Biomedical subjects

P Heldin

Publications and source records attributed to P Heldin.

34 records · Page 2Linked to original sources

PubMed1995

Functional hyaluronan receptors are expressed on a squamous cell lung carcinoma cell line but not on other lung carcinoma cell lines.

We investigated the production of hyaluronan and the presence of hyaluronan receptors in a panel of human lung carcinoma cell lines, consisting of small cell carcinomas (SCLC) and non-small cell carcinomas (non-SCLC). These transformed cell lines produced only minute amounts of hyaluronan, whereas normal lung fibroblasts synthesized high amounts. CD44 molecules (an integral membrane glycoprotein suggested previously to function as a hyaluronan receptor) were differentially expressed on non-SCLC cell lines but essentially not on the SCLC cell lines. In contrast, RHAMM molecules (receptor for hyaluronan-mediated motility) were preferentially expressed on SCLC cells. Although all the lung tumor cell lines expressed various amounts of CD44 and RHAMM, only the SCLC line U-1752 could bind [3H]hyaluronan. The binding sites were saturated with about 19,700 hyaluronan molecules (Mr 1.4 x 10(6)) bound per cell with a Kd of 0.16 x 10(-9) M. CD44 molecules were responsible for the binding activity since Hermes-1 antibodies that block the binding of hyaluronan to CD44 blocked the binding of [3H]hyaluronan to U-1752 cells. 4-Phorbol 12-myristate 13-acetate (PMA) treatment of U-1752 cells both increased the hyaluronan-binding activity in U-1752 cells as well as induced abrogation of cell-cell and cell-matrix interactions. Addition of hyaluronan inhibited the PMA-induced disassembly of the cells. The fact that CD44 molecules are able to bind [3H]hyaluronan only on the SCLC line U-1752 but not on other lung carcinoma cell lines may be of value as a marker for squamous cell carcinoma differentiation. Furthermore, the inhibitory effect of hyaluronan on the PMA-promoted cell disassembly suggest that hyaluronan surrounding squamous cell carcinoma cells may affect their migration and invasiveness.

Carcinoma, Non-Small-Cell Lung↗

PubMed1995

Stimulation of hyaluronan biosynthesis by platelet-derived growth factor-BB and transforming growth factor-beta 1 involves activation of protein kinase C.

The intracellular signal transduction pathways that mediate the stimulatory effects of platelet-derived growth factor (PDGF)-BB and transforming growth factor (TGF)-beta on hyaluronan biosynthesis in human fibroblasts were investigated. The stimulatory effects of both PDGF-BB and TGF-beta 1 were dependent on protein kinase C (PKC), since the PKC inhibitor calphostin C inhibited the stimulation by the growth factors. Direct activation of PKC by phorbol 12-myristate 13-acetate (PMA) also stimulated hyaluronan production, and the combination of either PDGF-BB or TGF-beta 1 and PMA gave an increased effect. One possible mechanism for activation of PKC is via induction of phospholipase C (PLC) activity; U-17322, an inhibitor of PLC-gamma, was found to inhibit partially PDGF-BB-stimulated hyaluronan synthesis. PDGF-BB is known to activate PLC-gamma through tyrosine phosphorylation; however, a PDGF beta-receptor mutant unable to interact with and activate PLC-gamma was still able to mediate induction of hyaluronan biosynthesis, indicating that PDGF-mediated stimulation is not entirely dependent on PLC-gamma. The stimulations by PDGF-BB and TGF-beta 1 were partly dependent on protein synthesis, since parts of the effects were inhibited by cycloheximide; in contrast, the effects mediated by PMA were not. Our results indicate that PKC is involved in the transduction of the effects of growth factors on hyaluronan biosynthesis, and that the effects involve direct or indirect activation of existing hyaluronan synthetase molecules, as well as induction of new enzyme molecules.

Becaplermin↗

PubMed1995

Chondroitin sulfate proteoglycan modulates the permeability of hyaluronan-containing coats around normal human mesothelial cells.

