Protein adsorption can be reversibly switched on and off on mixed PEO/PAA brushes.

Adsorption of proteins on surfaces placed in biological fluids is a ubiquitous and mostly irreversible phenomenon, desirable or not, but often uncontrolled. Adsorption of most proteins on poly(ethylene oxide) (PEO) brushes is very limited, while the amount of proteins adsorbed on poly(acrylic acid) (PAA) brushes varies with the pH and ionic strength (I) of the protein solution. Mixed brushes of PEO and PAA are designed here to reversibly adsorb and desorb albumin, lysozyme, collagen and immunoglobulin G, four very different proteins in terms of size, solubility and isoelectric point. Protein adsorption and desorption are monitored using X-ray photoelectron spectroscopy, as well as with quartz crystal microbalance for in situ and real-time measurements. Large amounts of protein are adsorbed and then nearly completely desorbed on mixed PEO/PAA brushes by a simple pH and I trigger. The mixed brushes thus nicely combine the properties of pure PAA and pure PEO brushes. These adsorption/desorption cycles are shown to be repeated with high efficiency. The high-performance smart substrates created here could find applications in domains as diverse as biosensors, drug delivery and nanotransport.

Design of mixed PEO/PAA brushes with switchable properties toward protein adsorption.

Adsorption of proteins at interfaces is an ubiquitous phenomenon of prime importance. Layers of poly(ethylene oxide) (PEO) are widely used to repel proteins. Conversely, proteins were shown to adsorb deeply into brushes of poly(acrylic acid) (PAA), and their subsequent partial release could be triggered by a change of pH and/or ionic strength (I). Mixed brushes of these polymers are thus promising candidates to tune protein adsorption onto new smart surfaces. In this work, the synthesis of such mixed brushes was performed based on a "grafting to" approach, the two polymers being either grafted sequentially or simultaneously. Detailed characterization of the obtained brushes using static water contact angle measurements, X-ray photoelectron spectroscopy, electrochemical impedance spectroscopy, and polarization-modulation reflection-absorption infrared spectroscopy is presented. While sequential grafting of the two polymers for different reactions times did not give rise to a broad range of composition of mixed brushes, simultaneous grafting of the polymers from solutions with different compositions allows for the synthesis of a range of mixed brushes (mass fraction of PEO in the mixed brushes from 0.35 to 0.65). A key example is then chosen to illustrate the switchable behavior of a selected mixed PEO/PAA brush toward albumin adsorption. The adsorption behavior was monitored with a quartz crystal microbalance. The mixed brush could adsorb high amounts of albumin, but 86% of the adsorbed protein could then be desorbed upon pH and I change. The obtained properties are thus a combination of the ones of PEO and PAA, and a highly switchable behavior is observed toward protein adsorption.

Tumor size of hepatocellular carcinoma in noncirrhotic liver: a controversial predictive factor for outcome after resection.

BACKGROUND: Hepatocellular carcinoma in noncirrhotic liver (NC-HCC) presents usually with large size, which is seen as a contraindication to liver transplantation (LT) or even resection. The objective of our single-center study was to identify prognostic factors following resection of large NC-HCCs and to subsequently devise a treatment strategy (including LT) in selected patients. METHODS: From 2000 to 2010, 89 patients who had hepatic resection for NC-HCC (large ≥ 8 cm in 52) were analyzed with regard to pathological findings, postoperative and long-term outcome. RESULTS: Five patients died postoperatively. After a mean follow-up of 35 ± 30 months, NC-HCC recurred in 36 patients (26/47 survivors in group 8 cm+, 10/37 in group 8 cm-; p = 0.007). Five-year overall (OS) and disease-free survival (DFS) rates were significantly worse for group 8 cm+ (43.4% vs. 89.2% and 39.3% vs. 60.7% for group 8 cm-, p < 0.05). Seven patients underwent re-hepatectomy and/or LT for isolated intrahepatic recurrence, with 5-year DFS of 57.1%. In a multivariate analysis, the factors associated with poor OS and DFS were vascular invasion and tumor size ≥ 8 cm in the overall population and vascular invasion, fibrosis and satellite nodules in group 8 cm+. Adjuvant transarterial chemotherapy was a protective factor in group 8 cm+. In 22 isolated NC-HCC cases with no vascular invasion or fibrosis, tumor size had no impact on five-year DFS (85%). CONCLUSIONS: Although patients with NC-HCC ≥ 8 cm had a poorer prognosis, the absence of vascular invasion or fibrosis was associated with excellent survival, regardless of the tumor size. In recurrent patients, aggressive treatment (including LT) can be considered.

