Loss Of Cell Adhesion Research Articles

Loss of cell adhesion in Xenopus laevis embryos mediated by the cytoplasmic domain of XLerk, an erythropoietin-producing hepatocellular ligand.

The erythropoietin-producing hepatocellular (Eph) family of ligands and receptors has been implicated in the control of axon guidance and the segmental restriction of cells during embryonic development. In this report, we show that ectopic expression of XLerk, a Xenopus homologue of the murine Lerk-2 (ephrin-B1) transmembrane ligand, causes dissociation of Xenopus embryonic blastomeres by the mid-blastula transition. Moreover, a mutant that lacks the extracellular receptor binding domain can induce this phenotype. The carboxyl-terminal 19 amino acids of the cytoplasmic domain of XLerk are necessary but not sufficient to induce cellular dissociation. Basic fibroblast growth factor, but not activin, can rescue both the loss of cell adhesion and mesoderm induction in ectodermal explants expressing XLerk. Collectively, these results show that the cytoplasmic domain of XLerk has a signaling function that is important for cell adhesion, and fibroblast growth factor signaling modulates this function.

Loss of Cell Adhesion to Substratum Up-Regulates p21Cip1/WAF1Expression in BALB/c 3T3 Fibroblasts

Cell adhesion to substratum is essential for the transition of G1 to S phase in mouse BALB/c 3T3 fibroblast cell cycle. Loss of cell adhesion in late G1 phase caused blockage of the G1/S phase transition and repression of cyclin E-associated cyclin-dependent kinase-2 (CDK2) activity. A CDK2 inhibitor abundant in quiescent cells, p27Kip1, was down-regulated by growth factors in serum, and this down-regulation was partially prevented by loss of cell adhesion. Another CDK2 inhibitor, p21Cip1/WAF1, which was undetectable in quiescent cells, was markedly induced by loss of cell adhesion. In exponentially growing cells, loss of cell adhesion also induced p21Cip1/WAF1expression but did not affect the abundance of p27Kip1. These results suggest that loss of cell adhesion to substratum up-regulates p21Cip1/WAF1expression, which plays an essential role for arresting the BALB/c 3T3 fibroblast cell cycle.

Patterns of alpha- and beta-catenin and E-cadherin expression in colorectal adenomas and carcinomas.

Previous in vitro and in vivo model studies have shown that when E-cadherin expression in carcinoma cells is reduced, invasive behaviour ensues. The situation in human cancer in vivo, however, appears to be more complex, as immunohistochemically determined E-cadherin expression in various carcinomas, including colorectal cancer, does not always correlate with invasive growth. Loss of cell adhesion during invasion in spite of E-cadherin expression might be associated with a defective cadherin-catenin complex. The expression of alpha- and beta-catenin in comparison with E-cadherin was therefore examined in colorectal adenomas and carcinomas and in lymph node and liver metastases. In normal colonic mucosa, alpha- and beta-catenin immunoreactivity occurred along the lateral plasma membranes of the epithelial cells, in a pattern identical to E-cadherin staining. A similar pattern was found in colorectal adenomas and in most malignancies. In general, in neoplastic epithelia, the majority of the cancer cells displayed a normal (matching) pattern of E-cadherin and catenin expression. It is concluded that the patterns of expression of E-cadherin and alpha- and beta-catenin are very similar in colorectal neoplasms. This observation indicates that invasion in colorectal cancer is not paralleled by consistent loss of expression of the components of the cadherin-catenin complex.

Coagulation Factor XIa Cleaves the RHDS Sequence and Abolishes the Cell Adhesive Properties of the Amyloid β-Protein

