Carme de Bolós

Detection of the MUC2 apomucin tandem repeat with a mouse monoclonal antibody

BACKGROUNDnThe MUC2 intestinal mucin gene contains tandem repeats of 23 amino acid length that are rich in threonine.nnnMETHODSnMouse monoclonal antibody LDQ10 was raised against chemically deglycosylated mucin isolated from LS174T colon cancer nude mouse xenografts.nnnRESULTSnLDQ10 reacts with deglycosylated colon cancer mucin and with a synthetic peptide encompassing the MUC2 tandem repeat sequence. In immunohistochemical assays, strong reactivity with goblet cells in colon, small bowel, and stomach is observed; weaker reactivity with mucin-producing cells in other epithelial tissues is shown. The epitope recognized by LDQ10 is localized in the rough endoplasmic reticulum of normal colonic goblet cells. LDQ10 also shows strong reactivity with colorectal and stomach cancers and weaker reactivity with pancreas, breast, and bladder cancers.nnnCONCLUSIONSnAntibody LDQ10 detects a peptide epitope of MUC2 that becomes cryptic on glycosylation. Altered synthesis of the MUC2 apomucin takes place in a variety of epithelial cancers.

α2,3-Sialyltransferase ST3Gal III Modulates Pancreatic Cancer Cell Motility and Adhesion In Vitro and Enhances Its Metastatic Potential In Vivo

Background Cell surface sialylation is emerging as an important feature of cancer cell metastasis. Sialyltransferase expression has been reported to be altered in tumours and may account for the formation of sialylated tumour antigens. We have focused on the influence of alpha-2,3-sialyltransferase ST3Gal III in key steps of the pancreatic tumorigenic process. Methodology/Principal Findings ST3Gal III overexpressing pancreatic adenocarcinoma cell lines Capan-1 and MDAPanc-28 were generated. They showed an increase of the tumour associated antigen sialyl-Lewisx. The transfectants E-selectin binding capacity was proportional to cell surface sialyl-Lewisx levels. Cellular migration positively correlated with ST3Gal III and sialyl-Lewisx levels. Moreover, intrasplenic injection of the ST3Gal III transfected cells into athymic nude mice showed a decrease in survival and higher metastasis formation when compared to the mock cells. Conclusion In summary, the overexpression of ST3Gal III in these pancreatic adenocarcinoma cell lines underlines the role of this enzyme and its product in key steps of tumour progression such as adhesion, migration and metastasis formation.

MUC6 expression in breast tissues and cultured cells: Abnormal expression in tumors and regulation by steroid hormones

Neoplastic transformation of epithelial cells is commonly associated with alterations in the expression of mucin genes. The mechanisms involved in this process are largely unknown. MUC6, isolated from a stomach cDNA library, is mainly expressed in stomach antral glands, as detected by using in situ hybridization and immunohistochemistry. We examined MUC6 expression in normal and pathological breast tissues using immunohistochemistry with MUC6‐specific antibodies and in cultured breast cancer cells using immunocytochemistry and Northern blotting. MUC6 was generally not detected in normal breast (1/11) but was detected in fibrocystic disease without atypia (7/17, 41%), in atypical fibrocystic disease (11/11, 100%) and in carcinoma (57/60, 95%). To study the mechanisms involved in mucin gene up‐regulation in breast cancer, we examined baseline, growth‐related and steroid‐induced levels of MUC1, MUC3 and MUC6 in 4 breast cancer cell lines, 2 of which express estrogen receptors. MUC6 levels were up‐regulated at post‐confluence in 2/4 cell lines, whereas no changes were detected for the other mucin genes examined. MUC6 and MUC3 were constitutively expressed, and steroid‐induced, in BT‐474 and MCF‐7 cells, respectively. As a control, pS2 was induced in both cell lines. Our results indicate that (1) MUC6 is overexpressed in breast cancer and in benign breast disease, (2) in vitro, MUC6 and MUC3 are up‐regulated by steroids and (3) abnormal expression of MUC6 in breast cancers may, in part, be explained by hormonal changes associated with tumor development. Int. J. Cancer 77:193–199, 1998.© 1998 Wiley‐Liss, Inc.

