Abstract
While ATM-dependent nuclear foci formation of checkpoint factors has been thought to be indispensable for the activation of DNA damage checkpoint, biological significance of the foci formation requires a quantitative analysis using the parameter which integrates foci number, foci area and density, and thus, reflects molecular number of foci forming factors. So far, no such foci study has been performed. Therefore, the aim of this study is to conduct a quantitative analysis of the foci parameter, which is necessary and sufficient for G2/M checkpoint induction. In the present study, the parameter is represented based on the digital images acquired from fluorescence microscopy, as the Sum Of Integrated Density (SOID), which is the sum of the Integrated Density (= foci area x average intensity) of each foci in each cell calculated by image analysis software.
First, we determined the minimum dose of X-rays at which foci formation could be observed but G2/M checkpoint was not induced in normal human diploid cells. To evaluate G2/M arrest mitotic index was compared, and we found the minimum dose was 0.08 Gy. The average and maximum of SOID of phosphorylated H2AX foci in the cells which progressed to prophase at two hours after irradiation of 0.08 Gy of X-rays were 3846 and 10987, respectively. The minimal Integrated Density of each focus was about 1000. These results showed SOID could be quantitative index to evaluate inducibility of checkpoint by foci. Our results also indicated that SOID of foci have threshold for induction of G2/M checkpoint, which is approximately 11000.
