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...

Superficial injury involving the mucosa of the gastrointestinal tract heals by a process termed restitution that involves epithelial sheet movement into the damaged area. The forces that drive epithelial sheet movement are only partially understood, although it is known to involve changes in the morphology of cells bordering the damage, such as the formation of large, flat, cytoplasmic extensions termed lamellae. We investigated the mechanism of epithelial sheet movement by following the response of the actin cytoskeleton and specific integrins (alpha6beta4, alpha6beta1, and alpha3beta1) to wounding. To model this event in vitro, monolayers of T84 cells, well-differentiated colon carcinoma cells, were damaged by aspiration and the ensuing response was analyzed by a combination of time-lapse video microscopy, fluorescence confocal microscopy and antibody inhibition assays. We show that wound healing begins with retraction of the monolayer. alpha6beta4 integrin is localized on the basal surface in structures referred to as type II hemidesmosomes that persist throughout this early stage. We hypothesize that these structures adhere to the substrate and function to retard retraction. Once retraction ceases, the wound is contracted initially by actin purse strings and then lamellae. Purse strings and lamellae produce a pulling force on surrounding cells, inducing them to flatten into the wound. In the case of lamellae, we detected actin suspension cables that appear to transduce this pulling force. As marginal cells produce lamellae, their basal type II hemidesmosomes disappear and the alpha6 integrins appear evenly distributed over lamellae surfaces. Antibodies directed against the alpha6 subunit inhibit lamellae formation, indicating that redistribution of the alpha6 integrins may contribute to the protrusion of these structures. Antibodies directed against the alpha3beta1 integrin also reduce the size and number of lamellae. This integrin's contribution to lamellae extension is most likely related to its localization at the leading edge of emerging protrusions. In summary, wounds in epithelial sheets initially retract, and then are contracted by first an actin purse string and then lamellae, both of which serve to pull the surrounding cells into the denuded area. The alpha6 integrins, particularly alpha6beta4, help contain retraction and both the alpha6 integrins and alpha3beta1 integrin contribute to lamellae formation.

The phorbol ester phorbol 12-myristate 13-acetate (PMA) inhibits Cl(-) secretion (short-circuit current, I(sc)) and decreases barrier function (transepithelial resistance, TER) in T84 epithelia. To elucidate the role of specific protein kinase C (PKC) isoenzymes in this response, we compared PMA with two non-phorbol activators of PKC (bryostatin-1 and carbachol) and utilized three PKC inhibitors (Gö-6850, Gö-6976, and rottlerin) with different isozyme selectivity profiles. PMA sequentially inhibited cAMP-stimulated I(sc) and decreased TER, as measured by voltage-current clamp. By subcellular fractionation and Western blot, PMA (100 nM) induced sequential membrane translocation of the novel PKC epsilon followed by the conventional PKC alpha and activated both isozymes by in vitro kinase assay. PKC delta was activated by PMA but did not translocate. By immunofluorescence, PKC epsilon redistributed to the basolateral domain in response to PMA, whereas PKC alpha moved apically. Inhibition of I(sc) by PMA was prevented by the conventional and novel PKC inhibitor Gö-6850 (5 microM) but not the conventional isoform inhibitor Gö-6976 (5 microM) or the PKC delta inhibitor rottlerin (10 microM), implicating PKC epsilon in inhibition of Cl(-) secretion. In contrast, both Gö-6976 and Gö-6850 prevented the decline of TER, suggesting involvement of PKC alpha. Bryostatin-1 (100 nM) translocated PKC epsilon and PKC alpha and inhibited cAMP-elicited I(sc). However, unlike PMA, bryostatin-1 downregulated PKC alpha protein, and the decrease in TER was only transient. Carbachol (100 microM) translocated only PKC epsilon and inhibited I(sc) with no effect on TER. Gö-6850 but not Gö-6976 or rottlerin blocked bryostatin-1 and carbachol inhibition of I(sc). We conclude that basolateral translocation of PKC epsilon inhibits Cl(-) secretion, while apical translocation of PKC alpha decreases TER. These data suggest that epithelial transport and barrier function can be modulated by distinct PKC isoforms.

In this study, we used clone A, a human colon carcinoma cell line, to characterize those integrins that mediate colon carcinoma adhesion to laminin. Monoclonal antibodies specific for the human fi1 subunit inhibited clone A adhesion to laminin. They also precipitated a complex of surface proteins that exhibited an electrophoretic behavior characteristic of a2fll and a3fi1. A monoclonal antibody specific for a2 (PIH5) blocked clone A adhesion to laminin, as well as to collagen 1. An a3-specific antibody (P1 B5) had no effect on clone A adhesion to laminin, even though it can block the adhesion of other cell types to laminin. Thus, the a2,61 integrin can function as both a laminin and collagen I receptor on clone A cells. Although these cells express a31, an established laminin receptor, they do not appear to use it to mediate laminin adhesion. In addition, the monoclonal antibody GoH3, which recognizes the a6 integrin subunit, also inhibited carcinoma adhesion to laminin but not to fibronectin or collagen 1. This antibody precipitated the a6 subunit in association with the fl4 subunit. There was no evidence of a6,B1 association on these cells. In summary, the results obtained in this study indicate that multiple integrin a subunits, in association with two distinct ,@ subunits, are involved in colon carcinoma adhesion to laminin. Based on the behavior of a3,#1 and a2,81, the results also suggest that cells can regulate the ability of a specific integrin to mediate adhesion.

