Expression of occludin in human rectal carcinoid tumours as a possible marker for glandular differentiation.

AIMS: To examine whether or not the tight junction-associated transmembrane protein occludin is expressed in rosette or gland-like structures in human rectal carcinoid tumours. The tight junction is crucial for the formation and maintenance of organized tubular structures in glandular epithelia. Previous studies have reported the presence of glandular structures in carcinoid tumours, though they are not believed to arise from glandular epithelium. METHODS AND RESULTS: The expression profiles of occludin in 40 carcinoid tumours were examined immunohistochemically, using an anti-occludin monoclonal antibody. In eight (20%) samples of typical carcinoid tumours, a small number of rosette-like tubular structures outlined by occludin were detected. CONCLUSIONS: Tight junction-associated molecules, including occludin, are thought to be one of the most characteristic structural markers of polarized glandular structures. The results of the present study provide supportive evidence that carcinoid tumour cells are capable of glandular differentiation.

Retinoid X receptor alpha and retinoic acid receptor gamma mediate expression of genes encoding tight-junction proteins and barrier function in F9 cells during visceral endodermal differentiation.

Retinoids are critical for differentiation of columnar epithelial cells and for preventing metaplasia of these cells into stratified squamous epithelial cells, in which tight junctions (TJs) are essentially absent. This implies that retinoids might play important roles in regulating the structures and functions of TJs of columnar epithelium. F9 murine embryonal carcinoma cells differentiate into epithelial cells resembling visceral endoderm bearing TJs, when grown in suspension as aggregates in the presence of retinoic acid (RA). We show that RA induces the TJ structure and expression of several TJ-associated molecules, such as ZO-1, occludin, claudin-6, and claudin-7, as well as a barrier function in the genetically engineered cell line F9:rtTA:Cre-ER(T) L32T2, which allows sophisticated genetic manipulations simply by addition of ligands (H. Chiba et al., 2000, Exp. Cell Res. 260, 334-339). Interestingly, our data indicate that a barrier for small substances is generated after that for large ones during de novo formation of TJs. We also compared the RA-induced expression of TJ components and barrier function in RXRalpha(-/-)-RARgamma(-/-) F9 cells with those in wild-type cells and show that the retinoid signals for transduction of these events are mediated by specific RXR-RAR pairs.

Expression of receptors for glial cell line-derived neurotrophic factor (GDNF) and neurturin in the inner blood-retinal barrier of rats.

The retina is protected from somatic circulation by the blood-retinal barrrier (BRB) composed of tight junctions between retinal vascular endothelial cells (the inner BRB) and those between retinal pigment epithelial cells (the outer BRB). Our recent studies showed that glial cell line-derived neurotrophic factor (GDNF) secreted from astrocytes regulates the permeability of the BBB. In the present study, we immunohistochemically examined the expression of GDNF, neurturin (NTN) and their receptors, GFRalpha1 for GDNF and GFRalpha2 for NTN, because the capillaries of the inner BRB show specialization very similar to the blood-brain barrier (BBB). GDNF and NTN were detected in glial fibrillary acidic protein (GFAP)-positive cells, including Müller cells. GFRalpha1 and GFRalpha2 were localized in von Willebrand factor-positive cells. GDNF and NTN enhanced the barrier function of endothelial cells derived from porcine brain cortex. These results strongly suggest that the barrier function of the BRB is regulated by GDNF and NTN secreted from glial cells, like the BBB.

Expression of GFRalpha-1, receptor for GDNF, in rat brain capillary during postnatal development of the BBB.

It is well known that the blood-brain barrier (BBB) matures at approximately 2 wk after birth in the rat. Recently, we showed that glial cell line-derived neurotrophic factor (GDNF) enhances the barrier function of porcine endothelial cells forming the BBB in culture. In the present study, we examined the relation between permeability of the BBB, using Evans blue as a tracer, and expression of the GDNF family receptor (GFRalpha-1) during postnatal development of the BBB. Morphometric analysis showed that exudation of Evans blue from capillaries of the cerebral cortex progressively decreased until postnatal day 21. Inversely, immunohistochemical examinations showed expression of GFRalpha-1 in the capillaries at postnatal day 3 and expression that reached the same levels as observed in adult rats by postnatal day 10. However, c-ret, which is thought to mediate a signal evoked by binding of GDNF to GFRalpha-1, was not expressed in the capillaries of the brain cortex in 3-mo-old rats. On the other hand, the tight junction proteins occludin and ZO-1 appeared to be fully expressed at birth. The reciprocal relation between GFRalpha-1 expression and the permeability of the BBB strongly suggests active participation of GDNF in postnatal development of the BBB, although the mechanism(s) involved is still veiled.

