The overall goals of the research are to understand the regulation and function of putrescine and the higher polyamines, spermidine and spermine in mammalian cells and to use this knowledge to manipulate cellular polyamine levels and evaluate the role of the polyamines in normal and neoplastiC growth. There is strong evidence that ornithine decarboxylase [ODC] and putrescine production may play a role in the action of carcinogens and tumor promoters and increased ODC may be needed for tumor initiation and development ODC may therefore be a potentially important target for synthesis of chemopreventive agents and its inhibitor DFMO has been proposed for this purpose. The proposed investigations will study two key enzymes in mammalian polyamine biosynthesis, S-adenosylmethionine decarboxylase (AdoMetDC) and ODC and how their relative activities affect polyamine levels and growth. There are 4 specific aims: (a) in order to investigate the importance of ODC activity, a stable dominant negative mutant form of ODC will be expressed in cells in culture and in transgenic mice. The expression of this dominant negative mutant from strong tissue specific promoters will be used to greatly reduce ODC activity and ablate increases in ODC in response to physiological stimuli such as those occurring during tissue regeneration, carcinogenesis and tumor promotion. The consequences of this reduction in ODC on cellular polyamines and response to these stimuli will be evaluated; (b) the importance of AdoMetDC activity and cellular putrescine levels will be examined by transfection of cells using a construct which produces large amounts of active AdoMetDC causing conversion of virtually all the cellular putrescine into the higher polyamines. The effects of this alteration in polyamine metabolism on cell transformation and growth will then be studied. The opposite situation in which cellular putrescine levels are very high compared to the content of spermidine and spermine will also be tested using a specific AdoMetDC inhibitor to block the formation of spermidine and spermine thus leading to a very high level of putrescine. The effectiveness of this treatment in transforming these cells will be compared to the known transformation via overexpression of ODC; (c) in order to study the structure and processing of AdoMetDC, large amounts of mammalian AdoMetDC and its proenzyme precursor will be produced for determination of the protein structure by X-ray crystallography and for investigations of the mechanism of proenzyme processing to generate the pyruvate prosthetic group and of the catalytic activity and its activation by putrescine; (d) the importance of the association and free exchange of ODC subunits in ODC function and rapid turnover and the effect of the protein antizyme on ODC association will be studied in detail using recombinant mouse ODC and appropriate mutants purified to homogeneity by affinity chromatography. Antibodies to the carboxyl region of ODC, which is known to be essential for its rapid turnover, will be used to investigate conformational changes in ODC as a result of dimerization or antizyme binding.
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