Galectin-3 regulates MUC1 and EGFR cellular distribution and EGFR downstream pathways in pancreatic cancer cells.

MUC1 is a transmembrane glycoprotein which is typically expressed at the apical membrane of normal epithelial cells. In cancer cells, the over-expression of MUC1 and its aberrant localization around the cell membrane and in the cytoplasm favours its interaction with different protein partners such as epidermal growth factor receptor (EGFR) and can promote tumour proliferation through the activation of oncogenic signalling pathways. Our aims were to study the mechanisms inducing MUC1 cytoplasmic localization in pancreatic cancer cells, and to decipher their impact on EGFR cellular localization and activation. Our results showed that galectin-3, an endogenous lectin, is co-expressed with MUC1 in human pancreatic ductal adenocarcinoma, and that it favours the endocytosis of MUC1 and EGFR. Depletion of galectin-3 by RNA interference increased the interaction between MUC1 and EGFR, EGFR and ERK-1,2 phosphorylation, and translocation of EGFR to the nucleus. On the contrary, silencing of galectin-3 led to a decrease of cyclin-D1 levels and of cell proliferation. The galectin-3-dependent regulation of MUC1/EGFR functions may represent an interesting mechanism modulating the EGFR-stimulated cell growth of pancreatic cancer cells.

Tumour growth and resistance to gemcitabine of pancreatic cancer cells are decreased by AP-2alpha overexpression.

BACKGROUND: Activator protein-2alpha (AP-2alpha) is a transcription factor that belongs to the family of AP-2 proteins that have essential roles in tumorigenesis. Indeed, AP-2alpha is considered as a tumour-suppressor gene in different tissues such as colonic, prostatic or breast epithelial cells. Moreover, AP-2alpha also participates in the control of colon and breast cancer cells sensitivity towards chemotherapeutic drugs. Despite its potential interest, very few data are available regarding the roles of AP-2alpha in pancreatic cancer. METHODS: We have developed a stable pancreatic CAPAN-1 cell line overexpressing AP-2alpha. Consequences of overexpression were studied in terms of in vivo cell growth, gene expression, migration capacity and chemosensitivity. RESULTS: In vivo tumour growth of CAPAN-1 cells overexpressing AP-2alpha was significantly decreased by comparison to control cells. An altered expression pattern of cell cycle-controlling factors (CDK-4, CDK-6, cyclin-G1, p27(kip1) and p57(kip2)) was observed in AP-2alpha-overexpressing clones by microarrays and western blot analysis. Promoter activity and ChIP analysis indicated that AP-2alpha induces p27(kip1) protein levels by direct binding to and transactivation of its promoter. Moreover, AP-2alpha overexpression increased the chemosensitivity of CAPAN-1 cells to low doses of gemcitabine and reduced their in vitro migration capacity. CONCLUSION: Our data suggested that AP-2alpha overexpression could be exploited to decrease in vivo tumour growth of pancreatic cancer cells and to increase their sensitivity to gemcitabine.

Autocrine induction of invasion and metastasis by tumor-associated trypsin inhibitor in human colon cancer cells.

