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Biomedical subjects

B Westermark

Publications and source records attributed to B Westermark.

At least 55 records · Page 3Linked to original sources
PubMed1994

Point mutations of the thyrotropin receptor determining structural requirements for its ability to bind thyrotropin and to stimulate adenylate cyclase activity.

The two cysteines C494 and C569, located in the first and second extracellular loop, respectively, of the thyrotropin (TSH) receptor, were mutated to serines to test the functional significance of the putative disulfide bond between these two cysteines. Single (C494S and C569S) and double (C494/569S) mutant receptors were generated, transiently expressed in COS cells, and compared with regard to the ability to bind ligand and to mediate stimulation of adenylate cyclase activity. The double mutant retained ligand binding capacity, in contrast to the single cysteine mutants that were essentially devoid of binding capacity. The ability of the mutated receptor variants to stimulate adenylate cyclase activity was lost or greatly reduced.

Adenylyl Cyclases↗
PubMed1994

Characterization of the retention motif in the C-terminal part of the long splice form of platelet-derived growth factor A-chain.

Platelet-derived growth factor (PDGF) A-chain is found in two different splice variants; the long A-chain variant differs from the short one in that it contains a stretch of basic amino acid residues in the C-terminal part that mediates retention of the growth factor inside the producer cell and to the cell matrix. By analyzing mutants in which different amino acid residues in the retention motif had been changed to alanine residues, we found that the total positive charge of the sequence is of importance for the function of the retention motif. Moreover, we showed that retention also occurs if only one of the polypeptides in the PDGF dimer carries the retention motif. Surface iodination and competition with a peptide having the sequence of the retention motif revealed that the long A-chain variant, in contrast to the short A-chain variant, is localized on the outside of the cells and is also associated to the cell matrix. The association is likely to be mediated partially through heparan sulfate proteoglycans since treatment of matrix with heparitinase released the long A-chain variant.

Alternative Splicing↗
PubMed1994

Activated platelet-derived growth factor autocrine pathway drives the transformed phenotype of a human glioblastoma cell line.

Human glioblastoma cells (A172) were found to concomitantly express PDGF-BB and PDGF beta-receptors. The receptors were constitutively autophosphorylated in the absence of exogenous ligand, suggesting the presence of an autocrine PDGF pathway. Neutralizing PDGF antibodies as well as suramin inhibited the autonomous PDGF receptor tyrosine kinase activity and resulted in up-regulation of receptor protein. The interruption of the autocrine loop by the PDGF antibodies reversed the transformed phenotype of the glioblastoma cell, as determined by (1) diminished DNA synthesis, (2) inhibition of tumor colony growth, and (3) reversion of the transformed morphology of the tumor cells. The PDGF antibodies showed no effect on the DNA synthesis of another glioblastoma cells line (U-343MGa 31L) or on Ki-ras-transformed fibroblasts. The present study demonstrates an endogenously activated PDGF pathway in a spontaneous human glioblastoma cell line. Furthermore, we provide evidence that the autocrine PDGF pathway drives the transformed phenotype of the tumor cells, a process that can be blocked by extracellular antagonists.

Antibodies, Monoclonal↗
PubMed1994

Platelet-derived growth factor (PDGF) in neoplastic and non-neoplastic cystic lesions of the central nervous system and in the cerebrospinal fluid.

The aim of this study was to determine the concentration of PDGF in vivo in neoplastic and non-neoplastic brain lesions. Fluid from cystic lesions and cerebrospinal fluid was tested in a radioreceptor assay that detects all described PDGF isoforms. High concentration of PDGF were found in cyst fluids from several astrocytomas, one metastatic melanoma, one metastatic lung adenocarcinoma and one intracerebral abscess. The PDGF concentrations were several times higher than the levels known to be required for maximal PDGF effects on cells in vitro. PDGF could also be detected in some non-neoplastic lesions, especially one intracerebral abscess. The finding of high amounts of PDGF in neoplastic lesions strongly supports the possibility that PDGF can be a mediator of tumour and stromal cell growth and motility in vivo. Comparison of PDGF and beta-thromboglobulin concentrations in the same fluids strongly indicates that the PDGF protein is locally produced rather than a result of platelet activation and derangement of the blood-brain barrier.

