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Biomedical subjects

G C Bagby

Publications and source records attributed to G C Bagby.

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

Retroviral transfer of the recombinant human erythropoietin receptor gene into single hematopoietic stem/progenitor cells from human cord blood increases the number of erythropoietin-dependent erythroid colonies.

To test whether an enforced expression of a lineage-specific cytokine receptor would influence the proliferation/differentiation of hematopoietic stem/progenitor cells, retroviral vectors containing the human erythropoietin receptor (hEpoR) gene were used to transduce the hEpoR gene into phenotypically sorted subsets of cells. CD34 , CD34++CD33-, and CD34++CD33+ populations of human cord blood, highly enriched for hematopoietic stem/progenitor cells, were sorted and plated as single cells per well in methylcellulose culture medium containing early acting growth factors in the presence or absence of Epo. The hEpoR gene was efficiently transduced into single high proliferative potential colony-forming cells (HPP-CFC) and multipotential (colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM]), erythroid (burst-forming unit-erythroid [BFU-E]), and granulocyte-macrophage (colony-forming unit-granulocyte-macrophage [CFU-GM]) progenitor cells. As expected in cultures grown in the absence of Epo, no BFU-E or CFU-GEMM colonies grew. In the presence of Epo, the hEpoR-gene transduced cells formed significantly more CFU-GEMM and BFU-E colonies than did the controls. A significant decrease in HPP-CFC colonies was also observed under these conditions. Little or no effect of hEpoR gene transduction was apparent in the numbers of CFU-GM colonies formed in the presence or absence of Epo. All of the above results were similar whether the cell populations assessed were CD34 or their CD33- or CD33+ subsets plated in the presence of growth factors at 200 cells/mL or after limiting dilution at 2 cells/well. These results suggest that the profile of detectable stem/progenitors can be altered by retrovirus-mediated expression of the hEpoR gene.

Antigens, CD↗
PubMed1995

Involvement of SH2-containing phosphotyrosine phosphatase Syp in erythropoietin receptor signal transduction pathways.

Erythropoietin (Epo) regulates the proliferation and differentiation of erythroid precursors. The phosphorylation of proteins at tyrosine residues is critical in the growth signaling induced by Epo. This mechanism is regulated by the activities of both protein-tyrosine kinases and protein tyrosine phosphatases. The discovery of phosphotyrosine phosphatases that contain SH2 domains suggests roles for these molecules in growth factor signaling pathways. We found that Syp, a phosphotyrosine phosphatase, widely expressed in all tissues in mammals became phosphorylated on tyrosine after stimulation with Epo in M07ER cells engineered to express high levels of human EpoR. Syp was complexed with Grb2 in Epo-stimulated M07ER cells. Direct binding between Syp and Grb2 was also observed in vitro. Furthermore, Syp appeared to bind directly to tyrosine-phosphorylated EpoR in M07ER cells. Both NH2-terminal and COOH-terminal SH2 domains of Syp, made as glutathione S-transferase fusion proteins, were able to bind to the tyrosine-phosphorylated EpoR in vitro. These results suggest that Syp may be an important signaling component downstream of the EpoR and may regulate the proliferation and differentiation of hematopoietic cells.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Evolutionary conservation of the AU-rich 3' untranslated region of messenger RNA.

AU-rich sequence motifs (specifically sequences containing reiterations of AUUUA) are found in the 3' untranslated region of mammalian mRNAs encoding cytokines, adhesion molecules, and protooncogenes. Because these AU-rich elements (3'AURE) have been observed to reduce the stability and translational efficiency of transcripts that contain them, and because many of these transcripts accumulate in cells exposed to inflammatory stimuli, we reasoned that mRNAs with 3'AURE may be highly conserved and that the AURE is a marker of mRNAs that are inducible by environmental stressors. To test this hypothesis, we developed a polymerase chain reaction (PCR) strategy to isolate specifically mRNAs with 3'AURE. We first validated the effectiveness of this approach by selectively amplifying two mRNAs containing 3'AURE from interleukin 1 (IL-1)-induced human endothelial cells, then used the same primers in reverse transcriptase-PCR of sea urchin RNA, and used the radiolabeled reaction products to screen a cDNA library prepared from endotoxin-exposed sea urchin coelomocytes. We identified 124 positive clones and isolated a 1608-base-pair fragment that contains an AU-rich consensus sequence upstream from a poly(A) tail. This sea urchin transcript hybridizes with immobilized poly(A)(+)-selected RNA prepared from living coelomocytes maintained in vitro for 8.5-13 h but not with RNA prepared from freshly harvested coelomocytes. Our results provide support for the growing body of evidence that 3' AURE are both conserved and functional and indicate further that isolation and short-term in vitro culture of sea urchin coelomocytes is sufficient to induce the expression of transcripts containing 3'AURE.

