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Biomedical subjects

B Westermark

Publications and source records attributed to B Westermark.

At least 37 records · Page 2Linked to original sources
PubMed1997

Cell scattering and migration induced by autocrine transforming growth factor alpha in human glioma cells in vitro.

In the present investigation, we have transfected a human malignant glioma cell line, U-1242 MG, and derived clones that produce transforming growth factor alpha (TGF-alpha) in an inducible manner using the tetracycline suppressible vector system. TGF-alpha expression was confirmed by Northern analysis, by ELISA, and by immunoprecipitation of metabolically labeled cells. The functional activity of the induced protein was proven by the finding of epidermal growth factor receptor (EGFR) tyrosine phosphorylation on induction of TGF-alpha. A clear effect on cell motility, i.e., cell scattering and an increased phagokinetic track area of individual glioma cells, was demonstrated. The fact that the EGFR tyrosine kinase activation was independent of cell density suggests that autocrine activation of the EGFR kinase occurred at the single-cell level. These findings are of interest, because increased cell motility is most likely a requirement for glioma cell invasion in vivo. The results imply that as a result of coexpression of EGFR and its ligand, individual glioma cells are capable of acting as independent autocrine locomotory units.

Antibodies, Monoclonal↗
PubMed1997

Induction of senescence in human malignant glioma cells by p16INK4A.

p16INK4A is a G1-specific cell cycle inhibitor which maps to human chromosome 9p21, a region frequently mutated or deleted in cancer cell lines and primary tumors. In glioblastomas the frequency of homozygous deletions is 40-70% making it one of the most common mutations in this tumor type. We have analysed the significance of the loss of this gene in gliomas by introducing the cDNA for p16INK4A into the human glioma cell line U-1242 MG which has a deleted CDKN2 locus. We used the tetracycline repressible vector system and obtained two stably transfected clones that expressed p16INK4A upon induction. p16INK4A expression caused a G1 arrest and enlargement of the cells similar to that of senescent cells. When staining for Senescence-Associated beta-galactosidase activity, described to be specific for senescent cells, we could show that the enlarged cells specifically gave a positive staining reaction. This senescence phenotype was dependent on the continuous expression of p16INK4A since it was reversed upon reintroduction of tetracycline suppression. Thus, the induced expression of p16INK4A in these glioma cells reverted their immortal phenotype and caused an immediate cellular senescence.

Apoptosis↗
PubMed1997

Localization of the prostaglandin F2 alpha receptor in rat tissues.

The localization of the prostaglandin F2alpha (FP) receptor was examined in rat tissues by immunohistochemistry and in situ hybridization. Immunohistochemistry on paraffin sections was performed with a rabbit polyclonal antiserum raised against a synthetic peptide derived from the rat FP receptor sequence. In situ hybridization on cryosections was done with 35S-labelled rat FP receptor antisense and sense riboprobes. The most intense FP receptor-like immunoreactivity was observed in granulosa luteal cells, muscle and epithelial cells, e.g. cardiac, skeletal and smooth muscle, and hepatocytes. Weaker immunoreactivity was found in connective tissue fibroblasts. In the eye, intense immunostaining was associated with the corneal and conjunctival epithelium and moderate staining with the ciliary body, retina, iris and connective tissues. In situ hybridization generally confirmed the results. The riboprobe hybridized weakly with the heart, skeletal muscle, uterus, liver, lung and corpus luteum. Thus, the prostaglandin FP receptor was found to be widely distributed in rat tissues.

Animals↗
PubMed1996

Production of cell-associated PDGF-AA by a human sarcoma cell line: evidence for a latent autocrine effect.

The alternative splicing of platelet-derived growth factor A-chain is known to result in 2 different protein products. One variant is encoded by transcripts containing the 69 nts representing exon 6 (PDGF-AA(L)), and one variant is encoded by transcripts in which exon 6 is excluded (PDGF-AA(S). Transfection assays have suggested that the long splice variant of the A-chain is mainly associated with membrane- and matrix-associated heparan sulphate proteoglycans, whereas the shorter variant is soluble. We describe a human sarcoma cell line (U-2197) that expresses a high level of PDGF-A transcripts. Immunoprecipitations revealed cell-associated protein products of mainly 24, 28 and 33 kDa and less abundant forms of 40-45 kDa, while no PDGF was found in the medium. Analysis of extracellular medium in a radioreceptor assay confirmed that PDGF was not secreted by the U-2197 cells. The addition to U-2197 cultures of a carboxy terminal peptide that specifically competes with the binding of the long splice variant of PDGF-AA to extracellular matrix and cell membranes resulted in the release of 3 PDGF-AA-specific dimeric proteins with molecular masses of 33, 37 and 45 kDa. Furthermore, polymerase chain reaction studies discriminating between the long and the short splice variants of the PDGF-A transcripts revealed that U-2197 expressed relatively higher amounts of the long splice variant compared with U-343 MGa Cl 2:6, which is known to secrete PDGF-AA. These cell-associated forms of PDGF, released to the medium by adding carboxy terminal peptide, increased the tyrosine kinase activity of the endogenous PDGF alpha-receptor.

