Our goal is to explore the molecular mechanisms and the cellular implications of cytokine mediated regulation of ferritin in cytokine sensitive and resistant cellular targets. We will approach this task by asking three questions. Because many aspects of cytokine pathways relate to the generation of oxidant stress in target cells, in the first Specific Aim we will seek to clarify the relationship between changes in ferritin subunit composition and content and the response to oxidative stress. We will design three experimental systems in which ferritin can be specifically and selectively altered: 1.) transfection of cells with vectors which over-express ferritin H, ferritin L, or mutant ferritins; 2.) creation of ferritin H """"""""knock-out"""""""" mutants by homologous recombination; 3.) direct introduction into cells of our ferritin recombinant proteins with different H/L subunit ratios and species specificity. In these systems, we will assess the effect of alterations in ferritin composition and content on the extent and character of cell injury induced by oxidative stress using two methods: assessment of DNA base damage using GCMS, and DNA strand scission using alkaline elution. In the second Specific Aim, we will identify key proteins and pathways involved in the regulation of ferritin H basal transcription through the ferritin transcriptional enhancer, FER-1, that we have recently identified. We will clone the dyad binding protein required for transcriptional enhancement of ferritin through FER-1. In addition, we will clarify the relationship between ferritin H and growth control using mutants of the adenovirus gene E1A to identify the E1A binding protein which interacts with FER-1. Because FER-1 regulates basal transcription of the ferritin H gene, this should allow us to explore fundamental mechanisms of tissue-specific regulation of ferritin composition. In the third Specific Aim, we will elucidate the differential effects of TNF on ferritin H regulation in normal and transformed cells, using the ferritin H enhancer targeted by TNF (FER-2). To achieve this, we will clarify the exact sequence specificity of the enhancer element and its binding proteins (NF-kB family members), and whether DNA sequences themselves or the binding proteins are different (or differentially regulated) in normal and transformed targets.
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