Enhanced expression of SPARC/osteonectin in the tumor-associated stroma of non-small cell lung cancer is correlated with markers of hypoxia/acidity and with poor prognosis of patients.
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Secreted Protein Acidic and Rich in Cystein (SPARC)/osteonectin is a nonstructural matricellular protein involved in cell-matrix interaction during tissue remodeling and embryonic development. Using a novel monoclonal antibody (10-255), we examined immunohistochemically the patterns of SPARC expression in non-small cell lung cancer (NSCLC). High levels of SPARC in normal lung were confined exclusively to the bronchial cartilage. In NSCLC tissues, cancer cells were unreactive in 107 of 113 cases analyzed (95%), whereas substantial production of SPARC by stromal fibroblasts was noted in 42 of 113 cases (37%). Stromal SPARC was linked with tumor necrosis (P = 0.01) and, marginally, with node metastasis (P = 0.07), as well as with high levels of carbonic anhydrase 9 and LDH in cancer cells (P = 0.0001 and P = 0.01, respectively). SPARC was also coincident with enhanced levels of cancer cell differentiated embryo-chondrocyte expressed gene 1, hypoxia inducible factor 2alpha, and thymidine phosphorylase (P = 0.01, P = 0.05, and P = 0.03, respectively). Although endothelial reactivity for SPARC was noted only in small, immature vessels, SPARC production by stroma cells supported a high degree of vascular maturation (indicated by the presence of subendothelial lamina lucida). Survival analysis revealed a significant association of stromal SPARC with poor prognosis (P = 0.006), a finding that was also confirmed in multivariate models. In NSCLC, SPARC is selectively synthesized by the cells of the tumoral stroma. The strong association of this feature with markers of intratumoral hypoxia and acidity indicates an interesting link between cancer cell metabolism and the induction of a supportive stroma that favors cancer cell invasion and migration that lead to an ominous clinical outcome. (+info)
SPARC inhibits epithelial cell proliferation in part through stimulation of the transforming growth factor-beta-signaling system.
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Secreted protein, acidic and rich in cysteine (SPARC) is a multifunctional secreted protein that regulates cell-cell and cell-matrix interactions, leading to alterations in cell adhesion, motility, and proliferation. Although SPARC is expressed in epithelial cells, its ability to regulate epithelial cell growth remains largely unknown. We show herein that SPARC strongly inhibited DNA synthesis in transforming growth factor (TGF)-beta-sensitive Mv1Lu cells, whereas moderately inhibiting that in TGF-beta-insensitive Mv1Lu cells (i.e., R1B cells). Overexpression of dominant-negative Smad3 in Mv1Lu cells, which abrogated growth arrest by TGF-beta, also attenuated growth arrest stimulated by SPARC. Moreover, the extracellular calcium-binding domain of SPARC (i.e., SPARC-EC) was sufficient to inhibit Mv1Lu cell proliferation but not that of R1B cells. Similar to TGF-beta and thrombospondin-1, treatment of Mv1Lu cells with SPARC or SPARC-EC stimulated Smad2 phosphorylation and Smad2/3 nuclear translocation: the latter response to all agonists was abrogated in R1B cells or by pretreatment of Mv1Lu cells with neutralizing TGF-beta antibodies. SPARC also stimulated Smad2 phosphorylation in MB114 endothelial cells but had no effect on bone morphogenetic protein-regulated Smad1 phosphorylation in either Mv1Lu or MB114 cells. Finally, SPARC and SPARC-EC stimulated TGF-beta-responsive reporter gene expression through a TGF-beta receptor- and Smad2/3-dependent pathway in Mv1Lu cells. Collectively, our findings identify a novel mechanism whereby SPARC inhibits epithelial cell proliferation by selectively commandeering the TGF-beta signaling system, doing so through coupling of SPARC-EC to a TGF-beta receptor- and Smad2/3-dependent pathway. (+info)
IL-1 induced release of Ca2+ from internal stores is dependent on cell-matrix interactions and regulates ERK activation.
