Biomedical subjects

D T Graves

Publications and source records attributed to D T Graves.

84 records · Page 5Linked to original sources

PubMed1989

Initial characterization of cells derived from human periodontia.

The studies presented in this report describe an initial characterization of cell types derived from explants of human periodontia. Cell cultures were established from human periodontal ligament (PL4, PL7), gingival tissue (GF2), and alveolar bone (BP1) by means of explant techniques and monolayer culture. Cells were studied at passage numbers 2-4 and were characterized on the basis of morphological, biochemical, and proliferative parameters. Subconfluent cells did not have distinct morphologies useful in distinguishing them from one another. At confluence, PL4 and BP1 cells formed multilayered cultures of randomly oriented cells, while PL7 and GF2 cells grew in a monolayer of parallel cells. Biochemically, PL4 and BP1 cells exhibited characteristics consistent with an osteoblast-like phenotype. These included a significant increase in PTH-stimulated cyclic AMP and high basal levels of alkaline phosphatase activity, which were decreased on exposure to PTH and increased after stimulation by 1.25 dihydroxyvitamin D3. In contrast, PL7 and GF2 cells exhibited basal alkaline phosphatase levels that were low, and cyclic AMP levels were not modulated by PTH stimulation. Cell populations PL7 and GF2 did not proliferate in culture medium supplemented with 3% platelet-poor plasma. After the addition of platelet-derived growth factor (PDGF) to this medium, the proliferation of these cell populations was equal to that in media supplemented with 10% fetal bovine serum. In contrast, PL4 and BP1 cells did proliferate in culture medium supplemented with 3% platelet-poor plasma. The addition of PDGF to the medium resulted in only a moderate increase in the proliferation of cell populations PL4 and BP1.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1989

The potential role of platelet-derived growth factor as an autocrine or paracrine factor for human bone cells.

Platelet-derived growth factor, PDGF, is a potent mitogen for cells of mesenchymal origin such as fibroblasts, smooth muscle cells and glial cells. PDGF is thought to have the potential to act as both a paracrine and an autocrine factor. Studies described here extend these observations to human bone-derived cells. Exogenous PDGF induces biologic activity in two human osteogenic sarcoma cell lines and in one of these, the two PDGF genes, PDGF-1 and PDGF-2/c-sis are expressed. In addition, PDGF stimulates proliferation of normal osteoblastic cells derived from adult human cancellous bone. The expression of the PDGF-1 gene but not the PDGF-2/c-sis gene is demonstrated in normal human adult bone-derived cells by Northern blot analysis and synthesis of PDGF is shown by immunoprecipitation with PDGF antisera. These studies indicate that PDGF has the potential to act as a paracrine or autocrine regulator of bone cells.

Blotting, Northern↗

PubMed1988

Purification of a monocyte chemotactic factor secreted by nonhuman primate vascular cells in culture.

A protein chemotactic for peripheral blood monocytes (SMC-CF) of potential importance in their recruitment to the arterial intima in atherogenesis was purified from serum-free medium conditioned by cultured baboon aortic medial smooth muscle cells. The purification of SMC-CF was monitored by a filter assay using human peripheral blood mononuclear cells and was achieved by batch separation on a cation-exchange gel followed by gel permeation chromatography, ion-exchange high-performance liquid chromatography (HPLC), and reversed-phase HPLC. The overall recovery was approximately 10% of the initial activity and yielded 0.5-1 microgram of SMC-CF/L of conditioned medium. On analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SMC-CF migrated as a monomeric protein with an apparent molecular weight of 14,500. A dose-dependent relationship was observed between SMC-CF concentration and monocyte chemotactic activity, with maximal and half-maximal biologic activity being observed at approximately 5 and 0.1 nM, respectively. Cultured baboon aortic smooth muscle cells also express the genes for both the A and B polypeptide chains of platelet-derived growth factor, which has been reported to be chemotactic for blood monocytes and neutrophils [Deuel, T. F., Senior, R. M., Huang, J. S., & Griffin, G. L. (1982) J. Clin. Invest. 69, 1046-1049]. Amino acid composition analyses indicate that SMC-CF is not derived either from polypeptide chain of this growth factor or from certain potentially chemotactic connective tissue proteins.

Amino Acids↗

PubMed1988

DNA synthesis in U-2 OS human osteosarcoma cells is independent of PDGF binding to functional cell surface receptors.

