Frequent expression of mucin core protein MUC1 in non-neoplastic gallbladder mucosa from patients with pancreaticobiliary maljunction

Abstract: Background: Gallbladder carcinoma is known to develop frequently in patients with pancreaticobiliary maljunction, though the causal relationship remains speculative. Methods: Histopathologic changes, expression of mucin core protein MUC1 and MUC2, and cell proliferative activities in the gallbladder mucosa from 27 patients with panceaticobiliary maljunction and 21 control gallbladders were examined. Three cases of pancreaticobiliary maljunction were associated with gallbladder carcinoma. Results: The lining epithelia of the non-neoplastic gallbladder mucosa of pancreaticobiliary maljunction showed frequently papillary hyperplasia and higher proliferative activities, when compared to the control. In 3 cases with carcinoma, MUC1 was expressed on the luminal border and in the cytoplasm of carcinoma cells, particularly in de-differentiated and invasive areas. MUC1 was variably expressed on the luminal surface of the lining epithelia of non-neoplastic gallbladder mucosa in babies, children, youths and adults with pancreaticobiliary maljunction. However, such expression was focally seen in 2 of the 21 control cases (p<0.01). MUC2 was scattered in the hyperplastic and carcinomatous epithelial cells appearing as goblet cells in pancreaticobiliary maljunction and control groups. Conclusions: This study suggests that persistent MUC1 expression and increased cell proliferative activities of non-neoplastic gallbladder epithelium of the patients with pancreaticobiliary maljunction after birth reflect an altered phenotype of epithelial cells and these abnormalities may be related to carcinogenesis in such patients.     

Frequent expression of mucin core protein MUC1 in non-neoplastic gallbladder mucosa from patients with pancreaticobiliary maljunction.

BACKGROUND: Gallbladder carcinoma is known to develop frequently in patients with pancreaticobiliary maljunction, though the causal relationship remains speculative. METHODS: Histopathologic changes, expression of mucin core protein MUC1 and MUC2, and cell proliferative activities in the gallbladder mucosa from 27 patients with panceaticobiliary maljunction and 21 control gallbladders were examined. Three cases of pancreaticobiliary maljunction were associated with gallbladder carcinoma. RESULTS: The lining epithelia of the non-neoplastic gallbladder mucosa of pancreaticobiliary maljunction showed frequently papillary hyperplasia and higher proliferative activities, when compared to the control. In 3 cases with carcinoma, MUC1 was expressed on the luminal border and in the cytoplasm of carcinoma cells, particularly in de-differentiated and invasive areas. MUC1 was variably expressed on the luminal surface of the lining epithelia of non-neoplastic gallbladder mucosa in babies, children, youths and adults with pancreaticobiliary maljunction. However, such expression was focally seen in 2 of the 21 control cases (p<0.01). MUC2 was scattered in the hyperplastic and carcinomatous epithelial cells appearing as goblet cells in pancreaticobiliary maljunction and control groups. CONCLUSIONS: This study suggests that persistent MUC1 expression and increased cell proliferative activities of non-neoplastic gallbladder epithelium of the patients with pancreaticobiliary maljunction after birth reflect an altered phenotype of epithelial cells and these abnormalities may be related to carcinogenesis in such patients.     

MUC1 and MUC5AC mucin expression in liver fluke-associated intrahepatic cholangiocarcinoma

AIM: To investigate the expressions of MUC1 and MUC5AC in intrahepatic cholangiocarcinoma (ICC). Association of expressions of mucins MUC1 and MUC5AC with clinical findings, metastasis, and survival of the liver fluke-associated ICC patients was determined. METHODS: The expressions of MUC1 and MUC5AC mucins were examined by immunohistochemical staining in 87 cases of histologically-proven ICC. The expressions of mucins in relationship between clinicopathological significance and prognosis of the patients were evaluated. RESULTS: Fifty-two patients (60%) exhibited both MUC1 and MUC5AC expressions, whereas 31% expressed either MUC1 or MUC5AC, and 9% expressed neither. High MUC1 immunoreactivity displayed a significant correlation with tumor progression as reflected by vascular invasion (P<0.01), whereas high expression of MUC5AC significantly correlated with neural invasion (P = 0.022) and advanced ICC stage (P= 0.008). Patients with high expression of MUC1 had a significantly shorter survival (P = 0.0002). According to multivariate analyses, MUC1 reactivity (P = 0.026), histological grading and stage of tumor represented the least probability of survival. CONCLUSION: MUC1 is overexpressed in liver fluke-associated cholangiocarcinoma and relates to vascular invasion and poor prognosis, whereas MUC5AC mucin is neoexpressed and relates to neural invasion and advanced ICC stage. High MUC1 expression in tumor may be useful for predicting the poor outcome of ICC patients.     