Aged↗
PubMed1996

Decreased growth rate and tumour formation of human anaplastic thyroid carcinoma cells transfected with a human thyrotropin receptor cDNA in NMRI nude mice treated with propylthiouracil.

The effect of the human TSH-receptor (TSHR) on the growth of human anaplastic thyroid carcinoma cells lacking the endogenous expression of TSHR, was studied both in vitro and in vivo in NMRI nude mice. Cells from a human anaplastic thyroid carcinoma cell line (C643) were transfected with a TSHR cDNA, and clones were isolated after neomycin selection. The expression of a functional receptor protein was ensured by analysis of the specific binding of 125I-TSH and measurement of TSH-induced cAMP. Incorporation of [3H]thymidine and increase in cell number was slightly inhibited by TSH in TSHR-expressing cells in vitro. In order to investigate whether the regained expression of a functional TSHR protein in C643 cells could influence the in vivo growth, cells were injected subcutaneously into NMRI nude mice. To manipulate the endogenous level of TSH, animals were given 6n-propyl-2-thiouracil (PTU; resulting in a high TSH level), T4 (a low TSH level) or no treatment (as a control). There seemed to be a TSH induced inhibition of tumour growth, since tumours in mice treated with PTU grew after a longer take rate and with a slower growth rate. The present results suggest a TSH-mediated growth inhibition in the TSHR-transfected C 643 anaplastic thyroid carcinoma cells.

Animals↗
PubMed1996

Escape from senescence in hybrid cell clones involves deletions of two regions located on human chromosome 1q.

Human normal cells have been shown to undergo a limited number of cell doublings, a phenomenon termed cellular senescence. Human chromosome 1 has been implicated in this process, and several lines of evidence indicate that there is a senescence-inducing gene or genes on human chromosome 1q. Our approach to analyze the senescence-inducing effect of chromosome 1 includes the use of somatic cell hybrid revertants. We show here that fusion of a hypoxanthine phosphoribosyl transferase-negative mouse cell line (A9) containing a human neo-tagged chromosome 1 with an immortal hamster cell line (10W-2) results in cell hybrids that senesce after a few population doublings. Rare revertants that had escaped senescence were obtained after one large fusion experiment. Thirty-five nonsenescent hybrids were obtained from a total of approximately 1 million hybrids, and 25 of these were subjected to further analysis. The presence of a single copy of human chromosome 1 in the revertant hybrids was confirmed by fluorescence in situ hybridization analysis using a chromosome 1-specific painting probe. No visible translocations or deletions of chromosome 1 were observed in any of the hybrids. Deletion mapping revealed that 11 (56%) of the hybrids analyzed had lost one or more markers on chromosome 1q. Two regions with deletions were detected, one of which has been shown to be implicated in the senescence-inducing effect exerted by chromosome 1 following monochromosome transfer (P. J. Vojta et al., manuscript submitted for publication). The present study suggests that two separate loci on human chromosome 1q may be of importance for the induction of senescence. Moreover, this set of nonsenescent revertants could be useful for future detailed analyses of the senescence-inducing loci.

Animals↗
PubMed1996

Association of loss of heterozygosity on chromosome 17p with high platelet-derived growth factor alpha receptor expression in human malignant gliomas.

