Regulation of the Aryl Hydrocarbon Receptor by P-Anilinoaniline in McF10a Cells

The AhR is a ligand-activated transcription factor. Following TCDD-induced activation the AhR stimulates the transcription of target genes such as i. Transcription is terminated following ubiquitination of the activated AhR. Ubiquitination requires the coordinate activities of a cascade of enzymes. In silico screening of a chemical database identified p-anilinoaniline (pAA) as a putative inhibitor of the E2 enzyme UbcH5a, which has been implicated in the ubiquitination of AhR. This dissertation's research was aimed to investigate whether pAA can modulate TCDD-mediated AhR degradation and consequently CYP1A1 expression. Concentrations of pAA between 10--25 muM pAA were cytostatic to cultures of the normal human mammary epithelial cell line MCF10A. Concentrations >=50 muM were cytotoxic, and induced protracted G 1 and S cell cycle phase arrests. pAA caused DNA damage as monitored by the Comet assay. Cytotoxic concentrations of pAA induced morphological changes consistent with cells undergoing apoptosis and/or autophagy. Enzymatic, western blot, and binding analyses of fluorescent labeled VAD-FMK, suggested that caspases were activated by pAA. Analyses revealed the conversion of LC3-I to LC3-II, a post-translational modification involved in the development of the autophagosome. ShRNA knockdown of ATG7 suppressed autophagosome formation, prevented pAA-induced vacuolization, enhanced the activation of pro-caspase-3, and increased susceptibility of ATG7-deficient cells to the cytostatic and cytotoxic activities of markedly lower concentrations of pAA. Collectively, these data suggest that MCF10A cultures undergo autophagy as a pro-survival response to cytotoxic concentrations of pAA. pAA (25 muM) accumulated ∼3-fold more CYP1A1 mRNA above that of TCDD alone. Analyses of CYP1A1 mRNA half-lives were not significantly different in cultures treated with TCDD or co-treated pAA and TCDD. However, analyses of CYP1A1 hnRNA indicated that pAA significantly enhanced TCDD-induced CYP1A1 transcription. Although pAA partially suppressed (15%) TCDD-mediated AhR turnover, the accumulation of TCDD-induced CYP1A1 mRNA by pAA was not totally due to AhR stabilization. pAA (25 muM) induced a transient G1 and S phase cell cycle arrest. pAA did not enhance TCDD-induction of CYP1A1 in cultures arrested in G0/G1. In contrast, the kinetics of pAA enhancement of TCDD-mediated CYP1A1 transcription correlated with the S phase arrest. Western blot indicated that TCDD-mediated AhR degradation was greater in G1 than S phase cells. ChIP assay indicated that AhR may reside longer at CYP1A1 XREs during the period cells were arrested in S phase. Real-time PCR of CYP1A1 hnRNAs and mRNAs levels indicated that CYP1A1 transcripts and CYP1A1 mRNA contents were the highest during the period the cells were arrested in S phase. The cumulative data suggest that pAA enhancement of TCDD-mediated CYP1A1 mRNA accumulation is cell cycle related.