COMPLEMENTATION OF AH RECEPTOR DEFICIENCY IN HEPATOMA-CELLS - NEGATIVE FEEDBACK-REGULATION AND CELL-CYCLE CONTROL BY THE AH RECEPTOR

The Ah receptor (AhR) is a ligand-dependent transcription factor subun it; that heterodimerizes with the AhR nuclear translocator (Arnt) and mediates the predominant biological effects of 2,3,7,8-tetrachlorodibe nzo-p-dioxin (TCDD). TCDD activates target genes in xenobiotica metabo lism in many cell lines and, more specifically, delays G1-S progressio n of 5L hepatoma cells. Here we describe transient and stable AhR-expr ession analysis in AhR-deficient subclones of the TCDD-sensitive 5L ce lls. We tested the integrity of the AhR-signaling system beyond the la ck of the receptor in the variant subclone and analyzed the role of Ah R in cell cycle regulation, Transiently expressed AhR has a high basal activity on promoters containing AhR-binding sites, so called XREs, w hen transfected into receptor-deficient variant cells compared to wild -type cells, Single- and double-hybrid analysis dissociates AhR Ligand responsiveness, transactivation, and heterodimerization with Amt from receptor binding to an XRE, Hybrid receptors also shaw the high basal activity in, the absence of exogenous TCDD in AhR-deficient variant c ells, indicating that the endogenous AhR-activating signal acts direct ly on the receptor rather than XRE-dependent promoters or DNA binding of the receptor Stable expression of AhR in variant cell clones by ret roviral infection fully reconstitutes TCDD responsiveness, including t arget-gene induction and delay of cell cycle progression. These AhR-re constituted cells, like AhR-containing wild-type cells, show low basal activity of the transiently expressed AhR hybrid, Thus, the increased basal activity in AhR-deficient cells suggests a negative feedback co ntrol of AhR activity. In vitro ligand-binding assays are compatible w ith the idea that the increased basal. activity is due to the. accumul ation of an AhR-binding endogenous ligand, In conclusion, AhR is causa lly responsible for TCDD-dependent cell cycle regulation and feedback control of AhR activity. (C) 1996 Academic Press, Inc.