RESUMEN
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.
Asunto(s)
Adenocarcinoma/metabolismo , Antígenos de Diferenciación/metabolismo , Núcleo Celular/ultraestructura , Colon/metabolismo , Neoplasias del Colon/metabolismo , Pólipos del Colon/metabolismo , Mucosa Intestinal/metabolismo , Lectinas/metabolismo , Adenocarcinoma/patología , Secuencia de Aminoácidos , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/genética , Secuencia de Bases , Northern Blotting , Núcleo Celular/metabolismo , Transformación Celular Neoplásica , Clonación Molecular , Colon/citología , Colon/patología , Neoplasias del Colon/patología , Pólipos del Colon/patología , ADN/genética , ADN/aislamiento & purificación , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Galectina 3 , Humanos , Inmunohistoquímica , Mucosa Intestinal/citología , Mucosa Intestinal/patología , Lectinas/biosíntesis , Lectinas/genética , Datos de Secuencia Molecular , Peso Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa/métodos , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificaciónRESUMEN
The interaction of tumor cells with laminin is thought to be critical in invasion and metastasis. We found that an endogenous lectin, carbohydrate-binding protein 35 (CBP-35), is the major laminin-binding protein on human colon carcinoma cells and that its surface expression suggests involvement in metastasis. We identified CBP-35 by laminin-affinity chromatography and immunoblotting. Surface expression of CBP-35 on eight human colon carcinoma cell lines was compared by flow cytometry. Poorly differentiated cell lines and DLD-2, a signet-ring carcinoma cell line, expressed more surface CBP-35 than well-differentiated cell lines. Poorly differentiated cell lines and DLD-2 are characterized as aggressive cell lines because they adhere to and invade through reconstituted basement membrane significantly better than well-differentiated cell lines. These data suggest that CBP-35 is involved in tumor cell-basement membrane interactions and that an increase in CBP-35 surface expression may facilitate metastatic potential of colon carcinoma cells.
Asunto(s)
Antígenos de Diferenciación/análisis , Carcinoma/química , Proteínas Portadoras/análisis , Neoplasias del Colon/química , Hemaglutininas/análisis , Proteínas de la Membrana/análisis , Proteínas de Neoplasias/análisis , Cromatografía de Afinidad , Citometría de Flujo , Galectina 3 , Humanos , Immunoblotting , Células Tumorales CultivadasRESUMEN
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 beta 1 subunit inhibited clone A adhesion to laminin. They also precipitated a complex of surface proteins that exhibited an electrophoretic behavior characteristic of alpha 2 beta 1 and alpha 3 beta 1. A monoclonal antibody specific for alpha 2 (PIH5) blocked clone A adhesion to laminin, as well as to collagen I. An alpha 3-specific antibody (P1B5) had no effect on clone A adhesion to laminin, even though it can block the adhesion of other cell types to laminin. Thus, the alpha 2 beta 1 integrin can function as both a laminin and collagen I receptor on clone A cells. Although these cells express alpha 3 beta 1, an established laminin receptor, they do not appear to use it to mediate laminin adhesion. In addition, the monoclonal antibody GoH3, which recognizes the alpha 6 integrin subunit, also inhibited carcinoma adhesion to laminin but not to fibronectin or collagen I. This antibody precipitated the alpha 6 subunit in association with the beta 4 subunit. There was no evidence of alpha 6 beta 1 association on these cells. In summary, the results obtained in this study indicate that multiple integrin alpha subunits, in association with two distinct beta subunits, are involved in colon carcinoma adhesion to laminin. Based on the behavior of alpha 3 beta 1 and alpha 2 beta 1, the results also suggest that cells can regulate the ability of a specific integrin to mediate adhesion.