Biomedical subjects

M Jalkanen

Publications and source records attributed to M Jalkanen.

At least 37 records · Page 2Linked to original sources

PubMed1997

Subtype specific recognition of human alpha2C2 adrenergic receptor using monoclonal antibodies against the third intracellular loop.

Monoclonal antibodies (Mabs) against human alpha2C2-adrenergic receptor (alpha2C2-AR) were raised in mice and characterized. Bacterially expressed fusion protein consisting a sequence from the putative third intracellular loop (amino acids 213-343) of human alpha2C2 and glutathione-S-transferase (GST) was used as antigen. Results from mass spectrometry of purified thrombin cleaved alpha2C2 polypeptide suggested that the epitope region would lie near the aminoterminal end of the 3rd intracellular loop of human alpha2C2-AR. Elevation of Mabs was detected with Western blotting from mouse blood samples. Three alpha2C2 specific cell clones were expanded to in vitro production in hollow fiber systems. The specificity of the Mabs was further determined by immunoprecipitation and immunocytochemistry. Scatchard analysis of thrombin digested, purified, Europium-labelled antigen (amino acids 213-343 of alpha2C2) revealed binding affinity constants of 0.4 x 10(9), 0.7 x 10(9) and 1.6 x 10(9) M(-1) and Kds of 2.6, 1.4 and 0.6 nM for the three Mabs 2B1, 3G3 and 7G1, respectively.

Amino Acid Sequence↗

PubMed1997

The mapping and visual ordering of the human syndecan-1 and N-myc genes near the telomeric region of chromosome 2p.

The chromosomal locations of the human and mouse syndecan genes have been shown to associate with the corresponding locations of members of the myc gene family, proto-oncogenes that code for transcription factors. Here, we demonstrate the precise localization and order of the human syndecan-1 and N-myc genes using fluorescence in situ hybridization techniques that provide different levels of resolution. By visualizing the actual centromere-telomere orientation of these two genes, the human syndecan-1 gene is localized to 2p23-24, just centromeric to the N-myc gene at 2p24.1.

Chromosome Mapping↗

PubMed1997

Activation of an enhancer on the syndecan-1 gene is restricted to fibroblast growth factor family members in mesenchymal cells.

Fibroblast growth factors (FGFs) induce a variety of biological effects on different cell types. They activate a number of genes, including immediate-early genes, such as the transcription factors Fos and Jun, which are also common targets for other tyrosine kinase receptor-activating growth factors. Here we describe a secondary far-upstream enhancer on the syndecan-1 gene that is activated only by members of the FGF family in NIH 3T3 cells, not by other receptor tyrosine kinase-activating growth factors (e.g., epidermal growth factor, platelet-derived growth factor, insulin-like growth factor, or serum). This FGF-inducible response element (FiRE) consists of a 170-bp array of five DNA motifs which bind two FGF-inducible Fos-Jun heterodimers, one inducible AP-2-related protein, a constitutively expressed upstream stimulatory factor, and one constitutive 46-kDa transcription factor. Mutational analysis showed that both AP-1 binding motifs are required, but not sufficient, for FiRE activation. Moreover, agents such as 12-O-tetradecanoylphorbol-13-acetate, okadaic acid, or forskolin, which are known to activate AP-1 complexes and AP-1-driven promoters, fail to activate FiRE. However, FiRE can be activated by the tyrosine kinase phosphatase inhibitor orthovanadate. Taken together, this data implies a differential activation of growth factor-initiated signaling on AP-1-driven regulatory elements.

3T3 Cells↗

PubMed1997

Syndecan-1: a new prognostic marker in laryngeal cancer.

