Immunohistochemical study of the expression of MUC6 mucin and co-expression of other secreted mucins (MUC5AC and MUC2) in human gastric carcinomas.

To investigate the expression of MUC6 mucin in gastric carcinomas, we generated a novel monoclonal antibody (MAb CLH5) using an MUC6 synthetic peptide. MAb CLH5 reacted exclusively with the MUC6 peptide and with native and deglycosylated mucin extracts from gastric tissues. MAb CLH5 immunoreactivity was observed in normal gastric mucosa restricted to pyloric glands of the antrum and mucopeptic cells of the neck zone of the body region. In a series of 104 gastric carcinomas, 31 (29.8%) were immunoreactive for MUC6. The expression of MUC6 was not associated with histomorphological type or with clinicopathological features of the carcinomas. Analysis of the co-expression of MUC6 with other secreted mucins (MUC5AC and MUC2) in 20 gastric carcinomas revealed that different mucin core proteins are co-expressed in 55% of the cases. MUC6 was co-expressed and co-localized with MUC5AC in 45% and with MUC2 in 5% of the cases. Expression of MUC2 alone was observed in 25% of the cases. All carcinomas expressing MUC2 mucin in more than 50% of the cells were of the mucinous type according to the WHO classification. The co-expression of mucins was independent of the histomorphological type and stage of the tumors. In conclusion, we observed, using a novel well-characterized MAb, that MUC6 is a good marker of mucopeptic cell differentiation and is expressed in 30% of gastric carcinomas, independent of the clinicopathological features of the cases. Furthermore, we found that co-expression and co-localization of mucins in gastric carcinomas is independent of histomorphology and staging. Finally, we observed that intestinal mucin MUC2 is expressed as the most prominent mucin of the mucins tested in mucinous-type gastric carcinomas.     

Immunoreactivity of Lewis blood group and mucin peptide core antigens: correlations with grade of dysplasia and malignant transformation in the colorectal adenoma-carcinoma sequence

Previous studies on the immunoreactivity of various mucin peptide and carbohydrate antigens in neoplastic colorectal tissues led to at least in part contradictory results. Therefore, we investigated a series of 42 adenomas and 44 carcinomas applying monoclonal antibodies (mabs) directed against Lewis blood group antigens (sialyl-Le(a), Le(x), sialyl-Le(x), Le(y)) as well as mucin peptide cores (MUC1, MUC2 and MUC5AC) by immunohistochemistry. A statistically significant positive correlation between the development of high-grade dysplasia in colorectal adenomas and the immunoreactivity of Le(y) and MUC1 epitopes was observed, whereas MUC2 exhibited a significant negative correlation. The reactivity of the other epitopes did not show an association with the progression of malignant transformation. Colorectal carcinomas were subdivided according to their histopathological subtype. The immunohistochemical staining resulted in a significantly stronger MUC2 reactivity of mucinous vs. tubular adenocarcinomas. Immunoreactivity of the MUC1-specific mab, which does not react with the fully glycosylated peptide core, showed a statistically non-significant inverse tendency, whereas all carbohydrate antigens displayed a strong expression in both tumor subtypes. Furthermore, correlations between mucin peptide and carbohydrate epitope labelling were evaluated. Progression of the adenoma-carcinoma sequence was accompanied by an increase of Le(y) as well as MUC1 antigen and an increase of all Lewis antigens compared to MUC2 immunoreactivity. On the other hand, mucinous carcinomas exhibited an inverse pattern. In conclusion, these results demonstrate that Le(y) and MUC1 immunoreactivity correlate with malignant transformation in the colorectum, whereas MUC2 represents a marker for low-grade dysplasia and the subtype of mucinous carcinomas.     