The aim of this study was to examine platelet-derived growth factor alpha receptor (PDGFR-alpha) expression in gliomas of various degrees of malignancy and to correlate the findings with genetic alterations present in the same tumor samples. We analyzed 83 tumors by in situ hybridization using a PDGFR-alpha cRNA probe. Increased PDGFR-alpha mRNA expression was observed in astrocytic tumors of all stages of malignancy, although the highest levels were found in glioblastoma multiforme. To evaluate the frequency of PDGFR-alpha gene amplification, differential PCR requiring less DNA than Southern analysis was used with fluorescence-labeled primers corresponding to the kinase insert region of the PDGFR-alpha. Only 7 of 43 glioblastomas and none of the other tumors tested showed amplification of the PDGFR-alpha gene, suggesting that a mechanism other than gene amplification is responsible for the overexpression of PDGFR-alpha in glial brain tumors. Comparison of the in situ hybridization data with genetic alterations in the same tumor material showed a significant correlation of loss of heterozygosity on chromosome 17p (Fisher's exact, P < 0.0002) with high expression levels of PDGFR-alpha. Because that was the case in both low- and high-grade astrocytomas, our data imply that PDGFR-alpha is actively involved in tumor cell proliferation in early and late stages of glioma development. The association of PDGFR-alpha expression with a distinct subset of glioblastomas characterized by loss of heterozygosity 17p further supports the differentiation of these tumors into molecular variants.

Base Sequence↗
PubMed1996

Suppression of platelet-derived growth factor alpha- and beta-receptor mRNA levels in human fibroblasts by SV40 T/t antigen.

It is known that down-regulation of cell surface platelet-derived growth factor (PDGF) receptors accompanies transformation by SV40. In this work human embryonic lung fibroblasts were used as a model system to study the effects of SV 40 on PDGF receptor expression. It is shown that transformation by SV 40 early region leads to a total loss of PDGF alpha-receptor and partial loss of beta-receptor mRNA. Microinjection experiments revealed that receptor down-regulation was a primary effect, and not only secondary to transformation and clonal selection. Total loss of PDGF alpha-receptor expression requires both large T and small t, and down-regulation of the PDGF alpha-receptor occurs independently of p53 and Rb binding to large T.

Antigens, Polyomavirus Transforming↗
PubMed1995

Detection of TP53 gene mutation in human meningiomas: a study using immunohistochemistry, polymerase chain reaction/single-strand conformation polymorphism and DNA sequencing techniques on paraffin-embedded samples.

Mutations in the TP53 tumor suppressor gene have been studied in different types of brain tumors. Little is known about this genetic event in human meningioma, a mostly benign tumor. To investigate the frequency of TP53 gene mutations in human tumors derived from meningeal tissues, paraffin-embedded tissues from 30 cases (including 2 malignant and 4 atypical meningiomas, as well as 2 hemangioblastomas and 3 hemangiopericytomas) were screened by immunohistochemistry. Polymerase chain reaction/single strand conformational polymorphism (PCR/SSCP) and direct DNA sequencing were thereafter performed in selected cases. Nuclear p53 staining was not seen in any of the 19 benign meningiomas tested, while atypical meningiomas, hemangioblastomas, and hemangiopericytomas displayed nuclear staining in a subpopulation of tumor cells in 4 out of 5, 2 out of 2, and 3 out of 3 cases, respectively. One malignant meningioma showed an intense nuclear staining and a band shift in SSCP. In this case, we identified a mutation in the TP53 gene at codon 161 changing GCC to ACC and resulting in an alteration of alanine to threonine in this position. Our results indicate that TP53 gene mutation may be considered as a marker for malignant transformation in meningioma. p53 immunoreactivity, even in the absence of detectable gene mutation, is also associated with atypia and does not appear in regular benign meningiomas.

Adult↗
PubMed1995

[Platelet-derived growth factor. A growth factor of great physiopathological importance].

Platelet-derived growth factor (PDGF) is a 30 kilodalton dimeric peptide comprising A and B chains. The two types of PDGF receptors (alpha and beta) that have been identified belong to the protein-tyrosine kinase family of growth factor receptors. PDGF is a potent mitogen for a variety of cell types, and in gene knock-out experiments has been shown to be required for the development of inter alia mesangial cells of renal glomeruli and smooth muscle cells of lung alveolar septa. PDGF is believed to fulfil important autocrine and paracrine growth-related functions, and its overproduction has been recognised in various malignant tumours and in non-neoplastic hyperproliferative disorders.

Arteriosclerosis↗
PubMed1995

Compartmentalization of autocrine signal transduction pathways in Sis-transformed NIH 3T3 cells.

