2,3,7,8,Tetrachlorodibenzo-p-dioxin (TCDD ) is a ubiquitous environmental contaminant that promotes tumor formation and modulates cellular differentiation via actions thought to involve the aryl hydrocarbon receptor (AHR) signaling pathway. Given that study of keratinocyte differentiation has been shown to lend important insights into the mechanism(s) by which tumor promoters exert their effects and that this model is amenable to a number of molecular techniques, we have initiated studies of differentiation to fulfill our long-term goal of understanding the role of TCDD in carcinogenesis. Using normal human keratinocytes (NHK), we have shown that administration of TCDD to NHK cells ultimately results in suppression of differentiation and that this effect is accompanied by decreased levels of the tumor suppressor protein, p27Kipl Additional studies using the ARR antagonist, 3'-methoxy-4'nitroflavone implied a role of the AHR in mediating TCDD's suppression of differentiation. Finally, it is apparent that TCDD suppresses differentiation by decreasing the number of differentiating cells while increasing the number of non-differentiating cells. These results and others, form the basis of our overall hypothesis that the ability of TCDD to suppress differentiation is mediated by a decrease in the expression levels of the tumor suppressor protein, p27Kipl thereby diminishing the p27Kipl differentiation signal. To test this hypothesis, we will use flow cytometry, western blot analysis, real time PCR and adenovirus-mediated over expression systems to determine whether: 1) the ability of TCDD to suppress differentiation requires suppression of the expression of p27Kipl, 2) TCDD regulates the p27Kipl protein at the transcriptional or post-transcriptional levels, 3) the ability of TCDD to suppress the expression of p27Kipl and differentiation requires the AHR and ARNT proteins, 4) suppression of differentiation by TCDD results in increased) proliferation, and 5) and the actions of TCDD on differentiation occur at the) genomic level.