Tetranectin expression in gastric adenocarcinomas

AIMS: The aim was to analyze the immunohistochemical localization of tetranectin in gastric adenocarcinomas and the adjacent tissues of the wall of the stomach. METHODS AND RESULTS: Forty cases of gastric adenocarcinomas were stained by the indirect immunoperoxidase method. Of the ten cases of mucinous signet ring cell carcinomas 5 showed high, 3 moderate and 2 low tetranectin expression. Of the ten cases of well-differentiated intestinal type adenocarcinomas (ITA) 4 showed moderate regional, 3 low regional and 3 negative tetranectin expression. Of the ten cases of moderately-differentiated ITA 3 showed moderate regional, 4 low regional and 3 negative tetranectin expression. Of the ten cases of poorly-differentiated ITA 4 showed focal low and 6 negative tetranectin expression. Overall, the mucinous signet ring carcinomas showed significantly higher tetranectin expression compared to ITA (chi2 = 3.95, p<0.05). In contrast, no significant relationship was found between tetranectin expression and the degree of differentiation in ITA (chi2 = 2.5, p>0.05). In all cases, the perineoplastic desmoplastic reactive stroma showed high expression of tetranectin intra- and extracellularly. The mast cells and goblet cells in the areas of intestinal metaplasia showed high tetranectin expression. CONCLUSIONS: This study shows that: a) tetranectin is produced and deposited extracellularly in the desmoplastic peritumoral stroma of infiltrating gastric adenocarcinomas; b) tetranectin is more highly expressed by the mucinous signet ring cell carcinomas compared to ITA; and c) the amount of tetranectin produced by the ITA is unrelated with the degree of tumor differentiation.     

Signet ring cells in gastric carcinomas are derived from neuroendocrine cells.

Adenocarcinomas are malignant tumors with glandular growth and/or supposed intracellular mucin as identified by periodic acid-Schiff (PAS) positivity. Gastric signet ring cell carcinomas are classified as diffuse type. A proportion of diffuse-type adenocarcinomas have previously been suggested to be of neuroendocrine origin. In the present study we examined gastric signet ring cell carcinomas for neuroendocrine differentiation. Of 11 gastric signet ring cell carcinomas, 8 contained areas with PAS-positive signet ring cells that also were immunoreactive for one or several neuroendocrine markers: synaptophysin, chromogranin A, and histidine decarboxylase, the latter an enterochromaffin-like (ECL) cell marker. Whereas PAS positivity was located in the central cytoplasm, neuroendocrine immunoreactivity was often located as a rim surrounding an otherwise non-immunoreactive cytoplasm, presumed to represent the area with PAS-positive material. These findings indicate that signet ring cell carcinomas could be of neuroendocrine origin. We propose that signet ring cell carcinomas develop by gradual dedifferentiation from ECL cells via signet ring cells with neuroendocrine immunoreactivity toward signet ring cells where the cytoplasm mainly consists of PAS-positive material. This finding could have implications for the classification and understanding of gastric carcinogenesis.     

Developmental mucin gene expression in the gastroduodenal tract and accessory digestive glands. I. Stomach. A relationship to gastric carcinoma.

Studies were undertaken to provide information regarding cell-specific expression of mucin genes in stomach and their relation to developmental and neoplastic patterns of epithelial cytodifferentiation. In situ hybridization was used to study mRNA expression of eight mucin genes (MUC1-4, MUC5AC, MUC5B, MUC6, MUC7) in stomach of 13 human embryos and fetuses (8-27 weeks' gestation), comparing these with normal, metaplastic, and neoplastic adult tissues. These investigations have demonstrated that MUC1, MUC4, MUC5AC, MUC5B, and MUC6 are already expressed in the embryonic stomach at 8 weeks of gestation. MUC3 mRNA expression can be observed from 10.5 weeks of gestation. MUC2 is expressed at later stages, concomitant with mucous gland cytodifferentiation. Normal adult stomach is characterized by strong expression of MUC1, MUC5AC, and MUC6, less prominent MUC2, and sporadic MUC3 and MUC4, without MUC5B and MUC7. Intestinal metaplasia is characterized by an intestinal-type pattern with MUC2 and MUC3 mRNA expression. Gastric carcinomas exhibit altered mucin gene expression patterns with disappearance of MUC5AC and MUC6 mRNAs in some tumor glands, abnormal expression of MUC2, and reappearance of MUC5B mRNAs. In conclusion, we have observed that patterns of mucin gene expression in embryonic and fetal stomach could show similarities with some gastric carcinomas in adults. Differences in mucin gene expression in developmental, metaplastic, and neoplastic stomach compared to normal adult stomach suggest a possible regulatory role for their products in gastric epithelial cell proliferation and differentiation.     

