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Title: Platelet-derived growth factor stimulation of (/sup 3/H)-glucosamine incorporation in density-arrested BALB/c-3T3 cells

Abstract

G/sub 0//G/sub 1/ traverse in density-arrested BALB/c-3T3 cells is controlled by multiple serum-derived growth factors. Platelet-derived growth factor (PDGF) initiates a proliferative response, whereas factors present in plasma facilitate progression through G/sub 0//G/sub 1/. In the absence of competence formation, progression factors are unable to stimulate cell cycle traverse. The authors have identified the stimulation of a biochemical process specific to competence formation in BALB/c-3T3 cells. PDGF treated BALB/c-3T3 cells incorporated 5-10 fold more (/sup 3/H)-glucosamine (GlcN) into acid-insoluble material as compared to platelet-poor plasma (PPP) treated cultures. Increased GlcN incorporation occurred in density-arrested BALB/c-3T3 cells in response to treatment with other competence factors, fibroblast growth factor, and Ca/sub 3/ (PO/sub 4/)/sub 2/ and was not due to cell-cycle traverse. Stimulation of (/sup 3/H)-GlcN incorporation by PDGF was time dependent, and increased incorporation of (/sup 3/H)-GlcN into protein required de novo protein synthesis. Several mechanisms through which PDGF could increase GlcN incorporation into cellular material were examined. Results of these studies suggest an increase in the cellular capacity to glycosylate proteins is a response to or a part of competence formation.

Authors:
; ;
Publication Date:
Research Org.:
Univ. of North Carolina, Chapel Hill
OSTI Identifier:
6525593
Resource Type:
Journal Article
Resource Relation:
Journal Name: J. Cell. Physiol.; (United States); Journal Volume: 130:1
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; FIBROBLASTS; CELL PROLIFERATION; GLUCOSAMINE; UPTAKE; TRITIUM COMPOUNDS; BLOOD PLATELETS; CELL CYCLE; GLUCOPROTEINS; GROWTH; METHIONINE; PROTEINS; SULFUR 35; AMINES; AMINO ACIDS; ANIMAL CELLS; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; BIOLOGICAL MATERIALS; BLOOD; BLOOD CELLS; BODY FLUIDS; CARBOHYDRATES; CARBOXYLIC ACIDS; CONNECTIVE TISSUE CELLS; DAYS LIVING RADIOISOTOPES; DRUGS; EVEN-ODD NUCLEI; HEXOSAMINES; HEXOSES; ISOTOPES; LABELLED COMPOUNDS; LIGHT NUCLEI; LIPOTROPIC FACTORS; MATERIALS; MONOSACCHARIDES; NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC SULFUR COMPOUNDS; RADIOISOTOPES; SACCHARIDES; SOMATIC CELLS; SULFUR ISOTOPES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Harrington, M.A., Wharton, W., and Pledger, W.J. Platelet-derived growth factor stimulation of (/sup 3/H)-glucosamine incorporation in density-arrested BALB/c-3T3 cells. United States: N. p., 1987. Web. doi:10.1002/jcp.1041300114.
Harrington, M.A., Wharton, W., & Pledger, W.J. Platelet-derived growth factor stimulation of (/sup 3/H)-glucosamine incorporation in density-arrested BALB/c-3T3 cells. United States. doi:10.1002/jcp.1041300114.
Harrington, M.A., Wharton, W., and Pledger, W.J. Thu . "Platelet-derived growth factor stimulation of (/sup 3/H)-glucosamine incorporation in density-arrested BALB/c-3T3 cells". United States. doi:10.1002/jcp.1041300114.