Amyloid beta-protein (A beta) is the major constituent of senile plaques and cerebrovascular amyloid deposits in Alzheimer's disease and is proteolytically derived from its transmembrane parent protein the amyloid beta-protein precursor (A beta PP). Although the physiological role(s) of secreted A beta PPs are not fully understood, several potential functions have been described including the regulation of hemostatic enzymes factors XIa and IXa and a role in cell adhesion. In the present study, we investigated the proteolytic processing of A beta PP by factor XIa (FXIa). Incubation of the human glioblastoma cell line U138 stably transfected to overexpress the 695 isoform of A beta PP with FXIa (2.5-5 nM) resulted in proteolytic cleavage of secreted A beta PP. Higher concentrations of FXIa (> 25 nM) resulted in loss in cell adherence. Coincubation of FXIa with purified, recombinant Kunitz protease inhibitor domain of A beta PP blocked both the proteolytic processing of A beta PP and the loss of cell adhesion. The RHDS cell adhesion site of A beta PP resides within residues 5-8 of the A beta domain. Incubation of synthetic A beta 1-40 peptide with increasing concentrations of FXIa resulted in cleavage of A beta between Arg5 and His6 within the cell adhesion domain of the peptide. FXIa-digested A beta 1-40 or A beta PP695 lost their abilities to serve as cell adhesion substrates consistent with cleavage through this cell adhesion site. Together, these results suggest a new potential biological function for FXIa in the modulation of cell adhesion. In addition, we have shown that FXIa can proteolytically alter A beta and therefore possibly modify its physiological and perhaps pathological properties.

Disruption of cell adhesion in renal epithelium without cadherin loss

Loss of cell adhesion is a critical event in the development of tumour invasiveness and metastases. Although loss of cadherin expression has been demonstrated to be associated with increased invasiveness and metastatic potential in some tumours, others, including renal carcinoma, show no such correlation. The aim of this study was to demonstrate that cell adhesion could be lost in phorbol ester-treated renal epithelial cells and renal tumour cells without loss of A-CAM expression. The model used has been shown previously to mimic changes that occur in the progression of renal carcinoma. We found that A-CAM expression persists on the lateral surfaces of phorbol ester-treated cells even though these cells lose cell-cell adhesion. Similar findings were seen in renal carcinoma cells in culture. We conclude that loss of cell adhesion between tumour cells may other either by loss of cadherins or as a result of loss cadherin function occurring as a consequence of cell transformation.

Extracellular Domain of Pemphigus Vulgaris Antigen (Desmoglein 3) Mediates Weak Homophilic Adhesion

Pemphigus vulgaris antigen is in the cadherin supergene family. We hypothesized that the extracellular domain of pemphigus vulgaris antigen might mediate homophilic cell adhesion because 1) the originally described cadherins (e.g., E-cadherin) mediate this type of adhesion, 2) pemphigus vulgaris antigen is localized in desmosomes that are cell adhesion junctions, and 3) autoantibodies in pemphigus vulgaris patients cause loss of cell adhesion. To test this hypothesis we used a system developed for E-cadherin that, when transfected into L cells (mouse fibroblasts), has been shown to cause aggregation. Because this aggregation requires the cytoplasmic domain of E-cadherin to bind to catenins, we made a chimeric cDNA construct that encodes the extracellular domain of pemphigus vulgaris antigen and the cytoplasmic domain of E-cadherin. Analysis by immunofluorescence and flow cytometry with pemphigus vulgaris sera indicated that the pemphigus vulgaris antigen extracellular domain of this chimeric molecule (PVEC) was expressed on the cell surface of transiently transfected cells and permanently transfected L-cell clones. Immunoprecipitation of the chimeric molecule from extracts of these clones showed that the E-cadherin cytoplasmic domain bound catenins. Surprisingly, these L-cell clones displayed only slight aggregation compared to an L-cell clone transfected with E-cadherin. This weak aggregation was, however, specific and homophilic, as determined by cell sorting of only PVEC transfectants into aggregates from mixtures of PVEC and neomycin resistance gene transfectants, one of which was labeled with a fluorescent dye. We conclude that the extracellular domain of pemphigus vulgaris antigen mediates weak homophilic adhesion and is not interchangeable in function with the extracellular domain of E-cadherin.

