CHRONIC EXPOSURE OF LLC-PK1 EPITHELIA TO THE PHORBOL ESTER TPA PRODUCES POLYP-LIKE FOCI WITH LEAKY TIGHT JUNCTIONS AND ALTERED PROTEIN-KINASE C-ALPHA EXPRESSION AND LOCALIZATION

Acute exposure (up to 4 h) of LLC-PK1 epithelial cell sheets to the ph orbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) causes a rapid decrease of transepithelial electrical resistance (TER) to less than 1 5% of initial value, As the TPA exposure period is continued by chroni cally passaging cells in the presence of TPA, TER begins to recover, B y 6 weeks of exposure, TER recovers to almost 50% of its initial value , suggesting that tight junctions (TJs) are recovering barrier functio n even in the continued presence of TPA. Between 6 and 8 weeks, TER th en decreases a second time to approximately 20 to 40% of initial value s, and TER values remain at this level for at least 18 Reeks of exposu re, Transepithelial (paracellular) fluxes of D-mannitol inversely corr espond with TER changes, Across chronically treated cell sheets, rates are higher than those across control cell sheets, but lower than thos e across acutely treated cell sheets. The decrease of TER at 6-8 weeks coincides with the appearance of multilayered, polyp-like foci (PLFs) on the otherwise one cell layer thick epithelium, Electron microscopy shows that the electron-dense dye ruthenium red cannot penetrate acro ss TJs of control cells but passes across all of the TJs of a cell she et treated acutely with TPA, In chronically treated cultures, rutheniu m red penetrates TJs between most cells of PLFs, but not TJs of adjace nt morphologically normal epithelium. A clonal subline derived from ce lls of a PLF (clone PLF-A) is multilayered almost throughout and exhib its ruthenium red penetration across nearly all of its tight junctions , monolayer or multilayer, Acute exposure of control cell sheets to TP A induces activation, translocation, and down-regulation of protein ki nase C-alpha (PKC-alpha), In chronically TPA-treated and clone PLF-A c ells, total PKC-alpha levels are reduced even further and almost all r emaining PKC-alpha is found in the membrane-associated and Triton-inso luble fractions, Immunofluorescence shows that PKC-alpha expression is re stricted to the PLFs in chronically TPA-treated cells and is more homogeneously distributed in clone PLF-A cultures, In summary, the dat a show that chronic treatment of epithelial cells with a tumor promote r induces the formation of abnormal cell architecture (PLFs) associate d with increased leakiness of TJs and membrane translocation of PKC-al pha. Recovery of barrier function in portions of chronically TPA-treat ed cultures does not correlate with up regulation of PKC-alpha nor tra nslocation back to the cytosolic compartment. (C) 1996 Academic Press, Inc.