@article{osti_6525593,
title = {Platelet-derived growth factor stimulation of (/sup 3/H)-glucosamine incorporation in density-arrested BALB/c-3T3 cells},
author = {Harrington, M.A. and Wharton, W. and Pledger, W.J.},
abstractNote = {G/sub 0//G/sub 1/ traverse in density-arrested BALB/c-3T3 cells is controlled by multiple serum-derived growth factors. Platelet-derived growth factor (PDGF) initiates a proliferative response, whereas factors present in plasma facilitate progression through G/sub 0//G/sub 1/. In the absence of competence formation, progression factors are unable to stimulate cell cycle traverse. The authors have identified the stimulation of a biochemical process specific to competence formation in BALB/c-3T3 cells. PDGF treated BALB/c-3T3 cells incorporated 5-10 fold more (/sup 3/H)-glucosamine (GlcN) into acid-insoluble material as compared to platelet-poor plasma (PPP) treated cultures. Increased GlcN incorporation occurred in density-arrested BALB/c-3T3 cells in response to treatment with other competence factors, fibroblast growth factor, and Ca/sub 3/ (PO/sub 4/)/sub 2/ and was not due to cell-cycle traverse. Stimulation of (/sup 3/H)-GlcN incorporation by PDGF was time dependent, and increased incorporation of (/sup 3/H)-GlcN into protein required de novo protein synthesis. Several mechanisms through which PDGF could increase GlcN incorporation into cellular material were examined. Results of these studies suggest an increase in the cellular capacity to glycosylate proteins is a response to or a part of competence formation.},
doi = {10.1002/jcp.1041300114},
journal = {J. Cell. Physiol.; (United States)},
number = ,
volume = 130:1,
place = {United States},
year = {Thu Jan 01 00:00:00 EST 1987},
month = {Thu Jan 01 00:00:00 EST 1987}
}
  • Addition of the mitogenic peptides bombesin and vasopressin to quiescent Swiss 3T3 mouse cells increased the cytosolic Ca/sup 2 +/ concentration without any measurable delay. In contrast, there was a significant lag period (16 +/- 1.2 s) before platelet-derived growth factor (PDGF) increased cytosolic Ca/sup 2 +/ concentration. This lag was not diminished at high concentrations of either porcine or human PDGF. Similar results were obtained in 3T3 cells loaded with quin-2 or fura-2. The differences in the effects of bombesin, vasopressin, and PDGF on Ca/sup 2 +/ movements were also substantiated by measurements of /sup 45/Ca/sup 2 +/ effluxmore » and of cellular /sup 45/Ca/sup 2 +/ content. Activation of protein kinase C by phorbol esters inhibited Ca/sup 2 +/ mobilization induced by either bombesin or vasopressin. In contrast, phorbol esters had no effect on PDGF-induced cytosolic Ca/sup 2 +/ concentration increase or acceleration of /sup 45/Ca/sup 2 +/ efflux. Finally, bombesin and vasopressin caused a rapid increase in the production of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate, whereas PDGF, even at a saturating concentration, exerted only a small effect. These results indicate that the signal transduction pathway activated by PDGF that lead to Ca/sup 2 +/ mobilization can be distinguished form those utilized by bombesin and vasopressin.« less
  • Platelet-derived growth factor (PDGF) treatment of density-arrested BALB/c-3T3 cells results in increased ({sup 3}H)glucosamine (GlcN) incorporation into cellular material. The enhanced GlcN incorporation is not due to a preferential increase in proteoglycan synthesis as measured by ({sup 35}S)H{sub 2}SO{sub 4} incorporation. Approximately 50% of the GlcN incorporated in PDGF or platelet-poor plasma (PPP)-treated cultures enters N-linked glycoproteins. Addition of dolichol-phosphate (dolichol-P), a required intermediate in N-linked glycosylation, did not alter ({sup 3}H)GlcN incorporation in PDGF-treated cells but did increase incorporation in PPP-treated cultures to a level comparable to that observed for PDGF-treated cultures. PDGF-treated cultures contained twofold greater quantities ofmore » ({sup 3}H)GlcN dolichol intermediates and lipid-free glycoprotein. Over a 12-h time course 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) activity was similar in cultures treated with PDGF or PPP. Results of these studies reveal that enhanced protein glycosylation in response to PDGF treatment is not the result of a direct effect on HMG CoA reductase.« less
  • Polypeptide growth factors rapidly induce the transcription of a set of genes that appear to mediate cell growth. The authors report that one of the genes induced in BALB/c mouse 3T3 cells encodes a transmembrane protein (mTF) homologous to human tissue factor, which is involved in the proteolytic activation of blood clotting. mTF mRNA is present in many murine tissues and cell lines. The authors' results raise the possibility that mTF may also play a role in cell growth.
