formation. In the presence of the NO.-generator diethylamine nonoate, the electron spin resonance spectrum of the PHS-2-derived tyrosyl radical is replaced by the spectrum of another free radical containing a nitrogen atom. The magnitude of the nitrogen hyperfine coupling constant in the latter species unambiguously identifies it as an iminoxyl radical, which is likely formed by the oxidation of nitrosotyrosine, a stable product of the addition of NO. to tyrosyl radical. Addition of superoxide dismutase did not alter the spectra, indicating that peroxynitrite was not involved. Western blot analysis of PHS-2 after exposure to the NO.-generator revealed nitrotyrosine formation. The results provide a mechanism for nitric oxide-dependent tyrosine nitration that does not require formation of more highly reactive nitrogen oxide intermediates such as peroxynitrite or nitrogen dioxide.

Arachidonic Acid↗
PubMed1996

Nitric oxide: a prostaglandin H synthase 1 and 2 reducing cosubstrate that does not stimulate cyclooxygenase activity or prostaglandin H synthase expression in murine macrophages.

Several recent reports have investigated the possibility that nitric oxide (-NO) can regulate prostaglandin H synthase (PHS) activity. The potential significance of -NO regulation of PHS is considerable, when one considers the numerous important biological processes that are influenced by PHS. In this study, we used microsomal and purified PHS to investigate the direct effect of -NO and -NO-generating compounds on PHS catalytic activity. We found that -NO neither significantly inhibited nor enhanced prostaglandin (PG) formation, despite the fact that -NO stimulated PHS peroxidase activity. We also investigated the effect of -NO and -NO generators on PHS product, protein, and mRNA levels in the RAW264.7 murine macrophage cell line. We found that -NO or -NO generators had little or no effect on PG formation, PHS expression, or PHS mRNA expression in unstimulated RAW264.7 cells. The same results were obtained with macrophages that were stimulated by 18 h pretreatment with lipopolysaccharide, a known inducer of PHS-2 in macrophages. These data clearly indicate that -NO acts as a cosubstrate for PHS peroxidase. However, -NO does not enhance or inhibit either cyclooxygenase activity or expression of PHS in the model systems used in this study.

Animals↗
PubMed1996

Role of hydroperoxyeicosatetraenoic acids in oxidative stress-induced activating protein 1 (AP-1) activity.

We have previously reported that hydrogen peroxide, an active oxygen species and a cellular oxidant, induces c-Fos and c-Jun mRNA expression and DNA synthesis in vascular smooth muscle cells and that these events require arachidonic acid release and metabolism through the lipoxygenase pathway. Here we have identified the eicosanoids that mediate the hydrogen peroxide-induced growth-related events in these cells. Hydrogen peroxide stimulated the production of 12- and 15-hydroperoxyeicosatetraenoic acids in vascular smooth muscle cells. Both 12- and 15-hydroperoxyeicosatetraenoic acids induced the expression of c-Fos and c-Jun protein and increased activating protein 1 (AP-1) activity, as measured by AP-1-DNA binding and AP-1-dependent human collagenase promoter-driven chloramphenicol acetyltransferase reporter gene transcription. Hydrogen peroxide and arachidonic acid also induced the expression of c-Fos and c-Jun protein and AP-1 activity. Nordihydroguaiaretic acid, an inhibitor of the lipoxygenase pathway, significantly inhibited both hydrogen peroxide and arachidonic acid-stimulated c-Fos and c-Jun protein expression and AP-1 activity. Together, these findings suggest that hydrogen peroxide induces the production of eicosanoids and that the eicosanoids are potential mediators of the oxidative stress-stimulated growth-related events in vascular smooth muscle cells.

Animals↗
PubMed1996

Relative activities of retrovirally expressed murine prostaglandin synthase-1 and -2 depend on source of arachidonic acid.