The transforming protein of simian sarcoma virus is homologous to the platelet-derived growth factor (PDGF) B-chain. Fibroblasts transformed with simian sarcoma virus constitutively produce a growth factor that stimulates the endogenous tyrosine kinase of PDGF receptors in an autocrine manner. Autophosphorylation of PDGF receptors upon ligand stimulation provides binding sites for Src homology 2 domains of intracellular signaling molecules, which thereby become activated. We have characterized the PDGF receptor-mediated signal transduction in NIH 3T3 cells transformed with a PDGF B-chain cDNA (Sis 3T3 cells) in the absence and presence of suramin, a polyanionic compound that quenches PDGF-induced mitogenicity and reverts the transformed phenotype of the Sis 3T3 cells. Our data show that in the presence of suramin the general level of tyrosine phosphorylation was decreased. Nevertheless, autophosphorylated receptors complexed with substrates persisted in the cells. Suramin had no effect on activation of phosphatidylinositol 3'-kinase or on tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein of Ras. On the other hand, kinase activation of Src and Raf-1, phosphorylation of protein-tyrosine phosphatase 1D/Syp and Shc, and complex formation with Grb2 were greatly diminished by suramin. A possible explanation for our findings is that different PDGF receptor-coupled signaling pathways are active in different structural or functional compartments in the cell. Those pathways that are not affected by suramin might elicit distinct cellular responses, which are not sufficient for growth and transformation.

3T3 Cells↗
PubMed1995

Mice lacking glial fibrillary acidic protein display astrocytes devoid of intermediate filaments but develop and reproduce normally.

Glial fibrillary acidic protein (GFAP) is the main component of the intermediate filaments in cells of astroglial lineage, including astrocytes in the CNS, nonmyelin forming Schwann cells and enteric glia. To address the function of GFAP in vivo, we have disrupted the GFAP gene in mice via targeted mutation in embryonic stem cells. Mice lacking GFAP developed normally, reached adulthood and reproduced. We did not find any abnormalities in the histological architecture of the CNS, in their behavior, motility, memory, blood-brain barrier function, myenteric plexi histology or intestinal peristaltic movement. Comparisons between GFAP and S-100 immunohistochemical staining patterns in the hippocampus of wild-type and mutant mice suggested a normal abundance of astrocytes in GFAP-negative mice, however, in contrast to wild-types, GFAP-negative astrocytes of the hippocampus and in the white matter of the spinal cord were completely lacking intermediate filaments. This shows that the loss of GFAP intermediate filaments is not compensated for by the up-regulation of other intermediate filament proteins, such as vimentin. The GFAP-negative mice displayed post-traumatic reactive gliosis, which suggests that GFAP up-regulation, a hallmark of reactive gliosis, is not an obligatory requirement for this process.

Animals↗
PubMed1995

Platelet-derived growth factor in human glioma.

Platelet-derived growth factor (PDGF) is a 30 kDa protein consisting of disulfide-bonded dimers of A- and B-chains. PDGF receptors are of two types, alpha- and beta-receptors, which are members of the protein-tyrosine kinase family of receptors. The receptors are activated by ligand-induced dimerization, whereby the receptors become phosphorylated on tyrosine residues. These form attachment sites for signalling molecules, which inter alia activate the Ras.Raf pathway. PDGF has important functions in development and is required for a proper timing of oligodendrocyte differentiation. The v-sis oncogene of simian sarcoma virus (SSV) is a retroviral homolog of the B-chain gene, and induces transformation by an autocrine activation of PDGF receptors at the cell surface. SSV induces malignant glioma in experimental animals, suggesting a role for autocrine PDGF in glioma development. PDGF and PDGF receptors are frequently coexpressed in human glioma cell lines. Specific and nonspecific PDGF antagonists block the growth of some glioma cell lines in vitro and in vivo, suggesting that autocrine PDGF is involved in transformation and tumorigenesis. In situ studies of human gliomas show overexpression of alpha-receptors in glioma cells of high-grade tumors. In a few cases, overexpression is caused by receptor amplification. Since high-grade glioma cells also express the PDGF A-chain, an autocrine activation of the alpha-receptor may drive the proliferation of glioma cells in vivo.

Brain Neoplasms↗
PubMed1995

Transforming growth factor-beta promotes epidermal growth factor-induced thyroid cell migration and follicle neoformation in collagen gel separable from cell proliferation.

