SU5416 to activate the AHRb and AHRd polymorphisms with similar efficacy. These two isoforms are present in different strains of mice, and have been well characterized for many ligands, particularly TCDD. For the majority of ligands studied, the AHRd isoform displays less than one-tenth the response of AHRb after binding. It has been proposed that a true endogenous ligand of the AHR would activate the two polymorphisms similarly, given the importance of the AHR in normal physiologic development, and that mice with either genotype do not display the abnormal phenotypes seen in AHR2/2 and hypomorphic mice. While we initially utilized the AHRd polymorphism to narrow our search for potent ligands of the AHR, we inadvertently found that SU5416 activates these two isoforms with similar potency. This not only confirms the importance of this property of the drug in humans, who harbor the AHRd polymorphism, but also will allow the structure of SU5416 to serve as a model in our search for clinically relevant endogenous ligands of the AHR. To prove that induction of the DRE was mediated through classic AHR signal transduction, and not through a VEGF-related mechanism, we employed mutant cell lines that lack expression of the AHR or ARNT. The C35 cell line, which contains a 75887-54-6 dysfunctional AHR, was utilized. It was transfected with vector containing the Tauroursodeoxycholate (Sodium) structure murine AHR gene, the lacZ gene, and the luciferase reporter gene driven by 3 upstream DREs, as described in the Methods section. Controls were mock transfected with reporter plasmids and the empty vector. Cells were treated with either 3 mM SU5416 or DMSO. As seen in figure 2A, cells transfected with the AHR plasmid generated significant luciferase activity when exposed to SU5416 compared to DMSO. The control cells generated minimal activity. In a similar experiment, the ARNT-deficient mouse hepatoma cell line C4 was transiently transfected with plasmids encoding human ARNT, the lacZ gene, and the same DRE-driven luciferase gene, and control samples received empty vectors for ARNT. As shown in figure 2B, after exposure to SU5416 or DMSO, activity was only seen when ARNT was transfected. An important role for the AHR in the immune system, and specifically T-cell differentiation, has been recognized and continues to be
