ABSTRACT
PURPOSE: Glioblastomas (GBMs) are the most common primary malignant brain tumors. Majority of GBMs has loss of heterozygosity of chromosome 10. The PAX6 encodes a transcription factor that involves in development of the brain, where its expression persists. We have reported that the expression of PAX6 was significantly reduced in GBMs and that a low level of PAX6 expression is a harbinger of an unfavorable prognosis for patients with malignant astrocytic glioma. Interestingly, PAX6 expression was increased in suppressed somatic cell hybrids derived from introducing a normal human chromosome 10 into U251 GBM cells. Thus it is interesting to determine if repression of PAX6 expression is involved in anti-tumor suppression function in GBM. EXPERIMENTAL DESIGN: We overexpressed PAX6 in a GBM cell line U251HF via either stable transfection or infection with recombinant adenovirus, and examined cell growth in vitro and in vivo. RESULT: Although we did not observe changes in the cell doubling time for PAX6-stable transfectants, significantly fewer numbers of PAX6-positive colonies grew in soft agar. Transient overexpression of PAX6 via adenovirus, however, suppressed cell growth by increasing the number of cells in G1 and by decreasing the number of cells in S-phase, and later on caused a dramatic level of cell death. Repeated subcutaneous and intracranial implantation experiments in nude mice using PAX6-stable transfectants provided solid evidence that PAX6 suppressed tumor growth in vivo and significantly extended mouse survival. CONCLUSION: Our data demonstrate that PAX6exerts a tumor suppressor function that limits the growth of GBM cells.
Subject(s)
Chromosomes, Human, Pair 10/genetics , Eye Proteins/metabolism , Gene Expression Regulation, Neoplastic/genetics , Glioblastoma/genetics , Homeodomain Proteins/metabolism , Nervous System Neoplasms/genetics , Repressor Proteins/metabolism , Animals , Cell Line, Tumor , Cell Proliferation , Eye Proteins/genetics , Genes, Tumor Suppressor , Homeodomain Proteins/genetics , Humans , Loss of Heterozygosity/genetics , Mice , Mice, Nude , PAX6 Transcription Factor , Paired Box Transcription Factors , Repressor Proteins/genetics , TransfectionABSTRACT
Inactivation of the tumor suppressor gene PTEN and overexpression of VEGF are two of the most common events observed in high-grade malignant gliomas. The purpose of this study was to determine whether PTEN controls VEGF expression in gliomas under normoxic conditions. Transfer of PTEN to human glioma cells resulted in the transduction of a functional PTEN protein as evidenced by the upregulation of p27 and modification of the phosphorylation status of Akt. Under normoxic conditions, enzyme-linked immunosorbent assay and Northern blot analyses showed downregulation of VEGF in PTEN-treated cells. Moreover, conditioned media from PTEN-treated glioma cells significantly diminished the ability of endothelial cells to grow and migrate. Western blot assays demonstrated that, in a normoxic environment, PTEN downregulates HIF-1 alpha. Finally, promoter activity assays showed that the VEGF promoter region containing the HIF-1alpha binding site is necessary and sufficient for PTEN-mediated downregulation of VEGF. Experiments with PI3-K inhibitors and kinase assays suggested that PI3-K is mediating the effect of PTEN on VEGF, and not the p42/p48 or p38 MAP kinases. These results indicate that restoration of PTEN function in gliomas may induce therapeutic effect by downregulating VEGF. Furthermore, this close functional relationship between PTEN and VEGF suggests that a better understanding of the transduction signal regulated by PTEN might enhance the knowledge of the cause and physiology of vascular and inflammatory diseases.
