α-Amylase, which plays an essential role in starch degradation, is expressed mainly in the pancreas and salivary glands. Human α-amylase is also detected in other tissues, but it is unclear whether the α-amylase is endogenously expressed in each tissue or mixed exogenously with one expressed by the pancreas or salivary glands. Furthermore, the biological significance of these α-amylases detected in tissues other than the pancreas and salivary glands has not been elucidated. We discovered that human α-amylase is expressed in intestinal epithelial cells and analyzed the effects of suppressing α-amylase expression. α-Amylase was found to be expressed at the second-highest messenger RNA level in the duodenum in human normal tissues after the pancreas. α-Amylase was detected in the cell extract of Caco-2 intestinal epithelial cells but not secreted into the culture medium. The amount of α-amylase expressed increased depending on the length of the culture of Caco-2 cells, suggesting that α-amylase is expressed in small intestine epithelial cells rather than the colon because the cells differentiate spontaneously upon reaching confluence in culture to exhibit the characteristics of small intestinal epithelial cells rather than colon cells. The α-amylase expressed in Caco-2 cells had enzymatic activity and was identified as AMY2B, one of the two isoforms of pancreatic α-amylase. The suppression of α-amylase expression by small interfering RNA inhibited cell differentiation and proliferation. These results demonstrate for the first time that α-amylase is expressed in human intestinal epithelial cells and affects cell proliferation and differentiation. This α-amylase may induce the proliferation and differentiation of small intestine epithelial cells, supporting a rapid turnover of cells to maintain a healthy intestinal lumen.

Gastric cancer (GC) is now one of the most common malignancies with a relatively high incidence and high mortality rate. The prognosis is closely related to the degree of tumor metastasis. The mechanism of metastasis is still unclear. Proteomics analysis is a powerful tool to study and evaluate protein expression in tumor tissues. In the present study, we collected 15 gastric cancer and adjacent normal gastric tissues and used the isobaric tags for relative and absolute quantitation (iTRAQ) method to identify differentially expressed proteins. A total of 134 proteins were differentially expressed between the cancerous and non-cancerous samples. Azurocidin 1 (AZU1), CPVL, olfactomedin 4 (OLFM4) and Villin 1 (VIL1) were upregulated and confirmed by western blot analysis, real-time quantitative PCR and immunohistochemical analyses. These results were in accordance with iTRAQ. Furthermore, silencing the OLFM4 expression suppressed the migration, invasion and proliferation of the GC cells in vitro. The present study represents a successful application of the iTRAQ method in analyzing the expression levels of thousands of proteins. Overexpression of OLFM4 in gastric cancer may induce the development of gastric cancer. Overall, suppression of OLFM4 expression may be a promising strategy in the development of novel cancer therapeutic drugs.

The aims of this study were to examine the expressions of endothelium specific VE-cadherin, intestine specific LI-cadherin, and vascular endothelial growth factor (VEGF), and to determine their relationships with the clinicopathological parameters of gastric cancer.

A total 47 patients with gastric cancer who underwent surgery were enrolled. Endoscopic biopsies were obtained from the cancer and normal mucosa, respectively. Using semiquantitative RT-PCR, the mRNA expression levels of VE-cadherin, LI-cadherin and VEGF were measured by tumor/normal (T/N) ratios. The protein expressions of VE-cadherin, LI-cadherin and VEGF were examined by Western blot and immunohistochemical stain in surgically resected tissues. The clinicopathological variables were reviewed and analyzed, retrospectively.

Twenty two cases (46.8%) of VE-cadherin, 25 cases (53.2%) of LI-cadherin and 27 cases (51.1%) of VEGF mRNA expressions were overexpressed in gastric cancer compared to normal tissue. There was a tendency for T/N ratio of VE-cadherin mRNA to correlate with the lymphatic invasion (p=0.07) and the lymph node metastasis (p=0.099) in advanced gastric cancer. The T/N ratio of LI-cadherin mRNA showed significant association with distant metastasis (p=0.031) and lymphatic invasion especially in advanced gastric cancer (p=0.023). There was a tendency for the T/N ratio of VEGF mRNA to correlate with the distant metastasis (p=0.073) in advanced gastric cancer.

As increased mRNA expression of LI-cadherin was associated with distant metastasis and lymphatic invasion especially in the biopsy specimen of advanced gastric cancer before surgery, it may provide useful preoperative information on tumor aggressiveness.

