Inhibition of p53 function, either through mutation or inhibition by viral transforming proteins, correlates strongly with the oncogenic potential of the cell. In the following report, we describe a unique mechanism of p53 inactivation that involves the interaction of p53 with the p65 subunit of NF-kB. The inactive p53 complex is induced in HTLV-1-transformed and ATL leukemic cells. This mechanism of p53 inhibition may occur in other human cancers. We initially demonstrated that wild-type p53 is stabilized and transcriptionally inactive in HTLV-transformed cells. The viral transcriptional activator Tax plays a role in both the stabilization and inactivation of p53. p53 is hyperphosphorylated at serines 15 and 392 in HTLV-1-transformed cells and phosphorylation of p53 at these specific residues inactivates p53 by blocking its interaction with basal transcription factors. In T-lymphocytes, Tax-induced p53 inactivation is dependent upon NF-kB activation. Analysis of Tax mutants demonstrated that Tax inactivation of p53 function correlates with the ability of Tax to induce NF-kB. Further, the p65 subunit of NF-kB is critical and uniquely involved in the Tax-induced p53 inhibition pathway. Using chromatin immunoprecipitation assays we have determined that in HTLV-1-transformed cells, p53 and p65 form a complex on the inactive MDM2 promoter. Consistent with reduced transcription activity, TFIID binding is not observed. These studies provide evidence that the divergent NF-kkB proliferative and p53 cell cycle arrest pathways may be cross-regulated at several levels which include post-translational modification of p53. Further studies determined that p53 inhibition by Tax required a unique IKKbeta complex that required AKT activation. Overexpression of AKT wild type (WT), but not a kinase dead (KD) mutant resulted in increased Tax-mediated NF-kB activation. Blocking AKT with the PI3K/AKT inhibitor LY294002 or AKT siRNA prevented NF-kB activation and inhibition of p53. Further, we show that LY294002 treatment of C81 cells abrogates in vitro IKKbeta phosphorylation of p65 and causes a reduction of p65 Ser-536 phosphorylation in vivo, steps critical to p53 inhibition. Given these attributes, we were interested in the activity of small-molecule inhibitor 9-aminoacridine (9AA), an anticancer drug that targets two important stress response pathways, NF-kappaB and p53. In the present study, we have examined the effects of 9AA on HTLV-1-transformed cells. Treatment of HTLV-1-transformed cells with 9AA resulted in a dramatic decrease in cell viability. Consistent with these results, we observed an increase in the percentage of cells in sub-G(1) and an increase in the number of cells positive by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay following treatment of HTLV-1-transformed cells with 9AA. In each assay, HTLV-1-transformed cells C8166, Hut102, and MT2 were more sensitive to treatment with 9AA than control CEM and peripheral blood mononuclear cells. Analyzing p53 function, we demonstrate that treatment of HTLV-1-transformed cells with 9AA resulted in an increase in p53 protein and activation of p53 transcription activity. Of significance, 9AA-induced cell death could be blocked by introduction of a p53 small interfering RNA, linking p53 activity and cell death. These results suggest that Tax-repressed p53 function in HTLV-1-transformed cells is """"""""druggable"""""""" and can be restored by treatment with 9AA. The fact that 9AA induces p53 and inhibits NF-kappaB suggests a promising strategy for the treatment of HTLV-1-transformed cells. Our laboratory has expertise in Affymetrix GeneChip technology and analysis of gene expression in eukaryotic cells. In addition to our studies on HTLV-1 associated adult T-cell leukemia, we have used the microarray technology to collaborate with CCR and academic investigators to analyze expression patterns in other human cancers. For example, our collaboration with Dr. Michael Birrer has yielded important results on ovarian cancer. Despite the existence of morphologically indistinguishable disease, patients with advanced ovarian tumors display a broad range of survival end points. To resolve survival-associated loci, gene expression profiling was completed for an extensive set of 185 (90 optimal/95 suboptimal) primary ovarian tumors using the Affymetrix human U133A microarray.The prognostic signature successfully classified the tumors according to survival for suboptimally but not optimally debulked patients. The suboptimal gene signature was validated using the independent set of tumors. To elucidate signaling events amenable to therapeutic intervention in suboptimally debulked patients, pathway analysis was completed for the top 57 survival-associated probe sets. For suboptimally debulked patients, confirmation of the predictive gene signature supports the existence of a clinically relevant predictor, as well as the possibility of novel therapeutic opportunities.
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