PERSISTENT EFFECTS OF PHORBOL 12-MYRISTATE 13-ACETATE - POSSIBLE IMPLICATION OF VESICLE TRAFFIC

Relative to their normal counterparts, transformed epithelial cells ha ve a distinctive and quantifiable three-dimensional shape. Biophysical and mathematical methods are used to distinguish these extremes in ce lls from two lines, cultured from rat liver and tracheal epithelium, r espectively. Cells adopted a more transformed-looking configuration tr ansiently when exposed to phorbol 12-myristate 13-acetate (PMA) (Plumm er and Heckman, [1990] Exp. Cell Res., 188:66-74). The purpose of the present work was to dissect the physiological processes involved in th e shape change. Ruffling activity, known to be PMA-stimulated in other cells, was investigated. Although the ruffles appeared less robust th an normal, PMA stimulated ruffling activity over a 5 h period. The num ber of sites where ruffling was initiated declined by 5 h, however, an d suppression was seen by 10 h. Cells from both lines adopted the tran sformed shape configuration when exposed for 2 h to monensin. When the subset of shape features changed by this treatment was compared with those originally changed during transformation, it was found that mone nsin-treated cells mimicked the features of transformed cells. Its eff ect on ruffling was, however, unlike PMA's. Thus, the phenotype was un likely to arise from ruffling itself but might be a process driven by ruffling. Chloroquine also stimulated cells to assume characteristics of transformed cells. Since both it and monensin could interfere with endosomes and with the processing of endocytosed contents, this was a likely site of action. Experiments were done to determine whether PMA also affected the processing of extracellular fluids. When the accumul ation of horseradish peroxidase (HRP) was measured, the rate was found to be higher in PMA-treated cells from 5 min, the earliest time assay ed, onward. The results suggest that the transformed type of cell in t hese cell lines showed a constitutive dilation and/or reorganization o f some portion of the endosomal pathway. (C) 1996 Wiley-Liss, Inc.