The composition and permeability of the pericellular coat surrounding normal human mesothelial (NHM) cells have been studied in vitro. NHM cells were grown in the presence of 3H-glucosamine and the amount of label recovered in hyaluronan and chondroitin sulfate was determined after selective enzymatic digestion of the polysaccharides in medium, pericellular, and intracellular pools. For comparison a similar analysis was carried out on mesothelioma cells (Mero-14). Of the labeled polysaccharides in the medium and pericellular pools of NHM cells about 80-90% could be ascribed to hyaluronan and only 3-5% to chondroitin sulfate. In contrast, Mero-14 synthesized only minute amounts of hyaluronan whereas chondroitin sulfate corresponded to 61% of the total glycosaminoglycans in the culture. The results exclude a structure of the pericellular layer of NHM cells similar to the hyaluronan-proteoglycan aggregates found in cartilage. The permeability of the pericellular layer was tested by the exclusion of polystyrene microspheres and bacteria of diameter 0.1-3.0 microns, as well as erythrocytes of diameter 7 microns. While the erythrocytes were excluded the smaller particles penetrated the coat. By adding 0.5 mg/ml of aggregating cartilage proteoglycan to the medium particles of 0.3 microns or larger were also excluded. Thus exogenous proteoglycans can reinforce the structure of the pericellular layer.

Bacterial Physiological Phenomena↗

PubMed1995

Characterization of the mechanism involved in bleomycin-induced increased hyaluronan production in rat lung.

The molecular mechanisms behind the accumulation of hyaluronan during bleomycin-induced lung injury in rats were investigated. The stimulatory effects of bronchoalveolar lavage fluid (BALF) and alveolar macrophage (AM)-conditioned media on hyaluronan synthesis in normal rat lung fibroblast cultures were studied as well as the hyaluronan binding activity on AM. BALF obtained on days 1 and 5 after bleomycin instillation exhibited hyaluronan stimulatory activity similar to that of 10% fetal serum; the activity returned to control values on day 14 after bleomycin treatment. Conditioned media from cultures of AM obtained from bleomycin-treated rats exhibited stimulatory effects higher than that of media from AM of control rats and equal to or higher than that of 10% fetal calf serum. The stimulatory activity in BALF was significantly inhibited by neutralizing antibodies against transforming growth factor-beta; the activity in AM-conditioned media was only partially affected. Neutralizing antibodies against platelet-derived growth factor-BB or -AA had no such inhibiting effect. Interestingly, AM from bleomycin-treated rats exhibited low hyaluronan binding activity. [3H]Hyaluronan binding by AM on days 1 and 5 after bleomycin administration was about 2-fold and 4-fold lower, respectively, compared with that by AM derived from saline-treated rats. This decrease was normalized 14 days after bleomycin treatment. In conclusion, our results indicate that factors with high potential to stimulate hyaluronan synthesis in rat lung fibroblasts are accumulated in BALF from bleomycin-treated rats and that AM are likely to be one source of such stimulatory factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Hyaluronan receptors are expressed on human malignant mesothelioma cells but not on normal mesothelial cells.

Hyaluronan-binding sites were demonstrated on the cell surface of three malignant mesothelioma cell lines derived from human tumors using either [3H]hyaluronan or fluorescein-tagged hyaluronan. No hyaluronan-binding activity was observed on normal human mesothelial cells. The absence of hyaluronan receptors on normal human mesothelial cells was not due to a down-regulation by endogenously synthesized hyaluronan, since no binding sites appeared when the cells were cultured under conditions known to suppress hyaluronan synthesis (in starvation medium containing either hydrocortisone or n-butyrate) or to degrade endogenously synthesized hyaluronan (in the presence of Streptomyces or testicular hyaluronidase). The binding of [3H]hyaluronan on mesothelioma cells could be partially inhibited by prior incubation of the cells with trypsin, indicating that the hyaluronan-binding site is a protein. The binding sites on human malignant mesothelioma cells were shown to be saturable with about 54,000 hyaluronan molecules (M(r) 1.4 x 10(6)) bound per cell with a Kd of 0.3 x 10(-9) M. The binding was specific for hyaluronan inasmuch as a number of other macromolecules gave negligible inhibition of the binding. High molecular weight preparations of hyaluronan inhibited the binding more effectively than low molecular weight preparations; hyaluronan oligosaccharides down to a length of six monosaccharide units showed competing activity. The hyaluronan receptor appeared to be related to CD44 (a cell surface glycoprotein previously suggested to function as a hyaluronan receptor) since Hermes-1 monoclonal antibodies which inhibit the binding of hyaluronan to CD44 blocked a major part of the binding of hyaluronan to the mesothelioma cells. However, there was no strict correlation between the hyaluronan-binding activity on the mesothelioma cell lines tested and the levels of CD44 molecules on their cell surface, suggesting that only a subfraction of the CD44 molecules bound hyaluronan or that other hyaluronan-binding proteins also exist on those cells. The presence of hyaluronan receptors on mesothelioma cells, but not on their normal counterparts, may be of importance for the migration of the transformed cells in hyaluronan-enriched matrices and for their ability to form metastases.

Antibodies, Monoclonal↗

PubMed1993

Human mesothelioma cells produce factors that stimulate the production of hyaluronan by mesothelial cells and fibroblasts.