We have developed derivatives of mouse embryonic fibroblasts (10T1/2) and Chinese hamster ovary (AS52) cells that stably express high levels of murine prostaglandin synthase-1 or -2 (PGHS-1 or -2). The cDNAs were transferred using retroviral vectors and the resulting G418-resistant clones were analyzed for prostaglandin E2 (PGE2) production. Specific expression was confirmed by Western and Northern analyses. Enzyme activities, protein, and message levels peaked 1 (10T1/2) or 2 (AS52) days after seeding but decreased as cells became density arrested. Upon subculturing, enzyme activities returned to their initial high levels. With 10 microM exogenous arachidonic acid (AA) as the substrate, PGHS-1 activities were approximately 3- to 5-fold higher than PGHS-2 activities. Conversely, when exogenous AA was left out of the medium and only endogenous AA was available as substrate, enzyme activities were lower; but PGHS-2 activities were 5-fold (10T1/2) or 1.5-fold (AS52) higher than PGHS-1 activities. Following phorbol ester treatment to stimulate endogenous AA release, PGHS-2 activities increased over time and by 6 hours, were 4-fold (10T1/2) or 2-fold (AS52) higher than PGHS-1 activities. However, when calcium ionophore A23187 was used to stimulate endogenous AA release, maximum PGHS activities occurred within 30 min of treatment; PGHS-1 activities were equal to (10T1/2) or 2-fold higher (AS52) than PGHS-2 activities. Because these cell lines allow us to measure specific PGHS activity in intact cells, we were able to demonstrate that the relative activities of the two PGHS isozymes depend on the source of AA (exogenous versus endogenous) or biochemical stimulus used to mobilize endogenous AA (A23187 versus phorbol ester). These data suggest that PGHS-1 and PGHS-2 preferentially utilize different pools of AA and may be modulated through different stimulus-initiated pathways.

Animals↗
PubMed1996

Prostaglandin synthase 2 expression in epidermal growth factor-dependent proliferation of mouse keratinocytes.

BALB/c mouse keratinocytes (BALB/MK) are nontumorigenic epithelial cells which are dependent on mouse epidermal growth factor (EGF) for maintaining proliferation in culture. In BALB/MK the oxygenation of both arachidonic acid and linoleic acid was dependent on EGF. EGF stimulated the formation of prostaglandin E2 and prostaglandin F2 alpha from arachidonic acid and 9- and 13-hydroxyoctadecadienoic acid (HODE) from linoleic acid. Analysis of the linoleic acid metabolites determined the ratio of 9-HODE to 13-HODE was approximately 6 to 4, and the 9-HODE was the (R) enantiomer, consistent with metabolism by prostaglandin G/H synthase (PGHS). The formation of these linoleic acid metabolites was sensitive to indomethacin, a PGHS inhibitor. EGF induced the expression of PGHS-2 mRNA after 30 min, which peaked after 1 h, and remained expressed for at least 24 h after the addition of EGF. A less significant increase in the expression of PGHS-1 mRNA occurred 4 h after EGF stimulation. Immunoblot analysis did not detect expression of PGHS-1 protein. However, PGHS-2 protein expression was increased 2 h after EGF exposure and was dependent on EGF. PGHS-2 protein was not transiently expressed as reported with other cell types, but was continually expressed in proliferating cells maintained with EGF at a subconfluent density. Indomethacin significantly attenuated EGF-dependent mitogenesis and cell proliferation. These results suggest that PGHS-2 activity contributes to the proliferative response of BALB/MK to EGF.

Animals↗
PubMed1996

Cloning, sequencing and expression of a 5-lipoxygenase from Syrian hamster embryo fibroblasts.

The hamster ortholog of human and rat 5-lipoxygenase (5-LO) was cloned from a Syrian hamster embryo (SHE) cell line. A combination of polymerase chain reaction (PCR) and 5' and 3' RACE (rapid amplification of cDNA ends) was used to isolate the complete cDNA for this gene. The cDNA sequence demonstrates the extreme sequence conservation found in this gene family, with a deduced amino acid sequence 95% identical to the rat 5-LO, and 90% identical to the human enzyme. The hamster 5-LO was expressed in E. coli. The expressed protein was detected by an antibody to human 5-LO, and had an apparent molecular weight of 75-80 kD. The products of the action of this enzyme on arachidonic acid are 5-HETE and the diHETEs resulting from the breakdown of LTA4, in a pattern similar to that produced by the recombinant human 5-LO. No oxidation of linoleic acid by this enzyme was detected.

Amino Acid Sequence↗
PubMed1996

Retinoid-induced differentiation regulates prostaglandin H synthase and cPLA2 expression in tracheal epithelium.

Rat tracheal epithelial cells cultured in vitro at an air-liquid interface can differentiate into a mucociliary or squamous phenotype depending on the presence or absence of retinoic acid (RA). The airway epithelium is known to produce a number of eicosanoids. We propose that eicosanoid biosynthesis is dependent on the differentiation status of the epithelium. Therefore, prostaglandin production and the expression of cytosolic phospholipase A2 (cPLA2) and the prostaglandin H synthase (PGHS) isoforms were investigated during differentiation to these two phenotypes. The major eicosanoid produced by both phenotypes was prostaglandin E2 (PGE2). Proliferating undifferentiated cultures produced low levels of PGE2 regardless of retinoid status. Differentiated mucociliary cultures produced high levels of PGE2 (50 ng/10(6) cells), whereas differentiated squamous cultures produced low levels of PGE2 (< 5 ng/10(6) cells). Mucociliary cultures expressed high levels of cPLA2 and PGHS-2 isoform mRNA and protein. In contrast squamous cultures expressed very low levels of cPLA2 and PGHS-2 transcript and protein. The PGHS-1 isoform was expressed in squamous but not in mucociliary cultures. We investigated changes in expression of these enzymes during retinoid treatment of established squamous cultures. Treatment with RA resulted in a rapid (24 h) downregulation of PGHS-1 mRNA expression. However, the cPLA2 and PGHS-2 genes were expressed in squamous cultures only after 3 days of RA treatment coincident with redifferentiation of the culture to a mucociliary phenotype. These studies reveal that retinoid-induced differentiation of airway epithelium into either a mucociliary or squamous phenotype results in profound changes in the expression of cPLA2 and PGHS isozymes that regulate prostaglandin formation.