The regulation of growth and migration of thyroid follicular cells by epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta 1) were studied in primary cultures of porcine thyroid follicles embedded in collagen gel. Cultures were exposed to growth factors and [3H]thymidine (1 microCi/ml) for 3 days and then examined by light microscopic autoradiography. EGF at 1 ng/ml increased the [3H]thymidine labeling index from approximately 1% (control value) to 60% and stimulated fivefold the number of cells invading the collagen matrix. Intermediate responses were seen after treatment with 0.1 ng/ml of EGF. EGF-stimulated [3H]thymidine incorporation was reduced by TGF-beta 1 at concentrations above 0.1 ng/ml. In contrast, TGF-beta 1, which alone only had a minor stimulatory effect on cell motility, markedly promoted the migratory response to EGF (1 ng/ml). Thus, 0.1 ng/ml TGF-beta 1 doubled the fraction of migrating cells without changing the level of [3H]thymidine incorporation. Moreover, cell migration was still fourfold over control values in cultures exposed to 1.0 ng/ml EGF and 1.0 ng/ml TGF-beta 1, despite a strongly inhibited [3H]thymidine labeling. The number of microfollicles located peripherally to the mother follicles was increased synergistically by EGF and TGF-beta 1. The epithelium of mother follicles became grossly discontinuous in regions of intense cell migration induced by EGF and TGF-beta 1 in combination. In conclusion, TGF-beta 1 modulates the response of porcine thyrocytes to EGF in collagen gel cultures by promoting cell migration along with inhibition of [3H]thymidine incorporation. This suggests that EGF stimulates cell motility independent of the mitogenic signal. The persistent loss of epithelial integrity during enhanced cell migration indicates that mechanisms of intercellular adhesiveness are down-regulated by TGF-beta 1 and EGF in cooperation.

Animals↗
PubMed1995

PDGF and its receptors following facial nerve axotomy in rats: expression in neurons and surrounding glia.

We investigated the expression of platelet-derived growth factor (PDGF) and its receptors in rat facial nuclei following axotomy by in situ hybridization and immunohistochemistry. Facial nuclei were examined on days 3, 6, 12, 19 and 26 postoperatively (p.o.). Strong immunoreactivity for PDGF was found in facial neurons and surrounding astrocytes on the ipsilateral side of the brainstem already after 3 days p.o. and persisted at a high level until day 26 p.o. in rats with a facial nerve cut injury. After crushing of the facial nerve, a similar increase was seen in PDGF immunoreactivity which, however, decreased after day 19 p.o., when reinnervation had occurred. Reactive gliosis appeared on the operated side and was confirmed by an increase in intensity of GFAP staining. The kinetics of PDGF A-chain mRNA expression corresponded to the PDGF immunoreactivity, whereas the B-chain mRNA was present only in the neurons. The PDGF alpha-receptor immunoreactivity as well as the mRNA were detected in scattered glial cells. The density of the PDGF alpha-receptor mRNA expressing glial cells was higher on the injured side, but the intensity of the expression per cell did not change after axotomy. An increase in PDGF beta-receptor immunoreactivity was seen in the ipsilateral facial nuclei after 3-6 days p.o., however, the increase in the mRNA could not be detected. The staining persisted until day 26 p.o., when transected facial neurons showed heavier staining than those that had been crushed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Functional analysis of a variant of the thyrotropin receptor gene in a family with Graves' disease.

Nucleotide sequence analysis of PCR fragments corresponding to the TSH-receptor (TSHR) amplified from genomic DNA collected from the four members of a family, two of which had Graves' thyrotoxicosis, revealed a nucleotide substitution in the first position of codon 36 of the TSH-receptor gene in the two patients. The nucleotide substitution was from G to C, leading to a 36D-->36H change (D36H) in the predicted amino acid sequence of the receptor. The altered sequence was also found in DNA obtained from their mother, but not in DNA from their father. We stably expressed the two receptor variants in NIH 3T3 cells, by transfection of cDNA encoding the wildtype (WT) and D36H variants of the TSHR. Neither the binding of 125I-TSH nor the responsiveness to TSH measured as cAMP formation, appeared to be different in the TSHR-D36H compared to the TSHR-WT. Furthermore, the D36H-receptor also became desensitized when exposed to TSH as did the WT-receptor.

3T3 Cells↗
PubMed1995

Molecular genetics of human glioma.

This review focuses on genes that have a proven or presumed role in the genesis of astrocytic tumors. A common theme in glioblastoma is the amplification of genes that code for growth factor receptors of the protein-tyrosine kinase family (epidermal growth factor receptor, platelet-derived growth factor receptor-alpha, met). The majority of glioblastomas also have alterations in genes that encode factors that are involved in cyclin-dependent kinase activity, which is a critical step in G1-S transition in the cell cycle. These alterations include deletions of negative regulatory elements (TP53, CDKN2, MTS2) and amplification of positive factors (MDM2, CDK4). In addition, there are loci on chromosomes 10 and 19q that seem to be involved in tumor progression.

Animals↗