Metastatic potential severely altered by changes in tumor cell adhesiveness and cell-surface sialylation.

Abstract

It has been shown recently that high metastatic tumor lines can be converted to low metastatic ones by mutagenization and selection for resistance to toxic concentrations of the lectin wheat germ agglutinin (1-3). Lectin-resistant variants were found to differ from the parental lines and respective revertants in terminal cell-surface carbohydrates. Here we describe for the first time a drug-independent selection procedure for the isolation of a low metastatic variant type cell from a high metastatic tumor line. The method is based on the selection of plastic-adherent cells from a suspension culture of highly malignant cells. Similar to the above situation, plasticadherent low-metastatic variant cells were found to differ from the parental cells and from metastatic revertants in terminal cell surface carbohydrates. The importance of terminal carbohydrates, in particular of sialic acid, for cell-surface adhesiveness and metastatic potential will be discussed. Materials and MethodsTumor Lines. Eb is the Heidelberg subline of the methylcholanthrene A-induced T cell lymphoma L5178Y of DBA/2 mice and ESb is a spontaneous high metastatic variant thereof (4). Details of these cells have been described elsewhere (for a review see ref. 5).Selection of a Plastic-adherent Variant. Both tumor lines Eb and ESb grow in suspension culture (6). A strongly plastic-adherent variant (ESb-M) was isolated from the ESb cells by continuously selecting the adherent subpopulation from the suspension cultures over a period of 2 mo and >20 selection steps. ESb-M cells which were released into suspension from monolayer cultures grown to confluency were taken for analysis.Flow Cytofluorography. Immunofluorescent staining was carried out by incubating 2 × 106 cells with appropriately diluted fluorescein isothiocyanate-(FITC) conjugated lectins for 45 rain on ice. The reaction was carried out in saline containing 1 mM Mg and Ca salts. Flow cytofluorographic analysis was performed on an Ortho H-50 cell sorter (Ortho Instruments, Westwood, MA). Fluorescence gains were kept at constant setting for each lectin analyzed. Cell surface differentiation antigens were determined by indirect immunofluorescenceand cytofluorography as described (7).Isolation of Tumor Cells from Organs. Spleen and brain of ESb-M tumor bearing mice were removed 40 d after tumor cell inoculation and pressed through a fine nylon mesh. The cells were suspended and cultured in the appropriate tissue culture medium (4). ResultsWhen inoculated into syngeneic DBA/2 mice, the plastic-adherent ESb-M variant type cells were found to be tumorigenic but not metastatic. The survival curves of * Supported as a guest by the Deutsches Krebsforschungszentrum.Supported by a grant from the Stiftung Meyenburg.J. Exe. MED.

Mobilization of sialidase from intracellular stores to the surface of human neutrophils and its role in stimulated adhesion responses of these cells.

“…Furthermore, there is substantial evidence that sialic acids are structural determinants for a variety of important cell-cell interactions and cellular functions, such as the adhesiveness and metastatic potential of neoplastic cells (2)(3)(4) and the phenomenon of contact inhibition in tissue culture (5).…”

Low expression of polypeptide GalNAc N-acetylgalactosaminyl transferase-3 in lung adenocarcinoma: impact on poor prognosis and early recurrence

“…In the past few years, O-linked carbohydrate antigens, such as mucin antigen (MUC1), carcinoembryonic antigen (CEA), sialyl LewisX (SLX), CA19-9, Sialyl Tn (STn), and Tn have been reported to be associated with altered adhesion (Fogel et al, 1983;Rice and Bevilacqua, 1989), invasion (Bolscher et al, 1988;Matsushita et al, 1991;Nakamori et al, 1994), recurrence, and prognosis (Itzkowitz et al, 1990;Niklinski et al, 1992;Ogawa et al, 1994;Cao et al, 1995;Diez et al, 1996;Ohgami et al, 1999) in lung cancer and other tumours. Mucin-type O-linked carbohydrates may constitute up to 80% of the total mass of these glycoproteins ; O-glycosylation has been shown to be important in the binding of cell adhesion molecules, cell differentiation, invasion, and metastasis in tumours (Nakamori et al, 1994;Clausen and Bennett, 1996;Sutherlin et al, 1997;Nomoto et al, 1999).…”

Overexpression of lysosomal-type sialidase leads to suppression of metastasis associated with reversion of malignant phenotype in murine B16 melanoma cells

“…1,2 Alterations in sialylation during malignant transformation have been observed to be closely associated with the malignant phenotype in terms of metastatic potential and invasiveness. [3][4][5][6][7] To understand how sialidases are involved in this aberrant sialylation, our study has focused on mammalian forms in cancer cells.Mammalian sialidases are classified into 3 to 4 forms based on their subcellular localization and substrate preference. Cytosolic, lysosomal and membrane forms of the sialidases have been cloned; their primary sequences revealed that they are proteins encoded by distinct genes with different major subcellular locations and enzymatic properties.…”

“…1,2 Alterations in sialylation during malignant transformation have been observed to be closely associated with the malignant phenotype in terms of metastatic potential and invasiveness. [3][4][5][6][7] To understand how sialidases are involved in this aberrant sialylation, our study has focused on mammalian forms in cancer cells.…”