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F X Real

Publications and source records attributed to F X Real.

At least 73 records · Page 4Linked to original sources
PubMed1994

Altered expression of MUC2, MUC4, and MUC5 mucin genes in pancreas tissues and cancer cell lines.

BACKGROUND/AIMS: Neoplastic transformation of epithelial cells is commonly associated with altered synthesis and structure of mucin glycoproteins. The aim of the study was to determine if altered mucin gene expression takes place in pancreas cancer. METHODS: To examine mucin gene expression in normal pancreas and pancreas cancer, antibodies detecting the MUC1, MUC2, MUC5B, and MUC5C apomucins were used in immunohistochemical techniques and complementary DNA probes specific for the MUC1-MUC5 genes were used in Northern blots. RESULTS: MUC1 is the major apomucin expressed in normal pancreas, whereas MUC2-MUC5 are weakly expressed or undetectable. In pancreas cancer tissues and cell lines, increased expression of MUC2, MUC4, and MUC5C is shown. The cytoplasmic expression of MUC2 and MUC5C in tumor cells suggests that these apomucins are underglycosylated and abnormally compartmentalized. CONCLUSIONS: Enhanced expression of MUC2, MUC4, and MUC5C genes is a frequent event in pancreas cancer and may contribute to the alterations in the biochemical structure of pancreas cancer mucins.

Cell Division↗
PubMed1994

Diagnostic certainty and potential for misclassification in exocrine pancreatic cancer. PANKRAS I Project Investigations.

Whereas over the last decade epidemiologic studies on exocrine pancreatic cancer (EPC) continued to show a remarkable heterogeneity in diagnostic criteria applied to define caseness, the actual magnitude and consequences of misclassification remain largely unexplored. The objectives were: (1) to estimate the degree of certainty with which cases of EPC are diagnosed in the two participating hospitals (to this end a diagnostic certainty classification (DCC) was developed; (2) to test whether characteristics of cases differed by degree of diagnostic certainty; and (3) to assess what influence different definitions of case might have on risk estimates for tobacco and alcohol. All cases with a discharge diagnosis of EPC who attended at the Hospital del Mar between 1980-90 and at the Hospital Son Dureta between 1983-90 were identified through their respective tumor registries, and their clinical records were reviewed. Only 52% of 140 cases were classified in the group with a higher probability of EPC (group H). Diagnostic certainty appeared somewhat greater among women (age-adjusted odds ratio [ORa] 1.60, p = 0.18). Group H showed a higher proportion of cases with an interval from first symptom to diagnosis < or = 1 month (ORa = 2.38, p < 0.05) and the proportion of adenocarcinomas was slightly higher than in less certain cases (group L) (p = 0.051). A radical treatment was exclusively attempted in group H (p < 0.001). DCC cut-off points had a significant effect on the proportion of smokers and of alcohol drinkers, as well as on the percent of cases with pathological (cytohistological) confirmation. The proportion of cases unlikely to be of pancreatic origin in spite of having pathological confirmation was high enough to cause significant misclassification bias. Because past exposure to certain risk factors may differ among cases with different diagnostic certainty, we suggest to initially include in the case group patients who in spite of lacking pathological confirmation have strong clinical evidence supporting the diagnosis of EPC; subsequently, risk estimates should be computed across strata of diagnostic certainty to assess whether heterogeneity exists. In exocrine pancreatic cancer the impact of misclassification of disease status upon etiologic and prognostic estimates deserves at least as much attention as misclassification of exposure.

Adenocarcinoma↗
PubMed1994

Normal human pancreas cultures display functional ductal characteristics.

BACKGROUND: Normal cell cultures are invaluable in the analysis of cell differentiation and neoplastic transformation. EXPERIMENTAL DESIGN: We have developed methods to reproducibly culture normal pancreas epithelial cells. The characteristics of the cultures were analyzed using ultrastructural methods, antibodies, and cDNA probes detecting epithelial differentiation markers. RESULTS: Normal pancreas tissue (N = 56) was obtained from organ donors; isolation of highly enriched exocrine fraction consistently yielded epithelial cultures. In vitro proliferation assays revealed a lag growth phase of 2 days followed by a proliferative phase until 8. Epithelial cells formed a polarized monolayer displaying apical microvilli, tight junctions, and desmosomes. Zymogen granules were not observed. A panel of mouse monoclonal antibodies detecting differentiation antigens of epithelial cells was used to determine the phenotype of the cultures: all cells expressed cytokeratin polypeptides of simple epithelial (CK 7, CK 8, CK 18, and CK 19), whereas polypeptides typical of stratified epithelial (CK 5, CK 10, CK 13, and CK 16) were not detected. Cultured cells expressed the MUC1 apomucin as well as mucin-associated carbohydrate epitopes. Expression of the cystic fibrosis transmembrane regulator was demonstrated at the RNA level. Secretin induced a very high stimulation of cAMP levels. CONCLUSIONS: The ultrastructural characteristics, molecular markers, and hormone responsiveness of the cultures suggest a ductal cell phenotype. These cultures should be useful in the analysis of pancreas growth and differentiation.

