This application requests continuation of aggregate funding for programs to study the role of arachidonic acid oxygenation in carcinogenesis. The two major research goals are evaluation of the potential of malondialdehyde (MDA) as an endogenous animal and human carcinogen and determination of the structure and function of the two major isoforms of the arachidonic acid oxygenase, PGH synthase. MDA is a dicarbonyl compound that is a product of lipid peroxidation and prostaglandin biosynthesis. It is mutagenic and carcinogenic which raises the possibility that it is an endogenous mediator of genetic disease and perhaps cancer. To evaluate this possibility, we will determine the ability of MDA-DNA adducts to induce mutations in random and site-specific mutagenesis experiments. We will also investigate the molecular basis of mutations induced by MDA adducts with spectroscopic studies of adducted DNA molecules free in solution and complexed to DNA polymerase and with kinetic studies of replication of adducted template-primers. Parallel experiments will be conducted with structurally related exocyclic deoxyguanosine adducts. Analytical studies will be performed to quantitate the extent to which MDA-DNA adducts exist endogenously in animals and humans and to identify unknown adducts that are reported to be present in animal DNA. PGH synthase catalyzes the committed step in the biosynthesis of prostaglandins, thromboxane, and MDA. Its cyclooxygenase activity is the in vivo target for aspirin and several other non-steroidal antiinflammatory drugs. The epidemiological association of aspirin intake with reduced colon cancer mortality suggests a significant role for PGH synthase in human carcinogenesis. Two genes (cox-1 and cox-2) code for PGH synthases. Cox-1 appears to be constitutively expressed whereas cox-2 is transcribed in response to mitogens. Although the protein products are 60% identical, it appears they may be differentially inhibited. We propose to overexpress the two human forms of PGH synthase in order to determine by X-ray crystallography their structural differences. Affinity labeling and mass spectroscopy will be used to identify catalytically important protein residues and residues irreversibly oxidized during enzyme-catalyzed autoinactivation. The importance of these residues will be tested by site- directed mutagenesis of cloned cDNA's followed by high-level expression in mammalian cells.
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