Overexpression of the MUC2 gene through promoter hypomethylation in mucinous cell carcinomas and signet ring cell carcinomas of gastric cancer

Gastric carcinoma (GC) remains an important cause of mortality and morbidity in East Asia and the histological classification is still controversial, despite considerable understanding of the molecular nature of this disease and its precursor lesions. Mucins play important roles in carcinogenesis or tumor invasion and their aberrant expression are also associated with pathophysiological conditions and clinical outcomes. To investigate if differences with MUC2 expression in GC are associated through changes in promoter methylation and to evaluate the relationship with the histological features of GCs, the expression and methylation status of MUC2 gene was examined in samples from 40 gastric mucosa of GC by immunohistochemistry (IHC) and by methylation-specific PCR (MSP). MUC2 was minimally and focally expressed in welldifferentiated (WD) and moderately-differentiated (MD) GC, while mucinous cell carcinomas (MCC) and signet ring cell carcinomas (SRC) displayed a uniform and strong staining intensity with a diffused cytoplasmic pattern. In addition, the MUC2 hypomethylation were found in 33% of the WD, 0% of the MD, 77% of the MCC, 75% of the MCC/SRC, and 80% of the SRC. Moreover, IHC and MSP analyses showed that MUC2 hypomethylation correlated with its overexpression. Collectively, these results suggest that MUC2 overexpression event in MCC and SRC types of GCs. However, further studies with large numbers of patients will be needed to confirm these findings.     

Heregulin β-1 induces loss of cell-cell contact and enhances expression of MUC1 at the cell surface in HCC2998 and MKN45-1 cells

Signal transduction and cell responses after stimulation with heregulin β-1 (HRG) are examined in HCC2998 and MKN45-1 cells, which have been used for a model system to study the formation of signet ring carcinomas, one of poorly differentiated adenocarcinomas. HRG stimulation causes rounding of the cells, responding to HRG. The adherens junction, which is present in the control cells, is disrupted and cell-cell interaction is lost after stimulation. Inhibition of phosphatidylinositol (PI)-3 kinase or p38 MAP kinase blocked this reaction, which indicates that the PI-3 kinase-p38 MAP kinase pathway is required for this reaction. Inhibition of the p38 MAP kinase pathway resulted in immediate restoration of cell-cell interaction. This result indicates that signaling for adherent molecules is strictly regulated by growth factor signaling. Expression of MUC1 at the cell surface is also observed and found to be expressed only after HRG stimulation. The total amount of MUC1 remains unchanged, suggesting that this amount is not due to induction of gene expression but to translocation of MUC1 from the inner membrane to the plasma membrane. This reaction is independent of the cytohesin pathway but dependent on PI-3 kinase activity. In addition to these reactions, HRG stimulates cell growth of both HCC2998 and MKN45-1 cells, depending on the ERK pathway given that the MEK inhibitor abolishes this effect. Therefore, HRG induces various reactions in HCC2998 and MKN45-1 cells by different pathways. These reactions are all related to characteristics of tumors, which implicates that HRG signaling can contribute to the formation of tumors.     

Variation of human mucin gene expression in gastric cancer cell lines and gastric mucous cell primary cultures

