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Proc. Nati Acad. Sci. USA Vol. 80, pp. 1362-1366, March 1983 Cell Biology Characterization of human serum spreading factor with monoclonal antibody (cell adhesion/WI-38 fibroblasts/MCF-7 breast carcinoma/platelet/plasma) D. W. BARNES*, J. SILNUTZER*, C. SEEt, AND M. SHAFFER* *Department of Biological Sciences and tDepartment of Psychology, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 Communicated by Sidney P. Colowick, November 22, 1982 ABSTRACT Serum spreading factor is a glycoprotein isolated prior to plating cells to the following series of 1-hr room tem- from human serum that promotes spreading of a variety of cell perature incubations (1 ml per plate), washing twice with phos- types on culture dishes. We developed mouse hybridoma lines se- phate-buffered saline (Pi/NaCI) after each: (i) serum spreading creting monoclonal antibody to serum spreading factor that mark- factor preparation or fibronectin (Bethesda Research Labora- edly inhibited the rate of serum spreading factor-promoted tories) at 4 Ag/ml in Pi/NaCl, (ii) bovine serum albumin at 1 spreading of both fibroblastic and epithelial cells in culture. Fi- mg/ml in Pi/NaCl, (iii) monoclonal antibody to serum spread- bronectin-promoted cell spreading was unaffected by monoclonal ing factor or nonspecific mouse IgG at 15 Atg/ml in Pi/NaCl. antibody to serum spreading factor, and the factor appeared to be For the experiment of Fig. 8, the pretreatment of plates with distinct by several criteria from fibronectin or laminin. Human serum-promoted cell spreading was partially inhibited by mono- human serum (1% in F12:DME medium) was carried out for 20 clonal antibody to serum spreading factor. The antibody recog- hr at 370C. Cell spreading was scored 1.5 hr after plating cells nizedprimarily two forms of serum spreading factor thatmigrated onto pretreated dishes in F12:DME medium. Incubation with in NaDodSO4/polyacrylamide gel electrophoresis in a manner bovine serum albumin after the initial incubation of the dishes consistent with molecular weights of 65,000-70,000 and 75,000- with spreading factor was necessary to prevent nonspecific ad- 78,000. In addition to being found in plasma, serum spreading fac- sorption of mouse IgG to the plastic culture dish and subse- tor was also found associated with washed human platelets. quent inhibition of cell spreading unrelated to a specific inter- action of serum spreading factor with monoclonal antibody. The Among the functions that serum serves for cells in culture is the bovine serum albumin treatment was not, however, essential to provision offactors that allow proper attachment and spreading show that serum spreading factor promoted cell spreading; ob- of cells on the plastic or glass surface of the culture vessel (1). vious spreading-promoting activitywas seen within 15 min after One such factor is fibronectin; forms of this cell spreading-pro- plating cells onto dishes pretreated with serum spreading factor moting protein are found in plasma and basal lamina and on cell only, while the rate of spreading of cells plated onto dishes re- surfaces (2). Barnes and co-workers have described another cell ceiving no pretreatment of any kind was considerably slower. spreading-promoting glycoprotein, whichhas been termed serum Preparation of Serum Spreading Factor, Monoclonal An- spreadingfactor (3-10). This activity was first reported by Holmes tibody, and Blood Cell Extracts. Serum spreading factor was to exist in a preparation isolated from human serum by glass bead partially purifiedfrom human serum by glass bead column chro- column chromatography (11). In addition to actingin serum-free matography (10) and concanavalin A-Sepharose affinity chro- cell culture in a manner similar to fibronectin on a variety ofcell matography. Monoclonal antibodywas isolated from serum-free types, preparations of human serum spreading factor are also conditioned medium from hybridoma cultures by protein A- capable of mediating effects that cannot be duplicated by fi- Sepharose affinity chromatography. For the preparation of bronectin on the growth, morphology, and differentiative ca- platelet extracts, platelets were centrifuged from outdated pacity of some cell types (3-6). To better define the nature of platelet-rich plasma and the platelet-poor plasma supernatant the activity in the serum spreading factor preparations, we de- was removed and assayed for serum spreading factor in the ex- rived mouse hybridomas secreting monoclonal antibody to hu- periment of Fig. 9. The pellet of platelets was washed twice man serum spreading factor. Here, we report the isolation of with Pi/NaCl/l mM EDTA, suspended in a small volume ofthe the hybridoma lines and describe studies using monoclonal an- same buffer, and lysed by freeze-thaw, and the lysate was cen- tibody for the characterization of serum spreading factor. trifuged at 100,000 X g for 1 hr. The supernatant from this