The cell adhesion molecule syndecan-1 expression is induced during keratinocyte differentiation and reduced during the formation of squamous cell carcinomas (SCCs). A significant correlation between decreased syndecan-1 expression in head and neck SCC measured from frozen sections with immuohistochemical methods and clinical outcome are reported. The clinical relevance of the cellular proliferation marker Ki-67 is controversial in SCC of the head and neck. The purpose of this study was to determine the expression of syndecan-1 and Ki-67 in SCC of the larynx and correlate the results with known prognostic factors and clinical outcome. Paraffin-embedded samples of 100 patients with laryngeal SCC (44 glottic, 36 supraglottic, 20 transglottic) treated at Turku University Central Hospital were re-examined and divided into four histological grades of differentiation, four grades of keratinisation, and four grades of 104-9 (syndecan-1) immunostaining. The mitotic index was analysed as the number of mitoses per volume corrected high power fields. The relative number of Ki-67 positive cells was evaluated. The patients mean age was 64 years and the 5-year survival was 69%. In univariate analysis, intermediate or strong staining for syndecan-1 was associated with higher overall survival than those tumours with no or little syndecan-1 expression (p = 0.048). Nodal status (p = 0.0001), tumour size (p = 0.0004) and localisation (p = 0.0008), general condition (p = 0.0001), histological grade (p = 0.02) and patient age (p = 0.03) correlated with overall survival whereas the Ki-67 index (p = 0.093), mitotic index (p = 0.23) and grade of keratinisation (p = 0.90) failed to do so. The results suggest that syndecan-1 could be a useful prognostic factor in SCC of the larynx.

Adult↗

PubMed1997

Is the sensitivity of cells for FGF-1 and FGF-2 regulated by cell surface heparan sulfate proteoglycans?

We have examined the importance of cell surface heparan sulfate proteoglycans (HSPG) in fibroblast growth factor (FGF) signaling. 3T3 cells grown under conventional conditions were much more sensitive to FGF-2 compared to FGF-1. However, cells were equally sensitive to FGF-1 and FGF-2 using conditions which reduced the effect of endogenous HSPG. Addition of heparin, or treatment with chlorate, an inhibitor of proteoglycan sulfation, resulted in enhanced or reduced growth factor response, respectively, and eliminated the differences between FGF-1 and FGF-2. HSPGs isolated from trypsin digests of 3T3 cells had a much higher affinity for FGF-2 compared to that for FGF-1 when analyzed by affinity chromatography. Glycosaminoglycan chains or core protein fragments derived from the HSPG failed to show the same high apparent affinity for FGF-2, suggesting that an intact proteoglycan structure was important for the high FGF-2 affinity. Addition of HSPG ectodomains, isolated from cultured 3T3 cells or produced as recombinant molecules, to chlorate-treated cultures of 3T3 cells inhibited the mitogenic activity of FGF-2 and eliminated the effect of heparin as a potentiator of either growth factor. These results support the idea that the cell-associated HSPG is an integral component of the FGF signaling system and in 3T3 cells contributes to the increased sensitivity of these cells to FGF-2 compared to FGF-1. Since isolated ectodomains of HSPG inhibited rather than stimulated the mitogenic response of the FGFs, the proper anchoring of the HSPG in the cell membrane appears to be important for a stimulatory effect.

3T3 Cells↗

PubMed1996

Suppression of syndecan-1 expression in endothelial cells by tumor necrosis factor-alpha.

Syndecan-1 is a cell surface proteoglycan that binds extracellular matrix components and modulates the activity of heparin-binding growth factors. The expression of syndecan-1 is modified during development, carcinogenesis, and tissue regeneration. During cutaneous wound healing, syndecan-1 expression is transiently induced in newly-formed capillaries of granulation tissue as well as in proliferating keratinocytes. To study the mechanisms underlying this regulation we investigated the effects of several growth factors/cytokines on syndecan-1 expression in two human cell lines: EA.hy 926 endothelial cells and HaCaT keratinocytes. None of these factors significantly altered syndecan-1 mRNA expression in cultured keratinocytes, but when given to endothelial cells, tumor necrosis factor-alpha (TNF-alpha) specifically and dose-dependently suppressed syndecan-1 expression at both mRNA and protein levels. TNF-alpha reduced the amount of syndecan-1 protein in EA.hy 926 cells in both the presence and absence of serum and, at the same time, induced the expression of intercellular adhesion molecule-1 (ICAM-1). The suppressive effect of TNF-alpha on endothelial syndecan-1 expression was reproducible in in vivo experiments in which TNF-alpha-coated beads were administered directly to healing skin wounds of mice. Data supporting these findings were further obtained by injecting TNF-alpha into an experimental rat granulation tissue model. In this tissue TNF-alpha suppressed syndecan-1 mRNA expression by approximately 80%. These results indicate that TNF-alpha is capable of down-regulating syndecan-1 expression in endothelial cells both in vitro and in vivo and suggest that similar mechanisms may be responsible for the changes in syndecan-1 expression observed during various regenerative, developmental, and malignant processes.