Uncoupling of gate and fence functions of MDCK cells by the actin-depolymerizing reagent mycalolide B.

The tight junction serves as a paracellular gate to seal the paracellular space of apposing cells and as a molecular fence to prevent diffusion of membrane proteins and lipids in epithelial cells. Although involvement of the actin cytoskeleton has been considered to be important in these two functions, it remains to be elucidated whether both functions are regulated in a coupled manner or differentially by actin. Treatment of highly polarized MDCK cells with mycalolide B (MB), a recently developed actin-depolymerizing reagent, induced a decrease of transepithelial resistance in a dose- and time-dependent manner with reversibility when the reagent was washed out. Changes in cytoskeletal actin, such as a reduction of cortical actin, irregularity of stress fibers, and punctated actin aggregates, were observed after MB treatment. However, the fence function, as studied by diffusion of apically labeled sphingomyelin/BSA complex, remained intact in the MB-treated MDCK cells. Localization of junctional molecules and apical marker proteins such as E-cadherin, ZO-1, and 114-kDa protein was shown to be unaffected. Furthermore, freeze-fracture study showed apparent tight junction strands. Collectively, MB treatment abolished the paracellular gate but not the fence function of MDCK cells, suggesting that cytoskeletal actin may play differential roles in the gate and fence functions of the tight junction.

Glial cell line-derived neurotrophic factor induces barrier function of endothelial cells forming the blood-brain barrier.

Since a deep involvement of astrocytes, a kind of glial cells, in differentiation of the blood-brain barrier (BBB) has been suggested, we examined the relation of glial cell line-derived neurotrophic factor (GDNF) to the BBB. First, immunohistochemical examination of the cerebral cortex of rats revealed that glial cell line-derived neurotrophic factor receptor (GFRalpha1) was preferentially expressed on the cell membranes of capillary endothelial cells. Second, to elucidate the effects of GDNF on the BBB, capillary endothelial cells isolated from the porcine cerebral cortex were cultured and then changes in tight junction function of the endothelial cells were examined after addition of GDNF, in terms of transendothelial electrical resistance (TER) and permeability. GDNF at concentrations of 0.1 and 1 ng/ml significantly activated the barrier function of the endothelial cells in the presence of cAMP. Since GDNF is secreted from astrocytes sheathing capillary endothelial cells in the brain cortex, our results strongly suggest that GDNF enhances the barrier function of tight junctions of the BBB on the one hand, and also supports the survival of neurons on the other hand.

Bacterial lipopolysaccharide reduced intestinal barrier function and altered localization of 7H6 antigen in IEC-6 rat intestinal crypt cells.

The intestinal epithelial barrier restricts the passage of potentially toxic substances into the systemic circulation and is considered to be mostly mediated by tight junctions, though the mechanisms involved in the regulation of intestinal tight junctions are not yet fully understood. In the present study, we examined whether bacterial lipopolysaccharide (LPS) altered the barrier function of tight junction and localization of tight junctional proteins, ZO-1 and 7H6 antigen, in IEC-6 intestinal cells. Administration of LPS to the basolateral surface of IEC-6 cells disrupted the barrier function and caused the disappearance of 7H6 antigen from the cell border, whereas LPS administered to the apical surface altered neither the barrier function nor the localization of 7H6 antigen in IEC-6 cells. On the other hand, the localization of ZO-1 was not influenced by these treatments of LPS. These results suggest that the interaction of LPS with the basolateral surface of intestinal epithelial cells disrupts the barrier function and 7H6 antigen take part in the maintenance of the barrier function in IEC-6 cells.

Cell density regulates crypticity of GM3 ganglioside on human glioma cells.

Human glioma cell line KG-1C contains GM3 ganglioside as its sole glycolipid. The degree of M2590 antibody binding to GM3 was found to be regulated by the cell density; the percentage of positive cells in FACS analysis decreased from approximately 20% to close to none as the cells increased their density from sparse to confluent. The contents of GM3 with different cell densities were consistent, being more than 0.4 micromol/g of the cellular weight, which was high enough to be recognized by the antibody. Trypsin treatment of the cells did not increase antibody reactivity. The extracted GM3 retained its antigenicity, being intensely stained with M2590 on a TLC plate; there was no change in chromatographic mobility either, indicating no modification of its chemical structure. The fluorescent microscope disclosed scattered dot-like staining of GM3, particularly at the periphery of the cells. We were able to expose cryptic GM3 fully within 12 h by dispersion of the cells to a sparse density. Surface labeling of GM3 with the use of limited sodium periodate oxidation of sialylated residue equally labeled GM3 either from the confluent cells or the sparse cells. Disassembly of actin filaments with cytochalasin B (10 microM) partially exposed cryptic GM3 of confluent cells, indicating reversibility of the crypticity. All together, the results indicate that cryptic GM3 actually exists on the cell surface, hidden from the surface not by other molecules but by other mechanisms associated with the cellular architecture. We are beginning to explore the possibility of selective localization of GM3 in small caves or folds of the cell membrane produced upon cell-to-cell contact.