The integrin ␣ 6 ␤ 4 , a receptor for members of the laminin family of basement membrane components, contributes to the function of epithelial cells and their oncogenically transformed derivatives. In our efforts to study ␣ 6 ␤ 4 -mediated functions in more detail and to assess the contribution of the ␤ 4 cytoplasmic domain in such functions, we identified a rectal carcinoma cell line that lacks expression of the ␤ 4 integrin subunit. This cell line, termed RKO, expresses ␣ 6 ␤ 1 but not ␣ 6 ␤ 4 , and it interacts with laminin-1 less avidly than similar cell lines that express ␣ 6 ␤ 4 . We expressed a full-length ␤ 4 cDNA, as well as a mutant cDNA that lacks the ␤ 4 cytoplasmic domain, in RKO cells and isolated stable subclones of these transfectants. In this study, we report that subclones that expressed the full-length ␤ 4 cDNA in association with endogenous ␣6 exhibited partial G 1 arrest and apoptosis, properties that were not evident in RKO cells transfected with either the cytoplasmic domain mutant or the expression vector alone. In an effort to define a mechanism for these observed changes in growth, we observed that expression of the ␣ 6 ␤ 4 integrin induced expression of the p21 (WAF1; CiP1) protein, an inhibitor of cyclin-dependent kinases. These data suggest that the ␤ 4 integrin cytoplasmic domain is linked to a signaling pathway involved in cell cycle regulation in the ␤ 4 transfected RKO cells.The integrin ␣ 6 ␤ 4 is a receptor for members of the laminin family of basement membrane components. Initial studies established that ␣ 6 ␤ 4 is a receptor for laminin-1 (1-3), and subsequent work has shown that it also functions as a receptor for other laminin isoforms (4, 5). In its capacity as a laminin receptor, ␣ 6 ␤ 4 is involved in the formation and maintenance of hemidesmosomes (6 -8) and in the dynamic adhesion and migration of carcinoma cells (2, 3). Most likely, other functions of epithelial and carcinoma cells are dependent upon ␣ 6 ␤ 4 because it plays such a pivotal role in mediating their interactions with laminin matrices. It is widely assumed that the unusually large and structurally unique cytoplasmic domain of the ␤ 4 integrin subunit associates with cytoskeletal and signaling molecules and that such associations provide the basis for the distinct functions associated with ␣ 6 ␤ 4 (5, 8, 9).In our efforts to study ␣ 6 ␤ 4 -mediated functions in more detail and to assess the contribution of the ␤ 4 cytoplasmic domain in such functions, we identified a rectal carcinoma cell line that lacks expression of the ␤ 4 integrin subunit. This cell line, termed RKO, expresses ␣ 6 ␤ 1 but not ␣ 6 ␤ 4 (2, 3). In this study, we report that RKO transfectants, which expressed the fulllength ␤ 4 cDNA in association with endogenous ␣ 6 exhibited G 1 arrest and a basal rate of apoptosis, properties that were not evident in RKO cells transfected with either a ␤ 4 cytoplasmic domain mutant or the expression vector alone. In an effort to define a mechanism for these observed changes in growth, we observed that e...

Matrix metalloproteinases (MMP) and a disintegrin and metalloprotease 12 (ADAM 12) can be detected in the urine of breast cancer patients and provide independent prediction of disease status. To evaluate the potential of urinary metalloproteinases as biomarkers to predict breast cancer risk status, urine samples from women with known risk marker lesions, atypical hyperplasia and lobular carcinoma in situ (LCIS), were analyzed. Urine samples were obtained from 148 women: 44 women with atypical hyperplasia, 24 women with LCIS, and 80 healthy controls. MMP analysis was done using gelatin zymography and ADAM 12 analysis was done via immunoblotting with monospecific antibodies and subsequent densitometric measurement. Positive urinary MMP-9 levels indicated a 5-fold risk of atypical hyperplasia and >13-fold risk of LCIS compared with normal controls. Urinary ADAM 12 levels were significantly elevated in women with atypical hyperplasia and LCIS from normal controls, with receiver operating characteristic curve analysis showing an area under the curve of 0.914 and 0.950, respectively. To assess clinical applicability, a predictive index was developed using ADAM 12 in conjunction with Gail risk scores for women with atypia. Scores above 2.8 on this ADAM 12-Gail risk prediction index score are predictive of atypical hyperplasia (sensitivity, 0.976; specificity, 0.977). Our data suggest that the noninvasive detection and analysis of urinary ADAM 12 and MMP-9 provide important clinical information for use as biomarkers in the identification of women at increased risk of developing breast cancer. (Cancer Epidemiol Biomarkers Prev 2008;17(5):1034 -42)