Extracellular Domain of Pemphigus Vulgaris Antigen (Desmoglein 3) Mediates Weak Homophilic Adhesion

Pemphigus vulgaris antigen is in the cadherin supergene family. We hypothesized that the extracellular domain of pemphigus vulgaris antigen might mediate homophilic cell adhesion because 1) the originally described cadherins (e.g., E-cadherin) mediate this type of adhesion, 2) pemphigus vulgaris antigen is localized in desmosomes that are cell adhesion junctions, and 3) autoantibodies in pemphigus vulgaris patients cause loss of cell adhesion. To test this hypothesis we used a system developed for E-cadherin that, when transfected into L cells (mouse fibroblasts), has been shown to cause aggregation. Because this aggregation requires the cytoplasmic domain of E-cadherin to bind to catenins, we made a chimeric cDNA construct that encodes the extracellular domain of pemphigus vulgaris antigen and the cytoplasmic domain of E-cadherin. Analysis by immunofluorescence and flow cytometry with pemphigus vulgaris sera indicated that the pemphigus vulgaris antigen extracellular domain of this chimeric molecule (PVEC) was expressed on the cell surface of transiently transfected cells and permanently transfected L-cell clones. Immunoprecipitation of the chimeric molecule from extracts of these clones showed that the E-cadherin cytoplasmic domain bound catenins. Surprisingly, these L-cell clones displayed only slight aggregation compared to an L-cell clone transfected with E-cadherin. This weak aggregation was, however, specific and homophilic, as determined by cell sorting of only PVEC transfectants into aggregates from mixtures of PVEC and neomycin resistance gene transfectants, one of which was labeled with a fluorescent dye. We conclude that the extracellular domain of pemphigus vulgaris antigen mediates weak homophilic adhesion and is not interchangeable in function with the extracellular domain of E-cadherin.

Binding modes of IgG from Pemphigus autoimmune sera onto guinea pig keratinocytes and the fate of bound IgGs

Pemphigus is an intraepidermal autoimmune blistering disease of humans caused by circulating IgGs. We have investigated the binding mode and the fate of bound antibodies from Pemphigus sera (P-IgG) on guinea pig keratinocytes in suspension in order to find clues to the loss of cell adhesion in vivo (acantholysis). Flow cytometry, following indirect immunofluorescent labeling of the keratinocytes, and dead cells' staining with ethidium bromide, demonstrated the specific surface binding of P-IgG onto living keratinocytes only. This was shown with several Pemphigus sera or purified P-IgG. This technique, used with various Pemphigus sera, showed that the specific binding is increased when the serum titer is higher, and "Km" values for P-IgG were roughly and inversely correlated to the titers. Upon saturation the same average number of Pemphigus IgG sites per cell were found for the sera of different patients. Analysis of the specific binding of [125I]-P-IgG onto Percoll-separated (living) keratinocytes showed the existence of two classes of sites: 2 x 10(6) sites/cell high-affinity sites (Kd = 1.5 x 10(-6) M total IgG) and 25 x 10(6) sites/cell low-affinity sites (Kd = 6 x 10(-5) M total IgG). Cell sorting and flow cytometry of individual cells allowed us to correlate the light-scattering signal, the RNA content, the size and morphology, and the P-IgG binding to the cells. The results indicated that P-IgG binding is homogeneous within the living keratinocytes and increases with cell size (cell maturity). Cell-sorter analysis of cells with membrane-bound P-IgG, coupled to direct determination of P-IgG released in the medium, revealed the fate of bound P-IgG: 40-60% of the P-IgGs were released in the medium within 30 minutes at 37 degrees C. This was accompanied and followed by a much slower, metabolic energy-dependent, internalization process of the membrane-bound P-IgG. The internalization has been confirmed by electron microscopy of bound P-IgG labeled with protein A-gold. Internalized IgGs were seen in the cells in coated membranous vesicles and other endocytic compartments. Similar behavior was also observed with two other membrane ligands: i.e., concanavalin A and multispecific rabbit "antisurface" antibodies.

Divalent antibodies to mouse embryonal carcinoma cells inhibit compaction in the mouse embryo