Characterization of bovine gallbladder mucin. Amino acid sequences of tryptic peptides from the glycosylated domain of the protein core

Gallbladder mucin is a densely glycosylated macro-molecule that promotes cholesterol gallstone formation in experimental animals and in humans. Bovine gallbladder mucin structure was studied after chemical deglycosylation by treatment with anhydrous hydrogen fluoride at 23 degrees C for 3 hours. Deglycosylated mucin contained less than 5% of the amino sugar and neutral hexose content of native mucin. Electrophoretic and molecular sieve chromatographic analyses indicated that significant cleavage of the mucin polypeptide core had occurred during deglycosylation. Deglycosylated mucin was separated into three major fractions by reverse-phase chromatography, one of which was enriched with respect to threonine and proline. Tryptic peptides prepared from this fraction were purified by molecular sieve and reverse-phase chromatography, and the amino acid sequences (8-20 residues) of the four principal tryptic peptides were determined. These peptides contained 65%-75% threonine and proline residues and demonstrated 80%-100% sequence similarity. These data provide the first information on the primary structure of gallbladder mucin and suggest that repeating amino acid sequences occur in this protein. Comparison of gallbladder mucin peptide structure with the consensus repeat sequence of human intestinal mucin showed approximately 60% sequence similarity. It was concluded that mammalian gastrointestinal mucins may be derived from a common ancestral gene.     

Detection of circulating anti-MUC1 mucin core protein antibodies in patients with colorectal cancer

MUC1 mucin has a unique immunogenic peptide epitope in the extracellular domain, which has been shown to induce humoral and cellular immune response. In this study, we evaluated the pathophysiological significance of circulating anti-MUC1 mucin core protein IgG antibodies (anti-MUC1 antibodies) in colorectal cancer by Western blot analysis and ⁵¹Cr release assay. Anti-MUC1 antibodies were detected in 5 of 31 (16.1%) healthy subjects and in 27 of 56 (48.2%) patients with colorectal cancer. The presence of circulating anti-MUC1 antibodies was not significantly correlated with the level of circulating antigen MUSE11 or with other clinicopathological parameters tested. The incidence of positivity for anti-MUC1 antibodies in stage I and II (staged according to the General Rules for Clinical and Pathological Studies on Cancer of the Colon and Rectum of the Japanese Research Society for Cancer of the Colon and Rectum) cancers was 45.5% and 58.8%, respectively, suggesting that positivity for these antibodies may be of use as an adjunct for the diagnosis of colorectal cancer in the early stages in the absence of serious complications such as liver diseases. Because of the epitope similarity, anti-MUC1 antibodies in the serum may function in a manner similar to that of anti-MUC1 peptide monoclonal antibodies (mAbs). We therefore observed antibody-dependent cell mediated cytotoxicity with anti-MUC1 peptide mAb using MUC1 cDNA-transfected colon cancer CHC-Y1 cells as the target. The decreased sensitivity of MUC1 transfectants to effector cells was restored to a level equivalent to that in control cells. These data suggest that the detection of circulating anti-MUC1 antibodies may be a useful adjunct for the early diagnosis and immunological analysis of colorectal cancer. (Received Aug. 22, 1997; accepted Nov. 28, 1997)     

Immune recognition of tumor-associated mucin MUC1 is achieved by a fully synthetic aberrantly glycosylated MUC1 tripartite vaccine

The mucin MUC1 is typically aberrantly glycosylated by epithelial cancer cells manifested by truncated O-linked saccharides. The resultant glycopeptide epitopes can bind cell surface major histocompatibility complex (MHC) molecules and are susceptible to recognition by cytotoxic T lymphocytes (CTLs), whereas aberrantly glycosylated MUC1 protein on the tumor cell surface can be bound by antibodies to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Efforts to elicit CTLs and IgG antibodies against cancer-expressed MUC1 have not been successful when nonglycosylated MUC1 sequences were used for vaccination, probably due to conformational dissimilarities. Immunizations with densely glycosylated MUC1 peptides have also been ineffective due to impaired susceptibility to antigen processing. Given the challenges to immuno-target tumor-associated MUC1, we have identified the minimum requirements to consistently induce CTLs and ADCC-mediating antibodies specific for the tumor form of MUC1 resulting in a therapeutic response in a mouse model of mammary cancer. The vaccine is composed of the immunoadjuvant Pam(3)CysSK(4), a peptide T(helper) epitope and an aberrantly glycosylated MUC1 peptide. Covalent linkage of the three components was essential for maximum efficacy. The vaccine produced CTLs, which recognized both glycosylated and nonglycosylated peptides, whereas a similar nonglycosylated vaccine gave CTLs which recognized only nonglycosylated peptide. Antibodies elicited by the glycosylated tripartite vaccine were significantly more lytic compared with the unglycosylated control. As a result, immunization with the glycosylated tripartite vaccine was superior in tumor prevention. Besides its own aptness as a clinical target, these studies of MUC1 are likely predictive of a covalent linking strategy applicable to many additional tumor-associated antigens.     

