Elevated cellular polyamine levels and increased activity of ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis, are hallmarks of tumor development in epithelial tissues. Our long-term research goal is to understand the role of polyamines in the promotion and progression of epithelial cancers in order to develop novel prevention and treatment strategies. The specific objective of this application is to determine the effect of temporal and spatial manipulation of polyamine biosynthesis on keratinocyte growth control as well as skin tumor promotion and maintenance. Our central hypothesis is that cellular putrescine levels, rather than overall polyamine content, regulate keratinocyte proliferation, differentiation and transformation. To explore this hypothesis transgenic mouse models were produced that enable temporal and spatial control of antizyme (AZ) or S-adenosylmethionine decarboxylase (AdoMetDC) expression that is regulated by doxycycline (Dox). Interestingly, both AZ and AdoMetDC-expressing mice exhibit a thin fur phenotype. AZ is a negative regulator of cellular polyamine content that inhibits putrescine synthesis by ODC, stimulates ODC degradation and suppresses exogenous polyamine uptake into the cell. AdoMetDC provides the aminopropyl groups that are necessary for synthesis of the higher polyamines spermidine and spermine from the diamine putrescine. Therefore, AZ suppresses putrescine biosynthesis while AdoMetDC activity can deplete putrescine through enhanced utilization.
The first Aim i s to determine the effect of altered putrescine levels on keratinocyte proliferation and differentiation during normal development and oncogene activation. First, skin sections will be analyzed to identify the developmental defects in hair follicle morphogenesis and cycling that are induced by AZ and AdoMetDC expression. Next, primary keratinocytes from our transgenic models will be utilized to measure proliferation, migration, and markers of differentiation and senescence in response to calcium or TPA- induced differentiation as well as activated HRAS expression in order to identify the molecular targets of putrescine regulation.
The second Aim i s to evaluate the ability of AZ and AdoMetDC to inhibit promotion of initiated cells and determine the cell-type and stage specificity of the tumor suppressive effects. First, chemical carcinogenesis will be utilized to demonstrate the ability of AZ and AdoMetDC to impair tumor promotion in mouse skin. Thereafter, transgene expression will be silenced to determine whether latent initiated cells persist in the skin of thes animals. Next, AZ expression will be targeted to suprabasal keratinocytes to determine if this cell type provides the putrescine that is required for promotion of initiated basal cells. Finally, expression will be induced in transgene-naive tumor-bearing animals to quantify the ability of AZ or AdoMetDC to induce regression or slow the growth of established tumors. The successful completion of these studies will lead to a more complete understanding of polyamine function in epithelial carcinogenesis and will enhance future development of polyamine-targeted therapies to prevent and treat cancer.
This research project will utilize conditional transgenic mouse models to manipulate skin polyamine metabolism in specific cellular locations. We will elucidate the regulatory functions exerted by polyamines, especially putrescine, on keratinocyte growth control as well as tumor promotion and maintenance in the two- stage chemical carcinogenesis protocol. These studies are essential to the continued development of preventive and therapeutic agents to target polyamine metabolism in cancer.
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