Previous studies have shown that suramin reveals specific PDGF binding sites on U-2 OS human osteosarcoma cells. Studies presented here indicate that U-2 OS cells pretreated with suramin internalize and degrade 125I-PDGF and respond to PDGF by increased tyrosine kinase activity and amino acid transport. However, DNA synthesis in these cells is not reduced by incubation with the PDGF blocking agent suramin and is not stimulated by exogenous PDGF. These data indicate that U-2 OS cells possess functional PDGF receptors but that high levels of DNA synthesis in these cells is unrelated to the binding of secreted PDGF to these cell surface receptors. Thus, it is unlikely that the PDGF mitogen produced by U-2 OS cells stimulates proliferation through an autocrine mechanism involving secretion and subsequent binding to PDGF receptors.

Amino Acids↗

PubMed1988

Cultured primate aortic smooth muscle cells express both the PDGF-A and PDGF-B genes but do not secrete mitogenic activity or dimeric platelet-derived growth factor protein.

Proliferation of smooth muscle cells (SMC) in the arterial intima of man and experimental animals is important in the pathogenesis of atherosclerosis. Vascular SMC proliferation in vitro is stimulated by a number of agents, including the potent protein mitogen, platelet-derived growth factor (PDGF). Recent studies on rat arterial SMC indicate that these cells may, under certain circumstances, synthesize PDGF protein mitogens, suggesting that the regulation of SMC proliferation in vivo may have an autocrine or paracrine component. In this study we demonstrate that cultured nonhuman primate (baboon) aortic SMC transcribe both the PDGF-A and PDGF-B genes but do not secrete detectable mitogenic activity characteristic of native PDGF. The absence of this activity was not due to the presence in the cell conditioned medium of factors inhibitory for PDGF-mediated mitogenic activity. Metabolic labeling of the cells and immunoprecipitation with specific antibodies to human PDGF did not detect a dimeric (30 kDa) PDGF protein in either the intracellular or extracellular compartments, but instead identified PDGF-related proteins of molecular weight 12 kDa and 100 kDa. These data suggest the presence in vascular SMC of a mechanism regulating the translation of PDGF mRNA that may play an important role in the control of SMC proliferation in vivo.

Animals↗

PubMed1988

Characterization of a high-molecular-weight protein immunoprecipitated by platelet-derived growth factor antisera.

We previously demonstrated that a high-molecular-weight glycoprotein could be immunoprecipitated from metabolically labeled U-2 OS cells with platelet-derived growth factor (PDGF) antiserum and that it appears to be derived from a different precursor than is the 30 kD PDGF-like mitogen produced by these cells. These findings were unexpected, since the molecular weight of this glycoprotein is too large to be encoded by the PDGF structural genes. From experiments with metabolically labeled U-2 OS human osteosarcoma, fibroblasts, and NRK cells, we report here that a 185 kD protein immunoprecipitated with PDGF antiserum has the following characteristics. 1) It is a PDGF binding protein that is unrelated to alpha 2-macroglobulin. 2) It is phosphorylated in response to PDGF stimulation. 3) It is immunoprecipitated by phosphotyrosine antibodies. 4) It is not a substrate of epidermal growth factor-induced tyrosine kinase activity. These studies indicate that high-molecular-weight proteins immunoprecipitated by antiserum to PDGF represent a complex between PDGF and a binding protein capable of being phosphorylated by a PDGF-induced tyrosine kinase. These characteristics are identical to those of the PDGF receptor.

Cell Line↗

PubMed1986

Monocyte-macrophage participation in atherogenesis: inflammatory components of pathogenesis.

This article has addressed the roles of the monocyte-macrophage in atherogenesis and factors influencing monocyte recruitment to the intima. The diversity of the secretory products of the macrophage and their putative participatory roles in pathogenesis have been reviewed and discussed. Additionally, we have presented summary data on the monocyte chemoattractant peptide SMC-CF and on the differentiation of the monocyte-derived foam cell. Discussion has centered on the concept of atherosclerosis as an inflammatory process.

Animals↗

PubMed1986

Identification of processing events in the synthesis of platelet-derived growth factor-like proteins by human osteosarcoma cells.

The human osteosarcoma-derived cell line U-2 OS expresses c-sis mRNA and synthesizes platelet-derived growth factor (PDGF)-like proteins. Pulse-chase experiments indicate that proteins of 23 kDa and 180 kDa are synthesized first. The 23-kDa protein undergoes dimerization and proteolysis, giving rise to the 30-kDa dimeric protein secreted by the cells. The 180-kDa protein is proteolytically cleaved in a complex series of steps that give rise to several intracellular species. It is also the likely precursor of high molecular mass PDGF-like or PDGF-associated proteins secreted by these cells. The processing and secretion of the 180-kDa protein is slower than that of the 23-kDa protein. Subcellular fractionation and studies with the antibiotic monensin indicate that the processing events occur in the Golgi-endoplasmic reticulum compartment of U-2 OS cells.

