ABSTRACT
The PTEN gene regulates multiple signaling pathways that influence cell proliferation, survival and differentiation. Loss of PTEN expression is closely linked with oncogenesis. Little is known regarding regulation of PTEN gene expression. The PTEN promoter region has been reported and is regulated in part by p53. In a previous study, we found that Notch-1 signaling resulted in increased PTEN protein expression. Herein, we tested the hypothesis that the PTEN gene is a direct target of Notch-1 signal transduction, through binding of the Notch-activated transcription factor CBF-1 to the PTEN minimal promoter. 293 cells expressing constitutively active Notch-1 exhibited increased PTEN gene expression and promoter transactivation. Overexpression of CBF-1 in 293 cells resulted in decreased PTEN gene expression. Mobility shift assays and supershift assays demonstrated that CBF-1 binds to the PTEN minimal promoter. These data indicate that the Notch-1 receptor pathway is a key regulator of PTEN gene transcription.
Subject(s)
Gene Expression Regulation/physiology , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , PTEN Phosphohydrolase/metabolism , Promoter Regions, Genetic/physiology , Base Sequence , Cell Line , Humans , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Molecular Sequence Data , PTEN Phosphohydrolase/biosynthesis , PTEN Phosphohydrolase/genetics , Protein Binding/geneticsABSTRACT
Endostatin is a 20-kDa endogenous angiogenesis inhibitor that has recently been shown to inhibit the expression of vascular endothelial growth factor (VEGF), an angiogenic growth factor that is up-regulated by hypoxia via the HIF-1 transcription factor complex. To determine if the anti-angiogenic activity of endostatin involves a modulation of the HIF-1/VEGF pathway in cancer cells, experiments were conducted to establish what effect endostatin has on HIF-1 activity, HIF-1alpha protein production, and cellular localization in prostate cancer cells and endothelial cells. Endothelial cell tube formation was inhibited by endostatin purchased from Calbiochem (San Diego, CA) but not endostatin obtained from EntreMed (Rockville, MD). Subsequent experiments using Calbiochem endostatin showed that it did not alter HIF-1alpha protein production or cellular localization in any of the cell lines tested, nor did it alter HIF-1 transactivational activity in hypoxia. Whether or not this is also true in vivo remains to be determined. Nevertheless, these data suggest that the anti-angiogenic activity of endostatin is independent of the HIF-1/VEGF pathway. Immunocytochemical staining results do not indicate a decreased production of VEGF in Calbiochem endostatin-treated LNCaP or human umbilical vein endothelial cells (HUVEC). Treatment of rat aortic cross sections with human endostatin from Calbiochem resulted in a dose-dependent inhibition of microvessel outgrowth. Importantly, inhibition of vessel outgrowth by Calbiochem endostatin in a human saphenous vein angiogenesis assay required early treatment. In view of this in vitro data, we suggest that clinical trials involving endostatin treatment of late-stage disease may not adequately represent the efficacy of this drug in early-stage cancer.
