Study On The Inhibitory Effects Of Antisense HTERT On Proliferation Of Malignant Glioma Cells

Objective: One of the most important characteristics of Glioblastoma Multiforme (GBM) is its rapid proliferation and progression. The prognosis for GBM, which is categorized as a grade IV gioma in WHO classification, has a survival time of less than 14 months. Malignant gliomas are considered incurable, thus, the search for a novel therapy measure is essential.The proliferation of malignant tumor cells is believed to correlate tight with abnormal activation of telomerase, a DNA polymerase. As observed in recent studies, the protein component of telomerase, human telomerase reverse transcriptase (hTERT), has been detected in malignant gliomas (MG), but also has been expressed in high levels. This protein subunit of telomerase may be the key to promote the cell proliferation process in aggressive cancers like GBM. One particular reason is that hTERT is a rate limiting component in telomerase formation, thus, it makes a very promising target of gene therapy. Another critical reason is that hTERT is one of two essential components of telomerase.In present study, we introduced a complimentary DNA of hTERT in antisense to block its post-transcriptional expression in malignant glioma cells, of which mRNA hTERT is found in abundance in TJ905 cells. In doing so, we expected a ripple effect that would obstruct the formation of telomerase. As a result of reducing the telomerase activity, the proliferation of malignant glioma cells would be inhibited.Methods: In order to validate the efficacy of this antisense technology, we chose four methods to identify cell proliferation. But first, the expression vectors of sense hTERT and antisense hTERT, and pcDNA3.0 (vector control) were transfercted into TJ905 GBM cell line (from Tianjin Neurology Institute, Branch of Neurooncology) by Lipofectamine 2000 and was selected by G418. After sufficient passage of these cells, we applied a clonogenic assay and MTT to assess the growth of these transfected cell groups. These experimentsexplore the growth potential of the transfected glioma cells against the control cell line. With the MTT experiment, we delved deeper in studying the kinetics of cell proliferation through immunohistochemical staining and flow cytometry. Ki-67(MIB-1) immunohistochemistry staining enables a visual of the cell proliferating activity. Flow cytometry would give an idea of what phase these various groups of cells were in.Results: After 14 day or so of selection by G418, TJ905 GBM cells with sense, antisense and pcDNA3.0 vector were ready to be used. Clonogenic study was assessed for 8 days for control group, Sense hTERT and antisense hTERT. The colony formation at day 4 for antisense hTERT was found on an average of 3.3 ±1.7, whereas for sense hTERT and control groups, 76.0 ±9.4 and 67.0 ±14.8 respectively (P<0.05 antisense vs sense, P<0.01 antisense vs control). From day 1 to day 5, we concluded by looking at metabolically active cells in the MTT study, antisense cells grew 1.5 times slower than that of control group (P<0.05). In immunohistochemical study, Ki-67 label index about 86.0% of control TJ905 GBM cells, which compared to 35.8% of antisense hTERT cells and 54.2% of sense hTERT cells;a 2.4 fold reduction of the proliferating cells in cell cycle (P<0.001). Our cell cycle test labeled on average 31.5% of control cells in active S phase, whereas a 0.0% of antisense hTERT cells were found. Antisense hTERT cells were found to have 75.8% in Go/G₁ phase and a 24.2% in G₂/M phase.Conclusion: These results indicated a significant reduction in the glioma cell growth by the blocking hTERT through antisense RNA. From these results, we concluded that with reduced hTERT mRNA expression, telomerase formation post-transcriptionally was also affected. Hence, glioma cell proliferation was impeded.