Astrocytoma↗
PubMed1994

Epithelial-stromal interactions in basal cell cancer: the PDGF system.

A proposed progenitor cell for basal cell carcinoma is a stem cell located in the bulge of the hair follicle. Previous investigations have shown that basal cell carcinoma has a specific stroma requirement for its growth. Likewise the development of a normal hair follicle requires the inductive force of a specialized structure with condensed mesenchyme that eventually forms the dermal hair papilla. Investigations in mouse embryos also strongly indicate that induction/growth of skin structures is dependent on platelet-derived growth factor (PDGF) alpha-receptor expression in the mesenchyme. We therefore investigated the expression of PDGF A and B chain and PDGF alpha and beta receptors in basal cell carcinoma and in normal skin by immunohistochemistry and in situ hybridization. alpha and beta receptors were found in the specific stroma components of basal cell carcinoma, dermal hair papilla, and sweat glands, but not in the epithelial structures. The A and B chains, on the other hand, were mainly found in basal cell carcinoma cells, in hair matrix, and in sweat gland epithelium. This "appositional" expression of PDGF/PDGF receptor closely resembles that found in epithelial/mesenchymal structures during normal development. The findings also suggest that PDGF receptor expression is one of the characteristics of the specific stroma that is necessary for basal cell carcinoma growth.

Capillaries↗
PubMed1994

Thyrotropin (TSH)-induced receptor internalization in nonthyroidal cells transfected with a human TSH-receptor complementary deoxyribonucleic acid.

TSH-induced desensitization was studied in nonthyroidal cells expressing functionally active TSH receptors (TSHR). Chinese hamster ovary (CHO) cells and mouse NIH 3T3 cells were stably transfected with a human TSHR cDNA. Stimulation of the CHO-TSHR and NIH-TSHR cells with 10 mU/ml TSH resulted in a decreased sensitivity to a second TSH stimulation only in the NIH-TSHR cells. A decrease in TSH-induced cAMP was present within 1 h and coincided with a decreased binding of [125I]TSH. The half-maximal effect was observed after a 3- to 4-h stimulation with TSH, and exposure of cells to TSH for 20 h led to a 70-80% inhibition of cAMP formation. After withdrawal of TSH, cells regained full responsiveness to TSH after 6 h. Moreover, the desensitization effect observed in NIH-TSHR cells was not mimicked by forskolin and, therefore, was not mediated by cAMP. Stimulation of the CHO-TSHR cells with TSH did not result in a desensitization toward a second TSH stimulation, nor did it reduce the binding of [125I]TSH. This difference between the two cell lines might be explained by a higher turnover rate of receptors in the CHO cells. Indeed, incubation of cells with [125I]TSH showed a more efficient internalization of ligand in the CHO-TSHR cells compared to the NIH-TSHR cells. In summary, the homologous desensitization observed in TSHR-transfected NIH 3T3 cells appears to be the result of ligand-induced receptor down-regulation.

3T3 Cells↗
PubMed1994

Differences in binding to the solid substratum and extracellular matrix may explain isoform-specific paracrine effects of platelet-derived growth factor.

We have studied the paracrine response of fibroblasts to the two homodimeric isoforms of platelet-derived growth factor (PDGF-AA and -BB). CHO-cells stably transfected with a B-chain cDNA expression vector (CHO-PDGF-B cells), were found to elicit a marked paracrine response when seeded at clonal density on preformed monolayers of human or murine fibroblasts; no such response was elicited by CHO-PDGF-A cells. Immunofluorescence microscopy of CHO-PDGF-B cell cultures showed the presence of a pericellular deposit of material reacting with antibodies against PDGF-BB; no corresponding PDGF-AA immunoreactive material was found in the CHO-PDGF-A cultures. The pericellular material, deposited by CHO-PDGF-B cells, was shown to have a growth promoting effect on target cells. Furthermore, we could show that 125I-PDGF-BB binds more efficiently than 125I-PDGF-AA to extracellular matrix prepared from foreskin fibroblast cultures, as well as to defined extracellular matrix components (fibronectin, laminin and collagen III). The results reveal a marked difference in the paracrine activity of PDGF-AA and PDGF-BB; the latter has a strong local growth enhancing effect, that is mostly likely to be ascribed to its association with components of the extracellular matrix.