Animals↗

PubMed1996

Functional characterization of mouse syndecan-1 promoter.

The members of the syndecan family are temporally and spatially expressed heparan sulfate proteoglycans of various tissues, where they mediate extracellular influences on cell morphology and behavior. Functional characterization of the mouse syndecan-1 promoter was carried out in order to elucidate the mechanisms involved in the maintenance of the high transcription levels of syndecan-1 gene in various epithelia. For that 9.5 kilobase pairs of the upstream region of mouse syndecan-1 gene were cloned, sequenced, and used to prepare chimaeric constructs with a reporter gene followed by transient or stable transfections into NMuMG cells, cultured either in the presence or absence of serum, the 2.5-kilobase pair promoter region resulted in the constitutive transcription activity, whereas in 3T3 cells the serum depletion decreased the promoter activity significantly. Deletion of the upstream sequences to -437 base pairs relative to the translation initiation site had little effect on this promoter activity. Further deletion to -365 base pairs removed three GT boxes and slightly increased the promoter activity, whereas the deletion of the next two GC boxes (to -326 base pair) reduced the promoter activity dramatically. All of the GC or GT box sequences bound the same set of Sp1-like nuclear protein in gel shift assays. Nuclear protein binding was also demonstrated around both of the most intense transcription initiation sites. Mutation of these regions separately resulted in total loss of transcription initiation from the deleted site and decreased the promoter activity in relation to the intensity of the abolished start site. This indicates that the transcription initiation of the syndecan-1 gene is directed through initiator-like elements directly overlapping the start sites, as shown for several TATA-less housekeeping and growth regulated genes. We assume that the constitutive high level gene expression in epithelial cells is achieved by the proximal promoter, which is controlled by members of Sp1 transcription factor family.

3T3 Cells↗

PubMed1996

Expression of Mad, an antagonist of Myc oncoprotein function, in differentiating keratinocytes during tumorigenesis of the skin.

The Myc oncoprotein is associated with cell proliferation and is often down-regulated during cell differentiation. The related Mad transcription factor, which antagonises Myc activity, is highly expressed in epidermal keratinocytes. Mad also inhibits cell proliferation in vitro. To study Mad expression in keratinocyte proliferation and differentiation, we have analysed Mad RNA expression in regenerating and hyperproliferative epidermal lesions and epidermal tumours of varying degrees of differentiation using the RNA in situ hybridisation and RNAase protection techniques. Mad was strongly expressed in differentiating suprabasal keratinocytes in healing dermal wounds and in benign hyperproliferative conditions, but also in squamous cell carcinomas, in which the keratinocytes retain their differentiation potential. However, Mad expression was lost in palisading basal carcinoma cells and poorly differentiated squamous cell carcinomas, which lacked the epithelial differentiation marker syndecan-1. We therefore suggest that Mad expression is closely associated with epithelial cell differentiation, and that this association is retained in epithelial tumours of the skin.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1996

Syndecan-1 expression in mammary epithelial tumor cells is E-cadherin-dependent.

E-cadherin is a Ca(2+)-dependent cell-cell adhesion molecule, which is mainly expressed in epithelial cells. Recent studies have shown that E-cadherin has an important role as an invasion suppressor molecule in epithelial tumor cells. Syndecan-1 is a cell surface proteoglycan that has been implicated in a number of cellular functions including cell-cell adhesion, cell-matrix anchorage and growth factor presentation for signalling receptors. Its suppression has also been shown to be associated with malignant transformation of epithelial cells. In order to better understand the coordinated regulation of cell-cell and cell-matrix interactions during malignant transformation, we have studied the expression of syndecan-1 in malignant mammary tumor cells genetically manipulated for E-cadherin expression. In invasive NM-e-ras-MAC1 cells, where E-cadherin was partially downregulated by specific antisense RNA, syndecan-1 expression was suppressed. Furthermore, transfection of E-cadherin cDNA into invasive NM-f-ras-TD cells resulted in the upregulation of syndecan-1 expression in association with decreased invasiveness. In both cases, regulation of syndecan-1 occurred post-transcriptionally, since syndecan-1 mRNA levels remained unchanged. Instead, a translational regulation is suggested, since syndecan-1 core protein synthesis was E-cadherin dependent. Another cell adhesion protein, beta 1-integrin was not affected by E-cadherin expression. The data provide an example of coordinated changes in the expression of two cell adhesion molecules, syndecan-1 and E-cadherin during epithelial cell transformation.

