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G R Vandenbark

Publications and source records attributed to G R Vandenbark.

15 recordsLinked to original sources
PubMed1997

Astrocyte apoptosis induced by HIV-1 transactivation of the c-kit protooncogene.

HIV-1 infection of the central nervous system (CNS) frequently causes dementia and other neurological disorders. The mechanisms of CNS injury in HIV-1 infection are poorly understood. Apoptosis of neurons and astrocytes is induced by HIV-1 infection in vitro and in brain tissue from AIDS patients, but the apoptotic stimuli have not been identified. We report herein that HIV-1 infection of primary brain cultures induces the receptor tyrosine kinase protooncogene c-kit and that high levels of c-Kit expression are associated with astrocyte apoptosis. Overexpression of c-Kit in an astrocyte-derived cell line in the absence of HIV-1 induces rapid apoptotic death. The apoptotic mechanism requires the c-Kit tyrosine kinase domain. The mechanism of c-kit induction by HIV-1 involves transactivation of the c-kit promoter by the HIV-1 Nef protein. These studies demonstrate that c-Kit can induce astrocyte apoptosis and suggest that this mechanism may play a role in CNS injury caused by HIV-1 infection. We propose that c-Kit can serve dual functions as a growth factor receptor or apoptosis inducer.

Apoptosis↗
PubMed1996

Complex regulation of human c-kit transcription by promoter repressors, activators, and specific myb elements.

The c-kit proto-oncogene is expressed in several tissues during development. To understand the mechanisms controlling the expression of this gene, we characterized the human c-kit promoter. Expression is controlled transcriptionally. The 5'-flanking DNA was used to make promoter deletion-reporter constructs that were tested in cells that were either positive or negative for endogenous c-Kit. The results demonstrate that DNA, to at least position -4100, directs transcription well in both positive and negative cells. Addition of DNA from position -4100 to -5500 causes a reduction in expression to near-basal levels in c-Kit-negative cells but has little effect in c-Kit-positive cells. The DNA from -4100 to -5500 was tested for repressor function. It inhibits transcription from some heterologous promoters in c-Kit-negative cells. Likewise, this segment inhibits transcription from the homologous proximal promoter in a cell-specific manner, but the entire promoter is necessary for complete repression in c-Kit-negative cells. Two Myb binding motifs were also identified, and their role in regulating transcription was examined by mutation and functional testing. One, MYB1, acts as a partial repressor, whereas the other, MYB2, is a positive element that appears essential for expression. Binding proteins to both sites were characterized by several methods. MYB1 binds and responds functionally to c-Myb, but MYB2 does not. The results of these studies indicate that the regulation of c-kit transcription is complex, involving interactions among several activators and repressors.

Base Sequence↗
PubMed1994

The c-kit proto-oncogene receptor is expressed on a subset of human CD3-CD4-CD8- (triple-negative) thymocytes.

The c-kit receptor is a tyrosine-kinase transmembrane receptor first identified as an oncogene in the HZ4-feline leukemia virus and later found to be important in hematopoiesis in mice. The ligand for this receptor (Steel factor) can stimulate hematopoiesis both in vitro and in vivo. To study the pattern of c-kit receptor expression in normal human hematopoietic progenitor cells, we prepared a monoclonal antibody (9B9) against human c-kit receptor by using a synthetic peptide (amino acids 476-501) from the extracellular domain of c-kit receptor to immunize Balb/c mice. Monoclonal antibody 9B9 bound to recombinant c-kit protein, the erythroleukemic line HEL, the megakaryocytic line MEG-01, and the murine mast cell line P815. Monoclonal antibody 9B9 also bound to the surface of the CD7+CD3-CD4-CD8- T cell lymphoid cell lines DU.528 and HSB2T, and also to 1 to 4% of normal bone-marrow cells. The majority (67 +/- 6%) of CD34+ bone-marrow progenitor cells coexpressed c-kit receptor. Flow-cytometry analysis of immature CD3-CD4-CD8- (triple-negative) thymocytes indicated 30 +/- 9.5% expressed the c-kit receptor, and thymidine incorporation assay revealed that the receptor is functional. Indirect fluorescent microscopy of human thymic tissue, using a monoclonal antibody against Steel factor, revealed its presence on scattered mononuclear cells within the intralobular septae and the subcapsular cortex, which are regions where the triple-negative thymocytes are also localized. These data provide evidence that the c-kit receptor is present on human hematopoietic bone marrow and intrathymic T cell progenitor cells, and that it likely plays a role in early T cell lymphopoiesis.

Antibodies, Monoclonal↗
PubMed1992

Cloning and structural analysis of the human c-kit gene.