Gastric cancer is the second leading cause of cancer death worldwide, both in men and women. A genomewide gene expression analysis was carried out to identify differentially expressed genes in gastric adenocarcinoma tissues as compared to adjacent normal tissues. We used Agilent's whole human genome oligonucleotide microarray platform representing ~41,000 genes to carry out gene expression analysis. Two-color microarray analysis was employed to directly compare the expression of genes between tumor and normal tissues. Through this approach, we identified several previously known candidate genes along with a number of novel candidate genes in gastric cancer. Testican-1 (SPOCK1) was one of the novel molecules that was 10-fold upregulated in tumors. Using tissue microarrays, we validated the expression of testican-1 by immunohistochemical staining. It was overexpressed in 56% (160/282) of the cases tested. Pathway analysis led to the identification of several networks in which SPOCK1 was among the topmost networks of interacting genes. By gene enrichment analysis, we identified several genes involved in cell adhesion and cell proliferation to be significantly upregulated while those corresponding to metabolic pathways were significantly downregulated. The differentially expressed genes identified in this study are candidate biomarkers for gastric adenoacarcinoma.

IL-15 is crucial for the development of intestinal intraepithelial lymphocytes (IEL) and delivery is mediated by a unique mechanism known as trans-presentation. Parenchymal cells have a major role in the trans-presentation of IL-15 to IELs, but the specific identity of this cell type is unknown. To investigate whether the intestinal epithelial cells (IEC) are the parenchymal cell type involved, a mouse model that expresses IL-15Ralpha exclusively by the IECs (Villin/IL-15Ralpha Tg) was generated. Exclusive expression of IL-15Ralpha by the IECs restored all the deficiencies in the CD8alphaalpha(+)TCRalphabeta(+)and CD8alphaalpha(+)TCRgammadelta(+) subsets that exist in the absence of IL-15Ralpha. Interestingly, most of the IEL recovery was due to the preferential increase in Thy1(low) IELs, which compose a majority of the IEL population. The differentiation of Thy1(high)CD4(-)CD8(-) thymocytes into Thy1(-)CD8alphaalpha IELs was found to require IL-15Ralpha expression specifically by IECs and thus, provides evidence that differentiation of Thy1(low) IELs is one function of trans-presentation of IL-15 in the intestines. In addition to effects in IEL differentiation, trans-presentation of IL-15 by IECs also resulted in an increase in IEL numbers that was accompanied by increases in Bcl-2, but not proliferation. Collectively, this study demonstrates that trans-presentation of IL-15 by IECs alone is completely sufficient to direct the IL-15-mediated development of CD8alphaalpha(+) T cell populations within the IEL compartment, which now includes a newly identified role of IL-15 in the differentiation of Thy1(low) IELs.

Polymeric immunoglobulin receptor (PIgR) expression has been found in gastric mucosa and gastric cancers, but it is not known whether PIgR expression is related to background intestinal metaplasia nor the patterns of PIgR expression in tumors arising in the distal esophagus and gastroesophageal (GE) junction.

To identify clinicopathologic features of tumors associated with PIgR expression and to determine whether PIgR expression is associated with intestinal differentiation of tumors and intestinal metaplasia in background mucosa in 3 groups of upper gastrointestinal adenocarcinomas. These groups are (1) gastric adenocarcinomas, (2) adenocarcinomas of the distal esophagus and GE junction with background intestinal metaplasia, and (3) adenocarcinomas of the distal esophagus and GE junction without background intestinal metaplasia.

Expression of PIgR and CDX2 in nonneoplastic mucosa, intestinal metaplasia, and adenocarcinomas was examined by immunohistochemistry in 42 cases: 14 gastric and 28 from the distal esophagus and GE junction, including 13 with esophageal or GE junction intestinal metaplasia.

PIgR and CDX2 were expressed in all cases of intestinal metaplasia. PIgR expression was positive in 40% of group 3 versus 77% of group 2 and 71% of gastric adenocarcinomas (P = .06), and the expression of CDX2 was similar in all tumor groups (80%-83%). Metastatic-positive lymph nodes were more frequent in PIgR-negative tumors (94% vs 58%, P = .01).

PIgR is uniformly expressed in intestinal metaplasia and in a subgroup of adenocarcinomas of the distal esophagus, GE junction, and stomach. Esophageal and GE junction adenocarcinomas that do not express PIgR show more frequent lymph node metastasis, suggesting that lack of expression of PIgR identifies a subgroup of more aggressive adenocarcinomas.

To compare the relative risk of antigen expression being detected immunohistochemically in ovarian and gastric carcinoma aggregated from studies performed for diagnostic purposes, with the relative risks of their expression in all patients in the English literature.

Both types of series indicated that cytokeratin (CK) 7 expression was greater and that of CK20 and carcinoembryonic antigen less in ovarian than in gastric carcinoma (P < 0.05). Synthesis of all data available for MUC-2 suggested it was more commonly expressed in ovarian carcinoma, whereas the relative risk in papers that directly compared its expression suggested that it was more common in the gastric carcinoma (P = 0.2, NS). Aggregating all possible data suggested villin was more likely to be expressed in ovarian cancers, whereas studies in which its expression was compared directly in both tumours suggested the opposite. Although statistically significant, patient numbers were small.

Provided sufficient numbers of cases are studied, analysis of studies comparing antigen expression for diagnostic purposes in tumours from two body sites is likely to be supported in the wider literature. The design of such comparative studies is informed by aggregating data from single tumour studies.