Formation of actin filament networks in cultured rat hepatocytes treated with DMSO and glucagon.

Actin filament organization may play an important role in the maintenance of differentiated functions in epithelial cells. We previously reported our success in inducing and maintaining gap junctions, which are two kinds of differentiated function, in primary rat hepatocytes cultured with 2% DMSO and 10-7 M glucagon. In the present study, we demonstrated the formation of actin filament networks in the hepatocytes cultured with 2% DMSO and 10-7 M glucagon. Actin filaments in hepatocytes cultured in medium with only 2% DMSO added from 96 h after plating were concentrated under the plasma membrane and were observed to be circumferential. In hepatocytes cultured in the medium with both 2% DMSO and 10-7 M glucagon added from 96 h, not only the circumferential actin filaments but also the formation of actin filament networks were observed and the networks developed well with time in culture. The networks were observed as a dome-like structure under the cell face and terminated at the circumferential actin filaments. They were composed of electron-dense star-like vertices connected by microfilament bundles of varying length and were also very sensitive to the actin disruptor cytochalasin B. However, during the network formation, there were no significant increases in the amounts of actin protein and mRNA. The actin filament networks of the hepatocytes in this culture system might be closely related to the maintenance of differentiated functions.

Expression of p21(waf-1/cip-1) is significantly induced in the livers of LEC rats with chronic liver injury.

It is reported that hepatocytes isolated from LEC rats with chronic liver injury show reduced growth activity in primary culture. To elucidate the molecular basis of this phenomenon, we examined expression of p21(waf-1/ciP-1) and p27, cyclin-dependent kinase inhibitors, by northern blot analysis. The expression of p21(waf-1/cip-1 ) in the LEC rat liver was 3-fold higher than that of age-matched SD rat liver, while there was no significant difference in p27 expression level. Western blot analysis also revealed a significant increase in p21(waf-1/cip-1) in the nuclear matrix fraction of the LEC rat liver. Immunohistochemically, p21(waf-1/cip-1) was detected in the nuclei of normal LEC rat hepatocytes, but not in those of hepatocellular carcinoma cells, suggesting selective growth of neoplastic hepatocytes.

Enhanced paracellular barrier function of rat mesothelial cells partially protects against cancer cell penetration.

To study pathophysiological roles of mesothelial barrier functions in protection against cancer cell invasion, we isolated mesothelial cells from the rat abdominal cavity and then cultured them with 10(-6)M all-trans-retinoic acid (RA) for 10 days. Mesothelial barrier function assessed by measuring transcellular electrical resistance (TER) and the expression of 7H6 tight junction-associated antigen at the cell border were induced by the treatment (10.01 +/- 0.8 vs 6.05 +/- 0.7 omega cm2, without RA; mean +/- s.e.m., n = 10). Then we quantified the attachment and penetration of rat mammary cancer cells (SST-2 cells) into the mesothelial cell monolayer by prelabelling of the cancer cells with fluorescent dye and by observing optical sections at different heights using a laser confocal scanning microscope. When SST-2 cells were overlaid onto the mesothelial cell monolayer treated with RA, the number of cancer cells found at the basal level of the monolayer was significantly reduced. These results showed that enhanced mesothelial barrier function at least partially prevents the penetration of cancer cells into mesothelial cells and suggested that 7H6 antigen serves as a reliable immunocytochemical marker for monitoring mesothelial barrier function.

Expression of component desmosomal proteins in uterine endometrial carcinoma and their relation to cellular differentiation.

