Telomere Binding Protein PTOP Affects The Radio Resistance Of MDA-MB-435s Breast Cancer Cell

Part I Establish radio resistant breast cancer cell mode of MDA-MB-435sObjective In order to study the affect of telomere binding protein factor PTOP and TRF1(Telomere repeat binding factor1) to the radio resistance of breast cancer cell, the first step was to establish an radio resistance breast cancer cell mode. After evaluation of the MCF-7, MDA-MB-231and MDA-MB-435s cells, we chose MDA-MB-435s as the parent cell line to establish the radio resistant mode.6Mv X-ray in total60Gy dose was given in different fraction to irradiate the MDA-MB-435s breast cancer cell, and2kinds of stable radio resistant breast cancer cell lines MDA-MB-435s R60/2and MDA-MB-435s R60/6were expected.Methods Irradiated the MDA-MB-435s cell in2ways, which were2Gy/fraction and6Gy/fraction respectively in a total60Gy dose by a Simens RPMIus Accelerator. All the cells were cultured in PRMI1640medium (Hyclone) with10%fetal bovine serum, all the cells were grown approximate80%-90%confluence until the next irradiation. After finishing the irradiation of60Gy dose, the irradiated cell lines called MDA-MB-435s R60/2and MDA-MB-435s R60/6, were passed at least8generation. In the meanwhile, we performed the clonogenic assay experiments for the1st,5th, and8th generation cell lines of MDA-MB-435s R60/2and MDA-MB-435s R60/6, compare with the parent cell line MDA-MB-435s to evaluate whether they had the stable radio resistance.Results MDA-MB-435s R60/2and MDA-MB-435s R60/6cell lines were accepted an irradiation of6Mv X-ray in total dose of60Gy and the passed8th generation. We performed the clonogenic assay in the1st,5th and8th generation of MDA-MB-435s R60/2and MDA-MB-435s R60/6cell lines. From the results of the experiments, we observed that, compare to the parent cell MDA-MB-435s, the survival fraction of each generation of MDA-MB-435s R60/6and MDA-MB-435s R60/2were much higher at each point than the MDA-MB-435s cell. The radiobiological parameters, such as DO, Dq and SF2, their values in MDA-MB-435s, MDA-MB-435s R60/6and MDA-MB-435s R60/2on average were significant higher than the parent cell line (p<0.05). From these data, MDA-MB-435s R60/2and MDA-MB-435s R60/6cell lines had much more stable radio resistant compare to the parent cell MDA-MB-435.Conclusion Based on the parent cell MDA-MB-435s, we gave different dose per fraction to establish2kinds of radio resistant cell lines MDA-MB-435s R60/2and MDA-MB-435s R60/6. They were not only processing the same biological characters as the parent cell, but also obtained a stable heritable radio resistance. The successful of the 2radio resistant cell modes laid the foundation for the further study of telomere binding proteins. Part Ⅱ The expression of Telomere binding protein PTOP and TRF1in the radio resistant cell lines and the changes of telomere activitiesObjective The telomere binding protein PTOP was highly expressed in the radio resistant cell in some studies. The function of TRF1was controversial, some research though it worked as a negative regulator of telomere length, however, the others claimed it was a positive regulator. In our study, we used the MDA-MB-435s R60/2and MDA-MB-435s R60/6cell lines to detect the expression of telomere binding factor PTOP and TRF1, the changes of telomere length and telomerase activity in highly expressed radio resistant cell lines.Methods We used Western blot and RT-PCR to detect the expression of telomere binding protein PTOP and TRF1in both protein and mRNA level in MDA-MB-435s R60/2and MDA-MB-435s R60/6radio resistant cell lines. We performed the telomerase activity assay by Telomerase activity ELISA kit and measured the telomere length by Southern blot Assay kit.Results Compare to the parent cell MDA-MB-435s, the expression of telomere binding protein PTOP and TRF1in MDA-MB-435s R60/2and MDA-MB-435s R60/6radio resistant cell lines were significant highly expressed in not only protein level but also mRNA level. In another hand, the telomerase activity in MDA-MB-435s R60/2and