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Biomedical subjects

B Westermark

Publications and source records attributed to B Westermark.

At least 145 records · Page 8Linked to original sources
PubMed1988

Induction of circular membrane ruffling on human fibroblasts by platelet-derived growth factor.

One of the earliest effects of platelet-derived growth factor (PDGF) on human fibroblasts in culture is an induction of membrane ruffling. The morphology of the ruffles induced by PDGF is unique in that they form circular arrangements on the dorsal side of the cells. Here we report that the induction of circular ruffle arrangements is an effect specific for PDGF, dose-dependent and inhibitable by anti-PDGF antibodies. We have attempted to utilize this effect to design a rapid and sensitive bioassay for PDGF. The "membrane ruffling assay" is compared with other methods to measure PDGF and its specificity with regard to the different dimeric forms of PDGF is discussed. Introduction of Ca2+ into the cells via the Ca2+ ionophore A23187 or the addition of the tumor-promor 12-O-tetradecanoylphorbol-13-acetate (TPA), which is a stimulator of protein kinase C, does not induce circular ruffle formations on human fibroblasts, neither does the addition of the combination of these two agents. However, addition of TPA almost completely inhibits PDGF-induced circular ruffle formations. Further, we find a shift in the time-course of the PDGF-induced circular ruffle formations by sodium orthovanadate, an inhibitor of protein tyrosine phosphatases. This may indicate the involvement of protein phosphorylation in the regulation of PDGF-induced membrane ruffling.

Calcium↗
PubMed1988

Structural and functional aspects of platelet-derived growth factor.

The finding that PDGF production is common in normal as well as transformed cells indicate that PDGF has a function in autocrine and paracrine stimulation of cells in several physiological and pathological conditions. The expression of mRNA for the two chains of PDGF are independently regulated. The fact that the different dimeric forms of PDGF have different functional effects, indicate that the cellular response to PDGF may be more complex than previously thought, and may involve binding to more than one receptor. The cellular effects ascribed to PDGF, growth stimulation, ruffling and chemotaxis, seems to be mainly associated with B chain containing dimers. The function of PDGF-AA remains to be elucidated.

Cell Transformation, Viral↗
PubMed1988

Endothelial cell hyperplasia in human glioblastoma: coexpression of mRNA for platelet-derived growth factor (PDGF) B chain and PDGF receptor suggests autocrine growth stimulation.

The genes for platelet-derived growth factor (PDGF) A chain, B chain/c-sis, and the PDGF receptor are expressed in human malignant glioma cell lines. In the present investigation we have studied the expression of these genes in biopsy specimens from human glioblastomas. Hyperplasia of the vascular endothelium is a prominent characteristic of human glioblastoma multiforme and simian sarcoma virus-induced gliomas in primates. RNA transfer blot analysis of biopsies from glioblastoma multiforme showed transcripts for PDGF A and B chains and the PDGF receptor. Tissue sections from this tumor examined by in situ hybridization techniques revealed that the proliferating vascular endothelial cells contained large quantities of mRNA for PDGF B chain/c-sis and its receptor and, to a lesser extent, for PDGF A chain. In contrast, the tumor cells expressed more mRNA for PDGF A chain than for PDGF B chain and PDGF receptor. The latter two were also expressed at higher levels in glioma cells than in glial cells of nontumorous human brain tissue. Thus, an autocrine stimulation by the PDGF B chain/c-sis product via its receptor, evoked by interaction with surrounding glioma cells, could be the mechanism behind the pathological proliferation of endothelial cells characteristically found in this type of malignancy.

Animals↗
PubMed1988

Aberrant expression of receptors for platelet-derived growth factor in an anaplastic thyroid carcinoma cell line.

Receptors for platelet-derived growth factor (PDGF) have previously only been found on cells of mesenchymal and glial origin. This study shows PDGF receptors on an anaplastic thyroid carcinoma cell line, C 643, that was found to express thyroglobulin mRNA, confirming its origin from thyroid epithelium. Northern blot analysis of poly(A)+ RNA hybridized with a human PDGF B-type receptor cDNA probe revealed a 5.4-kilobase transcript in the C 643 cells. The existence of receptor protein on the cell surface was shown by immunofluorescence microscopy with a PDGF receptor monoclonal antibody. Binding experiments with 125I-labeled dimeric forms of PDGF indicated that the cells contain B-, but not A-, type PDGF receptors. The addition of PDGF to C 643 membranes in the presence of [32P]ATP induced phosphorylation of the receptor. A polyclonal PDGF B-type receptor peptide antiserum was used to immunoprecipitate a receptor protein from metabolically labeled C 643 cells; the receptor was found to be 5-10 kDa larger than that in normal human fibroblasts. Removal of N-linked carbohydrates using endoglycosidase H resulted in deglycosylated receptor proteins of similar size in C 643 cells and fibroblasts, indicating differences in glycosylation patterns of the two receptor proteins. The aberrant expression of receptors might be crucial in tumor development by conferring a selective growth advantage to the cancer cells.

