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...

Four synthetic peptides related in amino acid sequence to the area of the fibronectin molecule thought to be involved in cell binding were tested for the ability to inhibit cell spreading promoted by several different attachment factors. Effects of the peptides were assayed on a number of cell types. For all cell types tested, peptides effective at inhibiting fibronectin-promoted cell spreading were found also to inhibit cell spreading promoted by human serum spreading factor (SF). These peptides were also capable of inhibiting to a lesser extent cell spreading promoted by other attachment factors, although this phenomenon was dependent on the cell type examined.

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NIH3T3 cells grow in a serum-free basal nutrient medium supplemented with fibronectin, transferrin, insulin, epidermal growth factor (EGF) and high density lipoprotein (HDL). The individual omission from the serum-free medium of insulin, EGF, or HDL results in greatly reduced cell growth. These growth-restrictive conditions can be used to select for cells transformed with SV40, the polyomavirus middle T antigen gene, the activated human ras gene, and the mouse c-myc gene.