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The cellular mechanisms that modulate interleukin-1 (IL-1) signaling are not defined. In fibroblasts, IL-1 signaling is affected by the nature of cell-matrix adhesions including focal adhesions, adhesive domains that sequester IL-1 receptors. We conducted studies to elucidate which steps of cellular Ca2+ handling are affected by focal adhesions and by which mechanisms focal adhesions modulate IL-1-induced Ca2+ signals and ERK activation in human gingival fibroblasts. Cells were plated on poly-l-lysine or fibronectin and treated with tenascin, Hep-I, or SPARC peptides to inhibit focal adhesion formation. These treatments blocked IL-1 and thapsigargin-induced Ca2+ release from the endoplasmic reticulum, indicating that the ER-release pathway is focal adhesion dependent. Focal adhesions were also required for Ca2+ entry through store-operated channels and for IL-1-induced ERK activation. Thus interactions with the extracellular matrix and focal adhesion formation regulate IL-1-induced generation of intracellular Ca2+ signals that in turn are required for ERK activation. (+info)
Differential SPARC mRNA expression in Barrett's oesophagus.
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Barrett's oesophagus (BE) is the precursor lesion to adenocarcinoma of the oesophagus. Understanding of the molecular alterations in this multistage process may contribute to improved diagnosis and treatment. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that modulates cell adhesion and growth. Alterations in SPARC expression have been observed in a variety of solid tumours. The aim of this study was to assess the prevalence and timing of SPARC mRNA expression in Barrett's multistage disease and to investigate the impact of SPARC alterations on the development and progression of this disease. SPARC mRNA expression was measured using a quantitative real-time RT-PCR method in 108 specimens from 19 patients with BE without carcinoma, 20 patients with Barrett's-associated adenocarcinoma (EA), and a control group (CG) of 10 patients without evidence of gastro-oesophageal reflux disease. The median SPARC mRNA expression was significantly upregulated in BE tissues compared to paired normal oesophagus (NE) tissues for the BE group (P=0.004) and for the EA group (P<0.001). The SPARC mRNA expression was significantly higher in adenocarcinoma of the oesophagus compared to matching NE tissue and compared to Barrett's tissues in the EA group (P<0.001). Furthermore, SPARC expression values were significantly different between metaplastic and dysplastic Barrett's tissues (P=0.014). In histologically normal squamous oesophagus tissues obtained from carcinoma patients (EA group), the SPARC mRNA expression was significantly higher compared to NE mucosa from the BE group and the CG group (P=0.03). These findings suggest that the upregulation of SPARC mRNA expression is an early event in the development and progression of BE and EA, and that high SPARC expression may be a clinically useful biomarker for the detection of occult adenocarcinoma, and that a widespread 'field effect' is present in the NE of patients with oesophageal adenocarcinoma. (+info)
SPARC affects glioma cell growth differently when grown on brain ECM proteins in vitro under standard versus reduced-serum stress conditions.
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Secreted protein acidic and rich in cysteine (SPARC) has a suppressive effect on U87 glioma cell proliferation when assessed in vitro and in vivo using parental U87T2 and U87T2-derived SPARC-transfected clones. Since SPARCinteracts with extracellular matrix (ECM) proteins, we examined the effect of SPARC secretion on proliferation, morphology, and cell density of glioma cells grown in vitro, in the absence and presence of ECM proteins under standard (10% fetal bovine serum [FBSI) and reduced (0.1% FBS) serum stress conditions. Under standard conditions, MTT (3-(4,5-cimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide) growth curves, morphology, and Western blot analyses demonstrated that SPARC had a suppressive and biphasic effect on growth that was not grossly modulated by the ECMs. The SPARC-induced changes in morphology observed at 24 h were not altered by the presence of ECMs. Under reduced-serum stress conditions, Western blot, morphological, and flow cytometric analyses indicated that the SPARC-induced suppressive growth effects were eliminated when the cells were grown on plastic. However, ECM-specific changes in growth were observed, some of which correlated with secreted SPARC levels. These results indicate that the differential effects of SPARC and ECMs on proliferation are dependent on culture conditions. Since the results obtained under standard conditions agree with our in vivo observations, we conclude that the ability of SPARC to suppress proliferation is regulated to a greater degree by the level of SPARC and that this suppressive effect is not influenced by the presence of any of the ECMs examined. (+info)
Leukocyte, rather than tumor-produced SPARC, determines stroma and collagen type IV deposition in mammary carcinoma.