Blotting, Northern↗
PubMed1988

Expression of recombinant platelet-derived growth factor A- and B-chain homodimers in rat-1 cells and human fibroblasts reveals differences in protein processing and autocrine effects.

The autocrine effects of platelet-derived growth factor (PDGF) A- and B-chain homodimers (PDGF-AA and PDGF-BB) on rat-1 cells and human fibroblasts have been investigated by using human PDGF A- and B-chain cDNA clones expressed in a retroviral vector. Infection with replication-defective virus carrying the B-chain cDNA resulted in a phenotypical transformation resembling that induced by simian sarcoma virus. The resulting cells were focus forming in monolayer cultures, grew to high saturation densities, and formed large colonies in soft agar. The PDGF A-chain transfectants showed no transformed morphology and lacked focus-forming activity but grew to high saturation density in monolayer culture and formed small colonies in soft agar. A similar but weaker effect was obtained with an A-chain cDNA variant containing a 69-base-pair insertion in the 3' end of the protein-coding domain. A- and B-chain transfectants released PDGF receptor-competing activity into the medium, but only the medium conditioned by the B-chain transfectants possessed potent mitogenic activity on human fibroblasts. Both types of transfectants had downregulated levels of PDGF receptors; however, the B-chain transfectants were downregulated to significantly lower levels. Metabolic labeling and immunoprecipitations with PDGF antiserum showed that the PDGF B-chain protein was processed to a 24-kilodalton cell-associated and a 30-kilodalton secreted dimeric protein. The A-chain protein was rapidly secreted as a 31-kilodalton dimeric protein. The present study shows a marked difference in the autocrine effects of PDGF-AA and -BB expressed under the control of a retroviral promoter and suggests that different biological properties may be assigned to these two PDGF isoforms.

Animals↗
PubMed1988

Epidermal growth factor, but not thyrotropin, stimulates the expression of c-fos and c-myc messenger ribonucleic acid in porcine thyroid follicle cells in primary culture.

Previous studies have shown that TSH stimulates functional parameters in cultures of porcine follicle cells, but has no stimulatory effect on their growth. Epidermal growth factor (EGF), on the other hand, is a potent mitogen in such cells. The aim of the present investigation was to study the effect of EGF and TSH on the expression of proto-oncogene mRNA in porcine thyroid follicle cells. The addition of EGF caused a marked increase in the c-fos and c-myc mRNA levels. Stimulation of cells with 10 mU/ml TSH, however, failed to stimulate expression of the proto-oncogenes. The increase in c-fos mRNA caused by EGF was transient, with a maximum after 30 min. The c-myc transcript level increased after 30-60 min; the maximum level was at 90 min, and it decreased thereafter but remained at an elevated level throughout the 24 h of investigation. Half-maximal effects on proto-oncogene expression were obtained using 0.5-1 ng (c-fos) and 0.2-0.5 ng (c-myc) EGF/ml. Our data clearly demonstrate that increased expression of c-fos and c-myc mRNA is an early event in the prereplicative phase of EGF-stimulated porcine thyroid follicle cells. Since c-fos and c-myc expression may be necessary events in the prereplicative phase, the lack of effect of TSH on these functions may be closely related to its inability in stimulating the growth of porcine thyrocytes.

Animals↗
PubMed1988

Density-dependent inhibition of cell growth by transforming growth factor-beta 1 in normal human fibroblasts.

We report here that transforming growth factor-beta 1 (TGF-beta 1) inhibits platelet-derived growth factor (PDGF)-induced DNA synthesis in normal human fibroblasts in a cell density-dependent manner; no inhibition was seen in sparse cultures, approximately 50% inhibition in confluent cell cultures, and an almost total inhibition in dense cultures. The PDGF-inducible genes c-myc and c-fos were induced also by TGF-beta 1. Simultaneous addition of TGF-beta 1 and PDGF resulted in sustained, rather than transient, expression of c-fos mRNA; c-fos mRNA was detected as long as 24 hr after addition of PDGF and TFG-beta 1. TGF-beta 1 also induced mRNA for the A chain, but not the B chain, of PDGF. Conversely, PDGF induced TGF-beta 1 mRNA in sparse but not in dense cultures. These data indicate the existence of a complex interdependent regulation of PDGF and TGF-beta mRNA expression which is influenced by the cell density.

Blotting, Northern↗
PubMed1987

Evidence for progressional changes in the human malignant glioma line U-343 MGa: analysis of karyotype and expression of genes encoding the subunit chains of platelet-derived growth factor.