The mammalian SWI/SNF chromatin remodeling complex, an essential epigenetic regulator, contains either a single Brm or BRG1 molecule as its catalytic subunit. We observed frequent loss of Brm expression but not of BRG1 in human gastric cancer cell lines. Treatment with histone deacetylase inhibitor rescued Brm expression, indicating epigenetic regulation of this gene, and an RNA interference-based colony formation assay revealed antioncogenic properties of Brm. Brm immunostaining of 89 primary gastric cancers showed an obvious reduction in 60 cases (67%) and a severe decrease in 37 cases (42%). Loss of Brm is frequent in the major gastric cancer types (well- or moderately-differentiated tubular adenocarcinoma and poorly-differentiated adenocarcinoma) and positively correlates with the undifferentiated state. Among the minor gastric cancer types, Brm expression persists in signet-ring cell carcinoma and mucinous adenocarcinoma, but a marked decrease is observed in papillary adenocarcinoma. Intestinal metaplasia never shows decreased expression, indicating that Brm is a valid marker of gastric oncogenesis. In contrast, BRG1 is retained in most cases; a concomitant loss of BRG1 and Brm is rare in gastric cancer, contrary to other malignancies. We further show that Brm is required for villin expression, a definitive marker of intestinal metaplasia and differentiation. Via regulating such genes important for gut differentiation, Brm should play significant roles in determining the histologic features of gastric malignancy.

The Goseki system is a new gastric carcinoma classification system that classifies gastric carcinomas as grade I (intestinal type), grade II (mucinous type), grade III (mucin-poor, diffuse infiltrating type), and grade IV (signet ring cell type). The main advantage of the Goseki classification may be to separate these distinct entities into different groups. CK7 is not observed in normal gastric mucosa but can be detected in chronically inflamed gastric mucosa and coexists with incomplete intestinal metaplasia. CK20 can be observed in the superficial foveolar epithelium and mature goblet cells. The aim of this study was to examine the cytokeratin expression profiles in gastric carcinomas that are classified according to both the Goseki and Lauren systems. CK7, CK8, CK19, and CK20 were applied to the paraffin sections of 66 gastric carcinoma cases. The cytokeratin expression patterns were grouped as CK20+/CK7+, CK20+/CK7-, CK20-/CK7+, or CK20-/CK7-. The results were examined statistically. CK20 immunoreactivity was observed in 18%, 24%, and 31% of grade I, III, and IV cases, respectively. The CK20+/CK7- pattern was observed in 20% of the grade IV and 66.7% of the grade II carcinomas and was not observed in grade I or grade III tumors (P<0.0001). Goseki grade III and IV carcinomas originate from superficial gastric mucosa, but grade III carcinomas are poorly differentiated. Goseki grade II carcinomas have a specific immunophenotype other than intestinal-type gastric tumors. The Goseki classification seems to be superior in identifying poorly and well-differentiated forms of diffuse infiltrating carcinomas and mucinous carcinomas.

CDX2 is a Drosophila caudal-related homeobox transcription factor that is expressed specifically in the intestine. In mice, ectopic expression of CDX2 in the gastric mucosa gives rise to intestinal metaplasia and in one model, gastric carcinoma. In humans, increased CDX2 expression is associated with gastric intestinal metaplasia and tubular adenocarcinomas. These patterns of expression have shown that CDX2 is important for the initiation of intestinal metaplasia in the gastric mucosa, but the role of CDX2 in established gastric cancer remains unclear. We sought to determine whether CDX2 contributes to tumorigenic potential in established gastric cancer. The CDX2 gene in MKN45 gastric carcinoma cells was disrupted using targeted homologous recombination. The resulting CDX2-/- cells are essentially identical to their parental cells, with the exception of CDX2 ablation. We found no significant differences in the proliferation of CDX2-/- cells compared to CDX2+/+ cells, in vitro or in vivo. Molecular analyses show that loss of CDX2 predominantly altered the expression of genes involved in intestinal glandular differentiation and adhesion. However, there were no microscopic differences in tumor differentiation. We conclude that disruption of CDX2 in MKN45 cells does not significantly affect their tumorigenic potential.