Animals↗
PubMed1994

Repression of Fanconi anemia gene (FACC) expression inhibits growth of hematopoietic progenitor cells.

Bone marrow failure is a consistent feature of Fanconi anemia (FA) but it is not known whether the bone marrow failure is a direct and specific result of the inherited mutation or a consequence of accumulated stem cell losses resulting from nonspecific DNA damage. We tested the hypothesis that the protein encoded by the FA group C complementing gene (FACC) plays a regulatory role in hematopoiesis. We exposed normal human lymphocytes, bone marrow cells, endothelial cells, and fibroblasts to an antisense oligodeoxynucleotide (ODN) complementary to bases -4 to +14 of FACC mRNA. The mitomycin C assay demonstrated that the antisense ODN, but not missense or sense ODNs, repressed FACC gene expression in lymphocytes. Treatment with the antisense ODN substantially reduced, in a sequence-specific fashion, cytoplasmic levels of FACC mRNA in bone marrow cells and lymphocytes. Escalating doses of antisense ODN increasingly inhibited clonal growth of erythroid and granulocyte-macrophage progenitor cells but did not inhibit growth of fibroblasts or endothelial cells. The antisense ODN did not inhibit growth factor gene expression by low density bone marrow cells or marrow-derived fibroblasts. We conclude that, while the FACC gene product plays a role in defining cellular tolerance to cross-linking agents, it also functions to regulate growth, differentiation, and/or survival of normal hematopoietic progenitor cells.

Base Sequence↗
PubMed1993

High-frequency monitoring for early detection of cisplatin ototoxicity.

Cisplatin can cause irreversible hearing loss initially detectable as impairment of high-frequency hearing with progression to lower frequencies. Many patients receiving cisplatin are too ill to tolerate lengthy audiometric testing. Therefore, a rapid and sensitive high-frequency monitoring strategy to detect cisplatin-induced ototoxicity is needed. Serial conventional (0.25 to 8 kHz) and high-frequency (> or = 8 kHz) threshold monitoring was performed in patients receiving cisplatin, resulting in 84% of ears showing hearing loss, of which 71% were detected first in frequencies of 8 kHz or greater. By analysis according to an individualized, specific high-frequency range, early identification of hearing loss occurred in 94% of ears showing change. This five-frequency procedure is a sensitive detector of ototoxicity and is proposed as an alternative monitoring protocol for patients receiving cisplatin who cannot tolerate extended testing.

Algorithms↗
PubMed1993

Constitutive and induced expression of hematopoietic growth factor genes by fibroblasts from children with Fanconi anemia.

The pathophysiological abnormalities leading to marrow failure and leukemogenesis in children with Fanconi anemia (FA) are not understood. We tested the hypothesis that the Fanconi anemia mutation results in insufficient production of hematopoietic growth factors by stromal cells by quantifying constitutive and induced production of interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and steel factor (SF) by untransformed fibroblasts from eight patients with FA from five different families. While no abnormalities were noted in SF or M-CSF production, we noted substantial variability in IL-6, GM-CSF, and G-CSF responses of cells obtained from different FA patients. Responses ranged from blunting to augmentation when compared to normal controls. Because there was variation between fibroblast strains from affected members of two multiplex sibships, however, it is clear that neither augmentation nor blunting is a direct effect of the FA mutations. In addition, because there was discordance between the G-CSF responses and the GM-CSF and IL-6 responses, the abnormalities noted in IL-1 responsiveness must lie distal to IL-1 receptor function and to stimulus-response coupling pathways shared between the three cytokines.

Cells, Cultured↗
PubMed1992

Specific repression of granulocyte-macrophage and granulocyte colony-stimulating factor gene expression in interleukin-1-stimulated endothelial cells with antisense oligodeoxynucleotides.

