ALTERATIONS IN THE GROWTH-FACTOR SIGNAL-TRANSDUCTION PATHWAYS AND MODULATORS OF THE CELL-CYCLE IN ENDOCERVICAL CELLS FROM MACAQUES EXPOSED TO TCDD

After more than a year had elapsed since a single oral exposure to 2 a nd 4 mu g 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg, there was an apparent dose-related increased incidence of significant endocervical squamous metaplasia in a group of cynomolgus macaques (Scott et al., 1 998). In the present experiments we investigated the mechanisms by whi ch chemicals like TCDD could induce epithelial cell transdifferentiati on in the primate endocervix. One focus of investigation was epidermal growth factor receptor (EGFR) and the key cytosolic signaling kinases , c-Src and protein tyrosine kinase (PTK), whose responses to TCDD are well characterized. A second focus was the distal kinase Erk2 that tr ansduces the cytosolic signal into a nuclear signal, and which in comb ination with nuclear casein kinase II (CKII), can lead to activation o f p53. Finally, we studied three key target proteins of activated p53 (wafl/p21, Cdc2 p34, and Cdk4), whose modulation could produce cell cy cle effects. The studies were carried out using primary cell cultures prepared from endocervical epithelium recovered at necropsy from TCDD- treated (2 and 4 pg TCDD/kg) and untreated macaques. There was a signi ficant decrease in EGFR binding activity in cells from TCDD-treated an imals as compared to controls. A marked increase in the protein amount of H-Ras and a significant increase in the activity of c-Src kinase, PTK, and Erk2 were found in cells from TCDD-treated animals. A signifi cant decrease in the activity of CKII and in the protein amount of p53 , wafl/p21, and Cdc2 p34 was found. On the other hand, a substantial i ncrease in the protein amount of Cdk4 and DNA binding activity of AP-1 was found in cells from TCDD-treated animals. In vitro experiments us ing primary cultures of endocervical cells from untreated macaques rev ealed that these cells have AhR, and that c-Src protein is functionall y attached to the AhR and is specifically activated upon ligand bindin g as judged by the following criteria. (1) A structure-activity relati onship study with TCDD and three dioxin congeners revealed a rank orde r for their potency in activation of AhR-associated c-Src kinase from cervical cells which was identical to that of previously determined to xicity indices. (2) TCDD-induced, AhR-associated c-Src kinase activity was abolished when an AhR immunoprecipitate from cervical cells was p reincubated with alpha-naphthoflavone (AhR blocker) or geldanamycin (S rc kinase inhibitor) prior to the addition of TCDD. (3) The analysis o f the AhR complex showed three proteins of molecular weights of 100 (A hR), 90, and 60 kDa. (4) The same protein with molecular weight 60 kDa was found when the immunoprecipitate with anti AhR-antibody was analy zed by SDS-PAGE, then transferred into nitrocellulose membrane followe d by immunobloting the membrane with anti c-Src-antibody. Our data sug gest that TCDD induced pathology in endocervical cells through changes in growth factor receptor signaling, other cytosolic signaling protei ns, tumor suppressor proteins, and cell cycle proteins, (C) 1998 Acade mic Press.