3T3 Cells↗
PubMed1993

Platelet-derived growth factor increases the turnover of GTP/GDP on ras in permeabilized fibroblasts.

The potent mitogen platelet-derived growth factor (PDGF) induced a rapid increase in Ras.GTP in permeabilized human and murine fibroblasts. The effect was initiated by both PDGF-AA acting exclusively through PDGF alpha-receptors, and by PDGF-BB interacting with both alpha- and beta-type receptors. The dose-response curves suggest that both receptor types mediate the response. PDGF-dependent Ras activation, measured as increased formation of Ras.GTP, was rapid and reversible. At 37 degrees C the effect had a duration of around 10 min. The PDGF-dependent increase in Ras.GTP was followed by a simultaneous increase in Ras.GDP. Under no experimental condition could a relative increase in Ras.GTP be detected. 0.5 microM GDP and 0.5 microM GTP were equally potent competing for the formation of Ras.[alpha-32P]GTP upon PDGF stimulation. Furthermore, when the basal nucleotide exchange rate on Ras was elevated by omission of Mg2+ from the medium, PDGF had no further effect on the formation of Ras.GTP. We therefore conclude that PDGF activates Ras through a mechanism leading to an increased nucleotide exchange on Ras.

3T3 Cells↗
PubMed1993

Expression of functional PDGF beta receptors in a human large-cell lung-carcinoma cell line.

In this study we have investigated a panel of lung-cancer cell lines, both of small-cell carcinoma and non-small-cell type, for the expression of receptors for platelet-derived growth factor. Although we found mRNA expression for the beta-type receptor on one small-cell and one non-small-cell line and alpha-type receptor mRNA expression on one small-cell-cancer cell line, only the beta receptors of the non-small-cell line (H-157) proved to be functional. Thus, the cell line H-157 displayed specific binding of 125I-PDGF-BB in addition to mRNA expression of the 6-kb transcript for the PDGF beta type receptor. Further evidence for the presence of functional PDGF beta receptors in H-157 cells was obtained from an in vitro kinase assay, which demonstrated a ligand-induced receptor autophosphorylation as well as the phosphorylation of a number of potential substrates associated with the activated receptor.

Carcinoma, Non-Small-Cell Lung↗
PubMed1993

Platelet-derived growth factor (PDGF) in oncogenesis: development of a vascular connective tissue stroma in xenotransplanted human melanoma producing PDGF-BB.

Human WM9 melanoma cells, previously shown to be devoid of PDGF expression, were stably transfected with a PDGF-B cDNA under the transcriptional control of a cytomegalovirus promoter. Northern blot analysis revealed high expression of an mRNA of the expected size in the PDGF-B-transfected cells. Synthesis and secretion of PDGF-BB was confirmed by immunoprecipitation. Furthermore, conditioned medium from PDGF-B-transfected cells contained a mitogenic activity for fibroblasts. For analysis of tumor growth in vivo, cells of each type were injected subcutaneously into BALB/c nu/nu mice. Tumors from mice injected with WM9 cells transfected with the vector only contained large necrotic areas; only scant blood vessels with narrow lumina were observed. No connective tissue was present. In the tumors from PDGF-B-transfected WM9 cells, nests of tumor were divided by connective tissue septa. An abundance of blood vessels was observed in the connective tissue septa and within the tumor cell nests. There was a complete absence of necrosis in these tumors. The present results suggest that tumor-derived PDGF-BB is a potent mediator of connective tissue stroma formation. The connective tissue framework that is generated in response to PDGF-BB may form a solid support for newly formed blood vessels and, thereby, facilitate the formation of a functional vascular system in the tumor.

Animals↗
PubMed1993

Pool of ligand-bound platelet-derived growth factor beta-receptors remain activated and tyrosine phosphorylated after internalization.