MDA-MB-435s R60/6radio resistant cell lines were statistically higher than the parent cell MDA-MB-435s (p<0.05). In the part of telomere length, the MDA-MB-435s R60/2and MDA-MB-435s R60/6cell lines were significant longer than the parent cell MDA-MB-435s. The mean TRF (terminal restriction fragment) of MDA-MB-435s R60/2cell was approximate5.3-6.3kbp, about1.3-1.75fold to the parent cell MDA-MB-435s (The TRF of parent cell line was about3.6-4.2Kbp), and the mean TRF of MDA-MB-435s R60/6cell was approximate7.2-8.6kbp, about2-2.4fold to the MDA-MB-435s cell. These results suggested the there might be some close relationship among the highly expressed telomere binding protein, radio resistance, telomerase activity and telomere length.Conclusion After successfully established the MDA-MB-435s R60/2and MDA-MB-435s R60/6radio resistant cell lines, we observed that the telomere binding protein PTOP and TRF1were significant highly expressed compare to the parent cell MDA-MB-435s. At the meanwhile, both the telomere activity and telomere length had some statistical changes. These results suggested the telomere binding protein PTOP and TRF1had some positive influence to the telomere length, telomerase activity, even to the radio resistance. Part III Silence the telomere binding protein PTOP and detected the influence on the radio resistance and telomerase activityObjective In the experiments of Part II, we confirmed the telomere binding protein PTOP and TRF1had some positive effect on radio resistance, telomere length and telomerase activity. In Part III, we decided to do some further study on PTOP, using the lentivirus technic to silence the telomere binding protein PTOP and detected the changes of radio resistance and telomerase activity in MDA-MB-435s R60/2and MDA-MB-435s R60/6radio resistant cell lines and gene silenced cell lines.Methods The lentivirus vector was constructed by Genechem(?) company, the target PTOP sequence was5’-GTGGTACCAGCATCAGCCT-3’. The identification work was performed as DNA sequences test. After transfected the lentivirus vector to the MDA-MB-435s R60/2and MDA-MB-435s R60/6radio resistant cell lines in the optimal multiplicity of infection (MOI), we obtained the new cell lines called60/2PTOP RNAi and60/6PTOP RNAi. We evaluated the transfect efficacy of these2new PTOP knock down cell lines by Western blot. The clonogenic assay was used to evaluate the changes of radio resistance among the60/2PTOP RNAi and60/6PTOP RNAi cell lines, the MDA-MB-435s R60/2and MDA-MB-435s R60/6radio resistant cell lines and the parent cell MDA-MB-435s. At last, we conducted the telomerase activity assay to find whether the silence gene would change the telomerase activity.Results DNA sequences test verified the correction of lentivirus vector. After transfected the lentivirus vector to the MDA-MB-435s R60/2and MDA-MB-435s R60/6radio resistant cell lines in the optimal multiplicity of infection (MOI), we collected the cells and performed the Western blot assay and RT-PCR assay. All these tests showed the transfected efficiency was as highly as90%, the silence efficiency was good to do the further research. In the results of the clonogenic assay, the new60/2PTOP RNAi and60/6PTOP RNAi cell lines were significant lower than the MDA-MB-435s R60/2and MDA-MB-435s R60/6radio resistant cell respectively, but still higher than the parent cell. In the telomerase activity assay, the60/2PTOP RNAi and60/6PTOP RNAi cell lines were observed less activity than the MDA-MB-435s R60/2and MDA-MB-435s R60/6radio resistant cell lines.Conclusion Using the lentivirus technic to silence the telomere binding protein PTOP to obtain new60/2PTOP RNAi and60/6PTOP RNAi cells. After knocking down the PTOP gene, the radio resistance was observed significant lower than the MDA-MB-435s R60/2and MDA-MB-435s R60/6radio resistant cell, also less active than the radio resistant cell lines in telomerase activity assay. In this case, we can conclude the telomere binding protein PTOP had some instinct relationship with radio resistance and telomerase activity.