The hyaluronan production by three human malignant mesothelioma cell lines and nine primary human mesothelial cell types was determined. The mesothelioma cell lines produced only minute amounts of hyaluronan (less than 0.1 microgram/10(6) cells/48 h) whereas mesothelial cells synthesized large quantities of hyaluronan (10-72 micrograms/10(6) cells/48 h). Conditioned media from the mesothelioma cell lines were investigated for their ability to stimulate hyaluronan production by fibroblasts and mesothelial cells in vitro, and in all cases stimulatory effects were found. The factor(s) in the conditioned medium of the mesothelioma cell line Mero-25 that were responsible for hyaluronan stimulation were heat stable and partially trypsin resistant. The stimulatory activity was partially inhibited by an antiserum against platelet-derived growth factor and basic fibroblast growth factor. Our data suggest that the increased hyaluronan synthesis seen in patients with mesothelioma is due to the release of factors from mesothelioma cells that stimulate other cells to produce hyaluronan.

Culture Media↗

PubMed1993

Synthesis and assembly of the hyaluronan-containing coats around normal human mesothelial cells.

In this study we examined the capacity of normal human mesothelial (NHM) cells and human malignant mesothelioma cells to form hyaluronan-containing pericellular matrices or "coats." The assembly of the pericellular coats was visualized by a particle exclusion assay. We found that large hyaluronan-containing coats were formed around NHM cells whereas their transformed counterparts had no or very limited coats. The coats were removed by treatment with Streptomyces hyaluronidase, which specifically degrades hyaluronan. NHM cells exhibited hyaluronan-containing pericellular matrix within 5 h after seeding. The formation of the coats was stimulated by platelet-derived growth factor and epidermal growth factor. Interestingly, the assembly of the hyaluronan-dependent pericellular matrices was inhibited by the addition of hyaluronan dodecasaccharides. The inhibitory effect on the formation of the coats was due to a destabilization of pericellular matrix and not due to an inhibitory effect of hyaluronan dodecasaccharides on hyaluronan synthesis. In contrast, hyaluronan hexasaccharides, an inhibitor of the interaction between polymeric hyaluronan and its cell surface receptors, had no effect on the size of the coat. Thus, our results are compatible with the possibility that the pericellular matrix surrounding NHM cells consists of newly synthesized hyaluronan which is extruded from the cell and independent of hyaluronan receptors on the cell surface. The coat seems to be stabilized by interactions (hyaluronan-hyaluronan or hyaluronan-protein bridges) which can be prevented by hyaluronan dodecasaccharides.

Cell Transformation, Neoplastic↗

PubMed1992

Characterization of the molecular mechanism involved in the activation of hyaluronan synthetase by platelet-derived growth factor in human mesothelial cells.

The molecular mechanism involved in the stimulation of hyaluronan synthetase in normal human mesothelial cells was investigated. Exposure of mesothelial cells to platelet-derived growth factor (PDGF)-BB stimulated hyaluronan synthetase activity, measured in isolated membrane preparations, as well as hyaluronan secretion into the medium. The effect on hyaluronan synthetase was maximal after 6 h of treatment. In contrast, the stimulatory effect of transforming growth factor-beta 1 reached a maximum after 24 h. The stimulatory effect of PDGF-BB was inhibited by cycloheximide. The phosphotyrosine phosphatase inhibitor vanadate was found to stimulate hyaluronan synthetase activity, and to potentiate the effect of PDGF-BB. The protein kinase C (PKC) stimulator phorbol 12-myristate 13-acetate (PMA) also stimulated hyaluronan synthetase; furthermore, depletion of PKC by preincubation of the cells with PMA led to an inhibition of the PDGF-BB-induced stimulation of hyaluronan synthetase activity. Thus the PDGF-BB-induced stimulation of hyaluronan synthetase activity is dependent on protein synthesis and involves tyrosine phosphorylation and activation of PKC.

Cell Membrane↗

PubMed1991

Differential expression of platelet-derived growth factor alpha- and beta- receptors on fat-storing cells and endothelial cells of rat liver.

