Biological Effects Of X-ray And Camptothecine On Different Subsets Of Peripheral Blood Lymphocytes

The tumor is a major disease which is a serious threat to human health and its incidence rate has a year-to-year rising tendency. At present, the malignant tumor is mainly treated with surgery combined with radiotherapy and chemotherapy after surgical treatment. Excessive cell proliferation and too little apoptosis has been considered to be an important reason of tumor development. Accumulating evidence suggest that tumor is a cell cycle disease. The law of cell death is an important basis for clinical design of chemotherapy and radiotherapy. Clinically, tumor cells at different cell cycles will respond to radiotherapy or chemotherapy very differently.Recent studies suggested that there might exist a subset of dormant period cells existing in the rapidly proliferating tumor populations, the ratio of apoptosis of these quiescent cells under chemoradiation were different from the responses of those constant proliferation of tumor cells.Also, side population (SP) cells and cancer stem cells (CSCs) attracting some other researchers were also quiescent. However, further clinical studies suggested that quiescent cells were closely associated with the development and progression of tumor, even the drug resistance and recurrence. Therefore, the study of the DNA damage response and the law of cell death in different subsets of tumor cells with chemoradiation are becoming a hot spot and focus of cancer research. We use normal human peripheral blood lymphocytes as a model to construct different cell cycle state and respectively to study the DNA damage repair response and the law of cell death with chemoradiation in different subset of tumor cells. Over the past three decades, few studies have compared the difference in the response to the external stimuli of resting and proliferating peripheral blood lymphocytes, and some studies just used the traditional alkaline comet assay to simply analyse DNA damage in epidemiological studies.In this study, yH2AX biomarker was used to examine DNA damage response in different cell cycle states of peripheral blood lymphocytes (PBLs) and its sensitivity to chemoradiation. The formation of yH2AX surely reflects DNA damage, when chemical drug camptothecin and X-ray function in the Go phase and the proliferation of PBLs induced by PHA, the results of immunoblotting and flow cytometry exhibited that proliferating lymphocytes treated with chemical or ionizing radiation had higher yH2AX formation compared to resting lymphocytes. The results indicated that stimulated PBLs are more vulnerable to DNA fragmentation and more sensitive to chemoradiation than their quiescent counterparts. Comparison of the apoptotic rates of quiescent and proliferating PBLs revealed increased apoptosis ratio of proliferating cells. The expressions of apoptosis-related proteins Bcl-2, caspase-3 and caspase-9 were also consistent with the findings. To further elucidate the roles of H2AX in the biological processes, RNA interference technique was used to reduce its expression in 293T cells. It was found that H2AX siRNA had no significant effect on the CPT- and X-ray-induced apoptosis. Therefore, H2AX is only the marked protein involved in DNA damage but not in the regulation of apoptosis pathway.This study aims to use PBLs as a model to simulate the different cell cycle status of tumor cells (Go phase and proliferative phase) and further to study the DNA damage repair and the influence of apoptosis by damage factors (CPT and X-ray)in different subset tumor cells. Furtherly, it reveals that the DNA damage response and apoptosis in different subsets have significant differences; quiescent cells may escape DNA damge attacking, which has important clinical significance for cancer treatment.