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Biomedical subjects

D V Faller

Publications and source records attributed to D V Faller.

At least 91 records · Page 5Linked to original sources
PubMed1992

Oncogenic ras induces an inhibitor of double-stranded RNA-dependent eukaryotic initiation factor 2 alpha-kinase activation.

The interferon-inducible 68-kDa dsRNA-dependent eIF2 alpha-kinase (dsI) is a potent cellular antiviral enzyme which is activated by autophosphorylation in response to double-stranded RNA (dsRNA). Activated dsI has also been implicated as a second messenger for gene induction by platelet-derived growth factor (PDGF) and interferon (IFN). We have shown previously that introduction of a transforming ras gene into BALB/c-3T3 fibroblasts blocks induction of responsive genes by PDGF and IFN. We therefore investigated the effect of transforming ras genes on dsI activity in these cells. We report here that dsRNA-mediated activation of dsI is blocked in v-ras-containing cells in a manner specific to ras and not attributable to the transformed phenotype since: 1) a dexamethasone-inducible v-Ha-ras gene produced the effect within 18 h of induction; 2) morphologic reversion of ras-transformed cells with cAMP or the Krev-1 gene restored potential for dsI activation; and 3) transformation by v-mos or v-abl had no effect on dsI activation. Latent dsI levels were unaffected by v-ras. A heat-sensitive dsI inhibitory activity could be demonstrated in v-ras-containing cells which functioned in trans when mixed with untransformed cell extracts prior to stimulation with dsRNA. The inhibitory activity, which was destroyed by phenol-chloroform extraction, did not bind dsRNA.

3T3 Cells↗
PubMed1992

Effects of cysteamine administration on somatostatin biosynthesis and levels in rat hypothalamus.

A single injection of cysteamine (CSH; 2-aminoethanethiol; 300 mg/kg, sc) into male rats produced a rapid decline in immunoreactive somatostatin (IR-SRIF) levels in the hypothalamus (to 20% of preinjection values within 12 h) which persisted for several days. The levels of both somatostatin-14 (SRIF-14) and somatostatin-28 (SRIF-28) were reduced. In contrast, the levels of somatostatin-28(1-12) were unaffected. Most (80-90%) of the lost SRIF molecules (both SRIF-14 and SRIF-28) could be recovered from CSH-injected rats by subjecting hypothalamic samples to denaturing, reducing, and reoxidizing conditions. These results suggest that CSH does not deplete the hypothalamus of SRIF molecules, but, instead, alters their chemical structures, rendering them undetectable using a SRIF-14-directed RIA. CSH injection also caused a rapid and complete suppression (within 1 h) of [35S]cysteine incorporation into SRIF-14 and SRIF-28. This reduction, however, was short-lived; normal incorporation rates returned within 10 h of drug administration. CSH did not influence [35S] cysteine incorporation into acid-precipitable protein or [35S]cysteine specific activity in the hypothalamus. In addition, [35S]SRIF molecules were not recovered from hypothalami of CSH-treated rats by subjecting samples to denaturing, reducing, and then reoxidizing conditions. These findings indicate that CSH injection causes a true, but short-lived (1- to 10-h), suppression of hypothalamic SRIF-14 and SRIF-28 formation. Finally, biosynthesis studies of longer duration revealed no prolonged effects of CSH. The drug produced no changes 4, 24, or 72 h postinjection in hypothalamic levels of the prepro-SRIF mRNA. Moreover, two injections of CSH, separated by 3 days, which continuously suppressed IR-SRIF levels for almost 1 week, caused only a transient suppression of [35S]SRIF-14 and [35S]SRIF-28 synthesis after each injection. These results indicate that the SRIF biosynthetic pathway is not activated by the prolonged CSH-induced depletion of IR-SRIF stores.

Animals↗
PubMed1991

Dissociation of platelet-derived growth factor (PDGF) receptor autophosphorylation from other PDGF-mediated second messenger events.