The current study tests the hypothesis that chronic atrophic gastritis from hypochlorhydria in the gastrin-deficient mouse predisposes the stomach to gastric cancer. Gross morphology and histology of 12-month-old wild-type (WT), gastrin-deficient (G-/-) and somatostatin-deficient (SOM-/-) mice were examined. Parietal and G cells, Ki67, TUNEL, villin and MUC2 expression were analysed by immunohistochemistry. RUNX3 and STAT3 expression was analysed by Western blot. Anchorage-independent growth was determined by cell cluster formation in soft agar. Compared to the WT and SOM-/- mice, hypochlorhydric G-/- mice developed parietal cell atrophy, significant antral inflammation and intestinal metaplasia. Areas of metaplasia within the G-/- mouse stomach showed decreased RUNX3 expression with elevated MUC2 and villin expression. Cells isolated from the tumor grew in soft agar. However, the cells isolated from WT, nontransformed G-/- and SOM-/- gastric tissue did not form colonies in soft agar. Consistent with elevated antral proliferation, tumor tissue isolated from the G-/- mice showed elevated phosphorylated STAT3 expression. We then examined the mechanism by which STAT3 was constitutively expressed in the tumor tissue of the G-/- mice. We found that IFNgamma expression was also significantly higher in the tumor tissue of G-/- mice compared to WT and SOM-/- animals. To determine whether STAT3 was regulated by IFNgamma, MKN45 cells were cocultured with IFNgamma or gastrin. IFNgamma significantly stimulated phosphorylation of STAT3 in the MKN45 cell line, but not gastrin. Therefore, we show here that in the hypochlorhydric mouse stomach, the chronic gastritis, atrophy, metaplasia, dysplasia paradigm can be recapitulated in mice. Moreover, neoplastic transformation of the antral gastric mucosa does not require gastrin.

Cyclooxygenase (COX)-2 has been causally implicated in carcinogenesis. The evidence for increased COX-2 in the malignant progression of Barrett's esophagus is contradictory. We hypothesize that COX-2 expression may be causally affected by the gastrin status via the cholecystokinin 2 (CCK(2)) receptor.

COX-2 and prostaglandin E(2) expression were evaluated by Western blotting and enzyme-linked immune assay in samples of squamous esophagus, Barrett's esophagus with varying degrees of dysplasia to adenocarcinoma, and normal duodenum. Differentiation status was evaluated by histopathology and villin expression. A longitudinal case-control study compared COX-2 in patients who progressed to adenocarcinoma with nonprogressors matched for age and length of follow-up. Messenger RNA levels of gastrin and CCK(2) receptor in biopsies and cell lines were evaluated by reverse transcription-PCR, and in vitro gastrin stimulation was conducted with and without inhibitors for CCK(2) (YM022) and COX-2 (NS-398). Cell proliferation was evaluated using minichromosome maintenance protein 2 (Mcm2) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays.

COX-2 expression is significantly increased in Barrett's esophagus before dysplasia development. Expression is highly variable within Barrett's dysplasia and adenocarcinoma samples independent of differentiation status. In a longitudinal case-control study, the expression levels within patients increased over time, regardless of the degree of malignant progression. Biopsies from nondysplastic Barrett's esophagus expressed increased gastrin mRNA levels compared with other biopsies. Gastrin significantly induced COX-2, prostaglandin E(2), and cell proliferation in biopsies and cell lines. Gastrin-induced proliferation can be inhibited by YM022 and NS-398.

COX-2 is up-regulated early in the Barrett's metaplasia sequence. During carcinogenesis, gastrin is a significant determinant of COX-2 activity levels via the CCK(2) receptor.

Cytokeratins (CK) are being extensively used as diagnostic markers for various malignancies and other diseases, including human oral precancer and cancer, due to their tissue specific expression. CK are epithelia specific intermediate filament (IF) proteins, which are expressed in a differentiation dependent and tissue specific manner. There are about 30 polypeptides of CK expressed by different human epithelia. Each type of epithelium expresses about 4-6 polypeptides. CK polypeptides share many common epitopes, due to which the antibodies developed against CK tend to cross react. Therefore, a large number of monoclonal and polyclonal antibodies have been developed to distinguish among these proteins. Many of these antibodies are not only monospecific but are also epitope specific. These antibodies are being used in pathology laboratories for routine diagnosis using immunohistochemistry. A number of fixatives are used for fixation of tissue sections prior to the use of these antibodies. Sometimes, this leads in epitope masking. Hence, it becomes necessary to use a battery of monoclonal antibodies (MAb) for accurate diagnosis. Apart from the use of these antibodies in diagnostics, they are also being used in basic research for the study of CK function and their interactions with associated proteins and membrane proteins. In the present communication an effort has been made to make a comprehensive list of MAb to CK giving information like cross-reactivity, epitope specificity, various fixatives used, etc. along with the source of the antibodies, which will provide useful information to the users.

Gastric adenocarcinoma is one of the major malignancies worldwide. Gastric cell lines have been widely used as the model to study the genetics, pharmacology and biochemistry of gastric cancers. Here we describe a comprehensive survey of the gene expression profiles of 12 gastric carcinoma cell lines, using cDNA microarray with 43 000 clones. For comparison, we also explored the gene expression patterns of 15 cell lines derived from lymphoid, endothelial, stromal and other epithelial cancers. Expression levels of specific genes were validated through comparison to protein expression by immunohistochemistry using cell block arrays. We found sets of genes whose expression corresponds to the molecular signature of each cell type. In the gastric cancer cell lines, apart from genes that are highly expressed corresponding to their common epithelial origin from the gastrointestinal tract, we found marked heterogeneity among the gene expression patterns of these cell lines. Some of the heterogeneity may reflect their underlying molecular characteristics or specific differentiation program. Two putative gastric carcinoma cell lines were found to be B-cell lymphoma, and another one had no epithelial specific gene expression and hence was of doubtful epithelial origin. These cell lines should no longer be used in gastric carcinoma research. In conclusion, our gene expression database can serve as a powerful resource for the study of gastric cancer using these cell lines.

