Phorbol ester (PE) treatment of myeloid leukemic cells induces their differentiation towards monocyte/macrophages irrespective of the stage of maturity of the cells. Organized differentiation requires that the PEs activate specific sets of proteins in a coordinated manner. Activation can occur through phosphorylation of key transcription factors or stabilization of mRNA. The p21WAF1 gene product plays a critical role in the inhibition of cell growth and differentiation. We have demonstrated that PEs increase the synthesis of p21WAF1 mRNA, and that this PE-mediated stimulation is transcriptionally mediated by the binding of the Sp1 protein to the p21WAF1 promoter. However, Sp1 is not phosphorylated by PE treatment. Rather, we have found that PEs regulate the transcription of p21WAF1 by controlling the phosphorylation and activity of proteins that interact closely with the RNA polymerase II complex. We find that PE treatment of U937 leukemic cells stimulates the phosphorylation and activity of the TATA-box binding protein, TBP; E1A transfection blocks the ability of PEs to stimulate p21WAF1 transcription demonstrating that p300 is important in PE function; and PEs stimulate the phosphorylation of BRG1, a member of the SWI/SNF complex. Based on this preliminary data, this proposal will investigate in detail the hypothesis that regulation by PEs of a specific set of genes, which leads to monocyte/macrophage differentiation, occurs through the modulation of novel proteins closely associated with the RNA polymerase II complex. These studies will further clarify mechanisms underlying differentiation towards monocytes/macrophages and should pin-point the most effective targets for therapeutic intervention in leukemia treatment.
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