Adult↗
PubMed1994

Cytokeratins and mucins as molecular markers of cell differentiation and neoplastic transformation in the exocrine pancreas.

Acinar and ductal cells of the normal pancreas express cytokeratin (CK) patterns which indicate a heterogeneity of ductal epithelia. On account of the CK of pancreatic adenocarcinoma, it would seem to be probable that tumor epithelia possess a considerable potential for squamous epithelium metaplasia. Comparative studies of CK expression by ductal carcinoma of the pancreas and carcinoma of bile ducts, stomach and large intestine as well in cases of chronic pancreatitis have demonstrated the usefulness of CK for differential diagnosis of these conditions. From the number of apomucins, mainly MUC1 is produced in the normal pancreas. This capacity is maintained in pancreas carcinoma and cell lines derived from it.

Amino Acid Sequence↗
PubMed1993

alpha GalNAc is essential for recognition of Exo-1 epithelial antigen by mouse monoclonal antibody Pa-G-14.

Mouse monoclonal antibody Pa-G-14 detects Exo-1, an antigen whose expression is regulated in the processes of epithelial-cell differentiation and transformation. The epitope recognized by Pa-G-14 is present both in glycosphingolipids and in mucin glycoproteins. To characterize the specificity of Pa-G-14, immuno-thin-layer chromatography, biochemical, and enzymatic treatment of glycosphingolipid extracts from human pancreas were used. The antibody bound to all blood-group-A substances; alpha GalNAc, but not fucose, was essential for reactivity. In ELISA, Pa-G-14 also reacted with ovine and bovine submaxillary mucins but not with porcine submaxillary mucin. Binding to ovine submaxillary mucin was resistant to neuraminidase treatment. In solid-phase absorption assays on synthetic carbohydrate structures, Pa-G-14 recognized broadly blood group A, Tn and sialyl-Tn. Using immuno-electron-microscopic techniques, reactivity with all Golgi cisternae and mucin droplets of mucous cells in ovine submaxillary gland was demonstrated. All these assays indicate that Pa-G-14 shows a novel specificity, since it binds blood group A, Tn and sialyl-Tn, the common structural feature of these epitopes being the presence of a terminal alpha GalNAc sugar unit.

Acetylgalactosamine↗
PubMed1993

Cytokines as adjuvants: effect on the immunogenicity of NeuAc alpha 2-6GalNAc alpha-O-Ser/Thr (sialyl-Tn).

Sialyl-Tn, defined by monoclonal antibody (MAb) B72.3, shows restricted normal-tissue distribution but is expressed in a wide variety of carcinomas. To analyze the immunogenicity of sialyl-Tn, mice were immunized with ovine submaxillary mucin (OSM) in combination with monophosphoryl lipid A (MPLA), liposomes, or adjuvants that activate macrophages (rIL-1, rIFN-gamma, rM-CSF, IL-1-derived peptides) or T cells (rIL-2). The level and specificity of the immune response were analyzed by ELISA. rIL-1 and rIFN-gamma induced a very high and specific antibody response, whereas the effect of rM-CSF was dose-dependent: at a low dose it induced a high-level specific antibody response and at the high dose level it induced a polyclonal non-specific response. These results indicate that cytokines are powerful adjuvants which modulate both the magnitude and specificity of the immune response. More studies are necessary to determine the optimal doses in animal models and in active specific immunotherapy of patients with cancer.

Adjuvants, Immunologic↗
PubMed1993

Intermediate filaments as differentiation markers of exocrine pancreas. II. Expression of cytokeratins of complex and stratified epithelia in normal pancreas and in pancreas cancer.

