Cell surface proteins of normal human, mouse, and rat cells in primary culture, of human basal cell carcinoma, and of carcinogen-transformed cell lines were examined by lactoperoxidase-catalyzed iodination. Autoradiography was used to record the distribution of label in the polypeptide subunits separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. There was no significant difference in the results for normal cells of human, mouse, and rat. On the other hand,

more »

... ed mouse cells had many more labeled polyp eptide bands of widely distributed molecular weights. The iodination profiles from human basal cell carcinoma cells were much more akin to those from normal cells than to those from carcinogen-transformed cells. Treatment of iodinated cells with proteolytic enzymes visibly altered the polypeptide bands. Cell membranes are associated with m a n y cell functions, such as cell recognition, adhesion, and antigenicity, which ofte n becom e altered in n eoplastic cells . Lactoperoxidase-cat alyzed iodinatio n h as been a m ajor tool for studying cell s urface proteins [1, 2] . This technique selectively labels with 125 1 the exposeCl tyrosine r esidues of membrane proteins . The high molecular weight of lactoperoxidase preve nts its pe netration through the cell membrane, so that only t h e external su1·face of t h e cell is labeled . Iodination h as no t h eretofore been applied to epidermal cells. Surface ch a nges in the epidermis have been s tudied by s p ecific lectin binding [3, 4] and incorporation of labeled sugars [5,6]; th ese m ethods, howevet, label cell constitutents different from those affected b y iodination. This pa per describes iodination exp erime nts with normal human, mouse, and rat epidermal cells, with human basal cell carcinomas, and with carcinogentransformed mouse e pidermal cells [7, 8] . MATER IALS AND METHODS Culture Procedures T issue cul tw·e media, trypsin (1:250), penicillin (100 IU / ml), stre ptomycin (100 f.Lg / ml), a nd salts were purchased from GIBCO (Grand Isla nd, N.Y.). Feta l bovine serum (FBS) was obtained from Flow Laboratories (Rockville, Md.). Newborn animals used for the preparation of primary rodent cult ures were Sprague-Dawley rats a nd Swiss-Webster mi ce. Normal adul t human skin was excised from surgical specimens with a der!l' atome. Epidermis was separated from dermis by the trypsin fl otation procedure [9] which consists of overnight incuba-Manuscrip t buffered saline FBS: feta l bovine serum PMSF: phenylmethylsulfonylfluoride SDS-PAGE: sodium dodecyl sulfate polyacrylam ide ge l electrophoresis tion with 0. 25% trypsin at 4 °C. Separated epidermal fragments were fine ly minced, then dissocia ted by stirring for 1 hr in Waymouth tissue culture medium containing 10% FBS. Dissociated cells were separated from stratum co rneum by filtration through a 125 f.Lm nylon mesh, plated on co llagen-coated plates [10] and grown in a Way mouth medium as modified by Marchok [ll] with antibiotics added. Cells we1·e mainta ined at 37° in a humidified 4% co" atmosphere. Biopsy sampl es of human basal cell ca1·cinomas were exposed to 0.25% trypsin for 16 hr at 4°, minced, then grown in the same fas hion as norma l epidermal cells on collagen-coated plates. Carcinogen-transformed cell lines of mouse epidermal cells (JB-8, Dlla a nd T -6272 ), generously supplied by Dr. Na ncy H . Colburn of NIH, were maintained on Corning plastic tissue culture vessels in Minimal Essential Medium containing 10% FBS, nonessential amino acids, and antibio tics. Lactoperoxidase Labeling Cells growing in tissue culture vessels, at 75-100% co nflu ency, were washed 3 times with Dulbecco's phosphate buffered saline (DPBS) , pH 7.2, then iodinated in a 1 ml mixtUl'e of DPBS containing 60 f.lg lactoperoxidase (Worthington, Millipore Corp., Free hold, N.J.) , 55 mU glu cose oxidase (Worthington), 5 mM glu cose, and 150 f.LCi carrier-free N a 12 " I (N ew England Nuclear, Boston, M ass.) . The reaction vessel were gently swirled for 10 min at room temperature, after which the cells were washed 3 times wit h DPBS containing 5 mM Nal. Then the cells were scraped in to 0.1 ml DPBS containing 2 mM phenylmethy lsulfonylfluoride (PMSF) at 4°, sonicated for 10 sec, and mixed with 0.1 ml sodium dodecyl sulfate (SDS) sample buffer (0.125 M Tris HCl, pH 6.8; 20% glyce rol; 4% SDS; 10% ,8-mercaptoethanol; 2 mM PMSF; 0.004% bromphenol blue). For an investigation of the impact of proteolytic enzy mes, cells were iodinated, swirled a nd washed as above. At this poin t, the cells were reacted with dispase, 100 f.Lg / ml (Sigma Chemical Co., St. Louis, Mo.), or trypsin, 10 f.Lg/ml (Sigma), for 10 min at room temperature, then washed 3 times with DPBS co ntaining 2 mM PMSF. Thereafter, the above procedure was resumed, starting with sc raping the cells off the reaction vessels. 24 Each iodination ex perim ent was carried out three or more times, except for the basal cell carcinoma which was done tw ice (with different tumor samples). Electrophoresis and Autoradiog raphy Samples in SDS buffer were heated at 100° for 3 min. Proteins were se parated by the method of Laemmli [12] on 1.5 mm vertical sla bs of 7.5% polyacrylamide below a 3% stacking ge l. E lectrophoresis was for 3 hr at 30 mA per slab (at constant current). Gels were stained for 16 hr in 0.1% Coomassie Blu e in 50% methanol, 10% acetic ac id, then destained in the same solvents without the dye and dried on Whatman 3 MM paper in a Biorad ge l drier. Sheets of Kodak SB-5 film were exposed to the ge ls for 3 days at -70°. Molecular weight calibration was achieved with the following standards: myosin (200,000 daltons), phosphorylase B (92,500), bovine se rum albumin (68,000) , ovalbumin (46,000) , and carb onic anhydrase (30,000). RESULTS Normal Epidermal Cells Collage n-coated plates were r e quired for t h e growth of human epidermal cells and were often also used for rodent cells to improve cell attac hment during initial plating. S ince collagen is composed of protein, it can be expected to become labeled during iodination. To separate out the contribution of the collagen, controls we re run with collage n-coated plates devoid