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Secreted protein, acidic and rich in cysteine (SPARC), also known as osteonectin or BM-40, is a Ca2+-binding matricellular glycoprotein involved in development, wound healing, and neoplasia. However, the role of SPARC in tumors is ill defined mostly because it is expressed by both tumor and stromal cells, especially inflammatory cells. We analyzed the respective roles of host- and tumor-derived SPARC in wild-type and congenic SPARC knockout (SPARC-/-) mice on a BALB/c genetic background injected into the mammary fat pad with SPARC-producing mammary carcinoma cells derived from c-erB2 transgenic BALB/c mice. Reduced tumor growth but massive parenchyma infiltration, with large areas of necrosis and impaired vascularization were observed in SPARC-/- mice. Immunohistochemical analysis showed a defect in collagen type IV deposition in the stroma of lobular tumors from SPARC-/- mice. Chimeric mice expressing SPARC only in bone marrow-derived cells were able to organize peritumoral and perilobular stroma, whereas reciprocal chimeras transplanted with bone marrow from SPARC-/- mice developed tumors with less defined lobular structures, lacking assembled collagen type IV and with a parenchyma heavily infiltrated by leukocytes. Together, the data indicate that SPARC produced by host leukocytes, rather than the tumor, determines the assembly and function of tumor-associated stroma through the organization of collagen type IV. (+info)
Characterization of human osteoblast and megakaryocyte-derived osteonectin (SPARC).
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Osteonectin is an adhesive, cell, and extracellular matrix-binding glycoprotein found primarily in the matrix of bone and in blood platelets in vivo. Osteonectins isolated from these two sources differ with respect to the complexity of their constituent N-linked oligosaccharide. In this study, osteonectin synthesized by bone-forming cells (osteoblasts) and platelet-producing cells (megakaryocytes) in vitro was analyzed to determine if the proteins produced were analogous in terms of glycosylation to those isolated from bone and platelets, respectively. Immunoblot analyses of osteonectin produced by the osteoblast-like cell lines, SaOS-2 and MG-63, indicated that secreted and intracellular forms of the molecule are structurally distinct. Endoglycosidase treatment and immunoblotting of osteonectin secreted from SaOS-2 and MG-63 cells, under serum-deprived conditions, suggested that the molecule possessed a complex type oligosaccharide unlike the high-mannose moiety found on bone matrix-derived osteonectin. Biosynthetic labeling of SaOS-2 cells and human megakaryocytes indicated that both cell types synthesize osteonectin de novo. Electrophoretic and glycosidase sensitivity analyses of [35S]-osteonectin isolated from lysates of metabolically labeled SaOS-2 cells and megakaryocytes indicated that these two cell types synthesize osteonectin molecules that are identical in oligosaccharide structure to the isolated bone and platelet proteins. These data suggest that the intracellular form of the osteonectin molecule is glycosylated differently in SaOS-2 cells and megakaryocytes but that the extracellular form which is secreted from platelets in vivo and osteoblasts in vitro is characterized by the presence of a complex type N-linked oligosaccharide. (+info)
Immunohistochemistry of matrix markers in Technovit 9100 New-embedded undecalcified bone sections.
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Trabecular bone is routinely analysed by histomorphological-histometrical and immunohistochemical techniques as means of assessing the differentiation status of bone deposition and growth. Currently few embedding resins exist for which both morphological and immunohistochemical analyses can be performed on mineralised tissue. Paraffin, the standard embedding medium for bone enzyme and immunohistochemistry, can only be used on demineralised tissue, but then trabecular structure may be badly preserved. Methyl methacrylate (MMA), the resin of choice for undecalcified bone histology can only be used for bone immunohistochemistry if the usual, highly exothermic polymerisation procedure is avoided which destroys tissue antigenicity. Consequently, most current practices involve cutting samples in half to be processed in separate resins when more than one type of analysis is required. Technovit 9100 New is a low temperature MMA embedding system that is purported to significantly improve tissue antigenicity preservation allowing polymerisation at -20 degrees C. In this study, Technovit 9100 New-embedded undecalcified trabecular bone samples (adult human, young bovine and ovine) yielded immunolabelling with several bone matrix markers and preserved morphological features in 7 microm sections when stained with Masson-Goldner, von Kossa, or toluidine blue. Bone samples from all resins used were immunolabelled with antibodies against osteocalcin, alkaline phosphatase, osteopontin, osteonectin, bone sialoprotein and procollagen type I amino-terminal propeptide. Technovit-embedded bone yielded more reliable immunolabelling of the matrix proteins when compared with heat or cold-cured LR White or standard embedded MMA samples. Technovit 9100 New provided better routine histology than LR White, and was comparable to MMA. Results demonstrated that Technovit 9100 New can be used as a low-temperature acrylic resin embedding method for routine undecalcified bone histology, as well as for immunohistochemistry. (+info)