Our results are con sistent with the finding that AF is able to induce AhR sig naling. We showed that AF could activate a DRE driven luciferase reporter and induce expression of CYP1A1 and CYP1B1 in AF sensitive, ER negative MDA MB 468 hu man breast cancer cells. Interestingly, we found that except AF sensitivity could be uncoupled from AhR responsiveness as exemplified in a human breast cancer cell line, Cal51. We first showed that Cal51 cells, while expressing high levels of endogenous AhR protein, lack CYP1A1 and CYP1B1 induction upon treatment with AhR activators. Cal51 cells are sensitive to AF, exhibiting a GI50 value in the nanomolar range. When AhR is knocked down in Cal51shAhR, inducibility of CYP1A1 was further attenu ated, yet AFs GI50 value was not greatly affected.

MDA MB 468 cells are relatively responsive to AhR activation, and like Cal51, they maintain sensitivity to AF after AhR knockdown. SULT1A1 has also been implied in the bioac tivation Inhibitors,Modulators,Libraries of AF, and we showed that both MDA MB 468 and Cal51 express a basal level of SULT1A1 mRNA. How ever, AF and BNF were unable to increase levels of this gene. The AhR independency Inhibitors,Modulators,Libraries of AF sensitivity in MDA MB 468 and Cal51 is in discrepancy with the finding that MCF7 cells are sensitive to AF while AhR100 MCF7 cells are AF resistant. Previous studies had shown that AhR100 cells exhibited diminished AhR protein levels, mRNA levels, and ability to induce AhR target genes, rendering the cell line resistant to AF. The difference in AF sensitivity with regards to AhR levels and activity could be cell type specific.

Nonetheless, our data from parental Cal51 as well as from Cal51shAhR and MDA MB 468shAhR provide strong evidence that AhR protein level, as well as downstream AhR signaling may not be directly predictive of AF sensitivity Inhibitors,Modulators,Libraries in all cell types. While our data suggests that the growth inhibitory effects of AF still occur in cells Inhibitors,Modulators,Libraries where the levels of AhR and AhR signaling are sig nificantly decreased, we have only examined the genomic activity of AhR in the context of AF Inhibitors,Modulators,Libraries signaling. It is import ant to note that AhR has been shown to have non genomic kinase activity, including interactions with Src and effects on Ca2 as a second messenger in inflammatory pathways. The potential role of AhRs extranuclear effects in the context of AF sensitivity has yet to be uncovered.

While MDA MB 468 and Cal51 human breast cancer cell lines exhibit similar GI50 the following site values for AF, graphing the log of AF concentration versus viable cell number shows that the two curves differ in shape. In the graphical representation of growth inhibition by AF in MDA MB 468, we see that higher concentrations of AF can eliminate viable cells com pletely. In contrast, some Cal51 cells can still survive even at the highest AF concentrations. Due to the different pro files of their GI50 graphs, we predicted that the mechanisms underlying AF mediated growth inhibition would vary.