The objective of this work is to determine the role of mitochondrial injury in cytotoxicity and multistep carcinogenesis induced by diverse polycyclic aromatic hydrocarbons and arylamines. Recently, we have purified four distinct forms of mitochondrial p- 450 and characterized them by in vitro reconstitution of monooxygenase activity and amino acid as well as cDNA sequencing. Further, a mitochondrial specific protein kinase has been partially purified and characterized in this laboratory. This N- myristoylated protein kinase is regulated bY tumour promoter phorbol ester, and shows 20 fold higher activity in tumor cells. Based on this, it is proposed to continue research to determine the immediate as well as the long term effects of mitochondrial genetic and metabolic injury to cellular processes and carcinogenesis as follows: 1) Search for additional forms of p-450 using various xenobiotic as well as physiological inducers and purify and characterize them, as well as the recently identified sex specific form of P-450, from hepatic tissue. 2) Determine the levels of constitutive and inducible p-450 in mitochondria from other target cells and tissues and correlate with the extent of carcinogen binding to mitochondrial DNA. 3) Further characterization of mitochondrial DNA deletions in Ehrlich ascites cells and elucidate the source of aberrant transcription initiation in these cells by in vitro run off assays using cloned DNA templates, partially purified RNA polymerase and factors. 4) Analyze DNA from chemically transformed and established tumor cells to identify mitochondrial mutations. 5) Further characterization of mitochondrial protein kinase with respect to catalytic function and regulation, and determine its primary structure by cDNA cloning and sequencing to understand the molecular basis of altered activity in tumor cells. The long term objective is to determine the function of this gene in cellular processes using the transgenic mouse system.
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