Targeting TPP1Enhances Radiosensitivity Of Human Cancer Cells

Part I The expression and significance of TPP1protein in human colorectal cancer tissuesObjective To investigate the role of telomere-binding protein TPP1in the occurrence and development of colorectal cancer.Methods The expressions of TPP1in78cases of colorectal cancer and20cases of normal colorectal mucous tissues were detected by using SP immunohistochemical staining assay; Data between two groups were analyzed using chi-square test.Results TPP1protein was localized in the nucleus of colorectal cancer cells in tissues. Expression of TPP1protein was significantly up-regulated in colorectal cancer tissue compared to the noncancerous tissue,P<0.05. In addition, the expression of TPP1protein was significantly associated with invasion depth, Duke's stage and lymph node metastasis, P <0.05. There was no significant difference between TPP1expression and age, gender and different grade of tumor differentiation, P>0.05.Conclusion Telomere-binding protein TPP1is overexpressed in colorectal cancer tissues and play a key role in the progression of colorectal cancer. TPP1is expected to become a new therapeutic target in colorectal cancer. Part II Protein levels of telomere-binding protein TPP1are associated with telomere length and radio sensitivity in colorectal cancer cellsObjective To investigate the relationship between TPP1protein levels, telomere length and radiosensitivity of colorectal cancer cell lines.Methods Five colorectal cancer cell lines(HCT116?HT29?SW480?LoVo and DLD1)were used in this experiment. Protein levels of telomere-binding protein TPP1were detected using Western blotting assay; radiosensitivity index SF2was measured by clonogenic assay; telomere length was measured by Southern blotting.Results In this study, we found that elevated TPP1expression significantly correlated with radioresistance and longer telomere length in human colorectal cancer cell lines. Firstly,TPP1protein expression was significantly correlated with telomere length (R=0.9783, P<0.05). In addition, TPP1expression was negatively correlated with intrinsic radiosensitivity(R=0.9792, P<0.05). In summary, radioresistant cells have longer telomeres and higher production of TPP1than that in radiosensitive cells.Conclusion These result indicated that there was a significant correlation between TPP1expression, telomere length and cell intrinsic radiosensitivity. Our study indicated that elevated expressions of TPP1in human colorectal cancer cells could regulate telomere length and confer radioresistance. These results suggested that TPP1may be a potential target in the radiotherapy of colorectal cancer. Part III Effects of TPP1overexpression on the radiosensitivity of human colorectal cancer cells to X-rays and its mechanismsObjective The telomere-binding protein TPP1is an important component of the shelterin complex at mammalian telomeres. Our previous reports showed that TPP1expression was elevated in radioresistant cells, but the exact effects and mechanisms of TPP1on radiosensitivity is unclear. To investigate the effects of TPP1overexpression on the radiosensitivity of human colorectal cancer cells to X-rays and its mechanisms.Methods HCT116cells stably overexpressing TPP1were established by Blasticidin. Radiosensitivity was analyzed using a clonogenic cell survival assay. Cell apoptosis and cell cycle distribution was measured by flow cytometry. Western blotting assay was performed to measure the expression of TPP1,ATM, ATR, Chk1and phosphorylated Chkl. Telomere length and telomerase activity were measured using Southern blotting and TRAP-PCR-ELISA assay, respectively. Telomere dysfunction(telomere dysfunction induced foci, TIF) was detected using immunofluorescence and monitored by co-localization of TRF2and y-H2AX in this study.Results In this study, we found that TPP1overexpression showed a significant decrease of radiosensitivity to X-rays and TPP1mediated radioresistance was correlated with a decreased apoptosis rate after IR exposure. In addition, TPP1overexpression showed increased telomere length in HCT116cells but no significant change in telomerase. Furthermore, TPP1overexpression showed prolonged G2/M arrest mediated by ATM/ATR-Chk1signal pathway after IR exposure. Moreover, TPP1overexpression accelerated the repair kinetics of total DNA damage and telomere dysfunction induced by ionizing radiation.Conclusion We demonstrated that elevated expressions of TPP1in human colorectal cancer cells could protect telomere from DNA damage and confer radioresistance. These results suggested that TPP1may be a potential target in the radiotherapy of colorectal cancer. Part IV Effects of TPP1knockdown on the radio sensitivity of human colorectal cancer cells to X-rays and its mechanismsObjective To investigate the effects of TPP1knockdown on the radiosensitivity of human colorectal cancer cells to X-rays and its mechanisms.Methods The stable transfected cell clones were selected by G418. Radiosensitivity was analyzed using a clonogenic cell survival assay. Cell apoptosis was measured by flow cytometry. Western blotting assay was performed to measure the expression of TPP1. Telomere length and telomerase activity were measured using Southern blotting and TRAP-PCR-ELISA assay, respectively. Telomere dysfunction(telomere dysfunction induced foci, TIF) was detected using immunofluorescence and monitored by co-localization of TRF2and ?-H2AX in this study. Telomere-ChIP assay (telomere-chromatin immunoprecipitation assay was performed to explore the impact of TPP1knockdown on the association between TRF2> y-H2AX?POT1?hTERT and telomeres.Results In this study, we found that knockdown of TPP1lead to increased radiosensitivity in human colorectal cancer cells. TPP1mediated radiosensitization was correlated with increased apoptosis rate after IR exposure. In addition, our study revealed that TPP1knockdown induced telomere dysfunction but increased telomere length in human colorectal cancer cell lines. Furthermore, TPP1knockdown decreased the repair kinetics of total DNA damage and telomere dysfunction induced by ionizing radiation. Moreover, we examined the interactions of TRF2??-H2AX?POT1?hTERT and telomeric DNA by using a telomere-chromatin immunoprecipitation (ChIP) assay. We found that TPP1knockdown dissociated POT1?hTERT from telomeres but had no impact on the association between TRF2and telomeres. Our research also revealed that TPP1knockdown increased the association between y-H2AX and telomeres which had been confirmed by TIF assay.Conclusions We demonstrated that knockdown of TPP1expressions in human colorectal cancer cells could result in telomere dysfunction and confer radiosensitization. These results suggested that TPP1is a potential target in the radiotherapy of colorectal cancer.