Cell Line↗

PubMed1985

Demonstration of receptors for a PDGF-like mitogen on human osteosarcoma cells.

U-2 OS human osteosarcoma cells synthesize, process and secrete a platelet-derived growth factor (PDGF)-like mitogen. Incubation of these cells with 1 mM suramin unmasks PDGF receptor sites which are normally occupied or down regulated by the secreted endogenous PDGF-like mitogen. Partially purified preparations of metabolically labelled U-2 OS conditioned medium binds to U-2 OS cells and binding is inhibited by excess PDGF. These findings suggest that U-2 OS cells are capable of autocrine stimulation.

Binding, Competitive↗

PubMed1984

Detection of c-sis transcripts and synthesis of PDGF-like proteins by human osteosarcoma cells.

Platelet-derived growth factor (PDGF) has been previously shown to be homologous to the transforming gene of simian sarcoma virus (v-sis), and inappropriate expression of the cellular counterpart of the v-sis gene (c-sis) has been implicated in the generation of mesenchymal tumors. The U-2 OS human osteosarcoma line was shown to contain multiple c-sis transcripts. Immunoprecipitation experiments with antiserum to PDGF identified a variety of polypeptides ranging in size from 18,000 to 165,000 daltons that were immunoprecipitated specifically from U-2 OS cell extracts. The osteosarcoma also was shown to secrete a 29,000-dalton protein having the serological and structural characteristics of PDGF.

Cell Line↗

PubMed1984

Evidence for functional platelet-derived growth factor receptors on MG-63 human osteosarcoma cells.

The specific interaction of platelet-derived growth factor (PDGF) with the human osteosarcoma cell line MG-63 was studied. Scatchard analysis of 125I-PDGF binding to MG-63 cells indicated there were 32,000 specific PDGF-binding sites per cell with a Kd of 2.4 X 10(-11) M. Unlabeled PDGF blocked the specific binding of labeled PDGF to MG-63 cells at concentrations greater than 1 ng/ml. When assayed for phosphorylation of MG-63 membrane vesicles, PDGF was shown to stimulate a dose-dependent phosphorylation of a protein (phosphoprotein with a molecular weight of 185,000) which was stable in 1 M NaOH. In the absence of PDGF, a prominent alkali-stable phosphoprotein with a molecular weight of 116,000 was noted. PDGF also stimulated a dose-dependent increase in [3H]aminoisobutyric acid uptake, [3H]thymidine incorporation, and cell proliferation. When tested for secretion of PDGF-like factors, the mitogenic activity of MG-63-conditioned serum-free medium was not blocked by anti-PDGF antiserum. Concentrated MG-63-conditioned medium did not compete with 125I-PDGF for specific receptor sites on diploid fibroblasts. Therefore, MG-63 osteosarcoma cells have functional PDGF receptors and do not secrete PDGF-like mitogens.

Amino Acids↗

PubMed1983

Evidence that a human osteosarcoma cell line which secretes a mitogen similar to platelet-derived growth factor requires growth factors present in platelet-poor plasma.

A human osteosarcoma-derived cell line, 2T, grows almost as well in medium supplemented with platelet-poor plasma (PPP) as it does in medium containing fetal bovine serum. Human diploid fibroblasts, in contrast, will not grow in medium containing PPP unless human platelet-derived growth factor (PDGF) is added. PPP treated with carboxymethyl-Sephadex at pH 7.4 was able to support 2T cell proliferation, although at a reduced rate compared to untreated PPP. Addition of PDGF to carboxymethyl-Sephadex-treated PPP did not restore the growth rate. However, insulin-like growth factor isolated from human plasma did partially restore the activity of carboxy-methyl-Sephadex-treated PPP. Medium conditioned by 2T cells was mitogenic for quiescent BALB/c3T3 cells and human diploid fibroblasts. Antiserum to human PDGF blocked the mitogenic activity of the conditioned medium. Partial characterization confirmed the biochemical similarity to PDGF. The data are consistent with the hypothesis that these osteosarcoma-derived cells have growth factor requirements similar to those of normal mesenchymal cells but are able to overcome the normal growth limitations by autocrine secretion of PDGF-like mitogens.

Animals↗