Fat-storing cells and endothelial cells of the liver sinusoids play important roles in the biosynthesis and degradation of hyaluronan (HYA). These cells were isolated from rat liver by a simple and rapid procedure involving: (1) cell separation by centrifugation on a Nycodenz gradient, after dispersion of the liver cells by collagenase perfusion; (2) further purification of the cells by centrifugation on a discontinuous Percoll gradient; and (3) culturing of the cells, taking advantage of the different time that seeded cells need for attachment to plastic surfaces. We have determined the effects of two isoforms of platelet-derived growth factor (PDGF), PDGF-BB and PDGF-AA, on HYA production by the original fat-storing cells, as well as by fat-storing cells which had changed in vitro to myofibroblast-like cells. PDGF-BB was found to stimulate HYA synthesis in both types of cells with a maximal response equal to that obtained with 10% fetal calf serum. PDGF-AA had no stimulatory effect on HYA production. Fat-storing cells and their modified myofibroblast-like phenotype bound specifically to 125I-PDGF-BB, but not to 125I-PDGF-AA, indicating that they had PDGF beta-receptors, but not alpha-receptors. In contrast, liver endothelial cells were found to have PDGF alpha-receptors, but not beta-receptors.

Animals↗

PubMed1989

Effect of growth factors on hyaluronan synthesis in cultured human fibroblasts.

The effect of various growth factors on the synthesis of hyaluronan in human fibroblasts was investigated. When tested in medium containing 0.5% fetal calf serum, platelet-derived growth factor (PDGF)-BB was found to stimulate hyaluronan synthesis; the maximal response was equal to or higher than that obtained with 10% fetal calf serum. PDGF-AA gave only a limited effect, indicating that the stimulatory effect of PDGF on hyaluronan synthesis was mainly transduced via the B-type PDGF receptor. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta 1 also stimulated hyaluronan synthesis; their effects were less than that of PDGF-BB, but combinations of factors produced potent stimulatory effects on hyaluronan synthesis. All factors stimulated hyaluronan synthesis in sparse as well as dense cultures. The effects of the factors on hyaluronan synthesis did not correlate with their mitogenic activities; PDGF-BB, EGF and bFGF are equipotent mitogens, but PDGF-BB had a much more potent effect on hyaluronan synthesis, and TGF-beta actually inhibits the growth of fibroblasts under the conditions of the assay.

Cells, Cultured↗

PubMed1987

Effect of phosphorylation in vitro of human fibrinogen with protein kinase C on thrombin-induced gelation.

Thrombin-induced gel formation of fibrinogen phosphorylated by protein kinase C yielded a transparent gel, whereas unphosphorylated fibrinogen yielded a coarse gel. The mass-length ratio was found to be one order of magnitude higher for the unphosphorylated than for the phosphorylated fibrinogen. Since the phosphorylated sites are located near the cross-linking sites in the A alpha-chain of fibrinogen, it is likely that the introduction of charged phosphate groups in this region prevent the lateral growth of the fibrin fibres.

Electrophoresis, Polyacrylamide Gel↗

PubMed1987

Phosphorylation of human fibrinogen in vitro with protein kinase C: characterization of the phosphorylated sites.

Phosphorylation of human fibrinogen in vitro by incubation with [gamma-32P]ATP and protein kinase C purified from pig spleen, led to incorporation of [32P]phosphate at serine residues located in the A alpha-chain. In order to identify the residues that were phosphorylated, the A alpha-chain of fibrinogen was isolated and subjected to consecutive cleavage by cyanogen bromide, trypsin, and chymotrypsin. The resulting radioactive phosphopeptides were purified by gel chromatography and high-performance liquid chromatography using a reversed-phase column. Subsequent amino acid analysis and manual Edman degradation of the purified phosphopeptides revealed that Ser557, Ser558, Ser559, and Ser599 were phosphorylated. These serine residues are located in the carboxy-terminal part of the A alpha-chain. This region also contains lysine residues participating in the cross-linking of fibrin and, possibly, a site involved in the binding of fibrinogen to receptors on platelets. In addition, peptides derived from the middle section of the polypeptide chain were found to contain [32P]phosphate; in these cases, however, the exact localization of the phosphate could not be determined, due to the low yield of radioactivity. Two glutamine residues, Gln328 and Gln366, in this portion of the A alpha-chain take part in the cross-linking of fibrin.

Amino Acids↗

PubMed1987

Phosphorylation in vitro of human fibrinogen with casein kinase TS and characterization of phosphorylated sites.

Human fibrinogen was phosphorylated by casein kinase TS. The [32P]phosphate incorporated varied between 0.5 and 1 mol of phosphate per mole of fibrinogen. The phosphate was localized to Ser523 and Ser590 and serine and threonine residues between amino acids 259 and 268 in the A alpha-chain. In addition, Thr416 and Ser420 were phosphorylated in the gamma'-chain, which is a variant of the gamma-chain, constituting 7-10% of the gamma-chain population. The functional significance of casein kinase TS-induced phosphorylation of fibrinogen remains unknown; however, a slight but consistent increase of the turbidity in a gelation assay was observed for phosphorylated compared to unphosphorylated fibrinogen.

