Role of activating transcription factor 3 on TAp73 stability and apoptosis in paclitaxel-treated cervical cancer cells.

Taxol (paclitaxel) is a potent anticancer drug that has been found to be effective against several tumor types, including cervical cancer. However, the exact mechanism underlying the antitumor effects of paclitaxel is poorly understood. Here, paclitaxel induced the apoptosis of cervical cancer HeLa cells and correlated with the enhanced activation of caspase-3 and TAp73, which was strongly inhibited by TAp73beta small interfering RNA (siRNA). In wild-type activating transcription factor 3 (ATF3)-overexpressed cells, paclitaxel enhanced apoptosis through increased alpha and beta isoform expression of TAp73; however, these events were attenuated in cells containing inactive COOH-terminal-deleted ATF3 [ATF3(DeltaC)] or ATF3 siRNA. In contrast, paclitaxel-induced ATF3 expression did not change in TAp73beta-overexpressed or TAp73beta siRNA-cotransfected cells. Furthermore, paclitaxel-induced ATF3 translocated into the nucleus where TAp73beta is expressed, but not in ATF3(DeltaC) or TAp73beta siRNA-transfected cells. As confirmed by the GST pull-down assay, ATF3 bound to the DNA-binding domain of p73, resulting in the activation of p21 or Bax transcription, a downstream target of p73. Overexpression of ATF3 prolonged the half-life of TAp73beta by inhibiting its ubiquitination and thereby enhancing its transactivation and proapoptotic activities. Additionally, ATF3 induced by paclitaxel potentiated the stability of TAp73beta, not its transcriptional level. Chromatin immunoprecipitation analyses show that TAp73beta and ATF3 are recruited directly to the p21 and Bax promoter. Collectively, these results reveal that overexpression of ATF3 potentiates paclitaxel-induced apoptosis of HeLa cells, at least in part, by enhancing TAp73beta's stability and its transcriptional activity. The investigation shows that ATF3 may function as a tumor-inhibiting factor through direct regulatory effects on TAp73beta, suggesting a functional link between ATF3 and TAp73beta.

Microtubule-associated protein 1B light chain (MAP1B-LC1) negatively regulates the activity of tumor suppressor p53 in neuroblastoma cells.

The tumor suppressor and transcription factor p53 is a key modulator of cellular stress responses and can trigger apoptosis in many cell types, including neurons. In this study, we have shown that the Microtubule-Associated Protein 1B (MAP1B) light chain can interact with the tumor suppressor p53. We also demonstrate that both p53 and the MAP1B light chain (MAP1B-LC1) alter their localization from the cytoplasm to the nucleus when neuroblastoma cells, SH-SY5Y, are treated with doxorubicin. Additionally, we demonstrate that the MAP1B-LC1 negatively regulates p53-dependent transcriptional activity of a reporter construct driven by the p21 promoter. Consequently, MAP1B-LC1 binds to p53 and this interaction leads to the inhibition of doxorubicin-induced apoptosis in SH-SY5Y cells.

p19(ras) amplifies p73beta-induced apoptosis through mitochondrial pathway.

p73 and p53 have been known to play an important role in cellular damage responses such as apoptosis. Although p73 is a structural and functional homolog of p53 tumor suppressor gene, much less is known about the mechanism of p73-induced apoptotic cell death. In this study, we demonstrate that p19(ras) interaction with p73beta amplifies p73beta-induced apoptotic signaling responses including Bax mitochondrial translocation, cytochrome c release, increased production of reactive oxygen species (ROS) and loss of mitochondrial transmembrane potential (DeltaPsi(m)). Furthermore, endogenous expression of p19(ras) and p73beta is significantly increased by Taxol treatment, and Taxol-enhanced endogenous p73beta transcriptional activities are further amplified by p19(ras), which markedly increased cellular apoptosis in p53-null SAOS2 cancer cell line. These results have important implications for understanding the molecular events of p19(ras) to p73 functions in cancer cells.

TIP60 represses transcriptional activity of p73beta via an MDM2-bridged ternary complex.

TIP60, a histone acetyl transferase, acts as a p53 coactivator by interfering with MDM2-mediated degradation of p53. However, little is known about its functional regulation of p73, which has structural features similar to p53. In this study we found that TIP60 represses apoptosis, which is induced by exogenous and endogenous p73beta. TIP60 also negatively regulated the expression of p73beta downstream target genes such as p21 and Bax. Moreover, the specific repression of p73beta-mediated transactivation by TIP60 was independent of p53 expression and not due to histone deacetylase recruiting transcriptional machinery. Transcriptional activities of both p73 splicing variants, p73alpha and p73beta, were also repressed by TIP60. Furthermore, TIP60 markedly enhanced p73beta binding affinity to MDM2 and physically associated with MDM2 through its zinc finger domain, which is specifically localized in the nucleus. Therefore, we demonstrate that TIP60 forms a ternary complex with p73beta, which is directly bridged by MDM2. It is important to note that our findings contribute to a functional linkage between TIP60 and p73beta through MDM2 in the transcriptional regulation of cellular apoptosis.

Functional cross-talk between p73beta and NF-kappaB mediated by p300.

p73beta is associated with induction of apoptosis or cellular growth arrest, while NF-kappaB is closely related with promotion of resistance to programmed cell death. These biologically opposing activities between p73beta and NF-kappaB propose a regulatory mechanism of critical turning on/off in cellular apoptotic or survival responses. In this study, we demonstrate that NF-kappaB-mediated transactivation is specifically downregulated by p73beta; conversely, p73beta-transactivation is negatively regulated by functional expression of p65, NF-kappaB RelA subunit. The p73beta transactivation domain (TA) and p65 NH2-terminus are crucial for their negative regulation of p65- and p73beta-mediated transactivation, respectively. Furthermore, p65- or p73beta-interaction with p300 is reciprocally inhibited by their competitive binding to p300 in a restrict amount-dependent manner. Likewise, both p73beta-activated apoptosis and p65-dependent increase of cell viability are reciprocally repressed by p65 and p73beta, respectively. These results have important implications for p300-mediated regulatory mechanism between p73beta- and p65-transactivation, by which both p73beta and NF-kappaB could mutually affect on their biological activities. Therefore, we propose that p300 is a transactivational regulator of competitively balanced cross-talk between p73beta and p65.

p19ras interacts with and activates p73 by involving the MDM2 protein.

p73beta is a structural and functional homologue of p53, a tumor suppressor gene. In this study, we identified a novel p73beta-binding protein, p19ras, by the yeast two-hybrid screening method. Alternative splicing of the proto-oncogene H-ras pre-mRNA has led to two distinct transcripts, p19ras and p21ras. In both endogenous and overexpressed systems, we confirmed that p19ras binds to full-length p73beta in vivo and in vitro. Coexpression of p19ras with p73beta stimulated the transcriptional activity of p73beta. Ras proteins are known to be small membrane-localized guanine nucleotide-binding proteins. However, unlike other Ras proteins, p19ras is localized in the nucleus and the cytosol and its interaction with p73beta occurred exclusively in the nucleus. Oncogenic MDM2 (mouse double minutes 2) is a known repressor of p73 transcriptional activity. In this study, when p19ras was bound to MDM2, it further inhibited the association of MDM2 to the p73beta protein. In addition, p19ras abolished MDM2-mediated transcriptional repression of p73beta. Therefore, this study presents a novel pathway of Ras signaling that occurs in the nucleus, involving p19ras and p73beta. Furthermore, a p19ras-mediated novel regulatory mechanism of p73 involving the MDM2 protein is described.