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Biomedical subjects

M Takeichi

Publications and source records attributed to M Takeichi.

At least 145 records · Page 8Linked to original sources
PubMed1992

Morphoregulatory activities of E-cadherin and beta-1 integrins in colorectal tumour cells.

The cadherin family of adhesion molecules are prime mediators of cell-cell interactions while the integrins predominantly mediate cell-matrix and to a lesser extent cell-cell binding specificity. We have recently shown that a human colon carcinoma cell line (SW1222) organizes into glandular structures, with well defined polarity when cultured in three-dimensional type I collagen gel. The current study indicates that SW1222 cells display high levels of E-cadherin (E-cd, epithelial cadherin) by western blotting and immunohistochemical staining. A monoclonal antibody (HECD-1) specific for human E-cd blocks cell-cell adhesion (100%) and inhibits (up to 75%) the glandular differentiation of SW1222 cells growing in collagen gel. Furthermore the anti-beta 1 integrin monoclonal antibody (mAb13) inhibits the glandular differentiation of SW1222 cells (61%) and their cellular binding to type I collagen (60%). However, no significant inhibition of cell-cell adhesion was demonstrated using mAb13 nor the anti-carcinoembryonic antigen monoclonal antibody (PR3B10). These results are consistent with E-cd being a cell-cell adhesion molecule expressed by SW1222 cells. These data indicate that E-cd and beta 1 integrins mediate cell-cell and cell-collagen interactions required for the induction and maintenance of the glandular differentiation of colorectal tumour cells. Thus the down-regulation or loss of E-cd and beta 1 integrins seen in poorly differentiated colorectal tumours may represent one of the abnormalities underlying their progression towards an undifferentiated phenotype in vivo.

Blotting, Western↗
PubMed1992

Altered expression of E-cadherin in gastric cancer tissues and carcinomatous fluid.

Expression of E-cadherin in 21 patients with various histological types of gastric carcinomas was studied by immunoperoxidase staining. Intercellular boundaries of almost all cancer cells in well and moderately differentiated adenocarcinomas stained as deeply for E-cadherin as normal gastric mucosa. However, singly infiltrating cells of those histological types were poorly stained. In poorly differentiated adenocarcinomas, cancer cells forming clusters stained lightly and those infiltrating singly stained even less. In signet ring cell carcinomas, hardly any staining was observed. In each histological type, the staining patterns and intensity at different layers of the gastric wall, were essentially the same. Cancer cells from carcinomatous ascites of gastric adenocarcinomas and pancreatic adenocarcinomas, and those from pleural effusion of lung adenocarcinomas were also studied by immunofluorescence staining. Of 11 specimens, ten were negative and only one from a lung adenocarcinomas was positively stained. By phase-contrast microscopic observations, none of these cancer cells including those from the lung adenocarcinomas, formed obvious cell-cell contacts. Cell aggregation assays confirmed the above results. The molecular weight of E-cadherin of cancer cells of lung adenocarcinomas was less than intact E-cadherin as revealed by Western blot analysis. These results suggest that depressed expression and/or impaired function of E-cadherin in cancer cells, facilitates their liberation from primary sites to infiltrate freely into tissue or fluid.

Adenocarcinoma↗
PubMed1992

Cadherin-mediated cell-cell adhesion is perturbed by v-src tyrosine phosphorylation in metastatic fibroblasts.

Rat 3Y1 cells acquire metastatic potential when transformed with v-src, and this potential is enhanced by double transformation with v-src and v-fos (Taniguchi, S., T. Kawano, T. Mitsudomi, G. Kimura, and T. Baba. 1986. Jpn. J. Cancer Res. 77:1193-1197). We compared the activity of cadherin cell adhesion molecules of normal 3Y1 cells with that of v-src transformed (SR3Y1) and v-src and v-fos double transformed (fosSR3Y1) 3Y1 cells. These cells expressed similar amounts of P-cadherin, and showed similar rates of cadherin-mediated aggregation under suspended conditions. However, the aggregates or colonies of these cells were morphologically distinct. Normal 3Y1 cells formed compacted aggregates in which cells are firmly connected with each other, whereas the transformed cells were more loosely associated, and could freely migrate out of the colonies. Overexpression of exogenous E-cadherin in these transformed cells had no significant effect on their adhesive properties. We then found that herbimycin A, a tyrosine kinase inhibitor, induced tighter cell-cell associations in the aggregates of the transformed cells. In contrast, vanadate, a tyrosine phosphatase inhibitor, inhibited the cadherin-mediated aggregation of SR3Y1 and fosSR3Y1 cells but had little effect on that of normal 3Y1 cells. These results suggest that v-src-mediated tyrosine phosphorylation perturbs cadherin function directly or indirectly, and the inhibition of tyrosine phosphorylation restores cadherin action to the normal state. We next studied tyrosine phosphorylation on cadherins and the cadherin-associated proteins, catenins. While similar amounts of catenins were expressed in all of these cells, the 98-kD catenin was strongly tyrosine phosphorylated only in SR3Y1 and fosSR3Y1 cells. Cadherins were also weakly tyrosine phosphorylated only in the transformed cells. The tyrosine phosphorylation of these proteins was enhanced by vanadate, and inhibited by herbimycin A. Thus, the tyrosine phosphorylation of the cadherin-catenin system itself might affect its function, causing instable cell-cell adhesion.

