gms | German Medical Science

Objective: We have previously shown that RNAi of survivin blocks proliferation of malignant glioma cells in vitro and significantly inhibited growth of subcutaneous as well as orthothopic glioma xenografts [1]. Survivin is involved in two main cellular processes, cell division (serving as Chromosomal Passenger Protein, CPP) and inhibition of apoptosis (inhibitor of apoptosis protein, IAP). Our previous results suggest that RNAi of survivin primarily affects the cell cycle but does not induce apoptosis in permanent glioma cell lines and primary glioblastoma cell preparations. However, the mechanism leading to the observed cell cycle disturbances remained elusive. In this project we sought to elucidate the role of p53 in the cell cycle regulation and chromosomal stability of U87-MG glioma cells following knock down of survivin.

Methods: P53 was stable knocked down in cell lines using a retroviral shRNA-targeting vector expressing hygromycin. After selection cell lines were tested for downregulated p53 and in following experiments were compared to wild type cells. Survivin was stably knocked down by a retroviral vector encoding the small hairpin shRNA #433. As control we used a vector encoding a shRNA targeting firefly luciferase. Cells were subjected to cell cycle analysis, apoptosis assays, Western blot analysis, flow cytometry, karyotype analysis and proliferation analysis.

Results: Knock down of p53 led to a significant increase in the amount of polyploid cells with defective mitosis after RNAi of survivin when compared to U87-MG wild type cells. However, an induction of p21waf/cip on the steady state protein level was only observed in U87-MG wild type cells expressing functional p53. Further FACS and Western blot analyses revealed neither functional cell cycle arrest nor an increase in activated Caspase 3 in U87-MG wild type cells. In summary, it appeared that a knock down of survivin had no fundamental impact on DNA synthesis rates and apoptosis.

Conclusions: Our results suggest that knock down of survivin severely affects proliferation and survival of glioma cells irrespective of p53 status confirming its role as CPP. RNAi therapies targeting survivin or other genes essential for proliferation and migration might represents a promising avenue for adjuvant local treatment of secondary and primary glioblastomas.