Tumor promoters induce a specific morphological signature in the nuclear matrix-intermediate filament scaffold of Madin-Darby canine kidney (MDCK) cell colonies

Abstract

Tumor promoters such as phorbol 12-tetradecanoate 13-acetate (TPA), mezerein, teleocidin, aplysiatoxin, and benzoyl peroxide, although structurally unrelated, induce similar, profound changes in morphology in differentiated epithelial Madin-Darby canine kidney (MDCK) cell colonies. The alteration is evident in the organization of intermediate filaments in intact cells and in whole mounts of the nuclear matrix-intermediate filament (NM-IF) scaffold of the epithelial sheet. This substructure, obtained by salt extraction of the cytoskeletal framework, represents only 5% of the total cell protein but contains all of the intermediate filaments, nuclear matrix, and desmosomal core proteins arranged essentially as in the intact cell. The NM-IF is profoundly reorganized after exposure to TPA and retains the morphological changes observed in intact cells. These include bundling of the intermediate filaments, disruption of cell-cell borders, and marked deformation of the polygonal geometry of epithelia. Thus, TPA and all other complete or second-stage tumor promoters examined have a characteristic morphological signature that is not induced by mitogens, metabolic inhibitors, or agents known to disrupt microtubules or microfilaments. This signature, characteristic of tumor promoters, occurs in the absence of both protein and RNA synthesis. These results suggest that this response is prior to and independent of other biochemical markers for tumor promoters. Of the major filament systems, the cytokeratin network is implicated as an early or possibly primary site of tumor-promoter action because characteristics of the promoted cytoskeletal signature are observed in epithelial colonies after prior exposure to colchicine or cytochalasin D. Despite the massive reorganization of cytoskeletal morphology induced by TPA, the distribution of prelabeled proteins into structural fractions (i.e., cytoskeletal, chromatin, and the NM-IF) remains essentially unchanged. The sensitivity and specificity of the epithelial cell response suggest its possible use as a screen for promoting compounds.