Animals↗
PubMed1992

Local and transient expression of E-cadherin involved in mouse embryonic brain morphogenesis.

We found that E-cadherin (uvomorulin) is transiently expressed in restricted regions of the metencephalon, mesencephalon and diencephalon of mouse embryonic brain. This expression first occurred in parts of the mesencephalon and diencephalon at around E9.5, and subsequently extended to the primordia of cerebellum, the dorsal midline of mesencephalon and some other regions of the embryonic brain. These E-cadherin expressions ceased by E15 except at the dorsal midline. Immunohistological analyses showed that E-cadherin-positive cells are radially arranged in the neural tube and the E-cadherin-positive regions are sharply demarcated from E-cadherin-negative regions. Axons extending from some of the E-cadherin-positive regions also expressed this molecule. When embryonic brains were dissociated into single cells and cultured as monolayers, E-cadherin-positive cells formed clusters that were segregated from E-cadherin-negative cells. E9.5 brain fragments containing metencephalon and mesencephalon were isolated, explanted on Nucleopore filters and cultured in the absence or presence of antibodies to E-cadherin. This antibody treatment removed most of the E-cadherin molecules from the explants and consequently affected their growth pattern. To analyze cellular events induced by the antibody treatment, we stained these explants with an antiserum to En whose distribution was found to overlap in part with that of E-cadherin and found that the pattern of En staining was altered by the anti-E-cadherin antibody treatment. These results suggest that the local and transient expression of E-cadherin in embryonic brain is involved in regional pattern formation in this organ.

Animals↗
PubMed1992

A chimeric N-cadherin/beta 1-integrin receptor which localizes to both cell-cell and cell-matrix adhesions.

To study the molecular mechanisms involved in formation of cell contacts, we have transfected cultured cells with a chimeric cDNA encoding the cytoplasmic and transmembrane domains of beta 1 integrin and the extracellular region of N-cadherin and determined the subcellular distribution of the chimeric molecule. We show that the chimeric receptor associates preferentially with cell-matrix focal contacts, suggesting that its distribution is directed by its beta 1 integrin segment, presumably via interactions of the cytoplasmic domain with cytoskeletal elements characteristic of focal contacts. Transfected cells which expressed relatively high levels of the cadherin/integrin chimera underwent an apparent epithelialization and contained the molecule both in cell-matrix and cell-cell contacts. Location in cell-cell contacts indicates competence of the cadherin extracellular domain to participate in formation of cell-cell junctions using a foreign cytoplasmic domain. Labeling of these cultures for talin, which is normally associated only with matrix adhesions, revealed specific labeling along the newly formed intercellular junctions. This suggests that the local association of talin with these sites is induced by the cytoplasmic tail of beta 1 integrin receptor presented by the chimeric protein. These results suggest that the formation of adherens-type junctions is driven by the cooperative interactions of the relevant adhesion molecules (cadherins and integrins) both with the respective extracellular ligands and with the cytoskeleton.

3T3 Cells↗
PubMed1992

Postnatal changes in development of serotonin-, neuropeptide Y-, Leu-enkephalin- and substance P- terminals in the rat locus coeruleus; a quantitative immunohistochemical study.