Animals↗

PubMed1996

Viable AC-2, a new adult bovine serum- and colostrum-based supplement for the culture of mammalian cells.

In this study we have shown that Viable AC-2, a medium based on the ultrafiltrate fraction of bovine colostrum and adult bovine serum, can be used successfully as a fetal bovine serum (FBS) substitute in the culture of several anchorage-dependent and independent cell lines. Of the 15 cell lines cultured in 8% Viable AC-2 in microplates, 10 reached the maximum cell density of 65%-123% of that in 10% FBS, 4 cell lines reached maximum cell density of 35%-65% of that in 10% FBS and only one cell line, a human osteosarcoma G-292, grew slowly in Viable AC-2. In a small-scale suspension culture, 8%-15% Viable AC-2 supports the growth of Chinese hamster ovary cells (CHO-K1) on microcarriers in spinner flasks significantly better than 10% FBS. Shionogi mouse mammary tumour cell line (S115) transfected with human alpha 2-adrenergic receptor subtype C2 was used as a model to study recombinant protein production in Viable AC-2-supplemented medium. The results showed that in cell culture flasks and in an ACUSYST-R bioreactor, the alpha 2-C2 receptor expression level per mg of total protein was similar in both Viable AC-2 and FBS.

Animals↗

PubMed1996

The role of syndecan-1 in malignancies.

Syndecans form a family of cell surface proteoglycans, which can interact with various effector molecules, such as extracellular matrix (ECM) molecules and growth factors. Syndecan-1 is the most extensively studied member of the syndecan family. It is found mainly in epithelial cells, but its expression is developmentally regulated during embryonic development. It has been shown to mediate cell adhesion to several ECM molecules, and to act as a coreceptor for fibroblast growth factors, potent angiogenic growth factors involved also in differentiation. Syndecan-1 expression is reduced during malignant transformation of various epithelia, and this loss correlates with the histological differentiation grade of squamous cell carcinomas, lacking from poorly differentiated tumours. In squamous cell carcinomas of the head and neck, positive syndecan-1 expression correlates with a more favourable prognosis. Recent experimental studies on the role of syndecan-1 in malignant transformation have shown that syndecan-1 expression is associated with the maintenance of epithelial morphology, anchorage-dependent growth and inhibition of invasiveness in vitro. This review will focus on the biological role of syndecan-1 in normal epithelial differentiation and malignant transformation.

Biomarkers, Tumor↗

PubMed1995

Syndecan family of cell surface proteoglycans: developmentally regulated receptors for extracellular effector molecules.

Syndecans are a family of integral membrane proteoglycans with conserved membrane-spanning and intracellular domains but with structurally distinct extracellular domains (ectodomains). They are known to function as heparan sulphate co-receptors in fibroblast growth factor signalling as well as to link cells directly to the extracellular matrix. These and other biological activities of syndecans involve specific interactions of the heparan sulphate side chains of syndecans with cytokines and extracellular matrix proteins. Four different vertebrate syndecans, designated as syndecans 1-4 (or syndecan, fibroglycan, N-syndecan and amphiglycan, respectively), are known. During embryonic development, syndecans have specific and highly regulated expression patterns that are distinct from the expression in adult tissue, suggesting an active role in morphogenetic processes. The developmental expression of syndecans is particularly intense in mesenchymal condensates and at epithelium mesenchyme interfaces, where a number of heparan sulphate-binding cytokines and matrix components are also expressed in a regulated manner, often spatially and temporally co-ordinated with the syndecan expression. Recent evidence indicates that the regulation of heparan sulphate fine structure (mainly the number and arrangement of sulphate groups along the polymer) provides a mechanism for the cellular control of syndecan-protein interactions. Furthermore, morphogenetically active cytokines such as fibroblast growth factor-2 and transforming growth factor-beta participate in the regulation of syndecan expression and glycosaminoglycan structure. This review discusses the developmental expression and binding functions of syndecans as well as the molecular regulation of specific heparan sulphate-protein interactions.