We have examined the state of tyrosine phosphorylation of ligand-bound, internalized platelet-derived growth factor (PDGF) beta-receptors. Analysis by immunofluorescence staining of cells stimulated with PDGF-BB at 37 degrees C indicated colocalization of phosphotyrosine, PDGF beta-receptors, and PDGF-BB in endosome-like vesicles. Treatment of cells with an acidic buffer, which removed cell surface-bound PDGF-BB, led to a considerable decrease in phosphorylation and kinase activity of cell surface localized PDGF beta-receptors, but not of internalized receptors. Immunoprecipitations using antisera against phosphotyrosine and the PDGF beta-receptor from metabolically labeled cells showed that a major fraction of the tyrosine-phosphorylated pool of receptors were still accessible to the acid buffer treatment after 10 min of incubation of the cells at 37 degrees C. Under these conditions, about 20-25% of the total pool of tyrosine-phosphorylated receptors were intracellular, since they remained tyrosine phosphorylated after the acid buffer treatment. A considerable pool of tyrosine-phosphorylated, internalized receptors, after 10 min of incubation of the cells at 37 degrees C, could also be detected by immunoblotting analysis, using antisera against the PDGF beta-receptor and phosphotyrosine. Analysis by in vitro kinase assays of immunoprecipitated PDGF beta-receptors, obtained from PDGF-BB-stimulated cells different times after acid wash, showed that the internalized receptors retained kinase activity. These data suggest that a pool of internalized PDGF beta-receptors remain active and may participate in signalling a considerable time after internalization.

Animals↗
PubMed1993

Density-dependent inhibitory effect of transforming growth factor-beta 1 on human fibroblasts involves the down-regulation of platelet-derived growth factor alpha-receptors.

We have previously found that transforming growth factor-beta 1 (TGF-beta 1) inhibits the mitogenic activity of platelet-derived growth factor (PDGF) in cultures of human neonatal fibroblasts in a density-dependent fashion. In the present investigation we determined the effect of TGF-beta 1 on the PDGF alpha-receptor, which binds all PDGF isoforms, as well as on the beta-receptor, which binds only PDGF-BB with high affinity. We found that the inhibitory effect of TGF-beta 1 on PDGF-AA-induced mitogenesis was density-dependent; when dense cell cultures were preincubated with TGF-beta 1, there was an complete inhibition of 3H-thymidine incorporation, whereas the effect was less in sparse cultures. A similar density-dependent effect of TGF-beta 1 was seen in PDGF-BB treated cells, although less pronounced. The binding of 125I-labeled PDGF-AA and PDGF-BB to the alpha-receptor was significantly reduced after treatment with TGF-beta 1 in dense cultures, whereas the sparse cultures were less affected. A decrease of alpha-receptor mRNA was also seen. The levels of beta-receptor protein and mRNA were unaffected. We conclude that the growth inhibitory effect of TGF-beta 1 is cell density-dependent and involves down-regulation of PDGF alpha-receptors.

Cell Count↗
PubMed1993

Negative trans-acting mechanisms controlling expression of platelet-derived growth factor A and B MRNA in somatic cell hybrids.

We have studied a human malignant melanoma cell line (WM115) which expresses high levels of PDGFA and PDGFB mRNAs, a hamster fibroblast cell line (Wq3H) which lacks PDGFB and has a very low expression of PDGFA mRNA, and hybrids between these cell lines. The PDGFA and PDGFB mRNA expression in the hybrid cells was markedly suppressed, indicating that the melanoma PDGF genes are controlled in the hybrid cells by negative factors/mechanisms contributed by the fibroblast genome. The melanoma but not the fibroblast PDGFB mRNA was induced by cycloheximide in the hybrid cells, indicating that the fibroblast and the melanoma PDGFB genes are regulated differently despite their presence in the same intracellular environment.

Animals↗
PubMed1993

Differential expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase in human lung carcinoma cell lines.

In the present investigation we have studied the expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) in ten different human lung carcinoma cell lines, four small cell carcinomas and six non-small cell carcinomas. None of the small-cell lung carcinoma cell lines demonstrated expression of PD-ECGF/TP mRNA. However, four of six of the non-small cell carcinoma cell lines expressed the 1.8 kb PD-ECGF/TP transcript. The cell lines derived from the single squamous cell carcinoma and the two adenocarcinomas expressed the PD-ECGF/TP mRNA, and were found to have the corresponding protein both in cell lysates and conditioned media as determined both by immunoblotting and measurement of thymidine phosphorylase activity. Only one of three studied large cell carcinoma cell lines expressed low levels of PD-ECGF/TP mRNA, but the corresponding PD-ECGF/TP protein was not demonstrated by immunoblotting.