Antisense oligodeoxynucleotides (ODNs) have been used to effect the specific inhibition of cellular gene expression. We have evaluated the application of this approach to the inhibition of interleukin-1 (IL-1)-induced granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) expression in cultured human umbilical vein endothelial cells. Antisense ODNs or control ODNs (sense ODNs or missense ODNs containing random base substitutions) were added to cultures of endothelial cells, the cells were induced with IL-1 alpha, and the conditioned media were assayed for GM-CSF and G-CSF by quantitative bioassays and for immunoreactive GM-CSF by enzyme immunoassay. Antisense ODNs complementary to the first 15 or 18 bases of the translation start sites of GM-CSF or G-CSF mRNAs inhibited, in a concentration-dependent fashion, the IL-1-stimulated expression of the corresponding factor, but did not affect expression of the other factor. Control ODNs did not affect GM-CSF or G-CSF expression. Exposure to a GM-CSF antisense ODN, but not a control ODN, substantially reduced cytoplasmic GM-CSF mRNA levels in IL-1-stimulated endothelial cells. Neither ODN affected levels of endothelial leukocyte adhesion molecule (ELAM)1 or glyceraldehyde-3-phosphate dehydrogenase mRNAs. We conclude that antisense ODNs complementary to the translation start sites of GM-CSF or G-CSF mRNAs inhibit expression of the corresponding factor in a sequence-specific fashion and this effect is mediated, at least in part, by reduction in the cytoplasmic level of the targeted mRNA. Moreover, IL-1-induced GM-CSF or G-CSF expression does not depend on expression of the other factor.

Animals↗
PubMed1992

c-myc antisense oligonucleotides inhibit the colony-forming capacity of Colo 320 colonic carcinoma cells.

Colo 320 cells are colonic carcinoma cells known to express abundant c-myc mRNA. Based on the response of several hematopoietic cell lines to chemical inducers of differentiation, we reasoned that such agents might have similar inductive activity in Colo 320 cells. Accordingly, we exposed Colo 320 cells to 5 mM sodium butyrate (NaBT) for 7 d. C-myc expression decreased threefold and self-replicative potential decreased (defined as a greater than 60% decrease in colony-forming capacity in soft agar that did not contain inducer). In an effort to demonstrate a direct cause and effect between myc expression and the colony-forming capacity of Colo 320 cells, we exposed these cells to a 15-base antisense c-myc oligonucleotide (complementary to the translation initiation region of exon II). Cells were also exposed to equimolar (20 microM) amounts of sense and missense oligonucleotides. Subsequently, cells were incubated at 10, 20, 30, and 40 microM antisense DNA for 16 h, then washed and plated in oligonucleotide-free agar medium. We demonstrated that: (a) the oligomers were stable in the extracellular medium and in the cell cytoplasm; (b) the uptake of the oligonucleotides was 0.7%; (c) sense and missense oligonucleotides had no effect on colony-forming capacity; and (d) the antisense c-myc oligonucleotide resulted in a 40-75% concentration-dependent decrease in colony-forming capacity. The specific inhibition of colony-forming capacity by antisense DNA suggests that the role of myc expression in Colo 320 cells is similar to its role in hematopoiesis, and that the failure to inhibit myc expression maintains colony-forming capacity. This system provides a new strategy for inducing differentiation and may provide further insight into the genetic factors that govern the process of colonic carcinogenesis.

Butyrates↗
PubMed1992

Magnesium-dependent thermostability of DNase I.

DNase I can be used to remove contaminating DNA from RNA samples. Heat treatment has been recommended as a method to inactivate DNase I enzymatic activity, thereby allowing subsequent reverse transcription and PCR amplification of DNase I treated RNA. We have found that inactivation of DNase I by heat treatment is strongly dependent on Mg2+ concentration. In addition, deoxyribonucleolytic activity of "inactivated" enzyme may be restored by changes in Mg2+ concentration following heat treatment. Caution should be exercised when using heat treatment alone as a method of DNase I inactivation.

Deoxyribonuclease I↗
PubMed1992

A method for rapid analysis of peripheral blood mononuclear leukocyte cytokine mRNA.

Interleukins, hematopoietic growth factors, and adhesion molecules mediate multiple components of the inflammatory response. Conventional methods for purification of cytokine-producing cells are time consuming and can, therefore, depending on the conditions of cell purification and the nature of the mRNA being purified, result in both over- and underestimates of mRNA content. Therefore, it has been difficult to quantify such factors in ways that are unambiguously reflective of in vivo cytokine production. We describe herein a method for such rapid preparation of peripheral blood mononuclear leukocytes (PBML) that amounts of cytokine mRNA derived from these cells, apart from readily quantifiable losses during RNA preparation, will be reflective of quantities in vivo. Small quantities (10(5) of normal PBML were isolated by density centrifugation of heparinized blood for 1.25 min in a capillary tube. Poly(A)+ RNA isolated by oligo(dT)-cellulose column chromatography was sufficient to reverse transcribe both antigen-specific T-cell-receptor beta-chain mRNA and interleukin 6 (IL-6) mRNA and subsequently amplify the cDNA with Taq polymerase in reverse transcription-polymerase chain reactions (RT-PCR). Although IL-6 mRNA was not present in normal PBML, PBML incubated in culture medium for only 3 h contained approximately one molecule per cell. Because of its rapidity this technique will permit quantification of cytokine mRNA in the steady state and in clinical setting of inflammation. Because the method requires only a small quantity of blood it can be applied to clinical research studies involving children.