The recent identification of the mouse White spotting and Steel loci as genes encoding the c-kit receptor and its ligand, respectively, has shed light on the importance of this ligand and receptor in embryogenesis, melanogenesis and hematopoiesis. In order to determine if the c-kit proto-oncogene is involved in human disease, we isolated seven overlapping lambda recombinants, using a fetal brain cDNA, and characterized the normal human gene (KIT). The longest mapped transcript is 5230 bp, is alternatively spliced and includes 21 exons that span more than 70 kb of DNA. From the exon-intron structure, we have localized an alternative splice site to the 3' end of exon 9. The overall c-kit gene structure closely resembles that found in the CSF-1R gene (c-fms). This similarity includes a large first intron, the same number of exons containing translated sequence and very similar exon-intron boundaries. Using pulsed-field gel electrophoresis, we have linked KIT to the platelet-derived growth factor receptor A gene, with both residing on a 700-kb BssHI fragment. These data will allow investigation into the control of KIT expression and the potential to identify mutations or altered expression of this gene in human disease.

Base Sequence↗
PubMed1989

A cAMP-responsive element regulates expression of the mouse steroid 11 beta-hydroxylase gene.

In Y1 mouse adrenocortical tumor cells, expression of steroid 11 beta-hydroxylase (11 beta-OHase) is stimulated by cAMP following a delay of 4-6 h. Our results demonstrate that a cAMP-responsive element (CRE) within the 11 beta-OHase promoter region is a major determinant of this induction. The 5'-flanking sequences from the mouse 11 beta-OHase gene were placed in front of a human growth hormone reporter gene and transfected into Y1 cells. Treatment of transfected cells with 8-bromo-cAMP increased expression directed by the 11 beta-OHase 5'-flanking region by 3.8-fold. In 5'-deletion analyses, 123 base pairs of 5'-flanking sequences were sufficient for cAMP induction, whereas cAMP treatment did not affect expression of a plasmid with only 40 base pairs of 5'-flanking sequence. Within these 123 base pairs, a region from -56 to -49 matched 7 of 8 bases comprising the consensus sequence for the CRE. 11 beta-OHase 5'-flanking sequences from -65 to -42, including the CRE-like sequence, conferred cAMP inducibility to promoters from the thymidine kinase and chorionic gonadotropin alpha-subunit genes. DNase I footprinting and Southwestern blotting analyses demonstrated that the protein which interacted with the CRE in the 11 beta-OHase promoter region was similar to the CRE-binding protein associated with other cAMP-regulated genes. Together, these results suggest that an interaction between the 11 beta-OHase CRE and CRE-binding protein mediates cAMP induction of the 11 beta-OHase gene.

Adrenal Cortex Neoplasms↗
PubMed1989

Identification of multiple protein binding domains in the promoter-regulatory region of the phosphoenolpyruvate carboxykinase (GTP) gene.

Transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) from the rat is acutely regulated by a number of hormones, including glucagon (acting via cAMP), glucocorticoids, and insulin. In this study we demonstrate by DNase I footprinting that a region of the PEPCK promoter, extending from -460 to +73, contained eight protein binding domains. Two nuclear proteins protected adjacent sites from -121 to -99 and -96 to -77, which have been previously shown to be involved in maintaining the level of basal gene transcription and conferring cAMP responsiveness, respectively. Oligonucleotide competition studies suggested that the protein(s) binding to the cAMP-responsive element (CRE) occupies a second site at -147 to -130, which has a high degree of sequence homology to the CRE, and also binds to two other elements that show partial sequence homologies. The protein(s) which bound to these four elements copurified through oligonucleotide affinity chromatography, suggesting that the PEPCK promoter has four binding sites for the CRE-binding protein(s). Potential tissue-specific elements in the PEPCK promoter were identified by footprinting with nuclear extracts prepared from rat liver, kidney, brain, and spleen. The multiple protein-binding sites in this promoter-regulatory region reflect the complex transcriptional regulation that is characteristic of this gene.

Animals↗
PubMed1988

Characterization of the cAMP responsive elements from the genes for the alpha-subunit of glycoprotein hormones and phosphoenolpyruvate carboxykinase (GTP). Conserved features of nuclear protein binding between tissues and species.

