Comparing the Microarray Gene Expression Profiles of DNA-PKcs Gene Knockout and WT Cells after Gamma-Ray Irradiation

Autor: Chien-Chu Liu, 劉建助
Rok vydání: 2004
Druh dokumentu: 學位論文 ; thesis
Popis: 92
DNA contains genetic information for processing and regulating the cell’s normal physiological mechanisms. DNA double-strand breaks (DSBs) are the most serious form of DNA damages inflicted by ionizing radiation. The repair mechanisms must be activated to repair the damage so as to avoid cell death and reduce mutations, and thus to preserve the integrity of the genome. DNA dependent protein kinase catalytic subunit (DNA-PKcs) plays an important role that deals with DNA double strand breaks in the non-homologous end rejoining (NHEJ) repair pathway. In order to further understand the role DNA-PKcs may play in NHEJ and other biological functions, we used a DNA-PKcs knockout mouse embryo fibroblast cell line (DNA-PKcs-/-) and its wild type counterparts (WT) as a study model and employed a high-throughput oligonucleotide microarray technique for investigating the global gene expression between the two cell lines under irradiated and non-irradiated conditions. In the first part of this study, survival curves showed a much higher radiosenstivity of the DNA-PKcs-/- to gamma irradiation, compared to WT. Flow cytometry results showed that both cell lines post exhibited dissimilar patterns of the cell cycle redistribution post 5-Gy irradiation (PI), with DNA-PKcs-/- showing much prolonged G2/M arrest. The second part of the study contained two different experimential designs. The first was to use real-time PCR to analyze the NHEJ related genes in a culture of plateau-phase cells during an early phase (0、15、30、60、120min) PI.The second was by means of olig microarray to analyze the differences in gene expression profiles of log-phase cells between the two cell lines at an extended period (0-12hs) PI. In the second design,differences in gene expression profiles between WT and DNA-PKcs-/- were also investigated. Real-time RT-PCR results showed that among the NHEJ related genes,Ku70、Ku80、Xrcc4, had no change in expression level PI；only Lig4 was up-regulated toward the end of the early phase PI. Analysis of the microarray data using relatively stringent criteria showed that in the cell line comparison experiment, among a total of over 18,000 unigenes there were 169 up-regulated genes and 258 down-regulated genes,with most of them distributed in the functional groups of morphogenesis、cell death and response to stress.At the extended period following irradiation, DNA-PKcs-/- exhibited 372 differentially expressed genes while WT showed 265 and the difference in altered genes between the two cell lines was essentially related to apoptosis and cell-cycle regulation. Here,in DNA-PKcs-/-,apoptotic genes were activated while anti-apoptotic genes were suppressed.In contrast,the microarray could not demonstrate altered expression for any repair genes related to NHEJ or HR repair pathways,in the extended period PI for both cell lines.These results support the contention that the knock out of DNA-PKcs prohibits the repair of radiation-induced DSB and increases the radiosensitivity of the host cells by promoting specifically apoptotic events.The inability to demonstrate activities of NHEJ and HR repair genes indicates that DSB repair could be regulated post-transcriptionally or at the translation level. illustrated that DSB repair could be regulated at protein level. Real-time RT-PCR has validated the microarray data for the NHEJ repair genes with a correlation of 0.8.
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