Human gastric mucous cells - gastric cancer cell lines mucin gene expression - TNFalpha - RT-PCR immunocytochemistry Little is known on the expression pattern of mucin genes in human gastric cancer cell lines in relation to mucin expression in normal gastric epithelial cells. Thus, the aim of this study was to compare gastric cancer cell lines and non-transformed epithelial cells in their expression of the different mucin genes, in order to use these cells as models for physiological MUC expression in human stomach. Human gastric mucous cell primary cultures which were obtained from surgical specimen by collagenase/pronase treatment and a panel of six human gastric cancer cells were screened for mRNA expression of the mucin genes MUC1, MUC2, MUC5AC, MUC5B, and MUC6. Mucin gene expression was analyzed by semi-quantitative RT-PCR, and by Western blotting and immunocytochemistry. Primary cultured human gastric mucous cells retained the stomach-specific pattern of mRNA expression found in gastric mucosal biopsies (MUC1, MUC5AC, MUC6), whereas any gastric cancer cell line exhibited an aberrant mucin gene expression. Mucin gene expression showed large variations in levels and patterns from cell line to cell line, but MUC2 was aberrantly expressed in all cancer cells. Immunocytochemistry confirmed aberrant MUC2 protein expression in cancer cells. The expression of the secretory mucin genes MUC2 and MUC5AC varied in relation to the length of cultivation of the cancer cell lines. Treatment of the gastric cancer cells with TNFalpha resulted in an enhanced mRNA expression of MUC1, MUC2, and MUC5AC (2-fold increase within 3 hours; p <0.05). In contrast, immunocytochemistry disclosed a decrease in MUC2 and MUC5AC staining intensity. Our results indicate that primary cultured human gastric mucous cells provide a physiological in vitro system for investigations of gastric mucin gene regulation. In gastric cancer cells marked changes in the mucin gene expression pattern are found with coexpression of non-gastric type mucins. Gastric mucin gene expression may be regulated by proinflammatory cytokines which could have implications in gastritis.     

PTEN、p57／Kip2和CK19在非小细胞肺癌中的表达及意义研究

探讨非小细胞肺癌中PTEN、p57/Kip2和CK19的表达情况和临床意义。选取58例非小细胞肺癌手术切除标本,采用SP法进行免疫组织化学染色。PTEN、p57/Kip2和CK19的阳性表达率分别为44.8%、56.9%和98.3%。PTEN和p57/Kip2在低分化肿瘤中表达显著降低或缺失,在腺癌中的表达强度高于鳞癌;CK19几乎在所有的肺癌组织中均表达,且在腺癌中的表达强度高于鳞癌。PTEN和p57/Kip2的低表达参与肿瘤的生长分化和进展,并提示预后不良。     

Developmental mucin gene expression in the gastroduodenal tract and accessory digestive glands. II. Duodenum and liver, gallbladder, and pancreas.

Studies were undertaken to provide information regarding cell-specific expression of mucin genes and their relation to developmental and neoplastic patterns of epithelial cytodifferentiation. In situ hybridization was used to study mRNA expression of mucin genes in duodenum and accessory digestive glands (liver, gallbladder, pancreas) of 13 human embryos and fetuses (6. 5-27 weeks' gestation), comparing these with normal and neoplastic adult tissues. These investigations demonstrated that the pattern of mucin gene expression in fetal duodenum reiterated the patterns we observed during gastric and intestinal ontogenesis, with MUC2 and MUC3 expression in the surface epithelium and MUC6 expression associated with the development of Brünner's glands. In embryonic liver, MUC3 was already expressed at 6.5 weeks of gestation in hepatoblasts. As in adults, MUC1, MUC2, MUC3, MUC5AC, MUC5B, and MUC6 were expressed in fetal gallbladder, whereas MUC4 was not. In contrast, MUC4 was strongly expressed in gallbladder adenocarcinomas. MUC5B and MUC6 were expressed in fetal pancreas, from 12 weeks and 26 weeks of gestation, respectively. Surprisingly, MUC3 which is strongly expressed in adult pancreas, was not detected in developmental pancreas. Taken together, these data show complex spatio-temporal regulation of the mucin genes and suggest a possible regulatory role for mucin gene products in gastroduodenal epithelial cell differentiation.     

Relationship between immunoexpression of mucin peptide cores MUC1 and MUC2 and Lauren's histologic subtypes of gastric carcinomas