Activated p21ras alters the platelet-derived growth factor (PDGF) signal transduction pathway in fibroblasts by inhibiting autophosphorylation of the receptor as well as by inhibiting the induction of the growth-related genes c-myc, c-fos, and JE. To elucidate the cause and effect relationships between receptor autophosphorylation and other second messenger events in the PDGF signaling pathway we created revertants of v-ras transformed cells by two methods: 1) the use of cAMP analogues, and 2) the introduction of a gene, Krev-1, which has been reported previously to revert ras transformed cells to normal morphology. Analysis of the revertants shows that the PDGF-mediated tyrosine phosphorylation of the 180-kDa PDGF receptor remains inhibited; however, the PDGF-mediated activation of phospholipase C and the induction of the growth-related genes c-myc, c-fos, and JE have been restored. These data suggest the presence of parallel pathways for PDGF signal transduction which are not dependent on autophosphorylation of the PDGF receptor.

1-Phosphatidylinositol 4-Kinase↗
PubMed1991

trans-Activation of genes encoding activation-associated human T lymphocyte surface proteins by murine retroviral sequences.

The mechanisms whereby RNA leukemia viruses cause T lymphocyte leukemias or lymphomas after a long latent period are not understood. We report here that infection of human T lymphocyte lines with a murine leukemia virus results in up-regulation of a number of lymphocyte-specific cell surface Ag. These proteins include CD2, CD3, CD4, the TCR, and MHC class I Ag. The expression of other cell surface proteins, such as LFA-3, are unaffected by the presence of the retrovirus. This up-regulation occurs at the level of the mRNA transcripts encoding these proteins, and is the result of increased transcription of the respective genes. The increases in transcription are the result of a trans-activation process by the leukemia virus. The transient introduction of chimeric genes consisting of MHC class I gene promoter sequences attached to the reporter gene CAT into human T cells containing murine retrovirus produces stimulated transcription of the reporter gene. Subgenomic portions of the murine leukemia virus containing the long terminal repeats and the 5' untranslated region are sufficient to produce transactivation of the same set of T cell genes as the whole leukemia virus. The finding that murine leukemia viruses enhance transcription and expression of a group of T cell surface proteins, all of which have been reported to be capable of transducing an activating signal to the lymphocyte, may be relevant to the pathophysiologic mechanisms whereby these viruses induce leukemias and lymphomas.

Antigens, CD↗
PubMed1991

Inhibition of platelet-derived growth factor-mediated signal transduction by transforming ras. Suppression of receptor autophosphorylation.

The function of ras protein and its relationship to growth-factor mediated signal transduction remain unclear. The demonstration that the expression of transforming ras (v-ras) can block the stimulation of growth-related gene expression and cell division mediated by the platelet-derived growth factor (PDGF) may provide a model for the functional interation of ras with growth factor receptors. In the current studies, we have demonstrated that this blockade by v-ras of PDGF-BB signal transduction occurs very early in signal transduction, at the level of PDGF receptor autophosphorylation. Although the expression of PDGF receptor as detected by Western blot with anti-PDGF receptor antibody was not diminished in v-ras-transformed murine Balb/c 3T3 fibroblasts, the autophosphorylation of PDGF receptor in response to ligand (recombinant PDGF-BB homodimer) stimulation was profoundly suppressed. This same phenomenon of v-ras-mediated PDGF receptor autophosphorylation inhibition was also demonstrated in normal rat kidney fibroblasts. Further, factor(s) present in v-ras-expressing fibroblasts found in the membrane fractions of these cells can dominantly inhibit the autophosphorylation of the PDGF receptor obtained from normal fibroblasts. These findings suggest a role for ras in one of the earliest steps of the signal transduction pathway.

Animals↗
PubMed1991

BALB/c-3T3 fibroblasts resistant to growth inhibition by beta interferon exhibit aberrant platelet-derived growth factor, epidermal growth factor, and fibroblast growth factor signal transduction.