Animals↗

PubMed1995

Expression of proteoglycans and hyaluronan during wound healing.

We investigated the expression of proteoglycans (PGs) and hyaluronan (HA) during healing of human mucosal wounds. Biopsy specimens of experimental wounds were taken 1, 3, and 7 days after wounding. Frozen sections were used for immunolocalization of CD44, syndecan-1, basement membrane-associated heparan sulfate proteoglycan (BM-HSPG), decorin, and biglycan. HA was localized in paraffin sections with a specific HA-binding probe. Epithelium showed first signs of migration on Day 1, more progressive migration on Day 3, and epithelial sheets confronted on Day 7. CD44 surrounded migrating keratinocytes at all stages of wound healing. In epithelium, CD44 and HA remarkably localized to the same region. Expression of syndecan-1 was switched from the suprabasal cell layer of unwounded epithelium to the basal cell layer of the migrating wound epithelium. BM-HSPG was absent under migrating keratinocytes. It started to reappear at the basement membrane zone on Day 7. The area under the wound epithelium containing newly synthesized collagen fibers first became positive for decorin on Day 7, whereas staining of biglycan was negative. Granulation tissue was also strongly positive for CD44 and hyaluronan. Our results indicate that migrating keratinocytes express both CD44 and syndecan-1 but not BM-HSPG. During differentiation of keratinocytes, expression of CD44 preceded that of syndecan-1. The results suggest that different HSPGs have multiple functions in keratinocyte migration and differentiation during reepithelialization.

Basement Membrane↗

PubMed1995

Expression of the heparin-binding cytokines, midkine (MK) and HB-GAM (pleiotrophin) is associated with epithelial-mesenchymal interactions during fetal development and organogenesis.

Midkine (MK) and heparin binding-growth associated molecule (HB-GAM or pleiotrophin), constitute a new family of heparin-binding proteins implicated in the regulation of growth and differentiation (T. Muramatsu (1993) Int. J. Dev. Biol. 37, 183-188). We used affinity-purified antibodies against MK and HB-GAM to analyze their distribution during mouse embryonic development. From 9 to 14.5 day post-coitum (dpc), both proteins were detected in central and peripheral nervous systems, facial processes, limb buds, sense organs, respiratory, digestive, urogenital, and skeletal systems. MK and HB-GAM were often localized on the surface of differentiating cells and in basement membranes of organs undergoing epithelial-mesenchymal interactions. The level of MK protein decreased considerably in the 16.5 dpc embryo, whereas HB-GAM staining persisted in many tissues. Our in situ hybridization results revealed a widespread expression of MK transcripts that was not always consistent with the distribution of MK protein in developing tissues. In many epithelio-mesenchymal organs MK and HB-GAM were codistributed with syndecan-1, a cell surface proteoglycan. In limb buds and facial processes, MK, HB-GAM, and syndecan-1 were localized to the apical epithelium and the adjacent proliferating mesenchyme. Both MK and HB-GAM bound syndecan-1 in solid-phase assays in a heparan sulfate-dependent manner. The biological effects of MK and HB-GAM on limb and facial mesenchyme were studied in vitro by application of beads preloaded with the proteins. Neither MK nor HB-GAM stimulated mesenchymal cell proliferation or induced syndecan-1 expression. Taken together these results indicate that MK and HB-GAM may play regulatory roles in differentiation and morphogenesis of the vertebrate embryo, particularly in epithelio-mesenchymal organs, and suggest molecular interactions with syndecan-1.

Animals↗

PubMed1995

Increased glycosylation of beta 1 integrins affects the interaction of transformed S115 mammary epithelial cells with laminin-1.