IL-6 induces MUC4 expression through gp130/STAT3 pathway in gastric cancer cell lines

The gastric mucosal levels of the pro-inflammatory cytokine Interleukin 6 (IL-6) have been reported to be increased in Helicobacter pylori-infected subjects and, in gastric adenocarcinomas, the up-regulation of intestinal mucin genes (MUC2 and MUC4) has been detected. To analyse the regulatory effects of IL-6 on the activation of intestinal mucins, six gastric cancer cell lines were treated for different times with several concentrations of IL-6, and the expression of MUC2 and MUC4 was evaluated. IL-6 induced MUC4 expression, detected by quantitative RT-PCR, Western blot and immunofluorescence, and MUC2 expression was not affected. MUC4 mRNA levels decreased after blocking the gp130/STAT3 pathway at the level of the receptor, and at the level of STAT3 activation using the AG490 specific inhibitor. MUC4 presents two putative binding sites for STAT factors that may regulate MUC4 transcription after a pro-inflammatory stimulus as IL-6. By EMSA, ChIP and site-directed mutagenesis we show that STAT3 binds to a cis-element at -123/-115, that conveys IL-6 mediated up-regulation of MUC4 transcriptional activity. We also demonstrated that p-STAT3 binds to MUC4 promoter and a three-fold increase in p-STAT3 binding was observed after treating GP220 cells with IL-6. In conclusion, IL-6 treatment induced MUC4 expression through the gp130/STAT3 pathway, indicating the direct role of IL-6 on the activation of the intestinal mucin gene MUC4 in gastric cancer cells.

Differences in the O-Glycosylation Patterns Between Lung Squamous Cell Carcinoma and Adenocarcinoma

Mucins are highly O-glycosylated proteins synthesized by epithelial cells, and their glycosylation patterns can be altered during neoplastic transformation. The 2 types of non-small cell lung cancer (NSCLC) display a similar pattern of mucin gene expression but different reactivity to periodic acid-Schiff diastase, suggesting that a higher number of carbohydrate chains are present in adenocarcinomas. We compared the expression of core (Tn, sialyl-Tn, T) and terminal fucosylated and sialylated (Lewis antigens) carbohydrate structures in lung tumors. Specific antibodies were usedfor immunohistochemical and Western blot assays. Results indicated that core and terminal structures are detected more frequently in adenocarcinoma than in squamous cell carcinoma, except Lewis y, which is expressed strongly in both types of NSCLC. These data suggest that in squamous cell carcinoma and adenocarcinoma, different sets of glycosyltransferases must be expressed and that different posttranslational modifications of the mucin genes can take place in these 2 tumor types.

Role of sialyltransferases involved in the biosynthesis of Lewis antigens in human pancreatic tumour cells

AbstractThe sialylated carbohydrate antigens, sialyl-Lewisx and sialyl-Lewisa, are expressed in pancreatic tumour cells and are related to their metastatic potential. While the action of the fucosyltransferases involved in the synthesis of these antigens has already been investigated, no studies have been carried out on the activity and expression of the α 2,3-sialyltransferases in pancreatic tumour cells. We describe the sialyltransferase (ST) activity, mRNA expression, and analysis of the cell carbohydrate structures in four human pancreatic adenocarcinoma cell lines of a wide range of neoplastic differentiation stages and in normal human pancreatic tissues. Total ST activity measured on asialofetuin, employing a CMP fluorescent sialic acid, varied among the pancreatic cell lines and could be correlated to the expression of their cell surface antigens. However, in some of the pancreatic cell lines, no relationship could be established with their ST3Gal III and IV mRNA expression. Human pancreatic tissues also showed ST expression and activity. However, it presented a much higher expression of neutral fucosylated structures than sialylated structures. In conclusion, ST activity levels in pancreatic cells could be correlated to their expression of sialylated epitopes, which indicates their involvement in the formation of the sialyl-Lewis antigens, in addition to fucosyltransferase activities.nPublished in 2005.

Changes in the invasive and metastatic capacities of HT-29/M3 cells induced by the expression of fucosyltransferase 1

Lewis antigens are terminal fucosylated oligosaccharides synthesized by the sequential action of several glycosyltransferases. The fucosyltransferases are the enzymes responsible for the addition of terminal fucose to precursor oligosaccharides attached to proteins or lipids. These oligosaccharides, defined as cell surface markers, have been implicated in different types of intercellular interactions and in adhesion and invasion processes. Transfection of HT‐29/M3 colon cancer cells with the full length of human fucosyltransferase (FUT1), induces the synthesis of H type 2 and Lewis y antigens, associated with a decrease of sialyl‐Lewis x. The capacity to develop primary tumors when cells were injected intrasplenically was similar in parental and FUT1‐transfected cells, but the capacity to colonize the liver after spleen removal was significantly reduced in M3/FUT1 transfected cells. These results indicate that the expression of FUT1 induces changes in the metastatic capacity of HT‐29/M3 colon cancer cells, as a consequence of the altered expression pattern of type 2 Lewis antigens. Also, an association between MUC5AC expression and the degree of gland differentiation in both primary splenic tumors and hepatic metastases was detected. (Cancer Sci 2007; 98: 1000–1005)

Cytotoxicity and enzymatic activity inhibition in cell lines treated with novel iminosugar derivatives