From the conditioned medium of the human colon carcinoma cells, HT-29 5M21 (CM-5M21), expressing a spontaneous invasive phenotype, tumor-associated trypsin inhibitor (TATI) was identified and characterized by proteomics, cDNA microarray approaches and functional analyses. Both CM-5M21 and recombinant TATI, but not the K18Y-TATI mutant at the protease inhibitor site, trigger collagen type I invasion by several human adenoma and carcinoma cells of the colon and breast, through phosphoinositide-3-kinase, protein kinase C and Rho-GTPases/Rho kinase-dependent pathways. Conversely, the proinvasive action of TATI in parental HT29 cells was alleviated by the TATI antibody PSKAN2 and the K18Y-TATI mutant. Stable expression of K18Y-TATI in HT-29 5M21 cells downregulated tumor growth, angiogenesis and the expression of several metastasis-related genes, including CSPG4 (13.8-fold), BMP-7 (9.7-fold), the BMP antagonist CHORDIN (5.2-fold), IGFBP-2 and IGF2 (9.6- and 4.6-fold). Accordingly, ectopic expression of KY-TATI inhibited the development of lung metastases from HT-29 5M21 tumor xenografts in immunodeficient mice. These findings identify TATI as an autocrine transforming factor potentially involved in early and late events of colon cancer progression, including local invasion of the primary tumor and its metastatic spread. Targeting TATI, its molecular partners and effectors may bring novel therapeutic applications for high-grade human solid tumors in the digestive and urogenital systems.

Cysteine and serine proteases of synovial tissue in rheumatoid arthritis and osteoarthritis.

OBJECTIVE: To compare the activities of cathepsin B (EC 3.4.22.1) and L (EC 3.4.22.15), calpain (EC 3.4.22.17), and dipeptidyl peptidase (EC 3.4.14.5 or DPP IV or CD26) in synovial membrane from patients with rheumatoid arthritis (RA), osteoarthritis (OA), and post-traumatic joint injury (PT). METHODS: Forty RA patients were divided into two groups on the basis of surgical procedure: the RAs group comprised 18 patients requiring surgical synovectomy; the RAr group comprised 22 patients requiring a total joint replacement or arthrodesis. A third group (the OA group) comprised 19 OA patients while six patients with post-traumatic joint injury were included in the fourth group (the PT group). Cathepsin and calpain activity was assessed using a Cobas Fara II centrifugal analyser. DPP IV activity was determined kinetically using a fluorogenic substrate. RESULTS: RAs patients were significantly younger than RAr patients, and the mean duration of RA was shorter in the RAs group than in the RAr group. Cathepsin and calpain activity in synovial membrane was higher in RA and OA patients than in the control group, but no statistical difference was observed between RA and OA. However, cathepsin, calpain, and DPP IV synovial activity was significantly higher in the RAs group than in either the OA or the PT group. CONCLUSION: Our results show that proteinase activity tends to be higher in joints with early synovitis in RA, and suggest that these enzymes are not all involved at the same stage of the disease.

Involvement of glycosylation in the intracellular trafficking of glycoproteins in polarized epithelial cells.

The surface of epithelial cells is composed of apical and basolateral domains with distinct structure and function. This polarity is maintained by specific sorting mechanisms occurring in the Trans-Golgi Network. Peptidic signals are responsible for the trafficking via clathrin-coated vesicles by means of an interaction with an adaptor complex (AP). The basolateral targeting is mediated by AP-1B, which is specifically expressed in epithelial cells. In contrast, the apical targeting is proposed to occur via apical raft carriers. It is thought that apically targeted glycoproteins contain glycan signals that would be responsible for their association with rafts and for apical targeting. However, the difficulty in terms of acting specifically on a single step of glycosylation did not allow one to identify such a specific signal. The complete inhibition of the processing of N-glycans by tunicamycin often results in an intracellular accumulation of unfolded proteins in the Golgi. Similarly, inhibition of O-glycosylation can be obtained by competitive substrates which gave a complex pattern of inhibition. Therefore, it is still unknown if glycosylation acts in an indirect manner, i.e. by modifying the folding of the protein, or in a specific manner, such as an association with specific lectins.

