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-I-transformed and ATL leukemic cells. It will be of interest to determine if this mechanism of p53 inhibition occurs in other human cancers. Only a small percentage of human T-lymphotropic virus type-I (HTLV-I)-transformed cells carry p53 mutations, and mutated p53 genes have been found in only one-fourth of ATL cases. We demonstrated that the 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 through a mechanism involving the first 52 amino acids of p53. We further demonstrated that p53 is hyperphosphorylated at serines 15 and 392 in HTLV-I-transformed cells and that 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 for Tax-induced p53 inactivation using wild-type and p65 knockout MEFs. While Tax can inactivate endogenous p53 function in wild-type MEFs, it fails to inactivate p53 function in p65 knockout MEFs. Tax-induced p53 inactivation can be restored by expression of p65 in the knockout MEFs. These studies provide evidence that the divergent NF-kB proliferative and p53 cell cycle arrest pathways may be cross-regulated at several levels which include post-translational modification of p53. Using MEF knockout cells and antisense oliogonucleotides to inhibit expression of NF-kB family members, we found that the p65 subunit of NF-kB is uniquely involved in p53 inhibition. Coimmunprecipitation assays demonstrate an interaction between p65 and p53 in HTLV-I and Tax-expressing cells. Further, in transient transfection assays, we demonstrate that Tax induces the interaction between p65 and p53 and that phosphorylation of p53 at serines 15 and 392 is critical for complex formation. Using a series of NF-kB mutants, we find that Tax-mediated p53 inhibition correlates with p65 and p53 interaction. Using chromatin immunoprecipitation assays we have determined that in HTLV-I-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 identify a unique mechanism for p53 regulation by the p65/RelA subunit of NF-kB. We have also analyzed the functional activity of the p53 tumor suppressor in HTLV-2-transformed cells. Abundant levels of the p53 protein were detected in both HTLV-2A and -2B virus-infected cell lines. The p53 was functionally inactive, however, in both transient transfection assays using a p53 reporter plasmid and in the induction of p53-responsive genes in response to gamma-irradiation. We further investigated HTLV-2A Tax and HTLV-2B Tax effects on p53 activity. Interestingly, although Tax-2A and -2B inactivate p53, the Tax-2A protein appears to inhibit p53 function less efficiently than either Tax-1 or Tax-2B. Of interest, the Tax-2B inhibition of p53 function is correlated with NF-kB activation and could be inhibited by overexpression of an IkBalpha dominant negative mutant. Comparison of the action of HTLV-1 and HTLV-2 Tax protein on p53 function will provide important insights into the mechanism of HTLV viral transformation. CD4+ T lymphocytes, the primary host for HTLV-I, undergo a series of changes which lead to T-cell activation, immortalization and transformation. To gain insight into the genetic difference between activated and HTLV-I-infected lymphocytes, we have performed Affymetrix GeneChip analyses of activated and HTLV-I-infected cells. Using the Hu6800 GeneChip, we identified approximately 763 genes whose expression is differentially regulated in at least three out of five HTLV-I cell lines. Classification of these genes into functional groups including cellular receptors, kinases, phosphatases, cytokines, signal proteins and transcription factors provides insight into genes and pathways that are differentially regulated during HTLV-I transformation. Current studies are underway to analyze the gene expression profile in control and drug treated leukemic cells.
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