丙型肝炎病毒核心蛋白结合蛋白激酶R的功能区域研究

目的 构建并表达丙型肝炎病毒(HCV)不同病毒株：癌中心株(BT)、癌旁珠(BNT)、HCV-J及BT不同截短片段谷胱甘肽(GST)-核心融合蛋白；寻找核心蛋白(Core)与蛋白激酶R(PKR)相互作用区域，探讨它们在HCV持续感染及肝细胞癌(HCC)发病机制中的作用。方法 用聚合酶链反应扩增不同片段HCV核心蛋白基因，并将7个不同的基因片段分别克隆到原核表达载体pGEX-4T-1，诱导表达并纯化表达蛋白，与两株细胞(HepG2和Huh-7)的PKR进行相互作用试验。结果7个不同片段Core在体外都得到相应表达，不同片段结合PKR的能力存在一定差异。BT、BNT、C191的Core N端1～172氨基酸(aa)3个片段均能与PKR发生直接结合，BT与PKR结合的区域在Core N端的1～58aa。结论 不同片段Core在原核细胞中获得较好的表达；Core／PKR相互作用，在HCV持续感染和HCC的发病机制中可能起重要作用。     

Isolation and characterization of peptides from the protein core of bovine gallbladder mucin

Gallbladder mucin may promote cholesterol gallstone formation by accelerating cholesterol monohydrate crystal nucleation in supersaturated bile. In this study, peptides were isolated from the mucin protein core by protease digestion and molecular-sieve high-performance liquid chromatography. Tryptic peptides were purified by anion exchange or reverse-phase high-performance liquid chromatography, and amino acid compositions were determined. Tryptic peptides were (a) nonglycosylated, (b) selectively enriched in serine, glutamic acid plus glutamine, and glycine, and (c) depleted in threonine and proline compared with native gallbladder mucin. Bilirubin derivatized with Woodward's reagent K covalently bound to purified mucin. Tryptic digestion of the mucin-bilirubin complex yielded low-molecular-weight nonglycosylated peptides with covalently bound bilirubin. These data indicate that the mucin protein core contains at least two distinct domains. One domain is rich in threonine and proline and contains the majority of covalently bound carbohydrate. A second domain, possibly internally located, is nonglycosylated, enriched in serine, glutamic acid plus glutamine, and glycine, and binds hydrophobic ligands such as bilirubin and 1-anilino-8-naphthalene sulfonate. Hydrophobic domains on the mucin protein core may contribute to the pathogenesis of cholesterol cholelithiasis.     

The C domain of HIV-1 gp41 binds the putative cellular receptor protein P62.

OBJECTIVE: To characterize the binding of gp41 with the putative receptor protein P62. DESIGN: HIV-1 gp41 binds several cellular proteins by two binding sites, one of which has been shown to bind to a putative receptor protein P45 (45 kDa). Based on this, an attempt was made to determine the relationship between the two binding sites and P62 (62 kDa). METHODS: Using surface plasmon resonance (SPR) measurement, the interaction was measured between recombinant soluble gp41 (rsgp41, Env aa539-684) and protein P62. Inhibition of this interaction was attempted by the use of synthetic peptides (P1, aa583-599; P2, aa646-674) corresponding to the two binding sites in gp41. In addition, the direct binding of P62 to peptide P2 was examined in an enzyme-linked immunosorbent assay. RESULTS: Using SPR measurement, the interaction between P62 and rsgp41 was confirmed, and the interaction was found to be inhibited by only the synthetic peptide P2 sequence that corresponds to the C domain of gp41; neither P1 nor a control peptide inhibited the interaction. Moreover, like rsgp41, P2 was able to bind P62 whereas P1 and another recombinant gp41 (aa567-648 that does not include the C domain) were not. CONCLUSIONS: P62 bound rsgp41 and the synthetic peptide P2. This interaction could be inhibited only by P2. These results indicate that the C domain of HIV-1 gp41 binds P62.     