Inhibition of the glycosylation and alteration in the intracellular trafficking of mucins and other glycoproteins by GalNAcalpha-O-bn in mucosal cell lines: an effect mediated through the intracellular synthesis of complex GalNAcalpha-O-bn oligosaccharides.

To address the function of carbohydrates in mucins, GalNAcalpha-O-bn has been used in in vivo experiments on several human mucosal cultured cells as a potential competitor of the glycosylation of N-acetylgalactosamine residues. GalNAcalpha-O-bn is metabolized by glycosyltransferases expressed in the cell, and give rise to different internal derivatives starting in particular from the formation of the disaccharide Galalpha1-3GalNAcalpha-O-bn. In this line, GalNAcalpha-O-bn exposure inhibits peripheral glycosylation according a cell-type specific manner. The metabolic alterations are very important in HT-29 cell line, leading to a massive accumulation of GalNAcalpha-O-bn oligosaccharide derivatives and to a strong inhibition of the terminal elongation of O-glycans by alpha2,3 sialyltransferase ST3Gal I. GalNAcalpha-O-bn treatment also induced alterations at the cellular level, exhibiting a large scale in HT-29 cells, i.e. 1) an inhibition of mucin secretion, 2) a blockade in the targeting of some membrane glycoproteins (brush border glycoproteins such as dipeptidylpeptidase IV, carcinoembryonic antigen and the mucin-like glycoprotein MUC1, and the basolateral cell adhesion molecule CD44), 3) an inhibition in the processing of lysosomal enzymes. Morphological abnormalities have been evidenced in GalNAcalpha-O-bn treated cells, in particular the accumulation of numerous intracellular vesicles in HT-29 cells. Taken together, these data suggest that O-glycosylation might be involved in the regulation of the targeting of O-glycosylproteins through carrier vesicles.

Heterozygous M3Mmalton alpha1-antitrypsin deficiency associated with end-stage liver disease: case report and review.

Alpha1-antitrypsin (alpha1AT) deficiency is an autosomal recessive disorder that can cause pulmonary emphysema and liver disease. We report here the case of a 59-year-old woman who was admitted to hospital for evaluation of jaundice. She had no history of hepatitis or childhood liver disease. She had never received a blood transfusion, nor had she abused drugs or alcohol. Transjugular liver biopsy was then performed and revealed a micronodular cirrhosis. Ten months later, because of persistent liver cell failure and ascites, she underwent an orthotopic liver transplantation. Investigation of alpha1AT system in the proband revealed a substantial decrease in serum alpha1AT associated with a low elastase inhibitory capacity. The Pi phenotype revealed a PiM-like profile. Sequencing of exons 1-5 demonstrated the presence of the M3 allele. Moreover, a triple nucleotide deletion was detected in exon 2 of one allele. This caused an "in-phase" frameshift, coding for a protein deficient in a single Phe residue, which corresponded to the Mmalton variant. After liver biopsy, periodic acid-Schiff-positive acidophilic bodies resistant to diastase digestion were observed in the cytoplasm of hepatocytes. These results demonstrated that our patient had a heterozygous M3Mmalton alpha1AT genotype related to a deficiency phenotype. This observation is the first of a patient with heterozygous Mmalton genotype associated with an alpha1AT deficiency that induced severe liver disease requiring orthotopic liver transplantation.

Specific secretion of gel-forming mucins and TFF peptides in HT-29 cells of mucin-secreting phenotype.

