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Heritable Membrane Alterations and Growth Associated with Enhanced Leupeptin-sensitive Proteinase Activity in Epithelial Cells Exposed to Dibutylnitrosamine in Vitro¹

Department of Biology, University of California, Los Angeles, California 90024

Viable epithelial cells derived from bullfrog or rabbit urinary bladders were treated in vitro with dibutylnitrosamine (DBN) in an effort to identify early and long-term changes in membrane properties during chemical-induced growth. At 5 x 10^-4 M, the carcinogen provoked maximal increments in [³H]thymidine incorporation and growth in cells maintained in serum-free, chemically defined medium for 14 days as compared to control cells not exposed to DBN or treated with the noncarcinogen diphenylnitrosamine. These DBN-induced effects were abolished by prior treatment of cells with liposome-entrapped leupeptin and reduced by ovomucoid, but not by soybean trypsin inhibitor. Extracellular release of cathepsin B1, but not alkaline phosphatase, activity was enhanced within 1 hr of DBN exposure to 280% of that of control cells. Concanavalin A-mediated binding of erythrocytes, but not concanavalin A binding, to bullfrog cells increased to 194% of the level in cells treated for 1 hr with diphenylnitrosamine. Moreover, fluorescence microscopy revealed that 20% of cells exposed to DBN showed a redistribution into clusters of concanavalin A-binding sites that are normally disposed at random at the external surfaces of control cells. The rate of intercellular adhesion was also enhanced among cells treated with DBN, but not diphenylnitrosamine. These carcinogen-induced membrane alterations were diminished by treatment with cathepsin B1 inhibitors and were propagated during 14-day culture after the initial exposure to DBN. Fractionation of cells by selection for enhanced adhesiveness due to DBN exposure for 30 min elicited a rapidly dividing cell fraction very active in cathepsin B1 secretion as compared to their less adhesive counterparts. These results indicate that heritable membrane alterations and enhanced cell growth induced by DBN treatment are associated with increases in the activity of a leupeptin-sensitive proteinase at the external cell surface.

¹ This investigation was aided by Postdoctoral Fellowship CA 5176 (USPHS), general funds of the UCLA Molecular Biology Institute, and a grant from the Eli Lilly Research Laboratories.

Received 8/22/77. Accepted 1/18/78.