Cytokeratin (CK) expression in tumors generally reflects the CK pattern of the corresponding normal epithelium. Pancreas cancers express CK of simple epithelia 7, 8, 18 and 19, as normal ductal cells. To analyze whether CK of complex or stratified epithelia are abnormally expressed in pancreas cancers, we have used polypeptide-specific mouse monoclonal antibodies (MAbs) detecting CK 5, CK 10, CK 13, CK 14 and CK 17, and an antibody detecting CK 13, CK 15 and CK 16. The streptavidin-peroxidase technique was applied on sections of fresh-frozen specimens of normal pancreas and of pancreas cancer. None of these polypeptides were expressed by normal acinar and centro-acinar cells. CK 5, CK 14 and CK 17 were expressed by less than 5% of cells in normal ducts, whereas CK 10, CK 13, CK 15 and CK 16 were not expressed at all. In tumors, CK 14, CK 15/16 and CK 17 were detected in the majority of cases studied; CK 5, CK 10 and CK 13 were present in a sub-population of pancreas cancers. CK of complex/stratified epithelia were detected in areas of glandular differentiation, but expression was more intense in areas of squamous differentiation. In pancreatitis adjacent to cancer, CK of complex/stratified epithelia were weakly detected or undetectable. These results suggest that up-regulation of these CK takes place in pancreas cancer. The CK phenotype may be of help in the differential diagnosis of this tumor.

Antibodies, Monoclonal↗
PubMed1993

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

BACKGROUND: The MUC2 intestinal mucin gene contains tandem repeats of 23 amino acid length that are rich in threonine. METHODS: Mouse monoclonal antibody LDQ10 was raised against chemically deglycosylated mucin isolated from LS174T colon cancer nude mouse xenografts. RESULTS: LDQ10 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. CONCLUSIONS: Antibody 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.

Animals↗
PubMed1993

Expression of epithelial antigens EPM-1 and EXO-1 in normal, transitional, inflammatory and neoplastic colorectal mucosa.

EPM-1 (a high molecular weight glycoprotein) and EXO-1 (a carbohydrate epitope expressed on polar neutral glycolipids and mucins) are two developmental antigens of normal and neoplastic human epithelia and were characterised by monoclonal antibodies. Their distribution was investigated in normal and pathological human colorectal mucosa. In normal mucosa, EPM-1 and EXO-1 showed characteristic expression patterns. EPM-1 was differentially expressed along the crypt villus axis with maximum at the crypt basis. EXO-1 was present throughout the whole mucosa. The characteristic gradient of EPM-1 expression along the crypt axis in normal mucosa was no longer detectable in benign polyps. Intact gradient of EPM-1 staining discriminated between neoplastic changes of the benign adenomatous polyp and mucosal inflammation. Neoplastic mucosa in benign polyps and especially atypical glands in highly differentiated tumours showed essentially identical expression patterns. In colorectal carcinomas the overall reactivities for EPM-1 and EXO-1 were independently associated with the histopathological grade of tumour differentiation.

Antigens, Neoplasm↗
PubMed1993

Glycosyl phosphatidylinositol membrane anchoring of melanotransferrin (p97): apical compartmentalization in intestinal epithelial cells.

Melanotransferrin (p97) is an iron-binding membrane glycoprotein with 40% homology to transferrin and lactoferrin. It was first identified on the basis of its high level of expression in melanoma cells, as compared to normal melanocytes. It is also present in many cultured cell types. In normal tissues, p97 is expressed in fetal intestine, umbilical cord, sweat gland ducts and liver sinusoidal lining cells. Kinetic studies in melanoma cells have suggested that p97 plays a role in iron metabolism. We have examined expression of p97 in cell lines derived from human colorectal carcinomas which express a differentiated phenotype. When polarized, these cells showed a preferred apical distribution of p97, as demonstrated by immunohistochemistry, immune electron microscopy and domain-selective biotinylation. Correspondingly, p97 was only found on the apical brush border of epithelial cells in the fetal intestine. p97 was shown to be anchored to the membrane through a glycosyl phosphatidylinositol moiety by treatment with phophatidylinositol-specific phospholipase C (PI-PLC) and labeling with [14C]ethanolamine. These observations provide a basis for the elucidation of the physiological role of p97 in iron metabolism and its possible role in cell proliferation and malignant cell transformation.

Antigens, Neoplasm↗
PubMed1993

cis-Golgi resident proteins and O-glycans are abnormally compartmentalized in the RER of colon cancer cells.