Trefoil factor family (TFF) peptides are typical secretory products of mucin-producing cells, e.g. of the gastrointestinal tract. Here, the expression and secretion of mucins and TFF peptides was studied in the HT-29 cell line throughout cellular growth and differentiation in relation to a mucin-secreting (HT-29 MTX) or an enterocyte-like (HT-29 G(-)) phenotype. mRNAs of several MUC and TFF genes were expressed in both cell subpopulations. However, for most MUC and TFF genes, the expression appeared strongly induced with the differentiation into the mucin-secreting phenotype. On the other hand, TFF2 was specifically expressed in the mucin-secreting HT-29 MTX cells. The differentiation of HT-29 MTX cells into the mucin-secreting phenotype was characterised by secretion of the gel-forming mucins MUC2, MUC5AC, and MUC5B, however, according to a different pattern in the course of differentiation. A significant amount of TFF1 and TFF3 was secreted after differentiation, also according to a different pattern, whereas TFF2 was only faintly detected. Secretagogues, known to induce the secretion of mucus, increased the secretion of all three TFF peptides. In contrast, neither a secretory mucin nor a TFF peptide was found in the culture medium of HT-29 G(-) cells. Overlay assays indicated that HT-29 MTX mucins bound to secretory peptides of HT-29 MTX cells with relative molecular mass similar to TFF peptides. TFF1 and TFF3 were specifically localised in the mucus layer of HT-29 MTX cells by confocal microscopy. Finally, the secretion of TFF peptides and mucins appears as a co-ordinated process which only occurs after differentiation into goblet cell-like phenotype.

Differential effect of GalNAcalpha-O-bn on intracellular trafficking in enterocytic HT-29 and Caco-2 cells: correlation with the glycosyltransferase expression pattern.

Our previous work has shown that long-term treatment of mucus-secreting HT-29 cells with 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (GalNAcalpha-O-bn), a competitive inhibitor of O-glycosylation, induced several phenotypic changes, in particular a blockade in the secretion of mucins, which are extensively O-glycosylated glycoproteins. Here, we have analyzed the effects of GalNAcalpha-O-bn upon the intracellular trafficking of basolateral and apical membrane glycoproteins at the cellular and biochemical levels in two types of cells, HT-29 G(-) and Caco-2, differentiated into an enterocyte-like phenotype. In HT-29 G(-) cells, but not in Caco-2 cells, DPP-IV and CD44 failed to be targeted to the apical or basolateral membrane, respectively, and accumulated inside intracytoplasmic vesicles together with GalNAcalpha-O-bn metabolites. We observed a strong inhibition of alpha2,3-sialylation of glycoproteins in HT-29 G(-) cells correlated to the high expression of alpha2,3-sialyltransferases ST3Gal I and ST3Gal IV. In these cells, DPP-IV and CD44 lost the sialic acid residue substituting the O-linked core 1 structure Galbeta1-3GalNAc (T-antigen). In contrast, sialylation was not modified in Caco-2 cells, but a decrease of alpha1,2-fucosylation was observed, in correlation with the high expression of alpha1,2-fucosyltransferases Fuc-TI and Fuc-TII. In conclusion, in HT-29 G(-) cells, GalNAcalpha-O-bn induces a specific cellular phenotype, which is morphologically characterized by the formation of numerous intracellular vesicles, in which are accumulated defectively sialylated O-glycosylproteins originally targeted to basolateral or apical membranes, and GalNAcalpha-O-bn metabolites.

Alkyl-lysophospholipid 1-O-octadecyl-2-O-methyl- glycerophosphocholine induces invasion through episialin-mediated neutralization of E-cadherin in human mammary MCF-7 cells in vitro.