Postnatal developmental changes were investigated in afferent terminals immunoreactive to serotonin (5-HT), neuropeptide Y (NPY), Leu-Enkephalin (ENK) and substance P (SP) within the locus coeruleus from postnatal rat from day 1 (1 D) to 9 week (9 W) adult using immunohistochemical techniques. Quantitative study using a light microscopic image analyzing system revealed that the number of immunoreactive terminals increased after birth to reach a peak at 5 W, then decreased by 26% of this value and stabilized at 7 W; terminals immunoreactive to 5-HT or NPY increased gradually after birth, while those immunoreactive to ENK or SP increased suddenly at 3 W. Electron microscopic analysis revealed similar changes in the total terminal number during development. Synaptic terminals, on the other hand, increased sharply from 1 D to 3 W, then gradually until 5 W, and remained stable thereafter. These results suggest that surplus afferent terminals are eliminated prior to the establishment of afferent innervation and that 5 W postnatal is the critical time for maturation of the afferent system. Electron microscopy also demonstrated morphological characteristics of terminals immunoreactive to each peptide or 5-HT and developmental changes in their characteristics.

Aging↗
PubMed1992

Restricted expression of N- and R-cadherin on neurites of the developing chicken CNS.

The expression of two cadherins, N- and R-cadherin, was mapped in the CNS of chicken embryos of 6-11 d incubation, focusing on the sensory and motor fiber systems. In the spinal cord, the laterally located fibers of the dorsal funiculus express N-cadherin while the medially located fibers do not. These two fiber systems have a different course within the CNS but associate to form the spinal dorsal roots. In the hindbrain, N-cadherin is expressed by the descending trigeminal (general somatic sensory) tract, which is contiguous with the N-cadherin-positive zone of the dorsal funiculus of the spinal cord. R-cadherin is not expressed by sensory fibers, but is expressed by the visceral motor system of the vagus and glossopharyngeal nerves, which are N-cadherin negative. The motor neurites expressing R-cadherin have a different course within the brain than the sensory neurites expressing N-cadherin, although they form the common sensory/motor roots of the vagus nerve at the surface of the brain. The possibility that N-cadherin provides a guidance cue for sensory axon migration within the CNS by a homophilic adhesion mechanism was investigated in vitro. Explants from sensory spinal ganglia expressing N-cadherin were placed on N-cadherin-transfected neuroblastoma cells, and axon outgrowth was visualized. Results showed that the sensory axons defasciculate and closely follow the cell-cell boundaries between transfected cells where high levels of N-cadherin are expressed. These results show that the two cadherins, like members of the immunoglobulin superfamily of molecules, are expressed in a topographically restricted fashion during chick brain development. They furthermore suggest that N-cadherin expression by neurites may play a role in guiding these neurites along CNS paths that express the same molecule.

Animals↗
PubMed1991

Genomic organization and chromosomal mapping of the mouse P-cadherin gene.

Cadherins are a family of Ca(2+)-dependent cell adhesion molecules, that includes P-cadherin, E-cadherin, N-cadherin and L-CAM. In this study, the genomic organization of the mouse P-cadherin gene was determined by analyzing overlapping DNA clones obtained from a mouse genomic library. The results showed that this gene spans over 45 kb and consists of 15 exons. A marked feature of this gene is that the first intron is 23 kbp long accounting for half its length. Comparisons of this structure with that of L-CAM, a chicken cadherin, revealed that the exon-intron boundaries are conserved between the two genes except that the P-cadherin first exon includes the correspoding first and second exons of the L-CAM gene. This gene was also similar to the other in that the second intron, which corresponds to the P-cadherin first intron, is exceptionally longer than other introns. These results suggest that the exon-intron pattern conserved in these genes is of significance for generation of domain structure of cadherin molecules or for their transcriptional regulation. We also determined the chromosomal localization of the P-cadherin gene by interspecific backcross analysis, and found that this gene is located in the central region of mouse chromosome 8 and linked with the E-cadherin locus. This is the first evidence for the linkage of different cadherin genes.

Amino Acid Sequence↗
PubMed1991

The 102 kd cadherin-associated protein: similarity to vinculin and posttranscriptional regulation of expression.

The E-cadherin cell adhesion molecule is associated with cytoplasmic polypeptides, and this association is essential for its cell-binding function. Using isolated adherens junctions of the liver, we purified a 102 kd protein that can associate with E-cadherin (CAP102) and isolated cDNAs encoding this protein. Sequence analysis of the cDNAs revealed that this protein has a similarity to vinculin. L cells not expressing endogenous cadherin express the mRNA for CAP102 but have only a trace amount of CAP102 protein. Introducing exogenous E-cadherin into these cells, however, induced a high expression of CAP102 protein without affecting the amount of its mRNA, suggesting that there is a posttranscriptional regulatory mechanism for this molecule. The same effect was observed by introducing N- or P-cadherin into L cells.

Amino Acid Sequence↗
PubMed1991

Cadherin cell adhesion receptors as a morphogenetic regulator.

