Protein kinase C (PKC) isozymes are a family of related serinethreonine kinases that play an important role in the control of cell proliferation, death and differentiation. PKC isozymes are the target for the phorbol esters and are implicated in the molecular mechanisms of tumor promotion. A main objective of this proposal is to evaluate whether PAH oquinones and the ROS they generate affect PKC activity and signaling events resulting in phenotypic consequences on cell growth. Our preliminary data shows that PKC isozymes are direct targets for PAH o-quinones, suggesting that these compounds have epigenetic effects that could explain why PAH may act as complete carcinogens. Our studies will assess effects of these PAH metabolites on PKC in vitro as well as in bronchoalveolar cells.
In Aim#1 we will evaluate the effect of DMBA-3,4-dione, BA-3,4-dione and BP-7,8-dione, which correspond to Class I, II, and III o-quinones, respectively on the activity of individual PKC isozymes expressed in baculovirus. We will assess whether the o-quinones or the ROS they generate affect PKC kinase activity and cofactor-dependent binding of phorbol esters.
In Aim#2 we will evaluate whether PAH o-quinones or ROS affect translocation of PKC and activate or inhibit PKC isozymes in cells. We will take advantage of AKR1A1 overexpressors to determine whether o-quinones or ROS generated intracellularly affect the phosphorylation of endogenous PKC substrates (myristoylated alanine-rich C kinase substrates) and PKC-mediated AP-1 activation.
In Aim # 3 we have shown that the phorbol ester, phorbol-12- myristate-13-acetate (PMA), inhibits the proliferation of bronchoalveolar cells by causing G1 arrest. Individual PKC isozymes will be infected into bronchoalveolar cells to identify the isoform responsible for this PMA effect. We have shown that PAH o-quinones inhibit PKC in vitro and stimulate S phase entry in bronchoalveolar cells. Therefore we will test the hypothesis that PAH o-quinones or ROS dysregulate components of the cell cycle controlled by PKC. We will correlate increased progression through G1 phase with changes in the expression of G1 phase cyclins and cdk inhibitors and the consequences for cdk activity and Rb phosphorylation. Importantly our studies will determine the effects of individual PKC isozymes on the proliferation of human lung epithelial cells and whether PAH o-quinones dysregulate PKC mediated control of the cell cycle. The Project is reliant on the Bioanalytical Core-B for quality control of PAH-metabolites and determining PAH o-quinone concentrations in cell culture. The Project is reliant on Core-A for biostatistical analysis of the in vitro and in vivo effects of PAH o-quinones and ROS on PKC.
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