Alterations in the growth factor signal transduction pathways and modulators of the cell cycle in endocervical cells...

After more than a year had elapsed since a single oral exposure to 2 and 4 microgram 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., 1998). In the present experiments we investigated the mechanisms by which chemicals like TCDD could induce epithelial cell transdifferentiation 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 transduces the cytosolic signal into a nuclear signal, and which in combination with nuclear casein kinase II (CKII), can lead to activation of p53. Finally, we studied three key target proteins of activated p53 (wafl/p21, Cdc2 p34, and Cdk4), whose modulation could produce cell cycle effects. The studies were carried out using primary cell cultures prepared from endocervical epithelium recovered at necropsy from TCDD-treated (2 and 4 microgram TCDD/kg) and untreated macaques. There was a significant decrease in EGFR binding activity in cells from TCDD-treated animals 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 significant 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 increase in the protein amount of Cdk4 and DNA binding activity of AP-1 was found in cells from TCDD-treated animals. In vitro experiments using primary cultures of endocervical cells from untreated macaques revealed that these cells have AhR, and that c-Src protein is functionally attached to the AhR and is specifically activated upon ligand binding as judged by the following criteria. (1) A structure-activity relationship study with TCDD and three dioxin congeners revealed a rank order for their potency in activation of AhR-associated c-Src kinase from cervical cells which was identical to that of previously determined toxicity indices. (2) TCDD-induced, AhR-associated c-Src kinase activity was abolished when an AhR immunoprecipitate from cervical cells was preincubated with alpha-naphthoflavone (AhR blocker) or geldanamycin (Src kinase inhibitor) prior to the addition of TCDD. (3) The analysis of the AhR complex showed three proteins of molecular weights of 100 (AhR), 90, and 60 kDa. (4) The same protein with molecular weight 60 kDa was found when the immunoprecipitate with anti AhR-antibody was analyzed by SDS-PAGE, then transferred into nitrocellulose membrane followed by immunobloting the membrane with anti c-Src-antibody. Our data suggest that TCDD induced pathology in endocervical cells through changes in growth factor receptor signaling, other cytosolic signaling proteins, tumor suppressor proteins, and cell cycle proteins.