Carcinoma, Non-Small-Cell Lung↗
PubMed1993

Reversion of autocrine transformation by a dominant negative platelet-derived growth factor mutant.

A non-receptor-binding mutant of the platelet-derived growth factor (PDGF) A chain, PDGF-0, was generated by exchanging 7 amino acids in the sequence. The mutant chains formed dimers that were similar to wild-type PDGF-AA with regard to stability and rate of processing to the mature 30-kDa secreted forms. Moreover, the mutant chains formed disulfide-bonded heterodimers with the PDGF B chain in NIH 3T3 cells heterodimer underwent the same processing and secretion as PDGF-AB. Transfection of c-sis-expressing 3T3 cells with PDGF-0 significantly inhibited the transformed phenotype of these cells, as determined by the following criteria. (i) Compared with PDGF-0-negative clones, PDGF-0-producing clones showed a reverted morphology. (ii) Clones producing PDGF-0 grew more slowly than PDGF-0-negative clones, with a fivefold difference in cell number after 14 days in culture. (iii) The expression of PDGF-0 completely inhibited the ability of the c-sis-expressing 3T3 cells to form colonies in soft agar; this inhibition was overcome by the addition of recombinant PDGF-BB to the culture medium, showing that the lack of colony formation of these cells was not due to a general unresponsiveness to PDGF. The specific expression of a PDGF-0/PDGF wild-type heterodimer in COS cells revealed that the affinity of the mutant heterodimer for the PDGF alpha receptor was decreased by approximately 50-fold compared with that of PDGF-AA. Thus, we show that a non-receptor-binding PDGF A-chain mutant neutralizes in a trans-dominant manner the autocrine transforming potential of the c-sis/PDGF B chain by forming low-affinity heterodimers with wild-type PDGF chains. This method of specifically antagonizing the effect of PDGF may be useful in investigations of the role of PDGF in normal and pathological conditions.

3T3 Cells↗
PubMed1993

Platelet-derived growth factor. Structure, function and implications in normal and malignant cell growth.

Platelet-derived growth factor (PDGF) is a potent mitogen for a variety of cell types. PDGF is made up as dimers of A and B polypeptide chains which are combined to generate the three isoforms of PDGF (AA, AB, BB). These bind with different specificities and affinities to two types of cell surface receptors (the alpha-receptor and the beta-receptor), both being members of the protein tyrosine kinase family of growth factor receptors. A number of human tumor cell lines, particularly those established from glioma and sarcoma, have been shown to produce PDGF and express the cognate receptor type. In these instances, tumor cell growth may be enhanced by an autocrine receptor activation. In other tumor cell types, where PDGF is produced in the absence of receptor expression, the growth factor may act in a paracrine fashion. This view is supported by our recent finding that human melanoma cells that have been stably transfected with a PDGF B-chain cDNA, elicit a stroma response when transplanted to nude mice.

Cell Division↗
PubMed1993

Constitutive and inducible expression of PDGF in the human basophilic cell line, KU 812.

The human basophilic cell line KU 812, that also has some mast cell characteristics, was found to express the PDGF-A gene and secrete PDGF-A like activity. After treatment with IL-6+ TNF-alpha, the PDGF-A mRNA expression increased as did cytoplasmic immunostaining with anti-PDGF antibodies. Secretion of PDGF-A was visualized by immunoprecipitation. An augmentation of non-secreted PDGF-like activity after IL-6+ TNF-alpha treatment was not accompanied by induction of the long splice variant of the PDGF-A-chain mRNA. Treatment with TPA caused an increase in PDGF-A expression and in addition, an induction of PDGF-B transcripts were seen. Staining of cytospin preparations with anti-PDGF antibodies visualized a substantial increase in immunostaining of the TPA treated cells and both intracellular and secreted PDGF-AA-like activity was substantially increased as compared to untreated control cultures. There was a concomitant induction of exon 6 specific mRNA, corresponding to a cellular retention signal after TPA treatment. Our results show that PDGF can be produced by a cell line of the basophilic/mast cell lineage, i.e. cells involved in allergic disorders and inflammation.

Basophils↗