In the adult gastrointestinal tract, the morphologic borders between esophagus and stomach and between stomach and small intestine are literally one cell thick. The patterning mechanisms that underlie the development of these sharp regional divisions from a once continuous endodermal tube are still obscure. In the embryonic endoderm of the developing gut, region-specific expression of certain genes (e.g., intestine-specific expression of the actin bundling protein villin) can be detected as early as 9.0 days post coitum, although the morphologic differentiation of the gut epithelium proper does not begin until 4 to 5 days later. By using a mouse model in which a beta-galactosidase marker has been inserted into the endogenous villin locus, we examined the development of the stomach/intestinal (pyloric) border during gut organogenesis. The data indicate that the border is not sharp from the outset. Rather, the initial border region is characterized by a decreasing gradient of villin/beta-galactosidase expression that extends into the distal stomach. A sharp epithelial border of villin/beta-galactosidase expression appears abruptly at day 16 and is further refined over the next 3 weeks to form the distinct one-cell-thick border characteristic of the adult. These results indicate that an important previously unrecognized patterning event occurs in the gut epithelium at 16 days; this event may define an epithelial compartment boundary between the stomach and the intestine. The villin/beta-galactosidase mouse model characterized here provides an excellent substrate with which to further dissect the mechanisms involved in this patterning process.

Intestinal-type gastric cancer is often preceded by intestinal metaplasia in humans. The genetic events responsible for the transdifferentiation that occurs in intestinal metaplasia are not well understood. Cdx2, a transcription factor whose expression is normally limited to the intestine, has been detected in gastric intestinal metaplasia. Cdx2 induces differentiation of intestinal epithelial cells in vitro; therefore, we sought to establish whether a causal relationship exists between Cdx2 activation and intestinal metaplasia.

Cdx2 expression was directed to the gastric mucosa in transgenic mice using cis-regulatory elements of Foxa3 (Hnf3gamma). Transgenic mice were analyzed for histologic and gene expression changes.

Histologic examination of the gastric mucosa of the Foxa3/Cdx2 mice revealed the presence of alcian blue-positive intestinal-type goblet cells, a hallmark of intestinal metaplasia. In addition, Cdx2 induced the expression of intestine-specific genes.

Gastric expression of Cdx2 alone was sufficient to induce intestinal metaplasia in mice. These mice represent a powerful tool to investigate the molecular mechanisms that promote intestinal metaplasia. Moreover, as gastric cancer in humans is often preceded by intestinal metaplasia, the phenotype described here strongly suggests involvement of Cdx2 in the initiation of the process leading to intestinal neoplasia of the gastric mucosa.

To study the differentiation of epithelial tissues within their histological context, and to identify hypothetically, on the basis of keratin pattern, the putative tissue origin of a (metastatic) carcinoma.

Using well characterised monoclonal antibodies against individual keratins 7, 8, 18, and 19, which are predominantly found in columnar epithelia, and keratins 4, 10, 13, and 14, predominantly expressed in (non)-keratinising squamous epithelia, the keratin patterns for a series of 45 squamous cell carcinomas and 44 adenocarcinomas originating from various epithelial tissues were characterised.

The predominant keratins in all adenocarcinomas proved to be 8, 18, and 19. In addition, these keratins were also abundantly present in squamous cell carcinomas of the lung, cervix, and rectum and, to a lesser extent, of the larynx, oesophagus, and tongue, but not in those of the vulva and skin. Keratins 4, 10, 13, and 14 were present in almost all squamous cell carcinomas, but also focally in some of the adenocarcinomas studied.

There is a limited differential expression of distinctive keratin filaments between squamous cell carcinomas and adenocarcinomas. Apparently, squamous cell carcinomas that originate from columnar epithelium by squamous metaplasia gain the keratins of squamous cells but retain the keratins of columnar epithelial cells. However, the simultaneous expression of two of three squamous keratins (4, 10, and 13) identifies a squamous cell carcinoma, and thus might be useful in solving differential diagnostic problems.