Cyclic AMP responsive elements (CRE) have been identified in several genes, including those encoding the alpha-subunit of glycoprotein hormones and the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK) from the rat. Common to the CRE in these genes is the palindromic sequence T(G/T)ACGTCA. Based upon the strong conservation of this element, we hypothesize that the CRE functions by binding a protein that has been conserved across tissue and species lines. Scatchard analysis of gel mobility shift assays indicate that a nuclear protein in extracts prepared from rat liver and from a human choriocarcinoma cell line binds with high affinity to the cAMP responsive element from either gene (Kd approximately 10(-10) M). In order to identify the critical nucleotides within the CRE from these two genes, a series of oligodeoxynucleotides containing systematic mutations was synthesized and tested for protein binding and transcriptional function. Mutations within the palindromic core of either CRE resulted in a marked loss of binding to the nuclear proteins. Sequences outside the 8-base pair element were less important for nuclear protein binding to the PEPCK CRE and were not important for the alpha-subunit CRE. The relative binding, as determined by gel shift assays, correlated with the ability to confer cAMP responsive transcription to a viral promoter in transfected choriocarcinoma cells. DNase I protection assays suggest that binding of the nuclear factor from rat liver to the PEPCK CRE is more efficient when the core sequence is present in the intact PEPCK promoter regulatory region as compared to the isolated CRE oligodeoxynucleotide. Collectively, these results indicate that the nuclear factors necessary for cAMP induction of transcription of the alpha-subunit and PEPCK genes are conserved between tissues and species. In addition to the conserved features of these cis- and trans-active elements, nonconserved sequences and other elements of the promoter regulatory region influence the affinity of the protein-DNA interaction.

Animals↗
PubMed1985

Successful immune reconstitution in severe combined immunodeficiency despite Epstein-Barr virus and cytomegalovirus infections.

Cytomegalovirus (CMV) and Epstein-Barr virus (EBV), frequently found in the acquired immune deficiency syndrome (AIDS), have been suspected of contributing to the latter immunodeficiency. The ability of normal HLA-identical sibling bone marrow to reconstitute an 8-month-old infant with severe combined immunodeficiency infected with these two viral agents is of interest. After presentation with severe mucocutaneous candidiasis, cavitary pulmonary disease, nodular cutaneous lesions, and hepatic abscesses containing acid-fast organisms, immunologic studies revealed lymphopenia, 1-3% T cells, and no lymphocyte responses to mitogens. Prior to transplantation, the infant's blood B lymphocytes grew spontaneously in culture, suggesting they were infected with EBV. Indeed, an appropriate antibody response to EBV was detected at 2 months post-transplantation. At 3 weeks postgrafting, neutropenia and cholestatic jaundice developed without other signs of graft versus host disease. Liver biopsy demonstrated CMV but no EBV by DNA hybridization. There was evidence of T- and B-cell function by 2 weeks postgrafting, including vigorous in vivo and in vitro responses to candida. Although the blood lymphocyte T4:T8 ratio was inverted at 2 weeks, it reverted to normal by 6 weeks post-transplantation. All clinical disease resolved by 8 months and karotyping revealed all T and B lymphocytes to be XX. Thus, despite infections with both CMV and EBV, complete immunologic reconstitution was achieved in this, the most severe of all genetically determined immunodeficiency conditions, arguing against these viruses having a major role in the failure of bone marrow transplantation in AIDS.

Bone Marrow Transplantation↗
PubMed1985

Diacylglycerols mimic phorbol diester induction of leukemic cell differentiation.

Activation of cellular protein kinase C appears to be involved in the mechanism by which phorbol diesters induce differentiation of human myeloid leukemia cells (HL-60). Protein kinase C is thought to be physiologically activated by diacylglycerol derived from receptor-mediated phosphatidylinositol hydrolysis. sn-1,2-diacylglycerols with short saturated acyl side chains (C4-C10) were synthesized and found to be potent activators of protein kinase C partially purified from HL-60 cells. These diacylglycerols were also competitive inhibitors of [3H]phorbol dibutyrate binding to the soluble phorbol diester receptor. The most potent diacylglycerol, sn-1,2-dioctanoylglycerol, displaced greater than 90% of [3H]phorbol dibutyrate from the phorbol diester receptor of intact HL-60 cells. Because of probable cellular metabolism of sn-1,2-dioctanoylglycerol, hourly doses were required to maintain persistent occupancy of the phorbol diester binding site. Treatment of HL-60 cells with either phorbol 12-myristate 13-acetate or sn-1,2-dioctanoylglycerol produced identical phosphoprotein changes. Finally, sn-1,2-dioctanoylglycerol induced differentiation of the HL-60 cells into cells with morphologic characteristics of macrophages. Substitution of the hydroxyl group at position 3 with a hydrogen, chloro, or sulfhydryl moiety inactivated sn-1,2-dioctanoylglycerol. These data strengthen the hypothesis that protein kinase C activation plays a role in macrophage differentiation.

Caenorhabditis elegans Proteins↗
PubMed1985

Role of protein kinase C in diacylglycerol-mediated induction of ornithine decarboxylase and reduction of epidermal growth factor binding.