Three cell samples in different passages of the line U-343 MGa, derived from a human malignant glioma biopsy, gave rise to clones with different amounts of platelet-derived growth factor (PDGF)-like activity secreted to extracellular medium, and of 125I-labeled PDGF binding. Sixteen clones were completely karyotyped with the G-banding technique. The unique markers 1p-q+, 16p- found in all clones, as well as in the parallel uncloned line, U-343 MG, provided evidence of their common origin. The deduced early, possibly partly primary, deviations had the formula 44, XY, 1p-q+, -14, 16p-, -22, where loss of one chromosome 22 is in accordance with previous reports on early chromosomal deviations in gliomas. Two clones, the hypodiploid 26L and 5H, represented early progressional changes. The other clones followed two patterns of late progressional changes, probably starting from the karyotype of 5H, with additional markers and doubling of the stemlines. In late progressional line I 12q+ and in II +7 were the most characteristic findings. Northern blot analysis using complementary DNA clones for the A and B chains of PDGF showed that both PDGF chains were expressed in 26L and 5H indicating that activation of the PDGF genes could have been an early event in the development of this glioma. Clones with late progression pattern II had been subjected to the highest selective pressure in vitro, and they secreted the highest amount of PDGF-like activity to the extracellular medium. Among them were the most rapidly and tightly growing cells and some clones with high 125I-labeled epidermal growth factor binding. Possibly these findings reflect progressional changes including defective regulation of the growth factor/growth factor receptor genes, selected for in vitro, without involving gross rearrangements or amplifications of the genes. The possible significance of extra chromosomes 7, with the PDGF A chain and epidermal growth factor receptor genes, and of the 12q+ marker, located near the gamma interferon gene is discussed.

Cell Line↗
PubMed1987

Frequent expression of growth factors for mesenchymal cells in human mammary carcinoma cell lines.

The expression of growth factors for mesenchymal cells was investigated in 10 different human mammary carcinoma cell lines. Expression of mRNA for platelet-derived growth factor (PDGF) A-chain, PDGF B-chain, transforming growth factor-alpha, and insulin-like growth factor II were found in eight, nine, five, and two of the cell lines, respectively. The production of PDGF-like growth factors by the mammary carcinoma cell lines was investigated in detail. PDGF receptor competing activity and mitogenic activity, which could be neutralized by PDGF antibodies, were found in the conditioned medium of almost all the cell lines. A PDGF-like growth factor was partially purified from the conditioned medium of one of the cell lines, MCF7. A Mr 31,000 component, which was reduced to Mr 17,000, was furthermore precipitated by a PDGF antiserum from the conditioned medium of metabolically labeled MCF7 cells. These results indicate that growth factors for mesenchymal cells are frequently expressed in human mammary carcinoma cell lines. This is of interest in relation to the fact that mammary carcinoma tumors often contain a high proportion of proliferating connective tissue cells.

Biological Assay↗
PubMed1987

Induction of platelet-derived growth factor gene expression during megakaryoblastic and monocytic differentiation of human leukemia cell lines.

Platelet-derived growth factor (PDGF) is one of the most important polypeptide growth factors in human serum. It is composed of two polypeptide chains linked by disulfide bonds. The B-chain is encoded by the c-sis proto-oncogene, which is expressed in several malignant and non-malignant cells including K562 cells differentiating towards megakaryoblasts. Expression of the A-chain has been reported to occur in human solid tumor cell lines independently of c-sis expression. We report here the non-coordinate expression of the A- and B-chains in human leukemia cell lines. The PDGF-A and B-chain (c-sis) RNA expression as well as secretion of PDGF polypeptides are induced in the K562 cell line upon induction of megakaryoblastic differentiation with 12-O-tetradecanoyl phorbol-13-acetate (TPA) whereas erythroid differentiation induced with sodium butyrate is accompanied by c-sis expression only. Simultaneously with megakaryoblastic differentiation the RNA level for another platelet protein, the transforming growth factor-beta was also increased, but in a complex manner. The promyelocytic leukemia cell line HL-60 does not express PDGF-A RNA, whereas the promonocytic cell line U937 does. Preferential induction of the A-chain RNA is obtained in both cell lines after treatment with TPA which causes monocytic differentiation. PDGF-A expression in HL-60 cells is also observed after treatment with the tumor necrosis factor-alpha but granulocytic differentiation of HL-60 cells induced with dimethyl sulfoxide or the granulocyte colony-stimulating factor is not associated with PDGF gene expression.

Cell Differentiation↗
PubMed1987

An angiogenic growth factor is expressed in human glioma cells.