Base Sequence↗
PubMed1992

In vitro hematopoiesis in myelodysplasia: liquid and soft-gel culture studies.

In vitro clonogenic assays of bone marrow have provided invaluable information about the complex regulatory mechanisms controlling hematopoiesis, both normal and abnormal, and have led to the discovery of colony stimulating factors. Our understanding of the abnormalities of growth and differentiation characterizing the MDS have been advanced largely through use of these assays. Abnormalities commonly seen in cultures of marrow from patients with MDS include decreased or absent colony growth, abortive cluster formation, and defective maturation of cells within the colonies. Although some investigators have defined growth patterns as "leukemic" and "nonleukemic" and have tried to correlate growth patterns with potential for evolution to acute leukemia, these methods are difficult to apply to any given case. Given the wide variety of techniques used to collect cells and the lack of standard sources for stimulating growth factors, the results, not surprisingly, have been inconclusive in predicting both prognosis and progression to acute leukemia. New methods by which clonogenic assays can be standardized, such as purification of the clonogenic cells and use of recombinant growth factors, should allow these assays to advance our understanding of MDS, develop new therapies, and predict responses to therapy in individual patients.

Cells, Cultured↗
PubMed1991

Vascular endothelial cells and hematopoiesis: regulation of gene expression in human vascular endothelial cells.

Vascular endothelial cells do not constitutively produce significant quantities of hematopoietic growth factors, interleukins, or adhesion molecules, but they can be induced to do so by a variety of stimuli including the inductive cytokines, interleukin-1 (IL-1), and tumor necrosis factor-alpha, as well as phorbol esters, bacterial proteins, endotoxin, certain viruses, and modified low-density lipoproteins. Recently, some groups have begun to investigate the molecular mechanism by which expression of these genes is regulated. Because induced expression of the gene products is always prefaced by an increase in mRNA the focus of attention has been largely upon the mechanisms involved in the process of transcript accumulation. Certain inducing agents drive transcription of these genes, others may induce both transcription and transcript stability. In the case of the inducing factor IL-1, it was recently demonstrated that accumulation of G-CSF, GM-CSF, and interleukin-1 beta mRNAs induced in vascular endothelial cells occurs as a result of transcript stabilization. Based on preliminary studies in a cell-free system, it is proposed that the inductive capacity of interleukin-1 results, at least in part, from its capacity to activate cytoplasmic inhibitors of selective ribonucleases and hypothesize that this may be a mechanism that has been conserved throughout evolution. Because other inducing agents also incite IL-1 gene expression, transcript stabilization may be a common ingredient of most inductive stimuli.

Endothelium, Vascular↗
PubMed1990

Interleukin-1 stimulation stabilizes GM-CSF mRNA in human vascular endothelial cells: preliminary studies on the role of the 3' AU rich motif.

To date, the notion that the AU rich motif in the 3' UT of GM-CSF mRNA can function as an IL-1 response element has not been adequately examined. We suspect that it must play some role because virtually all IL-1 inducible gene have AU rich 3' untranslated regions. Thus, we will continue to address these technical problems so that the hypothesis can be more clearly tested. Nonetheless, we can state with reasonable certainty that: 1) IL-1 induces expression of GM-CSF in EC by inducing accumulation of mRNA, 2) the GM-CSF gene is constitutively transcribed but the half life of the mRNA is short, 3) IL-1 induced mRNA accumulation results from stabilization of the transcript, 4) although most IL-1 responsive genes have AU-rich domains in their 3' untranslated regions, it is not yet clear that these AU-rich regions are sufficient to function as an IL-1 response element, and 5) murine L cells cannot be used for studies on the molecular biology of cytokine induction by IL-1.

Animals↗
PubMed1989

Interleukin-1, stromal cells, granulopoiesis, and the inflammatory response.