BACKGROUND: While the assessment of the malignancy of neoplasms is based on morphologic studies of cells and tissues, use of objective molecular markers is leading to a better understanding and more biologically meaningful classification of neoplasms. In recent years, changes in the expression of cell adhesion molecules, especially E-cadherin, catenin, and adenomatous polyposis coli (APC), in carcinomas have attracted the attention of researchers. However, little is known about desmosomes in the uterine endometrium or in endometrial carcinomas. In this study, we semiquantified the desmosomal components desmoplakin I and II and desmoglein, in tissue sections using confocal laser scanning microscopy (LSM), and examined their relationship to the pathological type, the occurrence of lymph node metastases, and the extent of myometrial invasion. METHOD: Frozen sections of 31 specimens of normal endometrium, 5 specimens of atypical hyperplasia, and 41 specimens of endometrial carcinoma were stained by the immunofluorescence method using antidesmoplakin I and II and antidesmoglein, and these markers were then semiquantified in tissue sections by LSM. RESULTS: The expression and location of desmoplakin I and II and desmoglein were similar, and their expression decreased with loss of differentiation. The expression was lower in cases of lymph node metastasis than in negative cases and was lower in the cases with > one-half myometrial invasion than in cases with < one-half myometrial invasion. CONCLUSIONS: Reduction of desmoplakin I and II and desmoglein expression may play an important role in the invasiveness and metastatic activity of human endometrial carcinoma. They can therefore be used as differentiation markers for endometrial carcinoma.

Changes in cellular distribution of connexins 32 and 26 during formation of gap junctions in primary cultures of rat hepatocytes.

In the adult rat hepatocyte, gap junction proteins consist of connexin 32 (Cx32) and connexin 26 (Cx26). Previously, we reported that both Cx32 and Cx26 were markedly induced and maintained in primary cultures of adult rat hepatocytes. The reappearing gap junctions were accompanied by increases in both the proteins and the mRNAs, and they were well maintained together with extensive gap junctional intercellular communication (GJIC) for more than 4 weeks. In the present study, we examined the cellular location of the gap junction proteins and the structures in the hepatocytes cultured in our system, using confocal laser microscopy and immunoelectron microscopy of cells processed for Cx32 and Cx26 immunocytochemistry and freeze-fracture analysis. In immunoelectron microscopy, the size of Cx32-immunoreactive gap junction structures on the plasma membrane increased with time of culture, and some of them were larger than those in liver sections in vivo. Freeze-fracture analysis also showed that the size of gap junction plaques increased and that the larger gap junction plaques were composed of densely packed particles. These results suggest that in this culture system, not only the synthesis of Cx proteins but also the size of the gap junction plaques was increased markedly. In the adluminal lateral membrane of the cells, Cx32-immunoreactive lines were observed and many small gap junction plaques were closely associated with a more developed tight junction network. In the basal region of the cells, small Cx32- and Cx26-immunoreactive dots were observed in the cytoplasm and several annular structures labeled with the antibody to Cx32 were observed in the cytoplasm. These results indicated the formation and degradation of gap junctions in the cultured hepatocytes.

Cloning and characterization of cell adhesion kinase beta, a novel protein-tyrosine kinase of the focal adhesion kinase subfamily.

A second protein-tyrosine kinase (PTK) of the focal adhesion kinase (FAK) subfamily, cell adhesion kinase beta (CAK beta), was identified by cDNA cloning. The rat CAK beta is a 115.7-kDa PTK that contains N- and C-terminal domains of 418 and 330 amino acid residues besides the central kinase domain. The rat CAK beta has a homology with mouse FAK over their entire lengths except for the extreme N-terminal 88 residues and shares 45% overall sequence identity (60% identical in the catalytic domain), which indicates that CAK beta is a protein structurally related to but different from FAK. The CAK beta gene is less evenly expressed in a variety of rat organs than the FAK gene. Anti-CAK beta antibody immunoprecipitated a 113-kDa protein from rat brain, 3Y1 fibroblasts, and COS-7 cells transfected with CAK beta cDNA. The tyrosine-phosphorylated state of CAK beta was not reduced on trypsinization, nor enhanced in response to plating 3Y1 cells onto fibronectin. CAK beta localized to sites of cell-to-cell contact in COS-7 transfected with CAK beta cDNA, in which FAK was found at the bottom of the cells. Thus, CAK beta is a PTK possibly participating in the signal transduction regulated by cell-to-cell contacts.

Monoclonal antibody specifically reacting against 73-kilodalton heat shock cognate protein: possible expression on mammalian cell surface.

