EFFECT OF TUMOR-PROMOTING STIMULI ON GAP JUNCTION PERMEABILITY AND CONNEXIN43 EXPRESSION IN ARL-18 RAT-LIVER CELL-LINE

The ARL 18 rat liver cell line has previously been used for screening tumor promoters in the metabolic cooperation assay (Williams 1980; Wil liams et al. 1981; Telang et al. 1982). These cells display high level s of gap junctional communication, as assessed functionally and immuno logically. Intracellularly injected Lucifer Yellow diffused extensivel y and there was rapid fluorescent recovery after photobleaching. Moreo ver, expression of connexin43 (Cx43) was high as evaluated by immunocy tochemisty of cell monolayers and Western blot analysis of total cell homogenates. Western blot analysis revealed multiple forms of Cx43, wh ich presumably correspond to known dephosphorylated and phosphorylated states of this protein. Gap junction permeability and Cx43 expression in ARL 18 cells were studied after exposure to the tumor promoters 12 -0-tetradecanoyl-phorbol-13-acetate (TPA), and 1,1,1-trichloro-2,2-bis (p-chlorphenyl)-ethane (DDT), and after wounding the cell monolayer. T PA and DDT strongly inhibited gap junction permeability; whereas monol ayer wounding did not affect the degree of fluorescent recovery after injury, either in the cells on the edge of the wound or in distal regi ons. No changes in the cellular distribution of Cx43 were observed aft er any of these treatments, although Western blots revealed a decrease in total Cx43 after 24-h exposure to DDT (10 mug/ml) and a slight inc rease after TPA treatment (30 min, 0.1 mug/ml). Relative abundance of different phosphorylated Cx43 forms was increased after 1 h exposure t o DDT (10 mug) and 30 min exposure to TPA (0.1 mug/ml). Thus, various changes in Cx43 are correlated with gap junction closure induced by tu mor promoters including changes in the total amount of Cx43 and increa se in the proportion of Cx43 that is in phosphorylated forms. The poss ible role that connexin phosphorylation might play in gap junction reg ulation is discussed.