1-O-octadecyl-2-O-methyl-glycerophosphocholine (ET-18-OMe) is an analogue of the naturally occurring 2-lysophosphatidylcholine belonging to the class of antitumor lipids. Previously, we demonstrated that ET-18-OMe modulates cell-cell adhesion of human breast cancer MCF-7 cells. In the present study, we tested the effect of ET-18-OMe on adhesion, invasion and localisation of episialin and E-cadherin in MCF-7/AZ cells expressing a functional E-cadherin/catenin complex. The MCF-7/6 human breast cancer cells were used as negative control since their E-cadherin/catenin complex is functional in cells grown on solid substrate but not in suspension. The function of E-cadherin, a calcium-dependent transmembrane cell-cell adhesion and signal-transducing molecule, is disturbed in invasive cancers by mutation, loss of mRNA stability, proteolytic degradation, tyrosine phosphorylation of associated proteins and large cell-associated proteoglycans or mucin-like molecules such as episialin. Episialin, also called MUC1, is an anti-adhesion molecule that by its large number of glycosylated tandem repeats can sterically hinder the adhesive properties of other glycoproteins. ET-18-OMe inhibited the E-cadherin functions of MCF-7/AZ cells as measured by inhibition of fast and slow aggregation and by the induction of collagen invasion. These effects were enhanced by MB2, an antibody against E-cadherin and blocked by monoclonal antibodies (MAbs) 214D4 or M8 against episialin. ET-18-OMe had no influence on tyrosine phosphorylation of beta-catenin and the E-cadherin/catenin complex remained intact. Transcription, translation, protein turnover and cell surface localisation of episialin were not altered. ET-18-OMe induced finger-like extensions with clustering of episialin together with E-cadherin and carcinoembryonic antigen but not with occludin. In cells in suspension, ET-18-OMe caused a shift in the flow-cytometric profile of episialin toward a lower intensity for MCF-7/AZ cells. In contrast with MCF-7/AZ cells, the adhesion-deficient and noninvasive MCF-7/6 cells showed neither morphotypic changes nor induction of aggregation nor invasion in collagen I upon treatment with ET-18-OMe. Co-localisation of episialin with E-cadherin was rarely observed. We conclude that in the human breast cancer cells MCF-7/AZ, E-cadherin and episialin are key molecular players in the regulation of promotion and suppression of cell-cell adhesion and invasion.

Expression of sialyl-Tn antigen in breast cancer cells transfected with the human CMP-Neu5Ac: GalNAc alpha2,6-sialyltransferase (ST6GalNac I) cDNA.

Sialyl-Tn antigen (STn) is a cancer associated carbohydrate antigen over-expressed in several cancers including breast cancer, and currently associated with more aggressive diseases and poor prognosis. However, the commonly used breast cancer cell lines (MDA-MB-231, T47-D and MCF7) do not express STn antigen. The key step in the biosynthesis of STn is the transfer of a sialic acid residue in alpha2,6-linkage to GalNAc alpha-O-Ser/Thr. This reaction is mainly catalyzed by a CMP-Neu5Ac GalNAc alpha2,6-sialyltransferase: ST6GalNAc I. In order to generate STn-positive breast cancer cells, we have cloned a cDNA encoding the full-length human ST6GalNAc I from HT-29-MTX cells. The stable transfection of MDA-MB-231 with an expression vector encoding ST6GalNAc I induces the expression of STn antigen at the cell surface. The expression of STn short cuts the initial O-glycosylation pattern of these cell lines, by competing with the Core-1 beta1,3-galactosyltransferase, the first enzyme involved in the elongation of O-glycan chains. Moreover, we show that STn expression is associated with morphological changes, decreased growth and increased migration of MDA-MB-231 cells.

Massive in vitro synthesis of tagged oligosaccharides in 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside treated HT-29 cells.