Several lines of evidence now exist to suggest an interaction between the platelet-derived growth factor (PDGF) growth-stimulatory signal transduction pathway and the beta interferon (IFN-beta) growth-inhibitory signal transduction pathway. The most direct examples are inhibition of PDGF-mediated gene induction and mitogenesis by IFN-beta and the effects of activators and inhibitors of the IFN-inducible double-stranded RNA-dependent eIF2 kinase on expression of PDGF-inducible genes. To further investigate the nature of this PDGF/IFN-beta interaction, we selected BALB/c-3T3 cells for resistance to growth inhibition by IFN-beta and analyzed the phenotypes of resulting clonal lines (called IRB cells) with respect to PDGF signal transduction. Although selected only for IFN resistance, the IRB cells were found to be defective for induction of growth-related genes c-fos, c-myc and JE in response to PDGF. This block to signal transduction was not due to loss or inactivation of PDGF receptors, as immunoprecipitation of PDGF receptors with antiphosphotyrosine antibodies showed them to be present at equal levels in the BALB/c-3T3 and IRB cells and to be autophosphorylated normally in response to PDGF. Furthermore, treatment with other peptide growth factors (PDGF-AA, fibroblast growth factor, and epidermal growth factor) also failed to induce c-fos, c-myc, or JE expression in IRB cells. All of these growth factors, however, were able to induce another early growth-related gene, Egr-1. The block to signaling was not due to a defect in inositol phosphate metabolism, as PDGF treatment induced normal calcium mobilization and phosphotidylinositol-3-kinase activation in these cells. Activation of protein kinase C by phorbol esters did induce c-fos, c-myc, and JE in IRB cells, indicating that signalling pathways distal to this enzyme remained intact. We have previously shown that IFN-inducible enzyme activities, including double-stranded RNA-dependent eIF2 kinase and 2',5'-oligoadenylate synthetase, are normal in IRB cells. The finding that the induction of multiple growth-related genes by several independent growth factors is inhibited in these IFN-resistant cells suggests that there is a second messenger common to both growth factor and IFN signaling pathways and that this messenger is defective in these cells.

Animals↗
PubMed1991

Hypoxia induces endothelin gene expression and secretion in cultured human endothelium.

Hypoxia in vivo is associated with constriction of the distal vasculature in the lung. Uniquely situated at the interface between blood and the vessel wall proper, the vascular endothelium may release vasoactive mediators in the setting of hypoxia. Endothelin-1 is a potent vasoconstrictor released by endothelial cells that could function as a paracrine regulator of vascular tone. We found that physiologic low oxygen tension (PO2 = 30 Torr) increased endothelin secretion from cultured human endothelial cells four to eightfold above the secretion rate at ambient oxygen tension. This increase in secretion was accompanied by a corresponding increase in the transcriptional rate of the preproendothelin gene resulting in increased steady-state mRNA levels of preproendothelin. In contrast, the transcription of a number of other growth-factor-encoding genes, including transforming growth factor-beta, was unaffected by hypoxia. Endothelin transcript production increased within 1 h of hypoxia and persisted for at least 48 h. In addition, the stimulatory effects of low oxygen tension on endothelin mRNA levels were reversible upon reexposure to 21% oxygen environments. These findings suggest a role for endothelin in the control of regional blood flow in the vasculature in response to changes in oxygen tension.

Cell Hypoxia↗
PubMed1990

Distinct effects of interferon-gamma and MHC class I surface antigen levels on resistance of the K562 tumor cell line to natural killer-mediated lysis.

Various investigators have examined the relationship between tumor cell susceptibility to natural killer (NK) cell lysis and the expression of HLA class I antigens on the tumor cell. There is controversy as to whether or not an inverse relationship exists, and if so, the basis of the relationship between these two phenomena remains undefined. To address these questions, the genomic clones for two HLA antigens were transfected into the erythroleukemia cell line K562, a cell line that is used as the standard to assess human NK and major histocompatibility complex (MHC) nonrestricted cytolysis. Susceptibility to NK lysis was not affected by the de novo expression of HLA antigens on the K562 after DNA mediated gene transfer. Interferon-gamma (IFN-gamma) treatment of K562 induced levels of MHC class I antigen surface expression comparable to those found on the transfected cells; however, the IFN-gamma-treated cells were resistant to NK lysis. When very high levels of surface HLA antigens were induced on the transfectants, a potential effect of class I MHC expression on K562 lysis could be discerned that was distinct from the resistance to NK lysis induced by IFN-gamma-treatment.