cen- trifugation was assayed for serum spreading factor in the ex- MATERIALS AND METHODS periment of Fig. 9. Erythrocyte extract was prepared in an identical manner. Extracts of fresh platelets contained specific Cell Culture. Stock cultures of WI-38 and MCF-7 cells were anti-serum spreading factor binding activity comparable with maintained in a 1:1 mixture of Ham's F12 and Dulbecco's mod- that found in the experiment of Fig. 9. ified Eagle's (DME) media supplemented with sodium bicar- Enzyme-Linked Immunosorbent Assay (ELISA). The pro- bonate at 1.2 g/liter, 10 mM Hepes (pH 7.4), and antibiotics cedures are based on techniques described previously (13-15). (F12:DME medium)/10% fetal calf serum. P3-X63-AG8 mouse Immunochemicals were obtainedfrom Bethesda Research Lab- plasmacytoma cells were maintained in DME medium supple- oratories and Cappel Laboratories. For the experiments of Figs. mented with bicarbonate and antibiotics, 1 mM sodium pyru- 5 and 6, 96-well microtiter dishes containing antigen prepara- vate, 0.1 mM 8-azaguanine, and 10% horse serum. Hybridomas were derived as described (12). For the experiments in Figs. 1, Abbreviations: ELISA, enzyme-linked immunosorbent assay; DME 2, and 3, cell culture dishes (35-mm diameter) were exposed medium, Dulbecco's modified Eagle's medium; F12:DME medium, 1:1 mixture of Ham's F12 and DME media supplemented with Hepes The publication costs ofthis article were defrayed in part by page charge and antibiotics; Inh-Mcl, monoclonal antibody to human serum spread- payment. This article must therefore be hereby marked "advertise- ing factor secreted by the Inh-Hyl hybridoma cell line; Pi/NaCl, phos- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. phate-buffered saline. 1362 Downloaded by guest on September 27, 2021 Cell Biol6gy: Bames et al. Proc. Natl. Acad. Sci. USA 80 (1983) 1363 tions in Pi/NaCl (100 /.d per well) at the concentrations nec- essary to give the indicated amount of antigen preparation per well were incubated overnight. Monoclonal antibody was used at a final concentration.of 10 /.g/ml in Pi/NaCl containing bo- vine serum albumin at 0.5 mg/ml. Rabbit antisera-were used at a final dilution of 1:200 in Pi/NaC1. Peroxidase-conjugated goat anti-rabbit IgG and anti-mouse IgG were used at a 1:1,000 dilution in P'/NaCl. Antibody incubations were 1 hr at room temperature. 2,2'-Azino-i-(3-ethylbenzthiazelinesulfonic acid) was the peroxidase-dependent chromogen. The reaction was in- hibited after 15 min, contents of the wells were diluted with water, and the extent of reaction was determined by measuring the absorbance at 415 nm. The experiment of Fig. 7 was carried out in a similar manner, except that 35-mm-diameter cell cul- ture plates and appropriately larger volumes of solutions were used. For the experiment of Fig. 9, the appropriate samples were diluted with containing bovine serum albumin FIG. 1. Inhibition by Inh-Mcl of serum spreading factor-promoted Pi/NaCl at spreading.of WI-38 fibroblasts. Cells were photographed 1.5 hr after 1 mg/ml to give the. indicated final concentrations of sample. plating onto dishes previously treated with the following: Pi/NaCl (A), These dilutions (100 1.l) were incubated for 20 hr in microtiter serum spreading factor at 4 pg/mi (B), serum spreading factor (4 pg/ml) wells with 100 ,1u of P1/NaCl containing bovine serum albumin followedbyInh-Mcl at 15 pHg/ml (C), or serum spreadingfactor(4 /Ag/ml) at 1 mg/mland monoclonal antibody at 0.5 pug/ml. The con- followed by nonspecific mouse IgG.at 15 jg/ml (D). (x 120.) tents of these wells were transferred to other wells previously saturated with serum spreading factor, and the mixtures were directly to the medium into which the cells are plated (5). Pre- incubated for 1 hr at room temperature. The contents of the wells vious treatment of plates with serum spreading factor resulted were removed and the amount of antibody bound to the serum in rapid spreading of WI-38 cells subsequently seeded on these spreading factor-coated wells was determined as described plates; cell spreading was decreased on serum spreading factor- above. Immunoperoxidase localization of monoclonal antibody treated plates exposed to an additional pretreatment with Inh- to serum spreading factor on nitrocellulose blots (immunoblots) Mcl but not on serum spreading factor-treated plates exposed, of 7.5% polyacrylamide gels after NaDodSO4/polyacrylamide instead, to an additional pretreatment with an equal amount of gel electrophoresis was carried out by adaptation of the pro- nonspecific mouse IgG (Figs. 1 and 2). Although the rate of cell cedure of Towbin (13), using. 3-amino-O-ethylcarbazole as the spreading in both control and Inh-Mcl-treated plates was mark- peroxidase-dependent chromogen. edly reduced compared with the rate of spreading in serum spreading factor-treated plates, the cells spread eventually un- RESULTS der all conditions.

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