A well-developed experimental model for two-stage carcinogenesis in mouse lung involves a single dose of an initiator followed by multiple doses of the phenolic antioxidant BHT. The dramatic enhancement of tumorigenesis in this system is due to the formation of quinone methides by pulmonary cytochromes P450. These metabolites are electrophilic and bind to cellular proteins producing a number of effects that eventually lead to enhanced tumor formation. Our goals for the next phase of this project are to identify the important targets of BHT-derived quinone methides and determine the roles such adducts in promotion. The resulting data will address our working hypothesis that electrophilic promoters function through the formation of covalent adducts with proteins involved in cell signaling. We will utilize a number of effective tools to detect and identify important adducts, including structural analogs of BHT with differing promotion potencies, Clara and type 2 cells from the lungs of promotion-sensitive (B+) and promotion-resistant (B-) mouse strains, non-tumorigenic cell lines derived from mouse lung epithelia and the tumorigenic siblings, polyclonal antibodies that recognize the phenol group, and a variety of mass spectrometric techniques. The results from this work will enable improved insights into the mechanisms of tumor promotion by electrophilic metabolites.
The specific aims are as follows.
Aim 1 : Identify selected protein targets of quinone methides in lung cells from promotion-sensitive (B+) and promotion-resistant (B-) strains of mice and in tumorigenic and non-tumorigenic epithelial cell lines, and assess their roles in promoting the selective clonal expansion of initiated cells. Protein adducts formed in differing amounts in the comparison cell groups will be flagged for identification.
Aim 2 : Investigate biochemical effects of relevance to promotion resulting from metabolically-generated quinone methides produced in epithelial cell lines and cells isolated from mouse lung. Oxidative stress and effects on antioxidant enzymes will be determined in cell lines expressing cytochrome P450 and in Clara and type 2 cells isolated from B+/B- mice. The effects of quinone methides on gap junctional intercellular communication and apoptosis will be determined; protein expression and protein phosphorylation will be examined in the cell lines and isolated cells using isotope-coded affinity tags and mass spectrometry.
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