Laurèn's system subdivides gastric cancers into an intestinal type and a diffuse type. This histological classification mirrors histogenetic hypotheses according to which the intestinal-type cancer derives from intestinal metaplasia and dysplasia, while the diffuse-type originates directly from gastric mucosa, with or without a preceding non-metaplastic dysplasia. Studies concerning mucins expression in gastric neoplastic and preneoplastic lesions have provided contradictory data concerning such histogenetic relationships. The aim of the present study was to verify whether a correlation between mucins phenotype and Lauren's classification subsists. 40 gastric adenocarcinomas, subdivided, according to Laurèn's classification, into 27 intestinal-type, 10 diffuse-type and 3 unclassified cases, were examined for MUC1 and MUC2 immunohistochemical expression. Intestinal-type carcinomas displayed a MUC1-positive staining in 23/27 cases and a MUC2-positive immunoreaction in 10/27 cases. Diffuse-type carcinomas expressed MUC1 in 3/10 and MUC2 in 8/10 cases, respectively. According to the mucins expression pattern, three phenotypes were identified: the gastric phenotype (MUC1+/MUC2-); the gastro-intestinal phenotype (MUC1+/MUC2+) and the intestinal phenotype (MUC1-/MUC2+). The gastric phenotype was significantly higherin intestinal-type adenocarcinomas, whereas cases showing an intestinal phenotype were significantly more frequent in diffuse-type adenocarcinomas. These findings provide evidence for a lack of correlation between Lauren's classification and MUC1 and MUC2 phenotypes. In particular, the term intestinal-type tumour as referred to gland-forming gastric cancer does not seem to reflect an immunohistochemical phenotype.     

呼吸道黏蛋白5AC基因转录表达的顺式调控元件分析

目的：探讨呼吸道黏蛋白（mucin,MUC）5AC基因5'上游序列顺式调控元件在中性粒细胞弹力酶(neutrophil elastase , NE)诱导MUC5AC基因转录表达的调控机制。方法：应用DNA重组技术,构建含萤光素酶报告基因和MUC5AC启动子不同长度片段的嵌合质粒。采用定点突变技术,在嵌合质粒的基础上构建MUC5AC启动子区特殊蛋白（specificity protein）-1和核因子(nuclear factor, NF)-κB结合位点单独突变体,并测定NE刺激的转染细胞荧光素酶相对活性。结果：成功构建了4种含有不同长度MUC5AC基因启动子序列的荧光索酶报告基因质粒。含有启动子序列-1330bp、-689bp、-324bp的嵌合质粒荧光素酶相对光强度较对照组均显著增加,而含有启动子序列-64bp的嵌合质粒荧光素酶相对光强度与对照组相比差异无统计学意义。NE可诱导含有MUC5AC启动子区NF-кB结合位点单独突变体（pGL3E-MUC5AC-NF-кB-MU）荧光素酶相对光强度增加,而NE不能诱导Sp-l结合位点单独突变体（pGL3E-MUC5AC-SP-1-MU）荧光素酶表达增加。结论：MUC5AC 5'上游序列中-324～-64位点存在参与NE诱导MUC5AC基因表达的重要调控元件,位于此区域的顺式作用元件Sp-1位点在NE诱导MUC5AC基因表达机制中起重要作用,该位点可能作为靶向性基因治疗的关键调控元件。     

Up-regulation of mucin secretion in HT29-MTX cells by the pro-inflammatory cytokines tumor necrosis factor-alpha and interleukin-6

The pro-inflammatory cytokines IL-6 and TNF-alpha have been implicated in the pathogenesis of otitis media with effusion (OME). A disease where goblet cells proliferate in a modified respiratory epithelium, leading to the accumulation of a mucin-rich effusion in the middle ear cleft. The MUC5AC and MUC5B mucin gene products have been identified as components of these effusions. To determine the effect of IL-6 and TNF-alpha on MUC5AC and MUC5B secretion we have used HT29-MTX goblet cells, which secrete both types of mucins. MUC5AC and MUC5B mucin secretion was measured by an enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody NCL-HGM-45M1 and polyclonal antiserum TEPA, respectively. Time response (0-72 hours) and dose response (1.5-150 ng/ml) studies were carried out. IL-6 and TNF-alpha stimulated MUC5AC and MUC5B mucin secretion in a time dependent manner, both in pre-confluent and post-confluent cells. IL-6 (15 ng/ml and 20 ng/ml) produced a low and prolonged stimulation of mucin secretion that persisted for 72 hours, with peak response at 24 hours after induction. The IL-6-mediated mucin secretion at 24 hours was concentration-dependent, with a maximal effect at 15 ng/ml. TNF-alpha (20 ng/ml) induced rapid stimulation of mucin secretion within the first 24 hours, with peak response at 7 hours after induction. IL-6 and TNF-alpha exposure significantly increased MUC5AC secretion, but not MUC5B secretion. Maximal levels of cytokine-induced mucin secretion were detected in pre-confluent cells that showed one and a half- and two-fold increases in MUC5AC secretion after IL-6 and TNF-alpha stimulation, respectively, in comparison with post-confluent cells. The results presented here suggest that IL-6 and TNF-alpha generate a differential up-regulation of mucin secretion and thus contribute to the expression of mucin genes in inflammatory responses.     

