Knowledge of the mechanisms by which tumor promoting agents, such as 12-0-tetradecanoylphorbol-13-acetate (TPA), cause alterations in specific gene expression is fundamental to an understanding of their biological and tumor-promoting properties. TPA, and other agents which stimulate protein kinase C, may induce transcription of specific genes through phosphorylation of chromatin-associated nuclear proteins. Ornithine decarboxylase (ODC) is rapidly induced by TPA in many different systems. We propose to study the mechanism by which TPA alters transcription of ODC, with a focus on the possible role of chromatin protein phosphorylation. This objective will be pursued through the following specific aims. (1) The relative contribution of changes in transcription and messenger stabilization in the accumulation of ODC mRNA following TPA treatment will be determined. A nuclear transcription assay will be used to determine the rates of transcription of ODC in TPA treated rat H35 hepatoma cells, and the stability of ODC mRNA in these cells will be determined through the use of inhibitors of transcription and DNA-RNA hybridization. The requirement for protein kinase C activation in this process will be evaluated using PKC activators (phospholipase C; diacylglycerols) and inhibitors (palmitoyl carnitine, H7, trifluoroperazine). (2) The distribution of nuclear phosphoproteins and ODC coding sequences will be determined in chromatin fractions separated on the basis of their nuclease accessibility and solubility. (3) A functional ornithine decarboxylase gene will be cloned from rat H35 cells selected for resistance to the ODC inhibitor, difluoromethylornithine. The gene will be manned with restriction endonucleases and by S1 transcript mapping, and regions likely to contain important regulatory sites will be sequenced. (4) DNA sequence elements of the ODC gene required for basal expression and TPA induction will be identified through the use of a transient transfection assay. The sites of DNA- protein interactions within these elements will be located using gel mobility shift and nuclease protection assays, and the role of protein phosphorylation in the specificity of these DNA-protein interactions will be determined.
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