Difference in Laminin Expression Between High and Low Metastatic Cell Clones Derived from Murine Lewis Lung Carcinoma.

Abstract

ABSTRACT. Weinvestigated the expression of laminin in two cell lines with different metastatic potentials established from murine Lewis lung carcinoma. Immunostaining of the cells with anti-laminin antibody and Northern blot analysis of laminin mRNA demonstrated that the high metastatic clone expressed less laminin than the low metastatic one. In contrast, expressions of 67 kDa-laminin receptor were at similar levels between these two lines. These findings showthe possibility that endogenous laminin may contribute to the difference in metastatic properties in the murine Lewis lung carcinoma cell lines examined.Laminin (LN), a high-molecular-weight glycoprotein (Mr= 850,000-1 ,000,000), is a major component of basement membraneand possesses binding sites in the molecule to cell surface receptors, type IV collagen and heparin (1). Laminin has been shown to play important roles in various cellular activities such as adhesion, spreading, migration, differentiation, neurite outgrowth and tumor cell metastasis. In a metastatic process, attachment of malignant cells to LNof basement membranes is thought to initiate a cascade of invasion and metastasis (3). In some cell lines, e.g., murine BL6 from B16 melanoma cell and PM2from PMTfibrosarcoma cell, it has been shown that LNpromotes attachment of metastatic tumor cell to type IV collagen (14). However, in murine macrophage-derived M5706 cell (2), murine glioma cell (4) and human squamous carcinoma cell (16), LNinhibits the attachment of the cells to type IV collagen.In an experiment of chemotaxis using a modified Boyden chamber, exogenous soluble LN stimulates the directional migration of B16 Fl and B16 F10 in a dosedependent manner (7). However, the function of soluble LNwhich may be autocrined by the cell has not been fully elucidated yet. Recently two cell lines with different metastatic potentials have been established from murine Lewis lung carcinoma (3LL) cells (8). LM60-D6is a cell line with high metastatic potential and P29 is one with low metastatic potential. In this study, using these cell lines we examined the levels of expression of endogenous LNtogether with 67 kDa-laminin receptor (67 kDa-LNR) (17) and fibronectin (FN) by immunostaining and Northern blot analysis. As a result, it was shown that the expression level of LN was markedly decreased in the highly metastatic cell line as compared with that in the low metastatic one. In contrast to LN, no significant difference was found in the levels of 67 kDa-LNR. MATERIALS AND METHODSCells. Two cell lines LM60-D6 and P29 were established from mouse 3LL as described previously (8). The cells were cultured in Eagle's minimal essential medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin G, 100 ptg/vnl streptomycin and maintained at 37°C in 5% CO2. Murine teratocarcinoma cell line F9 wasused as the positive control in someexperiments, since F9 differentiates into endodermlike form and synthesizes a large amount of LN (13). Hereafter in this paper, differentiating F9 (dF9) means the F9 cells cultur...