Divalent antibodies from two antisera to embryonal carcinoma (EC) cells, F9 line, inhibited compaction in the preimplantation mouse embryo. One of these antisera, AF9-2, completely inhibited compaction at the 8-cell stage when the embryos were continuously incubated in a 1 100 dilution of the antiserum in culture medium from the 4-cell stage. Cell divisions continued and no cellular degeneration occurred. When cultured control embryos (preimmune and nonimmune sera) were normal blastocysts, many of the AF9-2-treated embryos were characterized by vacuolated cells and rounded rather than squamous, trophoectodermal cells. Anti-mouse spleen serum ( 1 10 , 1 100 ) had no effect on development. Purified divalent IgGs from AF9-2 (ammonium sulfate precipitation and DEAE chromatography) also were inhibitory at 30 μg/ml. Inhibition of compaction by AF9-2 was reversible. When uncompacted midmorula-stage embryos in AF9-2 ( 1 10 ) were transferred to medium without serum, there was a threefold increase in the percentage (70%) of normal blastocysts at the end of culture. Fluorescence microscopy demonstrated that AF9-2 antibodies, unlike preimmune and nonimmune sera, were bound to the surface of 8-cell and early morula-stage embryos. Inhibition by AF9-2 does not occur merely by nonspecifically coating cell surfaces so that they no longer recognize each other, since antispleen antibodies show similar binding by immunofluorescence but no inhibition. This study provides strong evidence that AF9-2 has specific divalent antibodies which block morphogenesis. Since newly compacted embryos lost their compacted appearance in AF9-2, these divalent antibodies cause a loss of cell adhesion, do not extensively cross-link adjacent cell surfaces, and cannot cause the compacted phenotype by agglutination.

Action of 1,25-dihydroxycholecalciferol on cartilage mineralization and on endosteal lining cells of bone. Experimental in vitro studies.

The aim of our study was to answer the following question: 1) Does 1,25-dihydroxycholecalciferol influence the mineralization process directly? 2) How does 1,25-D3 act on the endosteal cell envelope of bone, which is thought to be engaged in the moment to moment regulation of calcium homeostasis? We studied the endosteal surface of calvaria and the epiphyseal cartilage of rachitic rats in a tissue culture system in order to avoid the secondary effects of endogenous hormones or mineral sources. Investigations were carried out using transmission and scanning electron microscopy. Compared with untreated rachitic control specimens, 1,25-D3 (4 X 10(-8) M) did not enhance the mineralization process. We therefore conclude that the established in vivo effect of 1,25-D3 on the mineralization process must be an indirect one. Within 2 h the flat-shaped surface lining cells of calvaria showed a rapid transformation into globiform cells and a loss of cell adhesion. Within two days a second slower transformation into spindle-shaped fibroblast-like proliferating cells occurred. All changes of cell shape were reversible within several days in the absence of 1,25-D3. Obviously, bone matrix fiber texture influences the spatial movement of proliferating cells. The endosteal cell envelope is considered to act as a functional membrane of bone. Starting from this consideration we conclude that the changes of cellular shape described are the morphological basis for the action of 1,25-D3 in the regulation of calcium homeostasis in bone at the cellular level.

The cytological diagnosis of breast lesions

From March 1965 to March 1971 the cytology smears of 340 breast lesions were examined. Of these smears 144 were diagnosed as malignant, 193 as benign and in 3 cases a definite diagnosis could not be established. Frozen sections confirmed that 145 cases were malignant and 194 were benign with, in one case, no definite diagnosis possible by either method. From March 1971 to March 1978 a further 695 breast lesions were diagnosed by cytology smears only. Of these 276 were malignant and 416 benign with 3 cases undiagnosable by the method. Of the total 1035 breast lesions examined 1029 were correctly diagnosed by examination of cytology smears and no false positive interpretations were made. Criteria for malignancy includes hypercellularity, loss of cell adhesion, cellular and nuclear pleomorphism, chromatin clumping and increased nuclear-cytoplasmic ratio. Difficulty was initially experienced in cases of mammary duct ectasia because of associated epithelial dysplasia; in densely sclerotic lesions because of the paucity of epithelial cells, and in well differentiated sarcomas. Advantages of the method include ease of preparation and speed of diagnosis, while disadvantages include inability to provide as precise a tissue diagnosis as frozen sections provide.

The correlation between plasminogen activator-stimulated DNA synthesis and cell morphology in 3T3 cells

3T3 cells when grown in co-culture with SV40 3T3 cells under varying conditions of plasminogen activation displayed an increase in thymidine incorporation, suggesting growth stimulation, a decrease in area of cell spread, and loss of cell adhesion as measured by trypsin sensitivity. Evidence is presented that the mechanism whereby plasminogen activator initiates DNA synthesis is by enhancing serum stimulation; this is related to the concomitant changes in cell morphology which may allow greater access of serum to the cells.