Effect of pro-inflammatory stimuli on mucin expression and inhibition by secretory leucoprotease inhibitor

Stimuli-induced expression of certain mucin genes has been demonstrated to occur as a result of ligand-dependent activation of the epidermal growth factor receptor (EGFR). In particular, MUC5AC expression can be induced by cigarette-smoke, neutrophil elastase and lipopolysaccharide (LPS) following activation of tumour necrosis factor alpha-converting enzyme. We now show that a large of number of stimuli relevant to the cystic fibrosis lung - neutrophil elastase, LPS, Pam3Cys-Ser-(Lys)4 Hydrochloride (a lipopeptide analogue), CpG DNA (which mimics bacterial DNA) and cystic fibrosis bronchoalveolar lavage fluid - can activate MUC1 and 2 expression as well as MUC5AC expression in lung epithelial cells via an EGFR-dependent mechanism. In addition, we demonstrate that the immunomodulatory anti-protease, secretory leucoprotease inhibitor, can inhibit stimuli-induced MUC1, 2 and 5AC expression via a mechanism that is primarily dependent on the inhibition of transforming growth factor type alpha release. Therefore, mucin gene expression, induced by cystic fibrosis respiratory stimuli, can be inhibited by secretory leucoprotease inhibitor indicating its potential importance as an anti-mucin agent in cystic fibrosis and other chronic lung diseases characterized by mucus hypersecretion.     

人中性粒细胞弹性蛋白酶诱导气道黏液高分 泌细胞模型的建立及其机制的初步研究

目的：以人中性粒细胞弹性蛋白酶(HNE)为诱导因素,研究建立黏蛋白(MUC)5AC和5B高表达的细胞模型,同时对黏蛋白高表达机制进行初步研究。方法：培养人肺腺癌细胞A549,以HNE为刺激因素,EGFR中和抗体、表皮细胞生长因子受体（EGFR）磷酸化阻断剂AG1478为干预因素,分组培养。采用四甲基偶氮唑盐光吸收法（MTT法）检测HNE对细胞活性的影响;逆转录-聚合酶链反应（RT-PCR）检测MUC5AC mRNA、MUC5B mRNA的变化;酶联免疫吸附测定法(ELISA)定量分析MUC5AC和MUC5B蛋白含量的差异;细胞免疫化学以及激光共聚焦技术进一步直观观察MUC5AC、MUC5B、p-EGFR蛋白表达的变化。结果：HNE对A549细胞活力的影响呈剂量依赖性;HNE刺激组的MUC5AC、MUC5B基因转录和蛋白表达水平均明显高于对照组,差异有统计学意义（均P<0.01）;HNE刺激组p-EGFR蛋白表达显著增多,EGFR中和抗体、AG1478能显著降低HNE诱导的MUC5AC高表达,但对MUC5B高表达无干预作用。结论：人肺腺癌细胞A549同时表达MUC5AC和MUC5B,HNE能有效刺激A549细胞高表达MUC5AC和MUC5B,黏蛋白高表达细胞模型的建立为研究气道粘液高分泌疾病提供了实验基础。HNE通过激活EGFR信号转导通路诱导MUC5AC的高表达,但MUC5B高表达机制与之不同,有待进一步研究。     

Protein kinase B (AKT) regulates SYK activity and shuttling through 14-3-3 and importin 7