Permanent exposure of differentiated HT-29 cells to the sugar analogue, 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (GalNAcalpha- O -bn) leads to marked effects upon the phenotypic properties of mucin-secreting or enterocyte-like HT-29 cells: an inhibition in the secretion of mucins, a blockade in the apical targeting of membrane brush border glycoproteins and a swelling of cells with intracellular accumulation of numerous vesicles. Folch extraction and partition of treated enterocyte-like HT-29 cells revealed a very important accumulation of orcinol and/or resorcinol reactive material in the upper phase (usually containing gangliosides), as compared with untreated HT-29 cells and with treated and untreated Caco-2 cells. Structural analysis indicated the accumulation of a series of GalNAcalpha- O -bn derived oligosaccharides, most of them with the common core Galbeta1-3GalNAcalpha- O -bn. These oligosaccharides contained residues of GlcNAc, Gal, Neu5Ac, or Fuc. In particular, the tagged sialyl-Lewis(x)was identified, as well as more complex sialylated derivatives, including the sialyl-Lewis(x)substituted by an additional Neu5Ac residue. The benzylated oligosaccharides were not significantly detected in the culture medium except for Galbeta1-3GalNAcalpha- O -bn. Upon reversion of the treatment, these derivatives dis-appeared from the cells within few days, however were not recovered as such in the culture medium. Intracellular degradation occurred with desialylation and defucosylation as the first steps. The spectacular accumulation of benzylated oligosaccharides in HT-29 cell, permanently exposed to GalNAcalpha- O -bn very likely plays an important role in the alterations of cellular processes previously described in this cell line. The HT-29 cell culture system also appears to be an efficient source of several tagged oligosaccharides.

Overexpression level of stromelysin 3 is related to the lymph node involvement in non-small cell lung cancer.

Proteases contribute to tumor invasion and metastasis via their potential to degrade basement membranes and extracellular matrix. Our aim was to compare the level of several proteases: urokinase-type plasminogen activator (u-PA), matrix metalloproteinase 2 (MMP-2; 72-kDa type IV collagenase, also known as gelatinase A), MMP-11 [also known as stromelysin 3 (STR3)], and cathepsins B and L in resected non-small cell lung cancer. Between June 1996 and March 1998, samples of lung tumor tissues were taken from 119 surgically treated patients. Thirty out of the 119 tumor samples were matched with corresponding adjacent normal tissue. u-PA was measured by a commercially available immunoluminometric assay. Metalloproteinases and cathepsins have been evaluated at the RNA level by Northern blot and quantified with a PhosphorImager. Expression of these proteases was compared to the following clinicopathological parameters: pathological diagnosis, tumor size, exposure to asbestos, radiotherapy, neo-adjuvant chemotherapy, tumor-node-metastasis stage, lymph node involvement, presence of metastasis. u-PA, MMP-2, MMP-11/STR3, and cathepsin B were significantly increased in tumor (the tumor:normal ratio was on average increased by 5.4-, 2.2-, 83.5-, and 2.2-fold, respectively). The tumor:normal ratio of MMP-11/ STR3 was found to be significantly linked to the lymph node involvement (P < 0.05). Our results suggest that several proteases are involved in the invasive potential of non-small cell lung cancer and that the quantification of MMP-11/ STR3 could represent an useful prognostic marker.

Specific expression of matrix metalloproteinases 1, 3, 9 and 13 associated with invasiveness of breast cancer cells in vitro.

Several matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) were studied in highly invasive (MDA-MB-231) and slightly invasive (MCF-7, T47D, BT-20) breast cancer cell lines. Investigations were carried out at the protein level and/or at the mRNA level, either in cells cultured as monolayers on plastic, or in cells seeded on a thin layer of Matrigel basement membrane matrix. Analysis of MMP expression by RT-PCR showed expression of MMP-1. MMP-3, and MMP-13 in highly invasive MDA-MB-231 cells, but not in slightly invasive cell lines. The extracellular secretion of MMP-1 and MMP-3 by MDA-MB 231 cells could be also shown by ELISA. TIMP-1 and TIMP-2 mRNAs were found in all cell lines, however, the extracellular secretion of both TIMPs was much higher in MDA-MB-231 cells than in the other cell lines. When the cells were cultured on Matrigel matrix, MMP-9 expression was induced in MDA-MB-231 cells only, as assessed by RT-PCR and zymography experiments. The invasive potential of MDA-MB-231 cells evaluated in vitro through Matrigel was significantly inhibited by the MMP inhibitor BB-2516, by 25% and 50% at the concentrations of 2 x 10(-6) M and 10(-5) M, respectively. In conclusion, our data show that highly invasive MDA-MB-231 cells but not slightly invasive T47D, MCF-7 and BT-20 cells express MMP-1, MMP-3, MMP-9 and MMP-13. MMP-9 which is specifically up-regulated by cell contact to Matrigel, may play a key role in the invasiveness of MDA-MB-231 cells through basement membranes.

