Image-based quantitative determination of DNA damage signal reveals a threshold for G2 checkpoint activation in response to ionizing radiation

Abstract

BackgroundProteins involved in the DNA damage response accumulate as microscopically-visible nuclear foci on the chromatin flanking DNA double-strand breaks (DSBs). As growth of ionizing radiation (IR)-induced foci amplifies the ATM-dependent DNA damage signal, the formation of discrete foci plays a crucial role in cell cycle checkpoint activation, especially in cells exposed to lower doses of IR. However, there is no quantitative parameter for the foci which considers both the number and their size. Therefore, we have developed a novel parameter for DNA damage signal based on the image analysis of the foci and quantified the amount of the signal sufficient for G2 arrest.ResultsThe parameter that we have developed here was designated as SOID. SOID is an abbreviation of Sum Of Integrated Density, which represents the sum of fluorescence of each focus within one nucleus. The SOID was calculated for individual nucleus as the sum of (area (total pixel numbers) of each focus) x (mean fluorescence intensity per pixel of each focus). Therefore, the SOID accounts for the number, size, and fluorescence density of IR-induced foci, and the parameter reflects the flux of DNA damage signal much more accurately than foci number. Using very low doses of X-rays, we performed a "two-way" comparison of SOID of Ser139-phosphorylated histone H2AX foci between G2-arrested cells and mitosis-progressing cells, and between mitosis-progressing cells in the presence or absence of ATM or Chk1/2 inhibitor, both of which abrogate IR-induced G2/M checkpoint. The analysis revealed that there was a threshold of DNA damage signal for G2 arrest, which was around 4000~5000 SOID. G2 cells with < 4000 SOID were neglected by G2/M checkpoint, and thus, the cells could progress to mitosis. Chromosome analysis revealed that the checkpoint-neglected and mitosis-progressing cells had approximately two chromatid breaks on average, indicating that 4000~5000 SOID was equivalent to a few DNA double strand breaks.ConclusionsWe developed a novel parameter for quantitative analysis of DNA damage signal, and we determined the threshold of DNA damage signal for IR-induced G2 arrest, which was represented by 4000~5000 SOID. The present study emphasizes that not only the foci number but also the size of the foci must be taken into consideration for the proper quantification of DNA damage signal.

The Efficiency of Homologous Recombination and Non-Homologous End Joining Systems in Repairing Double-Strand Breaks during Cell Cycle Progression

“…To verify whether DSB repair efficiency was dose-related, the kinetics of γ-H2AX cellular content after irradiation with increasing doses of γ-rays was assessed using different methods: manual counting of nuclear foci, fluorescence intensity of foci measured by the SOID parameter and flow cytometry. In agreement with Ishikawa et al’s findings, our results indicated that the SOID parameter is an accurate method to quantify even slight variations of γ-H2AX fluorescence [26]. By contrast, γ-H2AX kinetics after 5 Gy irradiation could not be evaluated by manual foci counting since the number of foci induced by that dose was too high for a reliable determination; nor does the method take into account the increase in foci size detected at later times after irradiation.…”

“…Nuclear fluorescence was calculated as the mean intensity of all the pixels included in the nuclear area. In accordance with Mistrik et al [25] and Ishikawa et al [26], the SOID parameter was calculated for each nucleus as the product of the sum of the area of the foci and the mean fluorescence intensity. An intensity threshold was set to calculate the SOID so that only foci were included in the analysis.…”

“…From our experiments, the Sum Of Integrated Density (SOID) parameter [25] results a valuable tool which takes into account the number and the size of IR-induced foci, allowing to accurately quantify DNA damage signal after exposure to high or low doses of ionizing radiation [26].…”

Effects of nano-second electrical pulses (nsPEFs) on cell cycle progression and susceptibility at various phases

“…Repair machinery is capable of handling some damage without requiring arrest, but once the extent of DNA damage exceeds a set threshold, checkpoints are activated 26 . Research has indicated that thresholds and response pathways differ between the G1, S and G2/M phases and that damage sensors are capable of differentiating between the type and extent of DNA damage 11,26 .…”

“…Repair machinery is capable of handling some damage without requiring arrest, but once the extent of DNA damage exceeds a set threshold, checkpoints are activated 26 . Research has indicated that thresholds and response pathways differ between the G1, S and G2/M phases and that damage sensors are capable of differentiating between the type and extent of DNA damage 11,26 . Response to single-stranded breaks is regulated by replication protein A (RPA), which has a high affinity for ssDNA and acts as a landing pad for checkpoint proteins such as ataxia telangiectasia and Rad3 related (ATR) and the MRN complex, which interact with RPA via their binding partners 27 and initiate a pathway resulting in activation of checkpoint kinase 1 (Chk1).…”