Study On The Effects Of Telomerase Activity Inhibition On The Cell Proliferation And Apoptosis In Human Gliomas And The Underlying Mechanisms

Objective:To observe the inhibition of specific telomerase inhibitior 3'-azido-2', 3'-dideoxythymidine(AZT) on telomerase activity(TA) in human glioblastoma cell line TJ905. then investigate it's effects on the cells proliferation and apoptosis when TA was inhibited. Detect the changes of the tumor suppressing gene, Inhibitor of growth 1(ING1) mRNA expression level when telomerase activity was inhibited by AZT and analyse the relationship between the change of ING mRNA expression level and cell proliferation and apoptosis of glioblastoma cells. This study was aim to investigate the downstream mechanisms of cells proliferation and apoptosis in TJ905 cell line when telomerase activity was reduced. To research the mechanisms of AZT can inhibit cells proliferation and induce cells apoptosis in the malignant glioma. We investigated the effects of P33^ING1b induce cells senescence before mature. The object of this study were aim to evaluate the effect and outlook of AZT to cure glioma, and to present the foundation of theory and experiment about the target by telomerase to cure malignant glioma.Methords:Human malignant glioma cell lines TJ905 were cultured in DMEM media by 1～5×10⁵ cells/ml. Exponentially growing cells were passaged then divided into four groups: (1)control: without AZT (2) AZT50μM group: treated with 50μM AZT; (3) AZT100μM group: group: treated with 100μM AZT; (4) AZT200μM group: treated with 200μM AZT. The first generation, the third generation and the sixth were selected for the following detections. Telomerase activity was assessed by using the telomerase repeat amplification protocol(TRAP). The effects of AZT on proliferative inhibition in human glioblastoma cell line TJ905 were assessed by means of colony formation assay, Ki-67 immunocytochemical stain, GFAP immunocytochemical stain, we use flow cytometry,TUNEL and single cell gel electrophoresis assay(SCGE), which is comet assay evaluating the effects of AZT on apoptotic induction in human glioblastoma cell line TJ905. P33^ING1b was amplified by RT-PCR, then we detected the changes of p33^ING1b mRNA in four groups.Results:The result of TRAP showed that the telomerase activity of three treatment groups were lower than the control group, especially AZT 200μM group (p＜0.001). This result demonstrated that AZT can inhibit the telomerase activity of TJ905 cells and the inhibition was dose dependent.Colony formation assay showed that compared to the control, the colony formation ratio of the treatment groups were reduced, the ratio of 200μM AZT group were lower than other treatment groups, and the difference was significant (p＜0.001). Compare to the first generation, three treatment groups Ki-67 labeling index reduced, but GFAP labeling index increased in the sixth generation, and the difference was significant (p＜0.001). These results confirmed that AZT can inhibite cells proliferation in TJ905 cell line by inhibiting telomeraseTheseusFrom Single cell gel electrophoresis assay, The comet assay, we found the three threatment groups had more apoptotic cells(p＜0.001) than the control group, and the apoptotic index(AI)was higher than the control(p＜0.001). In the sixth generation, the tail measurement of apoptotic cells increased obviousely, which indicated that the DNA were cutted severely. In TUNEL test, the apoptosis index was higher in the first generation than the control, when passaged to the sixth generation, the apoptotic index was higher than the first generation, the AI of the AZT 200μM group was the highest, and the differences were significant(p＜0.01), the results were similar to the comet assay. From the flow cytomety, we can saw apoptotic peaks in the three treatment groups, and the apoptosis ratio were lower. Until passaged the sixth generation. the ratio achieved 29ï¼…. The flow cytomety came to conclusion that AZT can induce cell apoptosis when the telomerase activity was inhibited. Although there was no apoptotic peak in other generations, the cell cycle changed. In treatment groups, the number of cells in the G0/G1 period were more than the control group, otherwise in the S Period. This result demonstrated that AZT blocked the cell to transit from G0/G1 period to S period.Compare to the control group, the relative gray scale value of P33^ING1b mRNA increased with the AZT concentration, moreover the value was higher in the sixth generation than in the first generation, and the differences were significant (p＜0.01). P33^INGIb mRNA expression level correlated negatively with colon formation ratio and KI-67LI(r=-0.994～-0.990, P＜0.01) but positively with Apoptotic Index. (r=0.996～0.999, P＜0.01).Conclusions:From the present study, we prove that:1. Abnormally reactived telomerase lead to malignant glioma infinite prolifation and apoptosis inhibition. AZT can inhibit the telomerase activity of TJ905 cells line, and the inhibitionwas dose dependment.2. The inhibition of the telomerase activity by AZT make P33^ING1b mRNA expression level up-regulated, and the level was directly relative to the concentration of AZT.3. When telomerase activity was inhibited, the effect of AZT on cells prolifation and cells senescence induce was carried out by blocking the cell transit from G0/G1 period to S period.4. P33^ING1b mRNA expression level correlated negatively with colon formation ratio and KI-67LI, but positively with GFAPLI and the apoptosis degree.5. These results demonstrated that telomerase reactivation inhibited the P33^ING1b mRNA express by direct or indirect means, which lead to proliferation improved and apoptosis inhibited in malignant glioma. AZT can effectively blocked telomerase activity reactivation, AZT had therapeutical effect on tumor by inhibiting cells proliferation and inducing cells apoptosis.6. To present the foundation of theory and experiment about the target by telomerase to cure malignant glioma.