Neoplastic transformation is commonly associated with altered glycosylation of proteins and lipids. To understand the basis for altered mucin glycosylation, we have examined the distribution of RER markers, a cis-Golgi resident protein, and the GalNAc alpha-O-Ser/Thr epitope (Tn) in human colon cancer cells and in normal colon. In cultured mucin-producing colon cancer cells, Gal-NAc alpha-O-Ser/Thr was found in mucin droplets and in RER cisternae. In addition, the Golgi apparatus was disorganized in a proportion of cells and a 130 kDa cis-Golgi resident protein was also abnormally redistributed to the RER. The distribution of the MUC2 intestinal apomucin, protein disulphide isomerase, Gal-NAc alpha-O-Ser/Thr, and the 130 kDa cis-Golgi resident protein was analysed in normal colon and in colon cancer tissues. In normal colon, MUC2 apomucin and protein disulphide isomerase were located in the RER, whereas the cis-Golgi resident protein and GalNAc alpha-O-Ser/Thr were detected only in the cis-Golgi compartment. In contrast, the two Golgi markers colocalized with the MUC2 apomucin and protein disulphide isomerase in the RER of colon cancer cells. On the basis of these results, we propose that in colon cancer cells a redistribution of molecules normally present in the Golgi apparatus takes place; this alteration may contribute to the abnormal glycosylation of proteins and lipids associated with neoplastic transformation.

Cell Compartmentation↗
PubMed1993

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate blocks differentiation of HT-29 human colon cancer cells.

Recently developed HT-29-derived cell lines, which display variable differentiated phenotypes provide an invaluable opportunity to analyze the mechanism by which cell differentiation is regulated in the intestine. We have studied the effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in the differentiation phenotype of mucus-secreting (HT-29 M6) and absorptive (HT-29 M3) cells. TPA prevented the accumulation of differentiation markers such as dipeptidylpeptidase IV, villin or mucins, down-regulated the expression of these molecules in post-confluent differentiated cell cultures and induced the loss of the functional integrity of the tight junction in the monolayer (i.e. decreased transepithelial resistance and inhibited dome formation). These effects were mediated by activation of protein kinase C (PK-C), as demonstrated using the specific inhibitor GF109203x. Analysis by immunoblotting of the PK-C isoforms present in HT-29 M6 cells revealed that the most abundant TPA-sensitive isoform was PK-C epsilon, although low levels of cPK-C were also detected. Further studies are necessary to elucidate the role of the different PK-C isoforms in the differentiation of HT-29 cells.

Biomarkers↗
PubMed1993

Differential expression of the human mucin genes MUC1 to MUC5 in relation to growth and differentiation of different mucus-secreting HT-29 cell subpopulations.

Mucin expression was analysed, in relation to cell growth, in parental HT-29 cells and in two populations of mucus-secreting HT-29 cells selected by adaptation to methotrexate (HT29-MTX) or 5-fluorouracil (HT29-FU). These two populations express mature mucins that differ in their immunoreactivity to antibodies against gastric (HT29-MTX) or colonic mucins (HT29-FU). In the parental population, at late confluency, only very few cells produce mucins or the MUC1 glycoprotein, this being consistent with the low level of expression of the mRNAs corresponding to the MUC1 to MUC5C mucin genes. In the HT29-MTX and HT29-FU populations, the appearance of mucus droplets, as shown by histochemistry and immunofluorescence, starts a few days after confluency, progressively involving a greater proportion of cells and reaching a steady state at late confluency. The MUC1 glycoprotein appears earlier, already being detectable in preconfluent cells. Its distribution is restricted to the apical surface of the cells and is distinct from that of the mucus droplets. In both populations the growth-related levels of MUC1 mRNA are concordant with the apparent levels of expression of the MUC1 glycoprotein. The levels of MUC2, MUC3, MUC4 and MUC5C mRNAs differ from one population to another and, within each population, according to the stage of the culture. The highest levels of MUC2 and MUC4 mRNAs are found in the HT29-FU cells, whereas the highest levels of MUC3 and MUC5C are found in the HT29-MTX cells, suggesting that the differences observed in the mature mucins expressed by either population may be related to which MUC genes are expressed. In both populations significant or even high levels of MUC mRNAs are already present in early cultures, i.e. at a stage when the mature mucins are not yet detectable, suggesting that mucin maturation is a later event.

Blotting, Northern↗
PubMed1992

Intestinal brush-border-associated enzymes: co-ordinated expression in colorectal cancer.

