Abstract. Cell-substratum adhesion strengths have been quantified using fibroblasts and glioma cells binding to two extracellular matrix proteins, fibronectin and tenascin. A centrifugal force-based adhesion assay was used for the adhesive strength measurements, and the corresponding morphology of the adhesions was visualized by interference reflection microscopy. The initial adhesions as measured at 4°C were on the order of 10 -5 dynes/cell and did not involve the cytoskeleton. Adhesions to fibronectin after 15 min at 37°C were more than an order of magnitude stronger; the strengthening response required cytoskeletal involvement. By contrast to the marked strengthening of adhesion to FN, adhesion to TN was unchanged or weakened after 15 min at 37°C. The absolute strength of adhesion achieved varied according to protein and cell type. When a mixed substratum of fibronectin and tenascin was tested, the presence of tenascin was found to reduce the level of the strengthening of cell adhesion normally observed at 37°C on a substratum of fibronectin alone. Parallel analysis of corresponding interference reflection micrographs showed that differences in the area of cell surface within 10-15 nm of the substratum correlated closely with each of the changes in adhesion observed: after incubation for 15 min on fibronectin at 37°C, glioma cells increased their surface area within close contact to the substrate by ",,125-fold. Cells on tenascin did not increase their surface area of contact. The increased surface area of contact and the inhibitory activity of cytochalasin b suggest that the adhesive "strengthening" in the 15 min after initial binding brings additional adhesion molecules into the adhesive site and couples the actin cytoskeleton to the adhesion complex. C ELL-SUBSTRATUM adhesion to fibronectin (FN) ~ orto glass often has been associated with several morphological and cytological changes including cell spreading and the formation of focal contacts, close contacts, and stress fibers (1, 9, 12, 19, 32-34, 40, 42, 45, 49, 51). While a spread morphology and focal contacts provide physical evidence that an adhesion has occurred, the actual adhesion and its relationship to the cell's behavioral changes that follow must be defined sequentially and on functional terms. As in cell-cell adhesion (39, 48), cell-substrate adhesion has been suggested to involve at least two measurable steps: the first involves initial contact between cell and substrate and the second step strengthens the adhesion through processes requiring metabolic energy (10,27,35,44,48).A number of extracellular matrix proteins including FN and laminin have been found to serve as adhesive substrates for cells. A more recently discovered extracellular matrix Dr. Lotz's present address is New England
Abstract. In this study, the putative laminin receptor function of the a6ß4 integrin was assessed . For this purpose, we used a human cell line, referred to as clone A, that was derived from a highly invasive, colon adenocarcinoma . This cell line, which expresses the a6ß4 integrin, adheres to the ES and not to the PI fragment of laminin . The adhesion of clone A cells to laminin is extremely rapid with half-maximal adhesion observed at 5 min after plating . Adhesion to laminin is blocked by GoH3, an a6 specific antibody (60% inhibition), as well as by A9, a 04 specific antibody (30% inhibition) . Most importantly, we demonstrate that a6ß4 binds specifically to laminin-
The Mac-2 lectin (carbohydrate binding protein 35) is a soluble, 32-to 35-kDa phosphoprotein that binds galactose-containing glycoconjugates. We report here that the colonic epithelium is a major site of Mac-2 expression in vivo based on immunohistochemistry of human tissue specimens. In this epithelium, proliferating cells at the base of the crypts do not express Mac-2 but its expression increases with differentiation along the crypt-to-surface axis. Mac-2 expression is concentrated in the nuclei of these differentiated epithelial cells.The progression from normal mucosa to adenoma to carcinoma is associated with significant changes in Mac-2 nuclear localization and expression. In all adenomas (9/9) and carcinomas (13/13) examined, Mac-2 was not present in the nucleus but was localized in the cytoplasm. Sequencing of Mac-2 cDNAs from normal mucosa and carcinoma revealed no specific mutations that could account for this loss of nuclear localization. We also observed a 5-to 10-fold decrease in Mac-2 mRNA levels in cancer compared to normal mucosa as well as a significant reduction in the amount of Mac-2 protein expressed. These observations suggest that Mac-2 exclusion from the nucleus and its decreased expression may be related to the neoplastic progression of colon cancer.The Mac-2 lectin is a soluble, 32-to 35-kDa phosphoprotein that binds galactose-containing glycoconjugates (1-5). Molecular cloning studies revealed that Mac-2 is identical to carbohydrate binding protein 35 (CBP 35), a lectin characterized initially in 3T3 fibroblasts (3,4,6). It is also identical to IgE binding protein (7) and a 32-kDa tumor-associated lectin (8, 9). The COOH-terminal domain of Mac-2, which contains a conserved carbohydrate binding motif, is homologous to a related family of 14-kDa galactose-specific lectins (reviewed in ref. 5). Collectively, Mac-2 and the 14-kDa lectins are referred to as S-lectins because their carbohydrate binding function was thought to be thiol-dependent (10), an assumption that has recently been questioned (2).The biological functions of Mac-2 remain elusive. Its putative role in cell adhesion has not been substantiated (11). Mac-2 may be associated with cell growth and differentiation because it is found in the nucleus of 3T3 fibroblasts and its nuclear localization may be related to the proliferative state of these cells (5). Also, the expression of Mac-2 occurs as a function of macrophage differentiation (12). These reports are consistent with the recent finding that a homologue of the 14-kDa S-lectin is cytostatic for embryonic fibroblasts (13). Other in vitro studies have suggested that the expression of Mac-2 increases in oncogenically transformed and metastatic cells (14-16).Progress in elucidating the function of Mac-2 has been hampered, in part, by a lack of data on its expression in vivo.In the present study, we found that Mac-2 is largely an epithelial-specific lectin with high expression seen in the colonic epithelium. The progression from normal colonic mucosa to adenoma to carcin...
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