Overproduction of MUC5AC core protein in patients with diffuse panbronchiolitis

BACKGROUND: The genetic identities of mucins secreted in the airways of patients with diffuse panbronchiolitis (DPB) have not been previously investigated, although hypersecretion is a common feature of this disease. OBJECTIVE: The purpose of this study was to determine the production of MUC5AC, a major core protein of mucin in airway secretion, and the effect of macrolide treatment on such production in patients with DPB. METHOD: We performed Western blot analysis for MUC5AC of bronchoalveolar lavage fluid patients. Cases consisted of four groups including patients with DPB, idiopathic pulmonary fibrosis (IPF), and sarcoidosis, and healthy volunteers. RESULTS:MUC5AC was detected in DPB patients but not in other groups. Macrolide treatment tended to inhibit such production in DPB patients. CONCLUSIONS: Our results suggest that overproduction of MUC5AC plays an important role in the pathogenesis of DPB.     

Clinicopathological implications of immunohistochemically demonstrated mucin core protein expression in hepatocellular carcinoma

Methods

We examined the expression of mucin core protein 1 (MUC1) immunohistochemically in 186 surgical specimens of histopathologically nonmucinous hepatocellular carcinoma (HCC) and compared the clinicopathological features in patients with MUC1-positive HCC (MUC1-positive group) with those in patients with MUC1-negative HCC (MUC1-negative group).

Results

MUC1 immunoreactively was present in 85 of the 186 HCCs. Of the clinicopathological variables examined, the serum concentration of α-fetoprotein, tumor differentiation, bile duct invasion, lymph node metastasis, and cytokeratin 19 expression exhibited significant associations with MUC1 expression. Although cumulative and tumor-free survival rates were not different between the two groups, the percentage of patients with first recurrence of HCC in distant organs (distant metastasis) within 2 years after surgery was significantly higher in the MUC1-positive group than in the MUC1-negative group (P = 0.0104). The risk ratio of MUC1 positivity for this type of distant metastasis was 3.156 (95% confidence interval, 1.064–9.358).

Conclusions

In patients with MUC1-positive HCC, careful follow-up is necessary, not only for intrahepatic recurrence but also for distant metastasis, after the resection of primary HCC.     

Entamoeba histolytica cysteine proteases cleave the MUC2 mucin in its C-terminal domain and dissolve the protective colonic mucus gel

In order for the protozoan parasite Entamoeba histolytica (E.h.) to cause invasive intestinal and extraintestinal infection, which leads to significant morbidity and mortality, it must disrupt the protective mucus layer by a previously unknown mechanism. We hypothesized that cysteine proteases secreted from the amoeba disrupt the mucin polymeric network, thereby overcoming the protective mucus barrier. The MUC2 mucin is the major structural component of the colonic mucus gel. Heavily O-glycosylated and protease-resistant mucin domains characterize gel-forming mucins. Their N- and C-terminal cysteine-rich domains are involved in mucin polymerization, and these domains are likely to be targeted by proteases because they are less glycosylated, thereby exposing their peptide chains. By treating recombinant cysteine-rich domains of MUC2 with proteases from E.h. trophozoites, we showed that the C-terminal domain was specifically targeted at two sites by cysteine proteases, whereas the N-terminal domain was resistant to proteolysis. The major cleavage site is predicted to depolymerize the MUC2 polymers, thereby disrupting the protective mucus gel. The ability of the cysteine proteases to dissolve mucus gels was confirmed by treating mucins from a MUC2-producing cell line with amoeba proteases. These findings suggest a major role for E.h. cysteine proteases in overcoming the protective mucus barrier in the pathogenesis of invasive amoebiasis. In this report, we identify a specific cleavage mechanism used by an enteric pathogen to disrupt the polymeric nature of the mucin gel.     

Expression of the human MUC1 mucin cDNA in a hamster pancreatic tumor cell line HP-1.

A full length cDNA for the human mucin gene, MUC1, under the control of human beta actin promoter, was transfected into a carcinogen induced hamster pancreatic ductal tumor cell line, HP-1. Transfectants were selected by resistance to geneticin. Integration of the foreign human MUC1 cDNA occurred at multiple sites in the genome of HP-1. Northern blot analysis showed MUC1 expression in cells transfected with MUC1 cDNA placed in the correct orientation, but not in control cells (HP-1 cells transfected with vector alone, or with the MUC1 cDNA placed in the antisense orientation). Western blot analysis using monoclonal antibody HMFG-2, which is reactive with the MUC1 protein, showed results consistent with the Northern blot data. Positive immunoperoxidase staining using HMFG-2 was seen in HP-1 cells transfected with MUC1 cDNA but not with untransfected or HP-1 control cells. The integration of human MUC1 mucin gene in HP-1 cells caused no significant change in the growth rate of HP-1 cells in vitro, but resulted in an enhanced growth rate for xenografts of MUC1 transfected HP-1 cells grown in nude mice.     

