The Mechanism Of Telomerase In Repairing And Modifying Apoptosis Of Tumor Cells

Recently it was shown that 85-90% tumors show very high levels of expression of telomerase. Radiotherapy is a main treatment for cancers. Apoptosis and potentially lethal damage repair serve as the determinal factors for curative effects of radiotherapy. Telomeres have been considered as the hotspots of high sensitivity to radiation damage. However, wether repairing of damaged telomeres is a mechanism of potentially lethal damage repair has not been clarified until now, and little is understood about the mechanism of telomerase performing repairing at damaged telomeres and modifying apoptosis. The viewpoint of telomerase modifying apoptosis by repairing damaged telomeres urgily requires the suprots delived from experiments. The study is intending to elucidate the mechanism of telomerase performing repair and modifying apoptosis and introducing telomerase into clinic for improving radiotherapy ultimately. The study has investigated the mechanism of telomerase in repairing and modifying radiation-induced apoptosis by TRAP, Southern blotting, RT-PCR, FCM and in situ telomerase-apoptosis double staining, and further researched recently adopted RNAi as a method of inhibiting telomerase to inhence radiation-induced apoptosis of tumor cells, the results indicating that telomerase plays an important role in repairing and developing of apoptosis. Both the technique of in situ telomerase-apoptosis double staining and the results gained by the method have not been seen in articles until now, and technique has been proved successful;either the exploring applications of RNAi in radiation damages has not been seen in articles . Based on the supports delived from the experiment, it is proposed that telomerase modifying apoptosis by adding telomere sequences to the ends of damaged telomeres(telomerelentherning) is an important mechanism of potentially lethal damage repair, wich further perfects the mechanism of potentially lethal damage repair, and offer theoretic supports for forecasting tumor cell' s sensitivities to radiation by detecting the expression of telomerase and inhencing tumor cell' s sensitivities to radiation by inhibiting telomerase.The main results are as follows:1. Telomerase activities and hTERT in A549 and L02 were assayed by RT-PCR based TRAP and in situ hybridization. A dose-dependent increases in activity of telomerase after exposure to lGy-5Gyyray were approximately 1. 3-2. 5 times of control unirradiated cell levels. These maxima were reached at 48h-72h after irradiation, demonstrating that radiation can up-regulate telomerase activity. Increased expressions of hTERT demonstrates exist of the mechanism of induced transcription of telomerase.2. For the first time directly conforming retained high levels of the enzymes in apoptotic cells first by in situ telomerase-apoptosis double staining and observing up-regulation of expression of telomerase in the development of apoptosis suggests that the killing effects of radiation on cells are not by means of depressing telomerase. The fact that higher levels of expression of telomerase and lower rates of apoptosis in A549 and opposition in L02 demonstrate that telomerase can repair DNA damages and suppress apoptosis.3. Southern blotting showed that TRF lengthened 1kb after exposure to radiation, and there were dosages and timing effects approximate in common between TRF lengthening and up-regulating of the activities of telomerase, testifying that telomere damages can be repaired mainly by means of de nova composition, and further, elucidating that telomerase can modify the development of apoptosis via altering the lengths of telomere and blocking out initiates of apoptosis. Theresults indicate that tumor cell' s sensitivities to radiation may be forecasted by detecting the expression of telomerase. Based above, a connection was introduced as: irradiation- telomere damage-telomerase activating - repairing damaged telomeres(potentially lethal damage repair) - blocking out initiates of apoptosis and modifying the sensitivities to apoptosis-forecasting...