Characterization of human serum spreading factor with monoclonal antibody.

Abstract

Serum spreading factor is a glycoprotein isolated from human serum that promotes spreading of a variety of cell types on culture dishes. We developed mouse hybridoma lines secreting monoclonal antibody to serum spreading factor that markedly inhibited the rate of serum spreading factor-promoted spreading of both fibroblastic and epithelial cells in culture. Fibronectin-promoted cell spreading was unaffected by monoclonal antibody to serum spreading factor, and the factor appeared to be distinct by several criteria from fibronectin or laminin. Human serum-promoted cell spreading was partially inhibited by monoclonal antibody to serum spreading factor. The antibody recognized primarily two forms of serum spreading factor that migrated in NaDodSO4/polyacrylamide gel electrophoresis in a manner consistent with molecular weights of 65,000-70,000 and 75,000-78,000. In addition to being found in plasma, serum spreading factor was also found associated with washed human platelets.Among the functions that serum serves for cells in culture is the provision of factors that allow proper attachment and spreading of cells on the plastic or glass surface of the culture vessel (1). One such factor is fibronectin; forms of this cell spreading-promoting protein are found in plasma and basal lamina and on cell surfaces (2). Barnes and co-workers have described another cell spreading-promoting glycoprotein, which has been termed serum spreading factor (3-10). This activity was first reported by Holmes to exist in a preparation isolated from human serum by glass bead column chromatography (11). In addition to acting in serum-free cell culture in a manner similar to fibronectin on a variety of cell types, preparations of human serum spreading factor are also capable of mediating effects that cannot be duplicated by fibronectin on the growth, morphology, and differentiative capacity of some cell types (3-6). To better define the nature of the activity in the serum spreading factor preparations, we derived mouse hybridomas secreting monoclonal antibody to human serum spreading factor. Here, we report the isolation of the hybridoma lines and describe studies using monoclonal antibody for the characterization of serum spreading factor. MATERIALS AND METHODSCell Culture. Stock cultures of WI-38 and MCF-7 cells were maintained in a 1:1 mixture of Ham's F12 and Dulbecco's modified Eagle's (DME) media supplemented with sodium bicarbonate at 1.2 g/liter, 10 mM Hepes (pH 7.4), and antibiotics (F12:DME medium)/10% fetal calf serum. P3-X63-AG8 mouse plasmacytoma cells were maintained in DME medium supplemented with bicarbonate and antibiotics, 1 mM sodium pyruvate, 0.1 mM 8-azaguanine, and 10% horse serum. Hybridomas were derived as described (12). For the experiments in Figs. 1, 2, and 3, cell culture dishes (35-mm diameter) were exposed prior to plating cells to the following series of 1-hr room temperature incubations (1 ml per plate), washing twice with phosphate-buffered saline (Pi/NaCI) after each: (i) serum spreading factor prepa...

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