Altered expression and allelic association of the hypervariable membrane mucin MUC1 in Helicobacter pylori gastritis

BACKGROUND & AIMS: Infection with Helicobacter pylori causes chronic gastritis, and this confers a risk of gastric cancer. Short alleles of the membrane-bound mucin MUC1, which has a large extracellular highly glycosylated domain and is highly polymorphic due to variation in the number of tandemly repeated (TR) 20-amino acid units, have been shown to be associated with gastric cancer. Our aim was to investigate the involvement of MUC1 in chronic gastritis and, by implication, gastric cancer. METHODS: Immunohistochemical analysis was performed on endoscopic biopsy specimens from 95 patients. Gastritis was classified using the Sydney System, and H. pylori status was determined. MUC1 was detected with antibodies against different epitopes of the TR region and the cytoplasmic tail. Southern blot analysis of the MUC1 gene was performed on 57 Northern European patients to determine TR allele lengths. RESULTS: With the TR antibodies, apical staining and some perinuclear staining was seen in 34 of 41 biopsy specimens classified as histologically normal and H. pylori negative. None of the 36 biopsy specimens with gastritis and current H. pylori infection showed apical staining. In contrast, the cytoplasmic tail antibody detected apical staining in both groups. Comparison of the MUC1 allele length distributions between Northern European patients with H. pylori infection and those without H. pylori gastritis showed a statistically significant difference in distribution, with shorter alleles associated with H. pylori gastritis. CONCLUSIONS: Our results suggest that H. pylori interacts with MUC1 and that there are functional allelic differences that affect susceptibility to gastritis.     

M1/MUC5AC mucin released by human airways in vitro.

A series of monoclonal antibodies which bind to a mucin known as M1 (anti-M1 MAbs) have also been shown to detect the product of the human gene MUC5AC. The aim of this investigation was to determine the concentration of the M1 mucin in the surface epithelium of human bronchial preparations by means of immunohistochemistry and in the bronchial fluid derived from human airways by means of an immunoradiometric assay. Human bronchial ring preparations from the resection material of 20 patients were challenged with methacholine, leukotriene D4, or anti-immunoglobulin E. Experiments were performed in preparations with an intact epithelium as well as in tissues in which the epithelium had been mechanically removed. The anti-M1 MAbs stained the goblet cells in the epithelium intensely and there was also light and less uniform staining in the submucosa. The M1/MUC5AC mucin in the fluids secreted by the bronchial preparations was not modified during either the experimental protocol or stimulation with the different secretagogues. However, in preparations in which the epithelium had been removed, there was a significant reduction in the amount of M1/MUC5AC mucin detected. These data suggest that the M1/MUC5AC mucin detected in the biological fluids produced by human airways in vitro may be released constantly, and principally from the goblet cells in the epithelial layer.     

Relevance of MUC1 mucin variable number of tandem repeats polymorphism in H pylori adhesion to gastric epithelial cells

AIM：To evaluate the influence of MUC1 mucin variable number of tandem repeats （VNTR） variability on H pylori adhesion to gastric cells.
METHODS： Enzyme linked immunosorbent assay （ELISA）-based adhesion assays were performed to measure the adhesion of different H pylori strains （HP26695 and HPTx30a） to gastric carcinoma cell lines （GP202 and MKN45） and GP202 clones expressing recombinant MUC1 with different VNTR lengths.
RESULTS： Evaluation of adhesion results shows that H pylori pathogenic strain HP26695 has a significantly higher （P 〈 0.05） adhesion to all the cell lines and clones tested, when compared to the non-pathogenic strain HPTx30a. Bacteria showed a significantly higher （P 〈 0.05） adhesion to the GP202 cell line, when compared to the MKN45 cell line. Furthermore, both strains showed a significantly higher （P 〈 0.05） adhesion to GP202 clones with larger MUC1 VNTR domains.
CONCLUSION： This work shows that MUC1 mucin variability conditions H pylori binding to gastric cells. The extent of bacterial adhesion depends on the size of theMUC1 VNTR domain. The adhesion is further dependent on bacterial pathogenicity and the gastric cell line. MUC1 mucin variability may contribute to determine H pylori colonization of the gastric mucosa.