A number of in vitro studies carried out in our laboratory over the past ten years have led to some clarification of the role of mononuclear phagocytes in hematopoietic regulation. The results of these studies have demonstrated that mononuclear phagocytes produce proteins, notably interleukin-1 (IL-1), that induce the expression of multilineage hematopoietic growth factors by human vascular endothelial cells, fibroblasts, T-lymphocytes, and thymic epithelial cells. More recently we and others have identified these induced factors as G-CSF, GM-CSF, IL-6, and IL-1. Although IL-1 seems to stimulate expression of these genes by inducing the accumulation of gene transcripts, interestingly the accumulation results from prolongation of mRNA half-life. We propose that the inductive capacity of IL-1 results from its activation of ribonuclease inhibitory activity in the cytoplasm of IL-1 induced cells and hypothesize that this may be a general mechanism by which IL-1 induces gene expression.

Extracellular Matrix↗
PubMed1989

Interleukin-1 and hematopoiesis.

Interleukin-1 alpha and interleukin-1 beta are 17.5 kDa peptides which exert a wide variety of biological activities. Both forms of interleukin bind to a common 60-70 kDa receptor expressed by most somatic cells. One major effect of interleukin-1 is to induce the expression of other genes, including genes for other interleukins (IL-6), colony stimulating factors (GM-CSF and G-CSF) and growth factors (PDGFA) and adhesion proteins (ELAM-1, ICAM-1). In vitro, cells of the hematopoietic stroma including thymic epithelium, endothelial cells, fibroblasts, T-lymphocytes, and macrophages, are all capable of responding to interleukin-1 by expressing most of these IL-1 inducible genes. Accordingly, because interleukin-1 has no direct effect on hematopoietic progenitor cells, its major effect on hematopoiesis is to regulate the expression of hematopoietic growth factor genes by other cells. The mechanism by which interleukin-1 induces gene expression is to first induce mRNA accumulation which accounts for an increase in translation of the mRNA. The accumulation of RNA, interestingly, does not result from increased transcription of these genes but by the stabilization of the mRNA. Ordinarily, the hematopoietic growth factor gene transcripts have a very short half-life but in an interleukin-1 induced cell, the half-life of these transcripts is markedly prolonged (greater than 24 h). This particular effect of IL-1 on transcript stability likely accounts for virtually all of the hematopoietic activities of interleukin-1 including: (1) induction of growth factors; (2) synergy with other factors; (3) priming; and (4) auto-induction. Three clinical models of hematopoiesis are presented which suggest that the interleukin-1 CSF network is operative in vivo. The ability of interleukin-1 to induce the expression of hematopoietic growth factor genes as well as genes whose products regulate cellular function and traffic suggest that interleukin-1 is an essential molecular master switch for a number of cellular responses occurring in organisms facing the vicissitudes of their environment.

Gene Expression Regulation↗
PubMed1989

Human vascular endothelial cells, granulopoiesis, and the inflammatory response.

We have carried out a series of in vitro studies designed to characterize the role of mononuclear phagocytes as regulators of hematopoiesis. The results of these studies have demonstrated that mononuclear phagocytes produce factors, including interleukin-1 (IL-1), that induce the expression of multilineage hematopoietic growth factors by human vascular endothelial cells. In more recent studies we and others have identified these induced factors as G-CSF, GM-CSF, IL-6, and IL-1. Interleukin 1 stimulates expression of these genes by inducing the accumulation of gene transcripts. Moreover, transcript accumulation, at least with GM-CSF, results from prolongation of mRNA half-life. Based on preliminary studies in a cell-free system, we propose that the inductive capacity of IL-1 results from its activation of ribonuclease inhibitors in the cytoplasm of IL-1-induced cells and hypothesize that this may be a general mechanism by which IL-1 induces gene expression.

Biomechanical Phenomena↗
PubMed1989

The hematopoietic microenvironment in the elderly: defects in IL-1-induced CSF expression in vitro.

The expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) genes by stromal cells of the hematopoietic microenvironment is regulated, in vitro, by interleukin 1 alpha (IL-1 alpha) and IL-1 beta. We reasoned that malfunction of this inductive mechanism in vivo might contribute to the intolerance of the aged to myelosuppressive therapy. We found that bone marrow fibroblasts from healthy elderly (ages 65-75 years) volunteers were less sensitive to the inductive effects of recombinant IL-1 beta than were marrow fibroblasts from younger volunteers, and we proposed that an age-related decline in sensitivity of marrow stroma to IL-1 may impair the ability of the elderly to increase production of hematopoietic growth factors under conditions of physiologic stress.

Adult↗