Protein phosphatase 2A (PP2A) is very versatile owing to a large number of regulatory subunits and its ability to interact with numerous other proteins. The regulatory A subunit exists as two closely related isoforms designated Aalpha and Abeta. Mutations have been found in both isoforms in a variety of human cancers. Although Aalpha has been intensely studied, little is known about Abeta. We generated Abeta-specific antibodies and determined the cell cycle expression, subcellular distribution, and metabolic stability of Abeta in comparison with Aalpha. Both forms were expressed at constant levels throughout the cell cycle, but Aalpha was expressed at a much higher level than Abeta. Both forms were found predominantly in the cytoplasm, and both had a half-life of approx. 10 h. However, Aalpha and Abeta differed substantially in their expression patterns in normal tissues and in tumour cell lines. Whereas Aalpha was expressed at similarly high levels in all tissues and cell lines, Abeta expression varied greatly. In addition, in vivo studies with epitope-tagged Aalpha and Abeta subunits demonstrated that Abeta is a markedly weaker binder of regulatory B and catalytic C subunits than Aalpha. Construction of phylogenetic trees revealed that the conservation of Aalpha during the evolution of mammals is extraordinarily high in comparison with both Abeta and cytochrome c, suggesting that Aalpha is involved in more protein-protein interactions than Abeta. We also measured the binding of polyoma virus middle tumour antigen and simian virus 40 (SV40) small tumour antigen to Aalpha and Abeta. Whereas both isoforms bound polyoma virus middle tumour antigen equally well, only Aalpha bound SV40 small tumour antigen.

Cardiac tissues contain cells susceptible to and cells resistant to apoptosis, and this difference is important for normal morphogenesis during development and for abnormal loss of cells during pathogenesis such as myocardial infarction and heart failure. While efforts have been made to understand the cellular and intercellular events of apoptotic cells, the signaling mechanisms in cells surviving from apoptotic injuries have been overlooked. Understanding signal transduction processes in cells with apoptosis resistance is of crucial importance to develop better strategies of preserving post-mitotic cells. To this end, we performed studies in neonatal rat ventricular myocytes using oxidative stress (H₂O₂) as an apoptotic inducer. We identified a population of cells bearing higher resistance to apoptosis and found that the cells that survived from apoptotic insults had markedly higher levels of AKT and STAT3. Inhibition of AKT activity by a dominant negative AKT construct or by a PI3K inhibitor reduced active NF-ĸB and STAT3 expression without significantly altering the activity of the latter. Activation of AKT by a constitutively activated AKT construct caused the opposite effects. Direct activation of NF-ĸB also enhanced STAT3 expression, an effect abrogated by NF-ĸB inhibitor. On the other hand, knockdown of STAT3 by siRNA or inhibition of STAT3 activity by decoy oligodeoxynucleotides or by JAK2 inhibitor diminished AKT expression. In conclusion, cardiomyocytes possess an apoptosis-resistant property as a cytoprotection mechanism which is likely conferred by mutual transactivation between AKT/NF-ĸB and JAK2/STAT3, a novel crosstalk between the two signaling pathways within the networking governing the cell fate.

