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. AKT activation enhances resistance to apoptosis and induces cell survival signaling through multiple downstream pathways which include NF-kB. We recently reported that AKT is activated in HTLV-1-transformed cells and that Tax activation of AKT is linked to NF-κB activation, p53 inhibition and cell survival. 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. Treatment of C81 cells with LY294002 resulted in an increase in the p53-responsive gene MDM2, suggesting a role for AKT in the Tax-mediated regulation of p53 transcriptional activity. 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. We suggest that AKT plays a role in activation of pro-survival pathways in HTLV-1-transformed cells, possibly through NF-kB activation and inhibition of p53 transcription activity. We have extended these observations to identify several regulatory pathways affected by AKT in HTLV-1-transformed cells. First, we demonstrate that inhibition of AKT reduces the level of phosphorylated Bad, an important member of the pro-apoptotic family of proteins. Consistent with the decrease of phosphorylated Bad, cytochrome c is released from the mitochondria and caspase 9 is activated. Pre-treatment of the cells with caspase-9 specific inhibitor z-LEHD-FMK or pan caspase inhibitor Ac-DEVD-CHO prevented LY294002-induced apoptosis. Second, LY294002 inhibits NF-κB activation in HTLV-1 transformed cells as measured by IκBα phosphorylation, NF-κB gel shift analysis and NF-κB transcription activity. Third, LY294002 decreased expression of cyclin D1 and increased expression of cyclin dependent kinase (CDK) inhibitor p27. Consistent with these findings, FACS analysis revealed an accumulation of cells in the G1 phase. Fourth, p53 siRNA prevents the apoptosis and cell cycle arrest induced by LY294002, suggesting that both pathways are p53-dependent. In conclusion, the AKT pathway is involved in targeting multiple proteins which regulate p53-dependent apoptosis and cell cycle progression in HTLV-1-transformed cells. Since AKT inhibitors simultaneously inhibit NF-κB and activate p53, these drugs should be promising candidates for HTLV-1-associated cancer therapy. 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. Comparison of the gene profiles between mucinous tumors and normal ovarian epithelial cells identified 1,599, 2,916, and 1,765 differentially expressed in genes in the cystadenomas, LMP tumors, and adenocarcinomas, respectively. Hierarchical clustering showed that mucinous and serous LMP tumors are distinct. In addition, there was a close association of mucinous LMP tumors and adenocarcinomas with serous adenocarcinomas. Binary tree prediction revealed increased heterogeneity among mucinous tumors compared with their serous counterparts. Furthermore, the cystadenomas coexpressed a subset of genes that were differentially regulated in LMP and adenocarcinoma specimens compared with normal ovarian surface epithelium. PathwayAssist highlighted pathways with expression of genes involved in drug resistance in both LMP and adenocarcinoma samples. In addition, genes involved in cytoskeletal regulation were specifically up-regulated in the mucinous adenocarcinomas. These data provide a useful basis for understanding the molecular events leading to the development and progression of mucinous ovarian cancer
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