Purpose:The BRAF gene encodes a serine/threonine kinase and plays an important role in the mitogen-activated protein kinase signaling pathway. BRAF mutations in sporadic colorectal cancer with microsatellite instability (MSI) are more frequently detected than those in microsatellite stable cancer. In this study, we sought to compare the frequencies of BRAF mutations in sporadic colorectal cancer with MSI with those in hereditary nonpolyposis colorectal cancer (HNPCC).Experimental Design: We analyzed BRAF mutations in 26 colorectal cancer cell lines, 80 sporadic colorectal cancers, and 20 tumors from HNPCC patients by DNA sequencing and sequence-specific PCR. The methylation status of the hMLH1 gene was measured by either sequencing or restriction enzyme digestion after NaHSO 3 treatment.Results: We observed a strong correlation of BRAF mutation with hMLH1 promoter methylation. BRAF mutations were present in 13 of 15 (87%) of the colorectal cell lines and cancers with methylated hMLH1, whereas only 4 of 91 (4%) of the cell lines and cancers with unmethylated hMLH1 carried the mutations (P < 0.00001). Sixteen of 17 mutations were at residue 599 (V599E). A BRAF mutation was also identified at residue 463 (G463V) in one cell line. In addition, BRAF mutations were not found in any cancers or cell lines with K-ras mutations. In 20 MSI؉ cancers from HNPCC patients, however, BRAF mutations were not detectable, including a subset of 9 tumors with negative hMLH1 immunostaining and methylated hMLH1.Conclusions: BRAF mutations are frequently present in sporadic colorectal cancer with methylated hMLH1, but not in HNPCC-related cancers. This discrepancy of BRAF mutations between sporadic MSI؉ cancer and HNPCC might be used in a strategy for the detection of HNPCC families.

Cystic fibrosis (CF) is an autosomal recessive disorder, the most common lethal genetic disease in Caucasians. Respiratory disease is the major cause of morbidity and mortality. Indeed, 95% of CF patients die of respiratory failure. Pseudomonas aeruginosa, an opportunistic pathogen, chronically infects the lungs of over 85% of CF patients. It is ineradicable by antibiotics and responsible for airway mucus overproduction that contributes to airway obstruction and death. The molecular mechanisms underlying this pathology are unknown. Here we show that P. aeruginosa activates a c-Src-Ras-MEK1͞2-MAPK-pp90rsk signaling pathway that leads to activation of nuclear factor NF-B (p65͞p50). Activated NF-B binds to a B site in the 5-f lanking region of the MUC2 gene and activates MUC2 mucin transcription. These studies bring new insight into bacterialepithelial interactions and more specifically into the molecular pathogenesis of cystic fibrosis. Understanding these signaling and gene regulatory mechanisms opens up new therapeutic targets for cystic fibrosis.Cystic fibrosis (CF) is the most common lethal genetic disease in the Caucasian population (1). The mutation responsible for the disease is in the gene encoding the cystic fibrosis transmembrane regulator (CFTR), a chloride channel. This mutation gives rise to a chain of events involving chronic bacterial lung infection and mucus overproduction. Although there have been significant clues regarding the link between the mutation and chronic infection (2-4), the nature of the link between the mutation and mucus overproduction is completely unknown.We recently showed that bacterial exoproducts up-regulate epithelial MUC2 mucin transcription (5). This suggests that CF-associated mucin overproduction occurs secondary to lung infection, a sine qua non of advanced disease. The molecular mechanisms underlying mucin induction are unknown. In the present study, we performed experiments to examine these mechanisms. Results showed that Pseudomonas aeruginosa, a common CF pathogen, activates the MUC2 mucin gene transcription by activation of a Src-dependent Ras-MEK1͞2-ERK1͞2-pp90rsk-NF-B pathway. These studies provide insight into bacterial-host epithelial interactions and open up new therapeutic targets for CF. MATERIALS AND METHODSReagents. P. aeruginosa lipopolysaccharide (LPS) from serotype 10 was purchased from Sigma. PP1, PD98059, and caffeic acid phenethyl ester (CAPE) were purchased from Calbiochem.Bacterial Strains and Culture Conditions. The P. aeruginosa strains used in these studies were grown in M9 medium with aeration at 37°C to late logarithmic phase. The broth cultures were then centrifuged at 10,000 rpm in a Sorvall RC5C for 50 min. The supernatants containing bacterial exoproducts were sterilized by passage through a 0.22-m polymer filter (Corning) and were then kept at Ϫ80°C until use. Bacterial culture supernatants were added to epithelial cell culture medium at a 1:4 dilution ratio.Cell Culture. Two MUC2-expressing epithelial cell lines were used in these...