The brush border of normal small-intestine epithelial cells is rich in enzymes that are involved in the digestive process. Such molecules can be used as markers to analyze cell lineages and differentiation properties of colorectal cancers. Monoclonal antibodies detecting dipeptidyl peptidase-IV, aminopeptidase N, endopeptidase F, sucrase-isomaltase, alkaline phosphatase, maltase-glucoamylase and lactase have been used to analyze the phenotype of colorectal cancers, adjacent mucosa and histologically normal distant mucosa. The avidin-biotin peroxidase complex method was used. Expression of dipeptidyl peptidase-IV, aminopeptidase N, sucrase-isomaltase and alkaline phosphatase was common in non-neoplastic mucosa adjacent to, and distant from, the tumor; in contrast, endopeptidase F, maltase-glucoamylase and lactase were rarely expressed in normal distant mucosa and more frequently expressed in mucosa adjacent to the tumor. Dipeptidyl peptidase-IV, aminopeptidase N, endopeptidase F, sucrase-isomaltase and alkaline phosphatase were frequently expressed in colorectal cancers, whereas maltase-glucoamylase and lactase were rarely expressed. Two general patterns of antibody reactivity were observed: diffuse cytoplasmic and apical; apical reactivity was generally associated with more differentiated tumors. A logistic predictive regression model indicated that enzyme expression in colorectal cancers followed a coordinate pattern, but was unrelated to the location of the tumor, Dukes stage or differentiation grade. In conclusion, expression of brush-border-associated enzymes occurs frequently in colorectal cancers and is regulated in a co-ordinated manner. These markers can be used for the phenotypic sub-classification of colorectal cancers.

Alkaline Phosphatase↗
PubMed1992

Intermediate filaments as differentiation markers of normal pancreas and pancreas cancer.

Expression of intermediate filaments (IF) is regulated during development and differentiation. The authors have studied the expression of vimentin and cytokeratins (CK) 4, 7, 8, 13, 18, 19 in normal pancreas, chronic pancreatitis, and pancreas cancer using monoclonal antibodies. Immunohistochemical assays were performed on fresh frozen tissue sections and on cultured pancreas cancer cells using the streptavidin-peroxidase method. In normal pancreas, acinar cells expressed CK 8 and 18, whereas ductal cells expressed CK 7, 8, 18, and 19. CK 4 was expressed by 5-10% of pancreas duct cells in all specimens of normal pancreas. CK 13 was not detected in any epithelial cells of normal pancreas or pancreatitis. CK 7, 8, 18, and 19 were homogeneously expressed in all pancreas cancers, whereas CK 4 was expressed only in 5-50% of cells in 10/16 tumors. Foci of squamous metaplasia expressed CK 13 but showed partial loss of expression of CK 7, 8, 18, and 19. Thirteen pancreas cancer cell lines examined showed homogeneous expression of CK 7, 8, 18, and 19; 2/11 lines expressed CK 4 weakly, and 6/11 expressed vimentin. CK 13 was not detected in any of the lines. These results indicate that pancreas cancer cells consistently express cytokeratin polypeptides characteristic of ductal epithelial cells and that this phenotype is retained in pancreas cancer cell lines. In addition, squamous metaplasia is associated with a coordinate change in the expression of CK polypeptides.

Biomarkers↗
PubMed1991

Mucin production by colon cancer cells cultured in serum-free medium.

Although many colon cancer cell lines are available for study, few of them exhibit differentiated properties. When cultured in medium containing fetal bovine serum, WiDr cells (WiDr-FBS) show an undifferentiated phenotype: growth as a multilayer of cells adherent to plastic and lack of polarization, brush border, and mucin vacuoles. In contrast, WiDr cells cultured in a chemically-defined serum-free medium containing insulin, transferrin and selenium (WiDr-ITS) grow as clusters of nonadherent cells with abundant desmosomes and tight junctions, microvilli and electron-lucid vacuoles. As WiDr-FBS cells, WiDr-ITS are not polarized. WiDr-ITS cells show a marked enhancement in mucin synthesis as demonstrated by: periodic acid-Schiff and Alcian blue stains, electron microscopy, immunohistochemistry using monoclonal antibodies (MAbs) reactive with mucin-associated epitopes, immune electron microscopy and immunochemical analysis using Western blots. In comparison with WiDr-FBS cells, WiDr-ITS cells showed strong expression of Tn, sialyl-Tn, blood group A and CEA. When mouse MAbs were used, higher levels of the MUCI gene product were detected in WiDr-ITS than in WiDr-FBS cells. The full spectrum of phenotypic changes was observed after I month of culture in ITS medium, and transfer of WiDr-ITS cells to FBS medium was accompanied by a partial phenotypic reversal, suggesting that these phenotypic changes result from an adaptative--rather than selective--process.

Alcian Blue↗