Villin (V) is a glycoprotein of microvilli associated with rootlet formation. Most colonic adenocarcinomas have a V positive (+), cytokeratin (CK) 20 (+), CK7-negative (-) immunophenotype; most lung adenocarcinomas have a CK20(-), CK7(+) immunophenotype. The reports of villin immunoreactivity in lung adenocarcinoma range from 6% to 68% in studies using various fixations and varied anti-villin antibodies. Some lung adenocarcinomas have microvilli with rootlets leading to possible diagnostic confusion with metastatic colonic adenocarcinoma to lung. Nine primary lung adenocarcinomas with rootlets on ultrastructure (including four bronchioloalveolar carcinomas [BAC]), four metastatic lung adenocarcinomas with rootlets, nine metastatic colon adenocarcinomas to lung, and 10 randomly selected lung adenocarcinomas without rootlets (including five BAC), were immunostained with monoclonal antibodies to villin (1D2C3), CK7 (OV-TL12/30), and CK20 (Ks20.8) using a streptavidin peroxidase technique with heat-induced epitope retrieval. All primary lung adenocarcinomas with rootlets were CK7(+) CK20(-), and six of nine (67%) were V(+). Cytoplasmic villin positivity occurred in a diffuse--five of nine (56%), focal--two of nine (22%), or brush border pattern--two of nine (22%). Two of four metastatic lung adenocarcinomas with rootlets were V(+). One metastatic lung adenocarcinoma had a CK7(+), CK20(+), V(-) phenotype. All metastatic colonic adenocarcinomas were V(+), CK20(+), CK7(-), and 1 of 10 (10%) lung adenocarcinomas without rootlets was V(+), and all 10 were CK20(-), and CK7(+). In summary, villin positivity is more common in lung adenocarcinoma with rootlets (67%) than those without rootlets (10%). AU primary lung adenocarcinomas were CK7(+), CK20(-). The combination of villin, CK 7, and CK 20 is helpful in differentiating metastatic colon adenocarcinoma from lung adenocarcinoma with rootlets.

To obtain biochemical evidence that Barrett's esophagus (BE) is the precursor of most adenocarcinomas (Adc) of the esophagus and cardia.

Based on morphologic data, BE was previously proposed as the precursor of most Adc of the esophagus. This hypothesis would receive strong support if biochemical evidence were found to demonstrate a pattern common to BE and Adc of the esophagus and cardia.

We studied the presence of intestinal-type proteins sucrase-isomaltase (SI) and crypt Cell Antigen (CCAg) in BE, Barrett's Adc, and esophageal-cardial Adc without BE. In each case specimens were collected from normal esophagus, stomach, tumor, and BE mucosa when present. To study related conditions, five specimens of peptic esophagitis and of squamous cell carcinoma were also analyzed. An indirect immunofluorescence technique was employed and sections were analyzed with laser confocal microscopy imaging.

Most Barrett's mucosa specimens stained positively for SI (93%) and CCAg (89%). These proteins were detected in BE independently of the type of metaplasia, the coexistence of dysplasia, or the presence of associated Adc. SI and CCAg were present in 25 (96%) and 24 (92%) of the cases of Adc respectively. No statistical difference was detected in SI and CCAg expression between Adc samples with and without BE, between BE and Adc samples with or without BE, and between tumors located in the esophagus versus the cardia. No staining for these proteins was detected in stomach or esophageal mucosa, in submucosal glands of the esophagus, in peptic esophagitis or squamous cell carcinoma.

These data show that BE and Adc of the esophagus and cardia have a similar phenotype and support the hypothesis that most of these tumors probably originate from preexisting BE.

To investigate differences in expression of keratin 20, a cytoskeletal protein in gastrointestinal epithelial cells, in completely differentiated intestinal metaplasia (type I) and incomplete metaplasia (types II and III).

Gastric biopsy specimens from 66 patients with intestinal metaplasia were analysed immunohistochemically. Expression of keratin 20 was quantified as the percentage of immunoreactive cells on the tips, the upper, and deep foveolae.

In all specimens keratin 20 was found on the tips and in the upper foveolae of intestinal metaplasia. Keratin 20 was not observed in the deep foveolae. No differences were seen between the antrum and the body. Expression patterns were comparable between types I and III. In type II, however, lower immunoreactivity was found. Both the differences between types I and II as well as between types II and III were significant (p < 0.05).

Keratin 20 is expressed in metaplastic mucosa as a result of intestinal differentiation. Positive staining found exclusively in juxtaluminal cells occurs only in mature cells containing keratin 20. Lowered immunoreactivity in type II compared with types I and III indicates the different nature of type II intestinal metaplasia. Further studies are needed to shed light on the basic fundamental mechanism responsible for this.