Animals↗
PubMed1996

Regulation of 13(S)-hydroxyoctadecadienoic acid biosynthesis in Syrian hamster embryo fibroblasts by the epidermal growth factor receptor tyrosine kinase.

Metabolism of arachidonic and linoleic acid can be regulated by polypeptide growth factors in a variety of cell types. In Syrian hamster embryo (SHE) fibroblasts, epidermal growth factor (EGF) stimulates the conversion of exogenous linoleic acid to 13(S)-hydroxyoctadecadienoic acid (HODE). Inhibition of 13-HODE biosynthesis blocks the EGF-mitogenic response in SHE cells, and 13-HODE and its hydroperoxy precursor are potent and highly specific enhancers of EGF-dependent DNA synthesis. We demonstrated that EGF stimulates a biphasic production and release of endogenous 13-HODE. Through development of a stable isotope-dilution GC/MS assay for 13-HODE, we observed 13-HODE production as early as 5 min after EGF stimulation, and this initial phase peaked at 1 hr. A second rise in 13-HODE formation was seen at 2-4 hr, and this phase plateaued at 4-6 hr at a level of 30-40 ng/10(6) cells. EGF stimulation of 13-HODE biosynthesis is not mediated by transcriptional or translational regulation of the inducible form of prostaglandin H synthase. Based on enzyme inhibitor studies and structural characterization of products, the linoleate metabolite is apparently formed by an n-6 lipoxygenase that remains to be characterized. EGF stimulation of 13-HODE formation is linked with activation of the EGF receptor tyrosine kinase. Inhibition of EGF receptor tyrosine kinase activity with methyl-2,5-dihydroxycinnamate blocked EGF-dependent linoleic acid metabolism and EGF-regulated DNA synthesis. Potentiation of the EGF receptor tyrosine phosphorylation cascade through treatment of SHE cells with the tyrosine phosphatase inhibitor vanadate resulted in a 3-fold increase in EGF-stimulated 13-HODE production and a corresponding enhancement of the EGF mitogenic response. The coupling of EGF-regulated linoleic acid metabolism with the EGF receptor tyrosine kinase activity suggests the importance of specific linoleate compounds in mediating mitogenic signal transduction.

Animals↗
PubMed1995

Prostaglandin H synthase-2 is induced in Syrian hamster embryo cells in response to basic fibroblast growth factor.

Metabolites of arachidonic acid and linoleic acid can serve as regulators of the epidermal growth factor signal transduction system in Syrian hamster embryo (SHE) fibroblasts. We have now investigated the possible role of these lipids in modulating the signal transduction of basic fibroblast growth factor (bFGF), a potent mitogen to SHE fibroblasts. The addition of bFGF (0.1 to 1.0 ng/ml) to serum-deprived SHE cells stimulated a six- to sevenfold increase in the incorporation of thymidine into DNA. Structural analysis indicated that bFGF stimulated the metabolism of exogenous and endogenous arachidonic acid to primarily PGE2, PGF2 alpha, and PGD2, with lesser amounts of uncharacterized prostaglandins observed. The metabolism of linoleic acid in SHE cells was not affected by bFGF. bFGF stimulated the expression of the inducible form of prostaglandin H synthase (PGHS-2) as determined by Northern analysis using murine PGHS-2 cDNA as the probe. PGHS-2 protein in the SHE cells was also increased by bFGF as determined by Western analysis using antibodies specific for PGHS-2. Levels of the constitutive (PGHS-1) enzyme and mRNA were not altered by bFGF. Preincubation of the cells with 1-2 microM dexamethasone significantly inhibited bFGF-stimulated expression of PGHS-2 protein and mRNA. Dexamethasone potently inhibited bFGF induced mitogenesis in these cells. Pretreatment of SHE cells with indomethacin inhibited bFGF-dependent mitogenesis, as well as endogenously produced PGE2. The data suggests that regulation of PGHS-2 expression may be an element of the bFGF mitogenic signal transduction pathway.

Animals↗