Progression to increased malignancy frequently occurs in human brain tumors of glial origin and usually involves neovascularization--a massive proliferation of endothelial cells into the tumor tissue. We have shown previously that subversion of a normal growth factor-related pathway is frequently associated with human gliomas. Here we show that human glioma cell lines express the gene encoding the angiogenic peptide endothelial cell growth factor (ECGF) or acidic fibroblast growth factor (a-FGF) and that an ECGF-like polypeptide is produced by these cells. The glioma-derived growth factor was partially purified from cell extracts by heparin-Sepharose affinity chromatography where it eluted at 1.5 M sodium chloride. On reversed-phase h.p.l.c., growth factor activity for endothelial cells was eluted at the same concentration of acetonitrile as found for bovine brain-ECGF, also a potent mitogen for endothelial cells. Moreover, human glioma cells possess specific cell surface receptors for ECGF and are mitogenically stimulated by exogenous addition of this growth factor. Glioma derived-ECGF may therefore have a dual influence: first, by autocrine growth-stimulation of human gliomas and, second, by paracrine-stimulation of endothelial cell proliferation which results in neovascularization of the tumor tissue.

Cell Line↗
PubMed1987

PDGF-like growth factors in autocrine stimulation of growth.

PDGF is a dimeric molecule consisting of A chains and B chains. All three dimeric combinations have been found in platelets, or produced by certain normal or transformed cell types; whether functional differences occur between different dimers is currently under investigation. Production of PDGF-like factors by cells carrying PDGF receptors may establish an autocrine loop, driving cell multiplication.

Cell Division↗
PubMed1987

Effects of epidermal growth factor and platelet-derived growth factor on c-fos and c-myc mRNA levels in normal human fibroblasts.

The mRNA levels of two proto-oncogenes, c-fos and c-myc, were determined in human foreskin fibroblasts exposed to epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) in a serum-free, defined medium (MCDB 104). Untreated, quiescent cells were found to have low or undetectable levels of c-fos and c-myc mRNA. Within 10 min after the addition of EGF or PDGF the c-fos mRNA level increased, reached a peak at 30 min, and then declined to the control level after 60 min. The level of c-myc mRNA increased somewhat later and peaked after 8 h in cultures treated with either of the growth factors. The c-myc mRNA level remained elevated throughout the 24 h of investigation. The concentrations of EGF and PDGF required for a maximal effect on c-fos or c-myc expression were found to be similar to those that give maximal effect on cell proliferation. Both c-fos and c-myc mRNA expression were super-induced by the addition of cycloheximide. The addition of neutralizing PDGF antibodies to cultures that had received PDGF 4 h earlier inhibited the subsequent increase in the c-myc mRNA level, indicating that the effect of PDGF on c-myc expression is not caused by a "hit and run," mechanism. Density-inhibited cells responded to EGF and PDGF by an increase in c-fos and c-myc mRNA levels in the absence of any mitogenic response. The present results conform to the view that the c-fos and c-myc proto-oncogenes may be important (or necessary) but not sufficient for the initiation of DNA synthesis. Moreover, the finding that both EGF and PDGF increase c-fos and c-myc expression supports our previous suggestion that these two growth factors may in part act via a common intracellular pathway in the prereplicative phase of human fibroblasts.

Antibodies↗
PubMed1987

Regulation of platelet-derived growth factor gene expression by transforming growth factor beta and phorbol ester in human leukemia cell lines.

We studied the expression of the genes encoding the A and B chains of platelet-derived growth factor (PDGF) in a number of human leukemia cell lines. Steady-state expression of the A-chain RNA was seen only in the promonocytic leukemia cell line U937 and in the T-cell leukemia cell line MOLT-4. It has previously been reported that both PDGF A and PDGF B genes are induced during megakaryoblastic differentiation of the K562 erythroleukemia cells and transiently during monocytic differentiation of the promyelocytic leukemia cell line HL-60 and U937 cells. In this study we show that PDGF A RNA expression was induced in HL-60 and Jurkat T-cell leukemia cells and increased in U937 and MOLT-4 cells after a 1- to 2-h stimulation with an 8 pM concentration of transforming growth factor beta (TGF-beta). PDGF A RNA remained at a constant, elevated level for at least 24 h in U937 cells, but returned to undetectable levels within 12 h in HL-60 cells. No PDGF A expression was induced by TGF-beta in K562 cells or in lung carcinoma cells (A549). Interestingly, essentially no PDGF B-chain (c-sis proto-oncogene) RNA was expressed simultaneously with PDGF A. In the presence of TGF-beta and protein synthesis inhibitors, PDGF A RNA was superinduced at least 20-fold in the U937 and HL-60 cells. PDGF A expression was accompanied by secretion of immunoprecipitable PDGF to the culture medium of HL-60 and U937 cells. The phorbol ester tumor promoter tetradecanoyl phorbol acetate also increased PDGF A expression with similar kinetics, but with a mechanism distinct from that of TGF-beta. These results suggest a role for TGF-beta in the differential regulation of expression of the PDGF genes.

Cell Differentiation↗