Two human diffuse gastric carcinoma cell lines were established in vitro from xenografted tumours serially passaged in nude mice. Of 12 primary diffuse gastric carcinomas, 7 were successfully xenografted in nude mice (58.3%). Short-term primary cultures were achieved in all the xenografted lines. However, only 2 of the 7 short-term primary cultures were established as long-term cultures (GP202 and GP220). GP202 cells are larger than GP220 cells, show less abundant intercellular junctions at the ultrastructural level and grow in culture as a compact thin monolayer. The GP220 cells grow preferentially in small clusters attached to the monolayer, with a subpopulation of floating cells. Both lines have cells containing small mucin vacuoles in the cytoplasm and cells displaying a typical signet-ring shape. GP202 cells grow as solid tumours in nude mice but GP220 cells do not give rise to tumours. The flow cytometry and karyotype analysis showed aneuploidy in GP202 cells, with many numerical and structural chromosomal abnormalities, and diploidy in GP220 cells, with several structural chromosomal abnormalities. The CDw75 and Tn antigens are more prominently expressed in GP202 cells than in GP220 cells. T antigen is only expressed in GP202 cells, whereas only GP220 cells express EGFR. Sialosyl-Tn is not expressed in either of the cell lines. The gastric cancer cell lines described in this paper represent a valuable addition to the small number of diffuse gastric cancer cell lines currently available and also provide a good model for further in vitro and in vivo studies of gastric carcinogenesis.

The development of esophageal adenocarcinoma is frequently associated with intestinal-type Barrett's metaplasia. Barrett's metaplasia and esophageal adenocarcinomas were examined for expression of the intestinal brush-border-associated hydrolase aminopeptidase N (APN). APN mRNA was detected by utilizing the reverse transcription polymerase chain reaction (RT-PCR) in 50% of Barrett's metaplasia specimens and in 26% of esophageal adenocarcinomas. APN protein was detected by utilizing immunohistochemistry in 84% of Barrett's metaplasia specimens and in 71% of adenocarcinomas, although a decrease or loss of APN protein was sometimes observed in dysplastic Barrett's metaplasia and adenocarcinomas. Alterations in the p53 tumor-suppressor gene have previously been found in both dysplastic Barrett's mucosa and esophageal adenocarcinomas. The same specimens analyzed for APN were examined for the nuclear accumulation of the p53 protein. Utilizing immunohistochemistry, p53 staining was detected in 42% of Barrett's metaplasia specimens, most of which were dysplastic, and in 58% of adenocarcinomas. In the samples positive for p53 protein, gene mutations in exons 5, 7 and 8 were detected by utilizing single-strand conformation polymorphism analysis (SSCP) in 1 Barrett's metaplasia specimen and 6 adenocarcinomas. In Barrett's metaplasia, there was an inverse correlation between APN protein expression and p53 protein accumulation (p < 0.05) suggesting a link between genetic alterations and loss of this marker. The analysis of markers of intestinal differentiation with markers of disease progression may prove to be a useful approach for studying carcinogenesis in Barrett's metaplasia.

Specialized Barrett's esophageal mucosa, characterized by incomplete intestinal metaplasia of the esophageal mucosa, is associated with the development of adenocarcinoma. Although the intestinal disaccharidase sucrase-isomaltase (SI) has been shown in incomplete intestinal metaplasia of the stomach, it is commonly believed that Barrett's mucosa does not express SI based on the lack of enzymatic activity. This study was undertaken to determine whether the SI gene is expressed in Barrett's epithelium and its associated adenocarcinoma at the level of messenger RNA (mRNA) and protein.

Reverse transcription polymerase chain reaction was used to determine the presence of SI mRNA in Barrett's esophagus and esophageal adenocarcinomas. Cellular localization of SI protein was determined by immunohistochemistry.

SI mRNA was identified in 76% of Barrett's epithelium and 82% of esophageal adenocarcinomas. The transcriptional initiation site for SI in these tissues was identical to that of the small intestine. Immunohistochemical localization showed that SI was directed to the apical membrane in Barrett's epithelium in contrast to a more diffuse cytoplasmic pattern in esophageal adenocarcinomas.

Columnar cells of specialized Barrett's epithelium express SI and are, therefore, phenotypically similar to those in incomplete intestinal metaplasia of the stomach with respect to intestinal gene expression.

The differentiation of the predominant cell types of the mucosal epithelium of the mammalian gastrointestinal tract is characterized by increasing amounts of an intermediate-sized filament (IF) protein designated cytokeratin (CK) 20 which is a major cellular protein of mature enterocytes and goblet cells. Here we report the isolation of the human gene encoding CK 20, its complete nucleotide sequence and the amino acid sequence deduced therefrom that identifies this polypeptide (mol. wt. 48553) as a member of the type I-CK subfamily. Remarkable, however, is the comparably great sequence divergence of CK 20 from all other known type I-CKs, with only 58% identical amino acids in the conserved alpha-helical 'rod' domain of CK 20 and, e.g. CK 14. Using riboprobes corresponding to exon 6 of the gene in Northern blot and ribonuclease protection assays, we show that the approximately 1.75 kb mRNA encoding CK 20 is specifically produced in cells of the intestinal and gastric mucosa, including tumors and cell lines derived therefrom. The appearance of CK 20-positive cells in human embryonic and fetal development and in adult tissues has been studied using immunohistochemistry with CK 20-specific antibodies. CK 20 synthesis has first been recognized at embryonic week 8 in individual 'converted' simple epithelial cells of the developing intestinal mucosa. In later fetal stages, CK 20 synthesis extends over most goblet cells and a variable number of villus enterocytes. The distribution of CK 20-positive cells in the developing gastric and intestinal mucosa is similar to--but not identical with--the pattern in the adult intestine in which all enterocytes and goblet cells as well as certain 'low-differentiated' columnar cells contain CK 20, whereas the neuroendocrine ('enterochromaffin') and Paneth cells are negative. In gastrointestinal carcinomas similarly examined, CK 20 has been detected in almost all cases (50/52) of colorectal adenocarcinomas, including all grades of differentiation and malignancy and also metastatic tumors, whereas CK 20 immunostaining in gastric carcinomas has been found less consistent and more heterogeneous. The possible biological meaning of the specific expression of the CK 20 gene in certain cells of the gastrointestinal tract and carcinomas derived therefrom and the regulatory mechanisms involved in the integration of the protein in the IF cytoskeleton are discussed.