Epidermal growth factor receptor (EGFR) plays a critical role in mediating ultraviolet (UV) irradiation-induced signal transduction and gene expression in human keratinocytes. EGFR activation results from increased phosphorylation on specific tyrosine residues in the C-terminal intracellular domain. It has recently been reported that following growth factor stimulation EGFR translocates from the surface membrane to the nucleus, where it may directly regulate gene transcription. We have investigated the ability of UV irradiation to induce EGFR nuclear translocation in human primary and HaCaT keratinocytes. UV irradiation caused rapid nuclear translocation of EGFR. Significant accumulation of EGFR in the nucleus was observed within fifteen minutes after UV irradiation exposure. Maximal translocation occurred at 30 minutes post UV irradiation, and resulted in a 10-fold increase in EGFR in the nucleus, as determined by Western blot analysis of nuclear extracts and confirmed by immunofluorescence. Inhibition of nuclear export by Leptomycin B did not alter UV irradiation-induced nuclear accumulation. EGFR tyrosine kinase inhibitor (PD169540) reduced UV irradiation-induced EGFR nuclear translocation 50%. Mutation of either tyrosine 1148 or tyrosine 1173 reduced nuclear translocation 70%, while mutation of tyrosine 1068 was without effect. In addition, over-expression of receptor type protein tyrosine phosphatase-kappa (RPTP-κ), which specifically dephosphorylates EGFR tyrosines, decreased UV irradiation-induced EGFR nuclear translocation in human keratinocytes. These data demonstrate that UV irradiation stimulates rapid EGFR nuclear translocation, which is dependent on phosphorylation of specific EGFR tyrosine residues. EGFR nuclear translocation may act in concert with conventional signaling pathways to mediate UV irradiation-induced responses in human keratinocytes.Approximately 90% of human skin cancers, the most common human malignancies, are thought to be caused by solar UV irradiation [Koh, 1995]. In animal models, UV irradiation has been shown to be both a tumor initiator and a tumor promoter [Ananthaswamy and Pierceall, 1990;Staberg et al., 1983;Strickland, 1986]. UV irradiation can induce permanent DNA damage due to imperfect repair. UV irradiation also induces signal transduction pathways that lead to aberrant regulation of oncogenes and tumor suppressor genes. Both nuclear and non-nuclear initiated events seem to contribute to UV irradiation-induced biological effects. Elucidation of the mechanisms by which UV irradiation regulates gene expression is crucial for the understanding of UV irradiation-induced tumorigenesis in human skin.One of the earliest cellular responses to UV irradiation is phosphorylation and activation of certain cell surface growth factor receptors [Rosette and Karin, 1996;Sachsenmaier et al., 1994]. Among these receptors, epidermal growth factor receptor (EGFR) [Yarden and Sliwkowski, 2001]. EGFR is composed of an extracellular ligand binding domain, a single transmembrane do...

Metastasis is the leading cause of death for non‐small cell lung cancer (NSCLC) patients. However, how lung cancer cells invade blood vessels during metastasis remains unclear. Here, based on bioinformatics analyses, we found that PLEK2 might regulate NSCLC migration and vascular invasion. As little is known about the function of PLEK2 in NSCLC, we aimed to clarify this. We demonstrated that PLEK2 was significantly upregulated in transforming growth factor beta 1 (TGF‐β1)‐treated NSCLC cells through ELK1 transcriptional activation, highly expressed in NSCLC tissues, and negatively correlated with NSCLC overall survival. Meanwhile, PLEK2 overexpression significantly promoted NSCLC epithelial‐to‐mesenchymal transition (EMT) and migration, human lung microvascular endothelial cells endothelial‐to‐mesenchymal transition (EndoMT), and the destruction of vascular endothelial barriers. Moreover, PLEK2 knockdown inhibited TGF‐β1‐induced EMT and EndoMT. Furthermore, PLEK2 was found to directly interact with SHIP2 and target it for ubiquitination and degradation in NSCLC cells. Next, we confirmed that SHIP2 overexpression inhibits NSCLC EMT, migration and invasion and showed that PLEK2 overexpression can activate SHIP2‐associated TGF‐β/PI3K/AKT signaling. Our results suggest that PLEK2 could be a novel prognostic marker and potential therapeutic target for NSCLC metastasis and vascular invasion.

Quercetin, a natural polyphenolic flavonoid compound, can inhibit the growth of several malignant cancers. However, the mechanism still remains unclear. Our previous findings have suggested that quercetin can significantly inhibit HepG2 cell proliferation and induce cell apoptosis in vitro. It can also affect cell cycle distribution and significantly decrease cyclin D1 expression. In this study, we investigated the anti-cancer effect of quercetin on HepG2 tumor-bearing nude mice and its effect on cyclin D1 expression in the tumor tissue. First, the nude murine tumor model was established by subcutaneous inoculation of HepG2 cells, then quercetin was administered intraperitoneally, and the mice injected with saline solution were used as controls. The daily behavior of the tumor-bearing mice was observed and differences in tumor growth and survival rate were monitored. The expression of cyclin D1 in isolated tumor sections was evaluated by immunohistochemistry. We found that HepG2 tumor became palpable in the mice one-week post-inoculation. Tumors in the control group grew rapidly and the daily behavior of the mice changed significantly, including listlessness, poor feeding and ataxia. The mice in quercetin-treated group showed delayed tumor growth, no significant changes in daily behavior, and the survival rate was significantly improved. Finally, we observed increased tumor necrosis and a lighter cyclin D1 staining with reduced staining areas. Our findings thus suggest that quercetin can significantly inhibit HepG2 cell proliferation, and this effect may be achieved through the regulation of cyclin D1 expression.