The heat shock proteins (hsp) are regarded as being immunogenic to the animal hosts. Although certain hsp are suggested to be expressed on the cell surface, further evidence for the cell surface expression of these proteins has been required. In this article we report the development of a MAb NT22. This antibody reacted with ATP-binding proteins (which contain a large amount of 70-kDa hsp family) of HeLa cells, and with purified bovine 70-kDa hsp. It did not react with the E. coli lysate, but clearly reacted with the recombinant rat hsc73. However, NT22 failed to react with hsp72. Furthermore, stress treatment of cells also indicated that considerable amounts of NT22-defined antigen translocated into the nucleus from the cell cytoplasm. These results suggest that NT22 is a novel MAb that reacts specifically to the mammalian hsc73. Moreover, this antibody could detect the constitutive and stress-induced cell surface expression of its relevant antigen. It is expressed preferentially on EBV-transformed B cell and certain epithelial cancer cell lines. However, resting B cells did not express this antigen on the cell surface. These data indicate that hsc73 could be expressed on the cell surface of certain cells, and suggest that hsc73 may interact with the host immune system.

Sequential decrease in tight junctions as revealed by 7H6 tight junction-associated protein during rat hepatocarcinogenesis.

A sequential decrease in the number of hepatocyte tight junctions during the course of rat hepatocarcinogenesis was demonstrated by immunohistochemistry with a new 7H6 monoclonal antibody generated in our laboratory. Semiquantitative analysis by confocal laser scanning microscopy revealed that the expression of 7H6 antigen was reduced in hyperplastic foci, hyperplastic nodules and hepatocellular carcinomas (HCC) to 43%, 28% and 25%, respectively, compared to corresponding normal liver tissues. 7H6 antigen was scarce in HCC with a trabecular pattern, whereas it was expressed intensely at the apical and basolateral membrane of HCC with a glandular pattern. Immunoblot analysis of 7H6 expression in hepatocellular carcinomas showed a decrease roughly coincident with that shown by immunohistochemistry. These results indicated, for the first time, that tight junctions decrease progressively during carcinogenesis, leading to disruption of cellular polarity and cellular adhesiveness.

Hexestrol residues and metabolites in the tissues of wethers injected with hexestrol dicaprylate or hexestrol.

Four young (23 kg body weight) and two mature wethers (52 and 92 kg body weight) were subcutaneously injected with hexestrol (HX) or HX dicaprylate (HX-D) and killed 41 d later. The HX residues, comprising free, glucuronide and KOH hydrolyzable forms plus metabolites were determined by gas chromatography after liquid-liquid extraction and silica gel chromatographic purification of tissue sample. The HX residues were observed to be at concentrations of .1 to 1.0 ppb in most of the tissues examined. Maturity of the animals and the two hormonal formulations resulted in little difference in residues. The KOH hydrolyzable fraction was hardly detected in the tissues examined. Free HX was a major residue in muscle, representing about 70% of HX residues. Glucuronide HX represented 70 to 80% of HX residues in liver and kidney. In lung, glucuronide and free HX were present in similar amounts. This study showed that the gross metabolic patterns of HX and its esters are similar to other estrogens and steroids.

Gas-liquid chromatographic determination of hexestrol residues in adipose tissue.

A gas-liquid chromatographic (GLC) method is described for determining hexestrol residues in adipose tissue. The extraction and purification procedures were based on a published method for determining diethylstilbestrol. To increase precision and sensitivity, the sample was further cleaned up by silica gel column chromatography. The heptafluorobutyric (HFB) derivative of hexestrol was used for GLC analysis with HFB-docosanol as an internal standard. A variety of acetone-benzene mixtures were compared to determine the optimum ratio for hexestrol acylation. Acetone-benzene (90 + 10) or 100% acetone provided 16% higher GLC response than did a 50 + 50 mixture (P less than 0.001) and was selected for use in the acylation procedure. A system for evaporating excess reagents was also studied. Overall percent recovery reached 72 +/- 5. The method can be used to determine residual hexestrol at the 0.1 ppb level.

Modified method for electron capture gas-liquid chromatographic determination of hexestrol residues in bovine tissues.

A gas-liquid chromatographic (GLC) method with electron capture detection was developed for determining hexestrol residues based on a GLC method for diethylstilbestrol residues. The extraction and purification procedures were based on a published procedure. Pentafluoropropionic (PFP) and heptafluorobutyric (HFB) anhydrides and other halogenated compounds were compared as acylation reagents. PFP anhydride was selected because it provided reproducible GLC responses. Of the 3 column packing materials tested, OV-17, OV-210, and QF-1, OV-17 was the most stable under the GLC conditions used. A pH 10.5--10.6 in the purification step gave higher recoveries than pH 10.3 or pH 10.8 and was selected for use. The method is suitable for determining residues of 0.5 ppb. The limit of sensitivity ranges from 0.1 to 0.2 ppb.