Brush cells represent a population of epithelial cells with unknown function, which are scattered throughout the epithelial lining of both the respiratory system and the alimentary system. These cells are reliably distinguished from other epithelial cells only at the ultrastructural level by the presence of an apical tuft of stiff microvilli and extremely long microvillar rootlets that may project down to the perinuclear space. In the present study we show that brush cells can be identified in tissue sections even at the light microscopic level by immunostaining with antibodies against villin and fimbrin, two proteins that crosslink actin filaments to form bundles. In brush cells, villin and fimbrin are not only present in the actin filament core bundles of apical microvilli and their long rootlets but, in addition, both proteins are also associated with microvilli extending from the basolateral cell surface of the brush cells. Basolateral immunostaining specific for villin and fimbrin does not occur in any other epithelial cell type of the respiratory and alimentary tract. Thus immunostaining with antibodies against both proteins allows unequivocal identification of individual brush cells even in sectional planes that do not contain the brightly stained apical tuft of microvilli and their long rootlets.

Human pancreatic tissue was investigated by immunohistochemistry using a polyclonal antibody against the actin binding protein villin, which participates in the formation of actin filament bundles in the microvilli. In cells of the different parts of the pancreatic duct system as well as in the acinar cells villin immunoreactivity was located mainly at the apical cell surface. This was confirmed by the ultrastructural demonstration of microvilli on the surface of duct and acinar cells, which exhibited the typical actin bundles. In chronic pancreatitis the staining for villin in duct-like structures of degenerative pancreatic tissue was irregular or even absent. This correlated with the electron microscopic observation of duct-like structures known as tubular complexes composed of cells devoid of microvilli at the apical cell surface. At the light microscopical level degenerative structures without lumen and of unknown origin showed a strong staining for villin at their basal cell surface.

Previously we reported the neo-expression of cytokeratin 7 in metaplastic and dysplastic stomach mucosa. In this study using immunohistochemistry and immunoblotting technique, we observed an cytokeratin 7 expression in the fetal and "infantile" human stomach, thus indicating an "oncofetal" behaviour of some simple epithelial type cytokeratins.

Seventeen gastric carcinomas (intestinal n = 12; diffuse n = 1; mixed type n = 4) and one Barrett's carcinoma were prospectively studied by immunohistochemistry for the expression of different keratin polypeptides and of the brush border markers villin, sucrase isomaltase and aminopeptidase N. All carcinomas expressed the keratin polypeptides 8, 18, and 19 and were stained by the broad specific keratin antibody KL1, irrespective of histologic type. Keratin 7, however, was expressed in only one carcinoma in most tumor cells and in two further carcinomas in some tumor cells. Thus, specific differentiation of the various histologic types of gastric carcinoma does not seem to be aided by the use of keratin antibodies. Villin was positive in 80% of the tumors and sucrase isomaltase and aminopeptidase N were positive in 67% respectively with no obvious histologic difference. The frequent positivity of the brush border markers, usually typical for intestinal epithelium, reflects the high degree of intestinal differentiation of gastric carcinomas, but again does not seem to be associated with a particular histologic type.

Villin, a 95-kD cytoskeletal protein selectively expressed in the microvilli of some absorptive cells was localized immunohistochemically in the oviduct and the seminiferous excretory ducts of the mouse. Villin was found in the proximal part of the oviduct, comprising the preampulla, ampulla, and part of the isthmus. Distal to the isthmus the oviductal cells lining the junctura and the intrauterine colliculus tubaris were devoid of villin. No villin could be detected in the uterine cells. Ductuli efferentes, connecting the rete testis with the epididymis were the only portion of the male seminiferous ductal system expressing villin. The cells lining the epididymis and the vas deferens were devoid of villin. These data show that villin is selectively expressed in male and female reproductive systems and that it is limited to anatomically defined proximal portions of the reproductive ducts.