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

Background: Proteins 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.Results: The 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.Conclusions: We 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

Biological Significance of DNA Damage Checkpoint and the Mode of Checkpoint Signal Amplification

It is generally accepted that DNA damage checkpoint is the mechanism that allows time for DNA damage repair. However, several lines of evidence challenge this paradigm, especially, in the case of G1 checkpoint. The first evidence is the complete difference between the repair kinetics of DNA double-strand breaks (very rapid) and the timing of G1 checkpoint induction (very slow) after ionizing radiation. The second evidence is that inactivation of p53, which is a central player of G1 checkpoint, does not render cells radiosensitive, rather, such cells become radioresistant. Moreover, it was shown that G1 arrest persists almost permanently after irradiation, until the time when most of the initial damage should be repaired and disappear. Therefore, cells should have a mechanism to maintain G1 checkpoint signaling by amplifying the signal from a limited number of damage. In this review, we discuss what is the bona fide role of G1 arrest and how G1 checkpoint signal is maintained long after irradiation

A Possible Role of Stress-Induced Premature Senescence, SIPS, as a Producer of the Stress-Resistant Microenvironment

The microenvironment is consisted both of soluble factors involving growth factors and of insoluble factors. Stroma cells contribute to form the microenvironment through a secretion of these factors. Fibroblast, which is known as stroma cells, also secretes various soluble/ insoluble factors, but the secretion level is significantly up-regulated when they reach to a finite replicative lifespan. Recent accumulating studies not only in vitro but also in vivo provide us that secreted proteins from senescent cells promote pro-survival pathway in bystander cells, especially tumor cells rather than normal cells. Since various stresses including ionizing radiation (IR) prematurely induces cellular senescent stage, called Stress-Induced Premature Senescence (SIPS), there is the possibility that the secretion pathway in cells undergoing SIPS is also activated. Here, we propose that pro-survival factor is secreted from SIPS cells to provide the stress-resistant microenvironment