Cadherins are a family of cell adhesion receptors that are crucial for the mutual association of vertebrate cells. Through their homophilic binding interactions, cadherins play a role in cell-sorting mechanisms, conferring adhesion specificities on cells. The regulated expression of cadherins also controls cell polarity and tissue morphology. Cadherins are thus considered to be important regulators of morphogenesis. Moreover, pathological examinations suggest that the down-regulation of cadherin expression is associated with the invasiveness of tumor cells.

Adenocarcinoma↗
PubMed1991

Interactions of Schwann cells with neurites and with other Schwann cells involve the calcium-dependent adhesion molecule, N-cadherin.

During embryogenesis, Schwann cells interact with axons and other Schwann cells, as they migrate, ensheath axons, and participate in organizing peripheral nervous tissues. The experiments reported here indicate that the calcium-dependent molecule, N-cadherin, mediates adhesion of Schwann cells to neurites and to other Schwann cells. Cell cultures from chick dorsal root ganglia and sciatic nerves were maintained in media containing either 2 mM Ca++ or 0.2 mM Ca++, a concentration that inactivates calcium-dependent cadherins. When the leading lamellae of Schwann cells encountered migrating growth cones in medium with 2 mM Ca++, they usually remained extended, and the growth cones often advanced onto the Schwann cell upper surface. In the low Ca++ medium, the frequency of withdrawal of the Schwann cell lamella after contact with a growth cone was much greater, and withdrawal was the most common reaction to growth cone contact in medium with 2 mM Ca++ and anti-N-cadherin. Similarly, when motile leading margins of two Schwann cells touched in normal Ca++ medium, they often formed stable areas of contact. N-cadherin and vinculin were co-concentrated at these contact sites between Schwann cells. However, in low Ca++ medium or in the presence of anti-N-cadherin, interacting Schwann cells usually pulled away from each other in a behavior reminiscent of contact inhibition between fibroblasts. In cultures of dissociated cells in normal media, Schwann cells frequently were aligned along neurites, and ultrastructural examination showed extensive close apposition between plasma membranes of neurites and Schwann cells. When dorsal root ganglia explants were cultured with normal Ca++, Schwann cells migrated away from the explants in close association with extending neurites. All these interactions were disrupted in media with 0.2 mM Ca++. Alignment of Schwann cells along neurites was infrequent, as were extended close apposition between axonal and Schwann cell plasma membranes. Finally, migration of Schwann cells from ganglionic explants was reduced by disruption of adhesive contact with neurites. The addition of antibodies against N-cadherin to medium with normal Ca++ levels had similar effects as lowering the Ca++ concentration, but antibodies against the neuronal adhesive molecule, L1, had no effects on interactions between Schwann cells and neurites.

Animals↗
PubMed1991

R-cadherin: a novel Ca(2+)-dependent cell-cell adhesion molecule expressed in the retina.

cDNAs encoding a novel member of the cadherin cell adhesion receptor family were cloned. This cadherin is expressed in the retina of the chicken and is termed R-cadherin. It is similar to other cadherins in its primary structure, but most resembles N-cadherin, showing 74% amino acid identity. Cells expressing R-cadherin can adhere to those expressing N-cadherin when mixed, but they form homotypic clusters within their chimeric aggregates. In the development of the neural retina, R-cadherin begins to be expressed around embryonic day 8 in both neuronal and glial cells, and this expression continues up to the hatching stage. The pattern of the expression of R-cadherin was different from that of N-cadherin, suggesting distinctive roles in retinal morphogenesis.

Amino Acid Sequence↗
PubMed1991

Regulation of connexin 43-mediated gap junctional intercellular communication by Ca2+ in mouse epidermal cells is controlled by E-cadherin.

Gap junctional intercellular communication (GJIC) of cultured mouse epidermal cells is mediated by a gap junction protein, connexin 43, and is dependent on the calcium concentration in the medium, with higher GJIC in a high-calcium (1.2 mM) medium. In several mouse epidermal cell lines, we found a good correlation between the level of GJIC and that of immunohistochemical staining of E-cadherin, a calcium-dependent cell adhesion molecule, at cell-cell contact areas. The variant cell line P3/22 showed both low GJIC and E-cadherin protein expression in low- and high-Ca2+ media. P3/22 cells showed very low E-cadherin mRNA expression. To test directly whether E-cadherin is involved in the Ca(2+)-dependent regulation of GJIC, we transfected the E-cadherin expression vector into P3/22 cells and obtained several stable clones which expressed high levels of E-cadherin mRNA. All transfectants expressed E-cadherin molecules at cell-cell contact areas in a calcium-dependent manner. GJIC was also observed in these transfectants and was calcium dependent. These results suggest that Ca(2+)-dependent regulation of GJIC in mouse epidermal cells is directly controlled by a calcium-dependent cell adhesion molecule, E-cadherin. Furthermore, several lines of evidence suggest that GJIC control by E-cadherin involves posttranslational regulation (assembly and/or function) of the gap junction protein connexin 43.