Antigens, Surface↗
PubMed1990

Effect of cellular density and viral oncogenes on the major histocompatibility complex class I antigen response to gamma-interferon in BALB-c/3T3 cells.

Cellular density in culture has profound effects on major histocompatibility complex (MHC) class I antigen expression in BALB/c-3T3 cells. Cells which have been confluent for greater than 24 h demonstrate a 2- to 6-fold increase in MHC class I antigen expression compared to subconfluent cells. These density-associated changes in MHC class I antigen expression occur both in untransformed and in v-mos or v-rasKi-transformed cells. The density-associated increases are specific for MHC class I antigens and do not occur with the cytoskeletal antigen actin. Transformation of the BALB/c-3T3 cells by either v-rasKi or v-mos has little or no direct effect on MHC class I expression under standard culture conditions. However, both oncogenes can indirectly alter the enhancement of MHC class I antigen expression in response to gamma-interferon. Incubation of untransformed BALB/c-3T3 cells with gamma-interferon leads to greater relative and absolute increases in MHC class I antigen expression in confluent cells than it does in subconfluent cells. In contrast, in v-rasKi- and v-mos-transformed cells, the subconfluent cells have a greater increase in MHC class I antigen expression in response to gamma-interferon than the cells which have exceeded monolayer confluence. The dense v-rasKi- and v-mos-transformed BALB/c-3T3 cell cultures are able to deplete their medium of the exogenous gamma-interferon, and this depletion of gamma-interferon causes the increase in the MHC class I antigen expression to be less sustained with lower peak expressions than the expression found in subconfluent cells. Supplementation with additional gamma-interferon can restore the full enhancement of MHC class I antigen in the transformed BALB/c-3T3 cells. The v-mos- and v-rasKi-transformed cells are more likely to deplete their medium of exogenous gamma-interferon because these cells can exceed monolayer confluence and thus achieve 10-fold higher densities than the untransformed BALB/c-3T3 cells. At high cellular densities, untransformed cells can partially deplete their medium of exogenous gamma-interferon, but this phenomenon is generally less pronounced than in the transformed cells.

Animals↗
PubMed1990

The regulation of exogenous and endogenous class I MHC genes in a human tumor cell line, K562.

Previous studies have implied the existence of a trans-dominant intracellular repressor able to down-regulate the expression of the entire family of class I MHC genes in the genome of the K562 erythroleukemia cell line. This study demonstrates, however, that the transfection of human or murine class I genes into K562 cells leads to the cell surface expression of the transfected MHC gene product in all situations, even when several kilobases of 5' flanking sequence were included in the transfected genes. The endogenous cellular class I MHC genes remained repressed in the transfected cells. These findings suggest that repression of class I MHC gene expression in K562 may not be mediated predominantly by a trans-dominant repressor of MHC gene expression; rather, other more complex regulatory influences might exist.

Gene Expression Regulation↗
PubMed1990

Interferon-gamma modulates fetal hemoglobin synthesis in sickle cell anemia and thalassemia.

Interferon-gamma (IFN-gamma) has been shown to influence globin gene expression in cord blood and normal adult progenitor-derived erythroblasts. To explore the influence of IFN-gamma on fetal hemoglobin (HbF) synthesis in the hemoglobinopathies, erythroid progenitors (BFU-E, burst forming unit-erythroid) from patients with sickle cell anemia (SCA) and thalassemia were co-cultured with or without IFN-gamma. Hemoglobin content in progenitor-derived erythroblasts was assessed by radioligand assay (RIA). Co-culture of erythroid progenitors from 12 SCA patients with 200-400 U/ml of IFN-gamma resulted in a significant decrease in picograms of HbF and percent HbF per BFU-E-derived erythroblast. The mean decrease (+/- SEM) of picograms of HbF per cell and percent of HbF was by 42 +/- 9% and 35 +/- 8% of control cultures, respectively. Co-culture of erythroid progenitors from 10 patients with thalassemia major or thalassemia variant (HPFH/thalassemia, sickle/beta 0-thalassemia) with 200 U/ml IFN-gamma also resulted in a significant decrease in picograms and percent of HbF per BFU-E-derived erythroblast. IFN-gamma treatment also inhibited the enhancement in gamma-globin synthesis induced in culture by butyric acid. Erythroid progenitors from 2 patients with SCA, 1 patient with sickle/beta 0-thalassemia, and 1 patient with HbE/beta 0-thalassemia were co-cultured with IFN-gamma, L-alpha-amino-n-butyric acid, or both. HbF content (expressed as picograms HbF/cell) was decreased in samples co-cultured with IFN, increased in cultures with L-alpha-amino-n-butyric acid, but remained at control values in cultures treated with IFN plus L-alpha-amino-n-butyric acid. These data demonstrate that IFN-gamma is an environmental factor that influences gamma-globin gene expression in the beta hemoglobinopathies in vitro.

