ALTERATIONS IN THE LOCALIZATION OF F-ACTIN, FIBRONECTIN, AND THROMBOSPONDIN OCCUR PRIOR TO NEOPLASTIC TRANSFORMATION IN RAT TRACHEAL EPITHELIAL-CELLS

Structural glycoproteins and cytoskeletal proteins play a major role i n the regulation of cellular organization and function. Changes in the structure and function of these proteins are involved in the cascade of events which lead to neoplastic transformation. We evaluated RNA le vels, protein localization, and organization of selected proteins in a n in vitro model system of respiratory carcinogenesis to examine alter ations in cell architecture. Localization of fibronectin (Fn), thrombo spondin (Tsp), and F-actin was examined in (1) primary rat tracheal ep ithelial (RTE) cells; (2) spontaneously immortalized nonneoplastic cel ls (SPOC-1); and (3) neoplastic cells (EGV5T) derived from tumors aris ing following transplantation of an N-methyl-N-'nitro-N-nitrosoguanidi ne-transformed RTE cell line into nude mice. Proteins were stained wit h fluorescein-labeled antibodies or phalloidin compound and analysis w as performed with a confocal laser scanning microscope. Primary RTE ce lls display organized F-actin stress fibers, perinuclear Fn and Tsp, a nd pericellular Fn in fibrillar arrays. In larger colonies, Tsp occurs between cells and occasionally in fibrillar arrays. SPOC-1 cells, unl ike primary RTE cells and neoplastic EGV5T cells, seldom form junction s and exhibit few cell surface extensions. F-actin stress fibers are r educed in these immortalized cells. F-actin in SPOC-1 cells occurs in the perinuclear region, scattered diffusely throughout the cell and in punctate adhesions. Fn and Tsp are localized to the perinuclear regio n with Fn staining more intensely. EGV5T neoplastic cells also display a dramatic loss of stress fibers and F-actin is concentrated mainly n ear the cell periphery. Perinuclear staining of Fn and Tsp occurs in s ome cells within the colony. Levels of Tsp RNA and Fn RNA and protein are significantly reduced in both cell lines compared to primary RTE c ells. We conclude that structural protein disruptions are early events in the transformation of these respiratory epithelial cells. (C) 1994 Academic Press, Inc.