Casein Kinases↗

PubMed1985

Phosphorylation in vitro of fibrinogen from three mammalian species with four different protein kinases.

Human, dog, and rabbit fibrinogen served as substrates for calcium-activated, phospholipid-dependent protein kinase, cAMP-dependent protein kinase, casein kinase TS, and casein kinase S. The chains of phosphorylated fibrinogen were separated by polyacrylamide gel electrophoresis and the phosphorylation patterns, obtained on autoradiography of the gels, were found to be characteristic for each of the four protein kinases. Dog, and even more so rabbit, fibrinogen was phosphorylated more rapidly than human fibrinogen by calcium-activated, phospholipid-dependent protein kinase and by casein kinase TS. Dog fibrinogen was not a good substrate for cAMP-dependent protein kinase. The rate of phosphorylation with casein kinase S did not differ very much between the fibrinogens of the three species. In most cases the A alpha-chain was most rapidly phosphorylated. However, in dog fibrinogen incubated with casein kinase TS the B beta-chain was most rapidly phosphorylated. A substantial part of this phosphate seemed to be incorporated as phosphorylthreonine into fibrinopeptide B. In human fibrinogen incubated with the casein kinase TS preparation the gamma-chain as well as the A alpha-chain appeared to be phosphorylated.

Animals↗

PubMed1984

Phosphorylation of human fibrinogen in vitro by calcium-activated phospholipid-dependent protein kinase from pig spleen.

Human plasma fibrinogen rapidly incorporated stoichiometric amounts of [32P]-phosphate when incubated with [32P]ATP and calcium-activated, phospholipid-dependent protein kinase purified from pig spleen. Half-maximal fibrinogen kinase activity was attained at less than 0.1 mM calcium acetate. The optimum concentration of phosphatidylserine was about 50 micrograms per ml. Diolein slightly potentiated the stimulatory effect of phosphatidylserine. The alpha-chain of fibrinogen, which is reported to contain endogenous phosphate (Blombäck, B., Blombäck, M., Edman, P., & Hessel, B. (1962) Nature 193, 883-884 and Doolittle, R.F., Watt, K.W.K., Cottrell, B.A., Strong, D.D., & Riley, M. (1979) Nature 280, 464-468) was phosphorylated by the protein kinase. The apparent Km value for the phosphorylation reaction (0.3-0.6 microM fibrinogen) was comparable with the Km values reported for the hitherto most effective substrate proteins for protein kinase C. Up to 5 mol phosphate per mol fibrinogen could be incorporated, indicating at least three phosphorylatable sites per half molecule.

Alkadienes↗

PubMedDate not supplied

Expression of the CD44 glycoprotein (lymphocyte-homing receptor) in untreated human breast cancer and its relationship to prognostic markers.

CD44 is a cell surface glycoprotein which has been suggested to be associated with aggressive histological features in breast cancer (BC). It has also been implicated in conferring metastatic potential to rat carcinoma cells. The aim of this study was to determine the potential value of CD44 as a prognostic/metastatic marker in BC by means of immunohistochemistry. The expression of the CD44 glycoprotein was investigated in tumours from 52 untreated female patients with BC, using the monoclonal antibody A3D8. 10 samples of normal breast tissue were randomly obtained and also investigated with respect to CD44 expression. DNA ploidy, the S-phase fraction (SPF) and oestrogen-(OR) and progesterone-receptor (PgR) contents in the tumours were determined and together with the prognostic markers of age, tumour size, tumour grade and lymph node status, correlated with CD44 expression in BC. Also, the distribution of CD44 tumour cell expression was compared with expression of the permeability drug resistance glycoprotein (P-gp) in this material. Expression of CD44 on carcinoma cells was observed in 21/52 cases (40%). Capillary endothelial reactivity of the tumours occurred in 42 cases (80%). Non-neoplastic epithelial breast tissue was positive in 2/10 (20%) samples and capillary vessels in 7/10 (70%). Carcinoma CD44 cell expression was not associated with age, tumour size, tumour grade, DNA ploidy, SPF, hormone-receptor contents or lymph node metastases. There was a statistical correlation between CD44 and P-gp expression in breast carcinoma cells which may suggest a connection between adhesion molecules and drug resistance. These findings do not support an association between CD44 expression and adverse prognostic features or lymph node metastases in BC. Capillary CD44 staining was a common feature in BC. There appeared to be an upward regulation in CD44 expression in BC compared with the normal breast tissue.

ATP Binding Cassette Transporter, Subfamily B, Mem↗