Acute inflammatory intestinal vascular lesions and in situ abnormalities of the plasminogen activation system in Crohn's disease.

OBJECTIVES: The distribution of the intestinal vascular lesions and their relation with the fibrinolysis process are poorly known in Crohn's disease (CD). The mediators of the plasminogen activator system, namely urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1), are a key complex involved in fibrinolysis. The aims of this study were: (1) to further define vascular lesions and their distribution in the intestine; and (2) to study concomitantly the qualitative in situ expression and the levels of u-PA, t-PA and PAI-1 in the ileum of patients with CD. PATIENTS AND METHODS: Histological, immunohistochemical and ultrastructural studies of vascular lesions in the resected ileum of 27 patients with CD were performed and compared with 36 control patients. Levels of u-PA, t-PA and PAI-1 measured by ELISA methods were compared in healthy and inflamed ileal tissues of 17 patients with CD. RESULTS: Acute vascular lesions involving mainly serosal venules and capillaries were present in 63% of patients with CD vs 3/36 controls and were associated with PAI-1 expression. They were prominent on the mesenteric border beneath macroscopically normal mucosa. In contrast, chronic vascular lesions were present in all layers beneath mucosal ulcerations, where a significant increase of PAI-1 levels was found. CONCLUSIONS: These results suggest that vascular involvement associated with abnormalities of PAI-1 expression is an early and widespread event in CD. Their prominence on the mesenteric border might explain the characteristic location of CD ulceration along the mesenteric margin.

Permanent exposure of mucin-secreting HT-29 cells to benzyl-N-acetyl-alpha-D-galactosaminide induces abnormal O-glycosylation of mucins and inhibits constitutive and stimulated MUC5AC secretion.

Previous work has shown that treatment of HT-29 methotrexate (MTX) cells with benzyl-N-acetyl-alpha-D-galactosaminide results in profound changes in mucin oligosaccharide chains. To analyse in depth the effect of this drug, we first determined the structure of mucin oligosaccharide chains synthesized by HT-29 MTX cells and the changes induced by permanent drug exposure. Mucins from untreated cells contained nine monosialylated structures (core types 1, 2, 3 and 4) and four disialylated structures (types 1, 2 and 4). Core 1 structures predominated, in particular NeuAcalpha2-3Galbeta1-3GalNAc-ol. Exposure of HT-29 MTX cells to benzyl-N-acetyl-alpha-D-galactosaminide from days 2-21 resulted in a decrease in intracellular mucins and both their sialic acid and galactose content, and an increased T (Galbeta1-3GalNAcalpha-O-Ser/Thr) and Tn (GalNAcalpha-O-Ser/Thr) antigenicity. A 3-fold increase in both Galbeta1-3GalNAc alpha2, 3-sialyltransferase activity and mRNA expression was detected. At the ultrastructural level, T-antigen was not detectable in mucin droplets in control cells, but was strongly expressed in intracytoplasmic vesicles in treated cells. In these cells, MUC1 and MUC3 transcripts were up-regulated, whereas MUC2, MUC5B and MUC5AC were down-regulated. Furthermore, constitutive and secretagogue-induced MUC5AC secretion was reduced and no mucus layer was detected. In conclusion, benzyl-N-acetyl-alpha-D-galactosaminide induces abnormal O-glycosylation and altered regulation of MUC5AC secretion.