  • Data indicating that the 21-kDa protein (p21) Harvey-ras gene product shares sequence homology with guanine nucleotide-binding proteins (G proteins) has stimulated research on the influence(s) of p21 on G-protein-regulated systems in vertebrate cells. Previous work demonstrated that NIH-3T3 mouse cells expressing high levels of the cellular ras oncogene isolated from the EJ human bladder carcinoma (EJ-ras) exhibited reduced hormone-stimulated adenylate cyclase activity. The authors now report that in these cells another enzyme system thought to be regulated by G proteins is inhibited, namely phospholipases A/sub 2/ and C. NIH-3T3 cells incubated in plasma-derived serum release significant levels of prostaglandin E/submore » 2/ (PGE/sub 2/) as determined by radioimmunoassay when exposed to platelet-derived growth factor (PDGF) at 2 units/ml. The lack of PDGF-stimulated PGE/sub 2/ release from EJ-ras-transfected cells is not due to a defect in the prostaglandin cyclooxygenase enzyme, since incubation of control cells and EJ-ras-transfected cells in 0.33, 3.3, or 33 ..mu..M arachidonate resulted in identical levels of PGE/sub 2/ release. The lack of PDGF-stimulated PGE/sub 2/ release from EJ-ras-transfected cells also does not result from the loss of functional PDGF receptors. EJ-ras-transformed cells bind 70% as much /sup 125/I-labeled PDGF as control cells and are stimulated to incorporate (/sup 3/H)thymidine and to proliferate after exposure to PDGF. Determination of total water-soluble inositolphospholipids and changes in the specific activities of phosphatidylcholine in control and EJ-ras-transfected cells demonstrated that PDGF-stimulated phospholipase C and A/sub 2/ activities are inhibited in the EJ-ras-transfected cells.« less
  • A cell growth inhibitor (GI), purified from BSC-1 cell-conditioned medium, has little if any effect on DNA synthesis when added alone to monolayer cultures of quiescent Swiss mouse 3T3 cells in serum-free medium. However, the inhibitor, which is closely related to transforming growth factor type ..beta.. (TGF-..beta..), exhibits a pronounced synergistic stimulation of DNA synthesis in combination with certain peptide (bombesin, vasopressin) or polypeptide (platelet-derived growth factor) mitogens. /sup 125/I-EGF binding was measured and the efflux of /sup 45/Ca/sup 2 +/ was measured in response to mitogen stimulation. A similar synergistic response has been demonstrated for TGF-..beta.. purified from humanmore » platelets. In the presence of 3 nM bombesin, a half-maximal stimulation of DNA synthesis was obtained at a GI concentration of approximately 60 pg/ml, with a maximal response at approximately 600 pg/ml. The synergistic interactions demonstrated by GI or TGF-..beta.. in stimulating Swiss 3T3 cells closely resemble those previously shown for insulin, and the authors have observed that GI does not synergize with insulin to stimulate DNA synthesis in these cells. Like insulin, and in contrast to bombesin, vasopressin, and platelet-derived growth factor, GI does not activate cellular inositolphospholipid hydrolysis, calcium mobilization, or cross-regulation of epidermal growth factor receptor affinity. These results raise the possibility that the biochemical pathways activated by GI/TGF-..beta.. and insulin converge at a post-receptor stage.« less