Animals↗
PubMed1991

Drosophila PS integrins recognize vertebrate vitronectin and function as cell-substratum adhesion receptors in vitro.

Using the Drosophila cell line MLDmBG-1, a monoclonal antibody aBG-1 that can inhibit not only cell clumping but also cell spreading was generated. This antibody immunoprecipitates a complex of molecules consisting of a major 120 x 10(3) Mr and other components. To characterize the 120 x 10(3) Mr component, we purified it, generated antibodies to it, and cloned its cDNA. Sequencing of this cDNA suggests that the 120 x 10(3) Mr molecule is identical to PS beta, a beta chain of Drosophila integrins. The other components immunoprecipitated included two alpha chains of Drosophila integrins, PS1 alpha and PS2 alpha, as revealed using specific antibodies to these molecules. These suggest that aBG-1 recognizes the PS beta associated with PS1 alpha or PS2 alpha. However, immunostaining of embryos and larvae with aBG-1 showed that the staining pattern is similar to that for PS2 alpha but not for PS beta, suggesting that the antibody preferentially recognizes the PS beta associated with particular alpha chains in situ. We then attempted to characterize the ligands for these integrin complexes, using culture dishes coated with various vertebrate matrix proteins. These cells spread very well on dishes coated with vitronectin and, to a lesser extent, on those with fibronectin. This spreading was partially inhibited by aBG-1, but not by other control antibodies or RGD peptides. The cell attachment to these substrata was not affected by the antibody. The cells also can attach to dishes coated with laminin but without spreading, and this attachment was not inhibited by aBG-1. Furthermore, they do not attach to dishes coated with collagen type I, type IV, and fibrinogen. These results indicate that Drosophila PS integrins can recognize vertebrate vitronectin, and also fibronectin with a weaker affinity, at sites other than RGD sequences, and thus can function in cell-substratum adhesion.

Animals↗
PubMed1991

Differential expression of R- and N-cadherin in neural and mesodermal tissues during early chicken development.

R-cadherin is a newly identified member of the cadherin family of cell adhesion receptors. The expression of R-cadherin in early chicken embryos was studied using affinity-purified antibodies to this molecule, comparing it with that of N-cadherin. Immunoblot analysis of various organs of 10.5-day embryos showed that R-cadherin is most abundantly expressed in the retina and brain. Immunostaining of the cervical and thoracic regions of embryos revealed that R- and N-cadherin are expressed in all neural tissues. In the neural tube, R-cadherin appears at around stage 21, although N-cadherin expression begins at a much earlier stage. The distribution of R-cadherin in the neural tube differs from that of N-cadherin; for example, some regions of the tube express only R-cadherin, and other regions only N-cadherin. In the peripheral ganglia, these two cadherins are also expressed in different patterns which change during development. Some mesenchymal tissues including the notochord, the myotome, myotubes and perichondria also express these cadherins, again in different patterns. Thus, R- and N-cadherin are differentially expressed in all the tissues examined, and they may contribute to the spatial segregation of heterogeneous cells in a tissue.

Animals↗
PubMed1991

Expression of immunoreactive E-cadherin adhesion molecules in human cancers.

E-cadherin (E-CD), a Ca(2+)-dependent adhesion molecule, plays a major role in the maintenance of intercellular junctions in normal epithelial cells in most organs. The expression of E-CD in human carcinoma samples (esophagus, stomach, and breast) was investigated using immunohistochemical staining, which was performed on surgical specimens using a monoclonal antibody for human E-CD. E-cadherin was strongly expressed in all normal epithelium examined. However E-CD expression in primary tumors of esophagus (11 of 15: 73%), stomach (5 of 20: 25%), and breast (9 of 20: 45%) was reduced, and 68% of these (esophagus: 8 of 11, stomach: 4 of 5, breast: 5 of 9) displayed heterogeneous E-CD expression. In some tumor cells with reduced E-CD expression, E-CD molecules were located in the cell cytoplasm. These results indicate that there are human cancer cells in which E-CD-related intercellular adhesion is impaired.

Adenocarcinoma↗