Our Science – Masison Website
Cynthia A. Masison, Ph.D.
Our research program is focused on understanding human T-cell lymphotropic virus type-1 (HTLV-1)pathogenesis. HTLV-1 is closely associated with the human cancer adult T cell leukemia (ATL) and the neurologic disease HTLV-1-associated myelopathy (HAM/TSP). The laboratory is interested in interactions between the virus and cell that influence viral gene regulation, viral pathogenesis, and oncogenic transformation. Highlights from recent research projects are given below.
INHIBITION OF p53 TUMOR SUPPRESOR BY HTLV-1 TAX PROTEIN
p65/RelA Inhibits p53 Transcription Activity by Direct Interaction and Inhibition of Preinitiation Complex Formation. NF-kB activation plays a critical role in oncogenesis by HTLV-I, the etiologic agent of ATL, and is indispensable for maintenance of the malignant phenotype. In T-lymphocytes, Tax-mediated p53 inhibition is dependent on Tax activation of the NF-kB pathway and is linked to p53 phosphorylation. Recently we reported that blocking NF-kB transcriptional activation in HTLV-I-transformed cells restores p53 activity. Further, using MEF null cells and antisense oligonucleotides to inhibit expression of NF-kB family members, we demonstrated that the p65 subunit of NF-kB is uniquely involved in p53 inhibition. Coimmunoprecipitation assays showed an interaction between p65 and p53 in HTLV-I-transformed cells. In transient transfection assays, Tax induced the p53-p65 interaction. Phosphorylation of p53 at serines 15 and 392 was critical for complex formation. Importantly, Tax-mediated p53 inhibition correlated with p65 and p53 interaction. Using ChIP assays in HTLV-I-transformed cells, we found that p53 and p65 form a complex on the inactive, p53-responsive, MDM2 promoter. Consistent with reduced transcriptional activity, TFIID binding was not observed. These studies identify a unique mechanism for p53 regulation by the p65/RelA subunit of NF-kB. (Blood, 2004)
A novel NF-kB pathway involving IKKβand p65/RelA Ser-536 phosphorylation results in p53 inhibition in the absence of NF-kB transcription activity. Nuclear factor kappa B (NF-kB) plays an important role in regulating cellular transformation and apoptosis. Human T-cell lymphotrophic virus type I (HTLV-I) Tax, which is critical for viral transformation, modulates the transcription of several cellular genes through activation of NF-kB. We have previously demonstrated that Tax inhibits p53 activity through the p65/RelA subunit of NF-kB. We now present evidence which suggest that upstream kinase IKKβ plays an important role in Tax-induced p53 inhibition through phosphorylation of p65/RelA at Ser-536. First, mouse embryo fibroblasts (MEFs) IKKβ-/- cells did not support Tax-mediated p53 inhibition while MEFs lacking IKKα allowed Tax inhibition of p53. Second, transfection of IKKβ wild type (WT), but not a kinase dead (KD) mutant, into IKKα-/- cells rescued p53 inhibition by Tax. Third, the IKKβ specific inhibitor SC-514 decreased the ability of Tax to inhibit p53. Fourth, we show that phosphorylation of p65/RelA at Ser-536 is important for Tax inhibition of p53 using MEF p65/RelA-/- cells transfected with p65/RelA WT or mutant (MT) plasmids. Moreover, Tax induces p65/RelA Ser-536 phosphorylation in WT or IKKα -/- cells but failed to induce the phosphorylation of p65/RelA Ser-536 in IKβ-/- cells, suggesting a link between IKKβ and p65/RelA phosphorylation. Consistent with this observation, blocking IKKβ kinase activity by SC-514 decreases the phosphorylation of p65/RelA at Ser-536 in the presence of Tax in HTLV-I transformed cells. Finally, the ability of Tax to inhibit p53 is distinguished from the NF-κB transcription activation pathway. Our work, therefore, describes a novel Tax-NF-κB p65/RelA pathway that functions to inhibit p53, but does not require NF-κB transcription activity. (J. Biol. Chem., 2005)
Activated AKT regulates NF-kB activation, p53 inhibition and cell survival in HTLV-1-transformed cells. AKT activation enhances resistance to apoptosis and induces cell survival signaling through multiple downstream pathways. We now present evidence 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 IKK2 phosphorylation of p65 and causes a reduction of p65 Ser-536 phosphorylation in vivo, steps critical to p53 inhibition. Interestingly, blockage of AKT function did not affect IKK2 phosphorylation of IkBa in vitro suggesting selective activity of AKT on the IKK2 complex. Finally, AKT pro-survival function in HTLV-1-transformed cells is linked to expression of the Bcl-xL gene. 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. (Oncogene, 2005)
PI3/AKT inhibition induces caspase-dependent apoptosis in HTLV-1 transformed cellsThe phosphatidylinositol-3-kinase (PI3K) and AKT (Protein Kinase B) signaling pathways play an important role in regulating cell cycle progression and cell survival. In previous studies, we demonstrated that AKT is activated in HTLV-1 transformed cells and that Tax activation of AKT is linked to p53 inhibition and cell survival. In the present study, we extend these observations to identify regulatory pathways affected by AKT in HTLV-1-transformed cells. 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. Of interest, p53 siRNA prevents LY294002-induced apoptosis in HTLV-1-transformed cells, suggesting that p53 reactivation is linked to apoptosis. In conclusion, the AKT pathway is involved in targeting multiple proteins which regulate caspase- and p53-dependent apoptosis 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. (Virology, In press, 2007)
HTLV-1 TRANSCRIPTION REGULATION
Tax relieves transcriptional repression by promoting HDAC1 release from the HTLV-I LTR. Expression of human T-cell leukemia virus type I (HTLV-I) is regulated by the viral transcriptional activator Tax. Tax activates viral transcription through interaction with the cellular transcription factor CREB and the coactivators CBP/p300. We have analyzed the role of histone deacetylase-1 (HDAC1) on HTLV-I gene expression in HTLV-I transformed cells and in a cell line that contains an integrated single copy of the HTLV-I LTR. TSA, a HDAC inhibitor, enhanced Tax expression in the HTLV-I transformed cells, whereas overexpression of HDAC1 repressed Tax transactivation in the cell line with the integrated template. ChIP analysis of the interaction of transcription factors, coactivators, and HDACs with the basal and activated HTLV-I promoter revealed that HDAC1 is associated with the inactive, but not the Tax transactivated HTLV-I promoter. Furthermore, in vitro and in vivo GST-Tax pull-down and coimmunoprecipitation experiments demonstrated a direct physical association between Tax and HDAC1. Importantly, biotinylated chromatin pull-down assays indicated that Tax inhibits and/or dissociates the binding of HDAC1 to the HTLV-I promoter. Our results provide evidence that Tax interacts directly with HDAC1 and regulates the binding of the repressor to the HTLV-I promoter. (J. Virol., 2005)
This page was last updated on 4/23/2014.