The effect of transformation on the expression and the functions of beta 1 integrins was studied using an in vitro cell transformation model. S115 mammary epithelial tumor cells undergo transformation into tumorigenic fibroblastoid cells in the presence of steroids. Transformation was found to reduce the attachment and the spreading of S115 cells on laminin-1 but not fibronectin. Adhesion of S115 cells to laminin-1 was inhibited in the presence of an antibody against the beta 1 integrin subunit. Both nontreated and transformed S115 cells expressed at least two putative laminin-1-binding beta 1 integrins at the same level. In transformed cells, however, the mature integrin subunits appeared to be structurally altered, showing a slower electrophoretic mobility. Treatment with N-glycosidase-F and tunicamycin abolished this mobility difference, suggesting that the presence of complex-type N-linked oligosaccharides was responsible. Detailed enzymatic analysis of the oligosaccharides present on the beta 1 subunits revealed that the difference in glycosylation is, at least partially, due to poly-N-lactosaminoglycan chains on beta 1 integrin from transformed cells. Removal of this difference in glycosylation by either cleavage of the polylactosaminoglycan chains with endo-beta-galactosidase or inhibiton of complex-type glycan formation with swainsonine repeatedly enhanced the spreading of transformed cells on laminin-1. Thus, the increased size of complex-type oligosaccharides on beta 1 integrin may affect cell-laminin-1 interactions. Similar changes may contribute to the altered adhesion of cancer cells during the invasion and metastasis.

Animals↗

PubMed1994

Suppression of tumor cell growth by syndecan-1 ectodomain.

Syndecans are integral membrane proteoglycans characterized by the similarity of cytoplasmic and transmembrane domains of the core proteins. Syndecans may regulate cell behavior by participating in matrix recognition and growth factor binding. Using syndecan-1 deletion mutants, we show that the extracellular part of the molecule (ectodomain) can suppress malignant growth, stimulate actin polymerization, and induce epithelioid morphology in mouse mammary tumor Shionogi 115 (S115) cells. Free ectodomain isolated from the culture medium of either syndecan-1-transfected S115 cells or normal murine mammary gland (NMuMG) cells can suppress the growth of S115 tumor cells at nanomolar concentrations. The ectodomain of syndecan-1 inhibited also the growth of other carcinoma cell lines, such as CarB and MCF-7, but not such inhibition was observed for contact-inhibited cell lines, including NIH 3T3 cells, NMuMG cells, and human HaCaT keratinocytes. Intact heparan sulfate structure of the ectodomain was required for the suppression because degradation of heparan sulfate chains completely abolished growth inhibition. These results suggest a tumor suppressor activity for the syndecan-1 ectodomain.

3T3 Cells↗

PubMed1994

Use of a hollow fiber bioreactor for large-scale production of alpha 2-adrenoceptors in mammalian cells.

Gene cloning has revealed the existence of receptors, which are structurally similar but pharmacologically distinct. One recent example is the alpha 2-adrenergic receptor (alpha 2AR) family with three members. Preparation of membrane-embedded G-protein coupled receptor subtypes in pure form is practically impossible from natural sources and only recombinant techniques have provided possibilities to study these receptors in great detail. In this respect, both yeast and insect cell hosts have been applied successfully but no good mammalian alternative has been described for large-scale production. We describe in this report the use of S115 mouse mammary tumor cells as an effective host for large-scale production of alpha 2-adrenoceptors. These cells can be easily adapted to grow in a hollow fiber bioreactor, with up to 2.8 g of total cellular protein produced in one 0.8 m2 casette. We also show that each recombinant alpha 2-subtype exhibits their expected ligand binding properties, and suggest therefore that this system could be generally applicable to other eukaryotic plasma membrane proteins.

Animals↗

PubMed1994

Immunohistochemical localization of syndecan-1 in normal and pathological human uterine cervix.

Expression of syndecan-1, a cell surface proteoglycan that binds growth factors and extracellular matrix components, was studied in normal and pathological human uterine cervix using immunohistochemical methods. Normal cervical squamous epithelium showed positive staining for syndecan-1 in all cell layers, except the basal cell layer, whereas endocervical columnar epithelium stained weakly. In non-neoplastic reactive lesions, metaplastic squamous cells were positive for syndecan-1, whereas columnar cells showed weak or negative staining. In cervical condylomas, cells showing koilocytotic atypia were positive for syndecan-1. The progression of cervical intraepithelial neoplasia (CIN) grade I to grade III was associated with reduced syndecan-1 expression and localization of syndecan-1 to more superficial cell layers. In squamous cell carcinomas (SCCs), syndecan-1 expression correlated with histological differentiation, being absent from most poorly differentiated tumours. The results suggest that loss of syndecan-1 from atypical cells is an early event during cervical carcinogenesis and show a close association of syndecan-1 expression with preserved epithelial morphology and differentiation.

Carcinoma, Squamous Cell↗