Appropriate regulation of ornithine decarboxylase (ODC) is crucial for cell proliferation. Tumor promoters, such as 12-O-tetradecanoylphorbol-13- acetate (TPA) and transforming oncogenes frequently cause aberrant ODC induction, and ODC levels are elevated in many types of cancer. Recent studies from this laboratory have defined regions of the 5'-flanking region of the ODC gene which mediate transcriptional responsiveness to TPA and to proto-oncogenes c-fos, c-jun and c-raf. In addition, we have found that ODC promoter activity is inhibited by over-expression of the retinoblastoma gene product. Based on these results, we hypothesize ODC transcription is regulated through multiple promoter elements and associated transcription factors which respond to both positive and negative growth signals, and that deregulation of these transcription factors during carcinogenesis leads to aberrant ODC transcription. In order to further characterize the regulation of ODC transcription, we propose to investigate the following specific aims: 1. Identify the DNA sequence elements which regulate ODC transcription - Transient expression assays using defined deletions or mutations of the rat ODC gene coupled to heterologous reporter genes (e. g. luciferase) will be performed to identify elements that are critical for basal and regulated expression of ODC. Elements required for induction by TPA, by serum growth factors, and by Rb will be mapped and characterized. 2. Identify transcription factors which regulate ODC transcription - We will be particularly concerned with proteins which bind elements involved in TPA or growth factor responsiveness, and those which may be involved in repression by Rb. These factors will be identified using in vitro techniques to study DNA-protein interactions, and when possible by co-transfection of the appropriate factors with ODC reporter vectors to demonstrate function. 3. Determine the role of TPA responsive transcription elements of ODC in carcinogenesis -In this specific aim, we will characterize the regulation of transcription factors which mediate ODC transcription in response to TPA or growth factors. 4. To determine whether there is an association between regulation of ODC transcription by Rb and the transformed phenotype. In this aim, we will investigate the regulation of ODC in RB- and RB+ cells through introduction of active RB into RB- cells and inactivation of RB by introduction of viral oncoproteins (e.g. E1A). The results of these studies will provide insight into the activity of transcription factors involved in responses to tumor promotion and to altered ODC expression in the neoplastic state.
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