Article
Inhibition of malignant glioma cell growth by retroviral shRNA-targeting of chromosomal passenger proteins Survivin and INCENP
Gezielte Inaktivierung der "Chromosomal Passenger"-Proteine INCENP und Survivin mittels retroviraler shRNA-Vektoren zur experimentellen Gentherapie des Glioblastomas multiforme
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Published: | April 11, 2007 |
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Objective: Glioblastoma multiforme, a malignant brain tumor, exhibits dysregulated apoptosis and enhanced cell growth due to genetic alterations and is resistant to conventional radiotherapy and chemotherapy. Thus, it is necessary to establish new approaches for a successful treatment. A new strategy for the destruction of glioma cells is the specific silencing of effector proteins of mitosis. By retroviral gene delivery we sought to introduce shRNAs for the inhibition of the “Chromosomal Passenger” proteins (CPPs) INCENP (Inner Centromeric Protein) and Survivin into glioma cells. Silencing of these proteins interferes with the cell cycle and leads to p53-independent cell death.
Methods: A retroviral vector system for delivery of shRNAs into glioma cells was established. RNAi-mediated down-regulation of protein expression was proven by Western Blot analyses and confocal laser scanning microscopy. The effects caused by the loss of Survivin and INCENP on the cell cycle were investigated by flow cytometry. Long-term survival of GBM cells with inactivated “Chromosomal Passenger” proteins was visualized by Giemsa staining.
Results: Transduction of cells with shRNA against either Survivin or shINCENP resulted in polyploidy and growth inhibition of glioblastoma cells. The high degree of polyploidy seen early after transduction was followed by apoptosis as detected by internucleosomal DNA fragmentation analysis. In addition, clonogenic assays verified the strong cytotoxic effects mediated by the transduced shRNA against Survivin and INCENP, respectively.
Conclusions: The inactivation of CPPs leads to mitotic defects and subsequently to apoptosis in glioma cells. This suggests a p53-independent inhibitory effect of shSurvivin and shINCENP on these cells, which might be beneficial for the treatment of malignant brain tumors.