The Protein kinase B (AKT) regulates a plethora of intracellular signaling proteins to fine-tune signaling of multiple pathways. Here, we found that following B-cell receptor (BCR)-induced tyrosine phosphorylation of the cytoplasmic tyrosine kinase SYK and the adaptor BLNK, the AKT/PKB enzyme strongly induced BLNK (>100-fold) and SYK (>100-fold) serine/threonine phosphorylation (pS/pT). Increased phosphorylation promoted 14-3-3 binding to BLNK (37-fold) and SYK (2.5-fold) in a pS/pT-concentration dependent manner. We also demonstrated that the AKT inhibitor MK2206 reduced pS/pT of both BLNK (3-fold) and SYK (2.5-fold). Notably, the AKT phosphatase, PHLPP2 maintained the activating phosphorylation of BLNK at Y84 and increased protein stability (8.5-fold). In addition, 14-3-3 was required for the regulation SYK⿿s interaction with BLNK and attenuated SYK binding to Importin 7 (5-fold), thereby perturbing shuttling to the nucleus. Moreover, 14-3-3 proteins also sustained tyrosine phosphorylation of SYK and BLNK. Furthermore, substitution of S295 or S297 for alanine abrogated SYK⿿s binding to Importin 7. SYK with S295A or S297A replacements showed intense pY525/526 phosphorylation, and BLNK pY84 phosphorylation correlated with the SYK pY525/526 phosphorylation level. Conversely, the corresponding mutations to aspartic acid in SYK reduced pY525/526 phosphorylation. Collectively, these and previous results suggest that AKT and 14-3-3 proteins down-regulate the activity of several BCR-associated components, including BTK, BLNK and SYK and also inhibit SYK⿿s interaction with Importin 7.     

Mixed gastric- and intestinal-type metaplasia is formed by cells with dual intestinal and gastric differentiation.

We have proposed to divide intestinal metaplasia (IM) into two categories, i.e., a mixed gastric and intestinal (GI) type, and a solely intestinal (I) type, based on the residual gastric phenotype cells. The GI-mixed-type IM can be identified by the presence of both cells with either gastric or intestinal phenotypes in a single gland. This study is conducted to elucidate whether cells in the GI-mixed-type IM glands can simultaneously present both gastric and intestinal phenotypes. MUC5AC, MUC2, CD10 and villin expressions were investigated in 20 samples from five gastric cancer cases, directly using either AlexaFluor 488- or 568-labeled specific monoclonal antibodies and observed by fluorescent microscopy and confocal laser-scanning microscopy. GI-mixed IM glands comprise a population expressing MUC5AC and MUC2, MUC5AC and villin, and MUC5AC and CD10. MUC2 and villin expressions were reciprocally increased with decreasing MUC5AC expression, while CD10 expression was limited to cells with only a residual MUC5AC expression or no expression. These results suggest that a heterogeneous cell population with both gastric and intestinal phenotypes would develop into a single intestinal phenotype, as reflected in the progression of intestinal metaplasia from GI-mixed-type- to I-type IM-type glands.     

Suppression of mucin 2 enhances the proliferation and invasion of LS174T human colorectal cancer cells

Altered expression of MUC2 (mucin 2) is related to tumour development in colorectal cancer. Colorectal mucinous carcinomas are positive for MUC2 expression, whereas MUC2 is down-regulated in non-mucinous adenocarcinomas. In the present study, we down-regulated MUC2 expression by RNAi (RNA interference) and investigated the in vitro and in vivo effects on the proliferation and invasion/migration potential of the LS174T human colorectal cancer cells. The LS174T cell line is a goblet-cell-like colorectal cancer cell line that continuously produces high levels of MUC2. Inhibition of MUC2 expression in vitro by transfection of LS174T cells with the recombinant plasmid pcDNA6.2-GW/EmGFP-miR-MUC2 led to the production of a stably transfected MUC2-RNAi LS174T cell line. The proliferation and invasion/migration of MUC2-RNAi cells in vitro were significantly higher than those in control cells, as assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], colony formation and transwell assays. Subcutaneous injection of MUC2-RNAi LS174T cells into nude mice resulted in the development of subcutaneous tumours visible to the naked eye after 1 week. The growth rate of tumours derived from MUC2-RNAi LS174T cells was greater than that of tumours derived from control cells. Ki67 and matrix metalloproteinase-9 proteins were detected by immunohistochemistry in the xenografts. The expression levels of these proteins were higher in the MUC2-RNAi-derived xenografts than in xenografts derived from control cells. Although the role of MUC2 in colorectal tumorigenesis is not fully understood, these results strongly suggest a relationship between the proliferation and invasion of LS174T cells and the expression of MUC2.