Tumor-promoting phorbol esters induce ornithine decarboxylase (ODCase) activity and reduce epidermal growth factor (EGF) binding in rat tracheal epithelial 2C5 cells. Phorbol esters activate protein kinase C by interacting at the same site as sn-1,2-diacylglycerols, the presumed physiological regulators. The effects of added sn-1,2-diacylglycerols and those generated by phospholipase C treatment of 2C5 cells on ODCase induction and EGF binding were investigated to establish a role for protein kinase C in these cellular responses. Treatment of 2C5 cells with phospholipase C induced ODCase activity and reduced EGF binding, whereas phospholipases A2 and D were inactive. When sn-1,2-diacylglycerols containing fatty acids 3-10 carbons in length were added to 2C5 cells, those diacylglycerols containing fatty acids 5-10 carbons in length caused ODCase induction and reduction in EGF binding. sn-1,2-Dioctanoylglycerol was one of the most active compounds tested. It induced ODCase in a dose- (50-500 microM) and time-dependent manner. The reduction of binding of 125I-labeled EGF by sn-1,2-dioctanoylglycerol was also time and dose dependent and appeared to result from a change in EGF affinity and not the number of receptor sites. This series of sn-1,2-diacylglycerols showed similar structure-function relationships in their ability to induce ODCase activity, to decrease EGF binding, to stimulate protein kinase C, and to inhibit [3H]phorbol dibutyrate binding to the phorbol ester receptor. These data demonstrate biological activities for a number of diacylglycerols and indicate that protein kinase C activation is implicated in ODCase induction and decreased EGF binding.

Animals↗
PubMed1984

Possible mechanism of phorbol diester-induced maturation of human promyelocytic leukemia cells.

The phorbol diesters are the most potent inducers of differentiation of the promyelocytic leukemia cell line, HL-60. Soluble phorbol diester receptors from HL-60 cells were obtained from the cytosolic fraction and from the particulate fraction by either divalent ion chelation or detergent extraction. The partially purified soluble phorbol diester receptors required exogenous Ca2+ and phospholipid for maximal binding and displayed a dissociation constant (KD) of 8.1 nM for [3H]phorbol 12,13-dibutyrate. Phorbol diester analogues inhibited [3H]phorbol 12,13-dibutyrate binding in a stereospecific manner consistent with their biologic potency. The soluble phorbol diester receptors prepared by all three methods copurified in a constant ratio with the Ca2+/phospholipid-dependent protein kinase C through ammonium sulfate precipitation, DEAE ion exchange, and gel filtration chromatography. Partially purified protein kinase C was directly activated by the phorbol diesters even in the absence of exogenous Ca2+. The ability of a series of phorbol analogues to activate the kinase correlated with their known activity as inducers of cell differentiation. In addition, phorbol diester stimulation altered the phosphate acceptor substrate profile of protein kinase C, at least in part, by alteration of the Michaelis constant (Km). These data suggest that protein kinase C is the phorbol diester receptor and that phorbol diester-induced macrophage maturation of HL-60 cells may be mediated by activation of intracellular protein kinase C.

Caenorhabditis elegans Proteins↗
PubMed1983

Phorbol diester receptor copurifies with protein kinase C.

The phorbol diester receptor present in the particulate fraction of rat brain was solubilized by divalent ion chelation in the absence of detergents. The soluble receptor was partially purified by (NH4)2SO4 precipitation, DEAE-cellulose, and gel filtration chromatography. The purified receptor required exogenous phospholipid for activity and displayed a Kd of 7 nM for [3H]phorbol 12, 13-dibutyrate. Biologically active phorbol analogs inhibited binding, whereas inactive analogs did not. The Ca2+-dependent, phospholipid-sensitive protein kinase C copurified with the phorbol receptor. Purified protein kinase C was activated directly by phorbol 12-myristate 13-acetate in the presence of phospholipid.

Animals↗
PubMed1979

Human monocyte killing of Staphylococcus aureus: modulation by agonists of cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate.

This study was designed to test whether cyclic nucleotides play a role in the regulation of bacterial killing by human monocytes. Agents were tested for their ability to activate monocyte adenylate or guanylate cyclase in cell-free preparations, to increase cyclic adenosine 3',5'-monophosphate (cAMP) or cyclic guanosine 3',5'-monophosphate (cGMP) in intact human monocytes, and to modulate monocyte-induced killing of Staphylococcus aureus in vitro. Prostaglandin E1 and cholera toxin activated monocyte adenylate cyclase and inhibited monocyte killing of S. aureus. An adenylate cyclase inhibitor, RMI 12330A, reversed the prostaglandin E1-mediated inhibition of bacterial killing, thus implicating cAMP as the intracellular mediator of this inhibition. In contrast, monocyte cGMP levels were increased 5- and 17-fold by 5-hydroxytryptamine and N-methyl-N' -nitro-N-nitrosoguanidine, respectively, but neither agent was effective in modulating monocyte bactericidal activity. Thus, modulation of bactericidal activity in human monocytes did not conform to the yin/yang theory of opposing actions by cAMP and cGMP, for although monocyte-mediated killing of S. aureus was inhibited by cAMP agonists, it was not enhanced by cGMP agonists.

Adenylyl Cyclases↗