Aminobutyrates↗
PubMed1990

Stopping the biologic clock for globin gene switching.

The developmental switch from production of fetal (gamma) to adult (beta) globin occurs on a normally set biologic clock which proceeds even if expression of the adult (beta) globin genes is defective and produces little or no protein, as in the beta-thalassemias. Preventing or reversing the globin gene switch could provide a way of keeping the abnormal globin genes "silent" and maintaining expression of the fetal globin gene. We have identified a class of agents which, when present in elevated plasma concentrations during gestation, inhibits the gamma----beta-globin gene switch in developing humans. Further investigation has shown that butyric acid and related compounds can increase gamma-globin and decrease beta-globin expression in cultured erythroid cells of patients with beta-thalassemia. Butyrate compounds were therefore infused in an in vivo fetal animal model, and the globin switch was inhibited and even reversed in some fetal lambs. Histone hyperacetylation, which maintains active chromatin structure, and an effect on the gamma-globin promoter appear to be mechanisms of action involved. These data suggest that inhibiting expression of abnormal beta-globin genes by pharmacologic means may in the future be possible for treatment of individuals with beta-globin disorders.

Anemia, Sickle Cell↗
PubMed1990

Role of protein kinase A in tax transactivation of the human T-cell leukemia virus type I long terminal repeat.

The human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) is inducible both by the retroviral tax gene product and by cyclic AMP in the murine thymoma S49 cell line. The cis-acting sequences that control transcriptional induction by tax and by cyclic AMP are in close proximity within the HTLV-I promoter. By using a protein kinase A (PKA)-deficient S49 mutant cell line, the response of the viral promoter to cyclic AMP was shown to depend on PKA, whereas the response to tax did not require the activity of this enzyme. Transactivation of the HTLV-I LTR by tax, however, decreased in PKA-deficient and adenylate cyclase-deficient cells. The evidence presented supports largely independent mechanisms of promoter induction by cyclic AMP and tax but also suggests a role for PKA-mediated phosphorylation in the regulation of HTLV-I LTR-driven gene expression by tax.

Adenylyl Cyclases↗
PubMed1990

Oxygen tension regulates the expression of the platelet-derived growth factor-B chain gene in human endothelial cells.

Hypoxic states are associated with abnormal proliferation and constriction of the smooth muscle cells surrounding the distal vessels of the lung. In hypoxic as well as in normal states, the endothelial cell layer may play a key role in controlling smooth muscle tone by secreting a number of vasoactive agents. Platelet-derived growth factor (PDGF), produced by endothelial cells, is a major growth factor for vascular smooth muscle cells and a powerful vasoconstrictor. It consists of a disulfide-linked dimer of two related peptides, A and B, that are products of two different genes. We found that hypoxic conditions (0-3% oxygen environments) significantly increased PDGF-B mRNA in cultured human umbilical vein endothelial cells by enhancing the transcriptional rate of this gene. This increase was inversely proportional to oxygen tension and was reversible upon reexposure of cells to a 21% oxygen atmosphere. mRNA levels of PDGF-A were not affected nor was the overall rate of cellular gene transcription increased in response to hypoxia. These studies indicate that endothelial cells are not only capable of sensing oxygen tension, but are also able to discriminate and respond to even small differences in oxygen tension resulting in dramatic upregulation of the PDGF-B chain gene.

Blotting, Northern↗
PubMed1990

Soluble and surface-bound immunoglobulin triggers human monocyte activation and hydrogen peroxide release.