Iminosugars are monosaccharide analogues that have been demonstrated to be specific inhibitors for glycosidases and are currently used therapeutically in several human disorders. N-alkylated derivatives of d-fagomine and (2R,3S,4R,5S)-2-(hydroxymethyl)-5-methylpyrrolidine-3,4-diol with aliphatic chains were tested in eight human cancer cell lines to analyze their cytotoxicity and the inhibitory effect in the activities of specific glycosidases. Results indicate that these compounds were more cytotoxic as the length of the alkyl chain increases. N-dodecyl-d-fagomine inhibited specifically the α-d-glucosidase activity in cell lysates, whereas no effect was detected in other glycosidases. The N-dodecyl derivative of (2R,3S,4R,5S)-2-(Hydroxymethyl)-5-methylpyrrolidine-3,4-diol induced specific inhibition against α-l-fucosidase in cell lysates. Our results indicated that the length of the alkyl chain linked to the iminosugars determine their cytotoxicity as well as the inhibitory effect on the enzymatic activities of specific glycosidases, in human cancer cell lines.

Quantitative analysis of N-glycans from human alfa-acid-glycoprotein using stable isotope labeling and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry as tool for pancreatic disease diagnosis.

In this work we demonstrate the potential of glycan reductive isotope labeling (GRIL) using [(12)C]- and [(13)C]-coded aniline and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry (μZIC-HILIC-ESI-MS) for relative quantitation of glycosylation variants in selected glycoproteins present in samples from cancer patients. Human α1-acid-glycoprotein (hAGP) is an acute phase serum glycoprotein whose glycosylation has been described to be altered in cancer and chronic inflammation. However, it is not clear yet whether some particular glycans in hAGP can be used as biomarker for differentiating between these two pathologies. In this work, hAGP was isolated by immunoaffinity chromatography (IAC) from serum samples of healthy individuals and from those suffering chronic pancreatitis and different stages of pancreatic cancer, respectively. After de-N-glycosylation, relative quantitation of the hAGP glycans was carried out using stable isotope labeling and μZIC-HILIC-ESI-MS analysis. First, protein denaturing conditions prior to PNGase F digestion were optimized to achieve quantitative digestion yields, and the reproducibility of the established methodology was evaluated with standard hAGP. Then, the proposed method was applied to the analysis of the clinical samples (control vs. pathological). Pancreatic cancer samples clearly showed an increase in the abundance of fucosylated glycans as the stage of the disease increases and this was unlike to samples from chronic pancreatitis. The results gained here indicate the mentioned glycan in hAGP as a candidate structure worth to be corroborated by an extended study including more clinical cases; especially those with chronic pancreatitis and initial stages of pancreatic cancer. Importantly, the results demonstrate that the presented methodology combining an enrichment of a target protein by IAC with isotope coded relative quantitation of N-glycans can be successfully used for targeted glycomics studies. The methodology is assumed being suitable as well for other such studies aimed at finding novel cancer associated glycoprotein biomarkers.

Regulation of glycosyltransferases and Lewis antigens expression by IL-1β and IL-6 in human gastric cancer cells.

Inflammation of stomach mucosa has been postulated as initiator of gastric carcinogenesis and the presence of pro-inflammatory cytokines can regulate specific genes involved in this process. The cellular expression pattern of glycosyltransferases and Lewis antigens detected in the normal mucosa changed during the neoplassic transformation. The aim of this work was to determine the regulation of specific fucosyltransferases and sialyltransferases by IL-1β and IL-6 pro-inflammatory cytokines in MKN45 gastric cancer cells. IL-1β induced significant increases in the mRNA levels of FUT1, FUT2 and FUT4, and decreases of FUT3 and FUT5. In IL-6 treatments, enhanced FUT1 and lower FUT3 and FUT5 mRNA expression were detected. No substantial changes were observed in the levels of ST3GalIII and ST3GalIV. The activation of FUT1, FUT2 and FUT4 by IL-1β is through the NF-κB pathway and the down-regulation of FUT3 and FUT5 by IL-6 is through the gp130/STAT-3 pathway, since they are inhibited specifically by panepoxydone and AG490, respectively. The levels of Lewis antigens after IL-1β or IL-6 stimulation decreased for sialyl-Lewis x, and no significant differences were found in the rest of the Lewis antigens analyzed, as it was also observed in subcutaneous mice tumors from MKN45 cells treated with IL-1β or IL-6. In addition, in 61 human intestinal-type gastric tumors, sialyl-Lewis x was highly detected in samples from patients that developed metastasis. These results indicate that the expression of the fucosyltransferases involved in the synthesis of Lewis antigens in gastric cancer cells can be specifically modulated by IL-1β and IL-6 inflammatory cytokines.