An unresolved question in cystic fibrosis (CF) research is how mutations of the CF transmembrane conductance regulator, a Cl ion channel, cause airway mucus obstruction leading to fatal lung disease. Recent evidence has linked the CF transmembrane conductance regulator mutation to the onset and persistence of Pseudomonas aeruginosa infection in the airways, and here we provide evidence directly linking P. aeruginosa infection to mucus overproduction. We show that P. aeruginosa lipopolysaccharide profoundly upregulates transcription of the mucin gene MUC 2 in epithelial cells via inducible enhancer elements and that this effect is blocked by the tyrosine kinase inhibitors genistein and tyrphostin AG 126. These findings improve our understanding of CF pathogenesis and suggest that the attenuation of mucin production by lipopolysaccharide antagonists and tyrosine kinase inhibitors could reduce morbidity and mortality in this disease.

Mucins are high molecular weight glycoproteins that are heavily glycosylated with many oligosaccharide side chains linked O-glycosidically to the protein backbone. With the recent application of molecular biological methods, the structures of apomucins and regulation of mucin genes are beginning to be understood. At least nine human mucin genes have been identified to date. Although a complete protein sequence is known for only three human mucins (MUC1, MUC2, and MUC7), common motifs have been identified in many mucins. The pattern of tissue and cell-specific expression of these mucin genes are emerging, suggesting a distinct role for each member of this diverse mucin gene family. In epithelial cancers, many of the phenotypic markers for pre-malignant and malignant cells have been found on the carbohydrate and peptide moieties of mucin glycoproteins. The expression of carbohydrate antigens appears to be due to modification of peripheral carbohydrate structures and the exposure of inner core region carbohydrates. The expression of some of the sialylated carbohydrate antigens appears to correlate with poor prognosis and increased metastatic potential in some cancers. The exposure of peptide backbone structures of mucin glycoproteins in malignancies appears to be due to abnormal glycosylation during biosynthesis. Dysregulation of tissue and cell-specific expression of mucin genes also occurs in epithelial cancers. At present, the role of mucin glycoproteins in various stages of epithelial cell carcinogenesis (including the preneoplastic state and metastasis), in cancer diagnosis and immunotherapy is under investigation.

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The N terminus of the human MUC2 mucin (amino acids 1-1397) has been expressed as a recombinant tagged protein in Chinese hamster ovary cells. The intracellular form was found to be an endoglycosidase H-sensitive monomer, whereas the secreted form was an oligomer that gave monomers upon disulfide bond reduction. The secreted MUC2 N terminus contained a trypsin-resistant core fragment. Edman sequencing and mass spectrometry of the peptides obtained localized this core fragment to the C-terminal end of the recombinant protein. This core retained its oligomeric nature with an apparent mass of ϳ240 kDa. Upon reduction, peptides of ϳ85 kDa were found, suggesting that the N terminus forms trimers. This interpretation was also supported by gel electrophoresis and gel filtration of the intact MUC2 N terminus. Electron microscopy revealed three globular domains each linked via an extended and flexible region to a central part in a trefoil-like manner. Immunostaining with gold-labeled antibodies localized the N-terminal end to the three globular structures, and the antibodies directed against the Myc and green fluorescent protein tags attached at the C terminus localized these to the stalk side of the central trefoil. The N terminus of the MUC2 mucin is thus assembled into trimers that contain proteolytically stable parts, suggesting that MUC2 can only be partly degraded by intestinal proteases and thus is able to maintain a mucin network protecting the intestine.Mucins are highly glycosylated proteins protecting the mucosal surfaces of the body. All mucins are characterized by their heavy O-glycosylation, which is clustered in mucin domains rich in the amino acids Ser, Thr, and Pro. Mucins can be structured in different ways, either as membrane-bound with a transmembrane domain or secreted as monomers or as polymers (1, 2). The latter mucins are also described as gel-forming, as these give the mucus its viscoelastic properties. Four human gel-forming mucins have been found, MUC2, MUC5AC, MUC5B, and MUC6, which are clustered on chromosome 11p15 (3). However, the best studied mucin is the porcine submaxillary mucin (PSM), 1 which has become a model also for the human mucins of this type (3-6).The MUC2 mucin is the main gel-forming mucin of the small and large intestines and is produced by the intestinal goblet cells (2, 7-10). It is a major structural component of the mucous barrier covering the epithelium, protecting the epithelial cells from microorganisms as well as digestive enzymes. In fact, one of the remarkable features of this mucin is that it can withstand the pancreatic digestive enzymes. That the highly glycosylated mucin domains are totally resistant to these enzymes is easy to understand. However, the globular, less glycosylated ends must also be relatively proteolytically resistant, as the polymers must remain intact to maintain the mucous gel. The MUC2 mucin is difficult to solubilize, and it is insoluble in 4 M guanidinium chloride (8, 10), a property that might be related to the appearance of nonreducible ...

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