Human mononuclear phagocytes (MO) can mediate the destruction of a variety of foreign and tumor target cells. In most circumstances, however, peripheral blood MO are noncytotoxic and must acquire cytotoxic activity. To investigate the cell surface molecules that participate in the acquisition of MO-mediated cytotoxicity, we used a panel of monoclonal antibodies (MAbs) recognizing a variety of membrane molecules. MAbs to eight different MO surface molecules did not trigger the killing of tumor target cells. To determine if these cell surface molecules triggered an activated but incomplete lytic mechanism, the ability of these molecules to trigger hydrogen peroxide (H2O2) release was assessed. Several MAbs triggered H2O2 release from MO isolated from the peripheral blood. The pattern of MAb-triggered H2O2 release correlated not with the MO surface antigen, but with the immunoglobulin (Ig) isotype. Isotype-specific H2O2 release was abrogated with the enzymatic removal of the Fc portion of the Ig and enhanced by interferon-gamma pretreatment, indicating that the membrane signal was mediated by cell surface Fc receptors. H2O2 release was independent of the presentation of the Ig molecule. Comparable H2O2 release was observed whether the Ig was in surface-bound or soluble form. These data support an important role for Fc receptors in the acquisition of cytotoxic potential by human MO.

Antibodies, Monoclonal↗
PubMed1989

Platelet-derived growth factor production by human umbilical vein endothelial cells is regulated by basic fibroblast growth factor.

Human umbilical vein endothelial cells express the B chain gene (c-sis) of platelet-derived growth factor (PDGF). PDGF acts as a mitogen for mesenchymal cells and has recently been reported to be a potent vasoconstrictor (Berk, B. C., Alexander, R. W., Brock, T. A., Gimbrone, M. A., Jr., and Webb, R. C. (1986) Science 232, 87-90). Regulation of c-sis therefore, may play an important role in maintaining and controlling the integrity of the vasculature. We report here that basic fibroblast growth factor (bFGF), a growth factor for endothelial cells in culture, significantly decreases the amount of PDGF-like protein secreted by these cells. Levels of c-sis mRNA increase more than 10-fold in the absence of bFGF. This effect is specific for the B chain of PDGF as transcript levels of other growth factors synthesized by endothelial cells, such as transforming growth factor beta are not affected by bFGF. c-sis mRNA levels increase 12 h after bFGF removal as the cells begin to accumulate in G0 stage of the cell cycle and remain elevated for at least 96 h. Transcript levels fall again upon re-exposure of the cells to bFGF. bFGF may regulate c-sis mRNA levels and PDGF protein production by endothelial cells either directly, or indirectly via cell cycle arrest at G0. This effect is not due to nonspecific cessation of cell growth because arresting the cells at S phase or in metaphase does not result in increased c-sis transcript levels.

Cell Cycle↗
PubMed1989

Butyric acid modulates developmental globin gene switching in man and sheep.

The developmental switch from production of fetal (gamma) to adult (beta) globin occurs on a normally set biologic clock which proceeds even if the adult (beta) globin genes are defective. Preventing or reversing the globin gene switch would be beneficial for subjects with abnormal beta globin genes. We have now identified a class of agents which, when present in elevated plasma concentrations during gestation, appears to inhibit the gamma beta globin gene switch in developing humans. Further investigation has shown that butyric acid and related compounds can increase gamma globin and decrease beta globin expression in erythroid cells cultured from subjects with diseases of abnormal beta globin. Butyrate compounds were therefore infused in an in vivo fetal animal model, and the globin switch was inhibited in most and reversed in some fetal lambs. These data suggest that inhibiting expression of abnormal beta globin genes may be possible in future generations. Histone modification may be a mechanism of action involved. The developmental switch from production of gamma globin to beta globin results in significant morbidity when the beta globin genes are defective. The globin switch has therefore been extensively studied, appearing to be set on a biologic clock and proceeding despite the site of blood production and solely on the basis of gestational age. We previously found that this developmental gene switch is delayed in human fetuses developing in the presence of maternal diabetes. A number of metabolites present in abnormal concentrations in these infants were therefore tested for effects on globin expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