| Summary: | 碩士 === 國立陽明大學 === 放射醫學科學研究所 === 91 === In this study, we investigated the effects of 10-Gy -irradiation on cell-cycle arrest, apoptosis and clonogenic death in the p53-mutated human U138MG (malignant glioblastoma) cell line. In order to evaluate time-dependent events in cellular responses to radiation, we did a time course study by incubating cells ranging from 0.5 to 48 hours after irradiation. Cell-cycle distribution and apoptosis were evaluated by flow cytometry using propidium iodide (PI) staining. Cell viability and proliferative capacity were studied by colony formation assay. Dual fluorescence cDNA microarray technique was used to examine the differential expression patterns of the irradiated cells at five time points (0.5, 6, 12, 24, and 36 h) post-irradiation. The cDNA microarray chips (Agilent Human I_clonesetB2) used contained DNA sequences corresponding to 12,814 human genes. From the flow cytometry data, we observed that after 10-Gy irradiation, cell-cycle checkpoint for U138MG cells were predominantly at G2/M phase, reached a maximum of about 86% at 36 h and there was no appearent arrest at G1/S or S phase. Late apoptosis was more evident after irradiated cells released from G2/M arrest. Microarray analyses revealed changes in the expression of a number of cell-cycle-related genes (p21, cyclin B1, etc.) and cell-death genes (tumor necrosis factors, BBC3, etc.) suggesting their involvement in radiation-induced cell-cycle arrest and apoptosis. Validation of the microarray results was made using semiquantitative RT-PCR technique. Based on our transcription results and related established knowledge, we have proposed a p53-independent pathway model to explain the extensive G2/M arrest observed in irradiated U138MG cells.
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