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  • Here we investigate the ATM

    2023-01-28

    Here, we investigate the ATM to ATR switch in normal human fibroblasts (82-6 cells) after irradiation with γ-rays, or high LET 56Fe and 28Si and moderate LET 16O particles. Our data provide important evidence of LET dependence on the transition of ATM to ATR as well as end resection at the sites of DNA damage. We show an early transition (2h) from ATM to ATR kinase post irradiation with γ-rays, however this transition continues for prolonged times after high LET 56Fe and 28Si particle irradiation, indicated by sustained resection. Our results support previous studies which reported differential ATM kinase activation with varying radiation qualities [35]. It has been previously reported that DSB repair capacity of cells decrease with increasing LET [36]. The higher biological effectiveness of high LET radiation compared to γ-rays is often explained theoretically by increased clustered ionizations within a small volume of the scale of the DNA helix and across the path of the particle increasing with LET [37]. It has been shown that DSB repair kinetics correlates with complexity of damage induced [38] and that DNA damage of higher complexity are induced after high LET radiation exposure than X-/γ-rays [39]. Clonogenic survival and chromosomal aberration data confirm that high LET-induced DNA damage leads to increased cell lethality [38], [39], [40]. Processing of the broken DNA ends by nucleases provide distinct substrates for activation of specific repair pathway proteins. The importance of end processing in the repair of DNA damage induced by IR was discussed extensively in a study [23]. It was shown recently that Exo1 plays an important role in transition from ATM to ATR signaling and it is inhibited by NHEJ protein Ku80 [23], [41]. Absence of Exo1 leads to a higher level of ATM and lower level of ATR activation. Our results show LET-dependent effects on activation of ATM and ATR. Interestingly, the kinetics of ATM activation was similar for γ-rays and moderate LET 16O particle irradiation. We also observed a persistent increase in pATR signal for both 56Fe and 28Si irradiated samples until 24h post IR (2A and 3A), although a decline in intensity was observed at 48h post IR (suppl. Fig. 3). This persistent activation of ATR kinase after high LET radiation-induced damage has not been reported elsewhere. One possible reason may be that complex SSBs with associated PF 4800567 hydrochloride damage may cause continuous formation of DSBs and stalled replication forks in S-phase which perhaps leads to a persistent activation of ATR [42], [43], [44]. The occurrence of complex SSB's would be expected to increase with increasing LET. This persistence of ATR activation does not lead to more or persistent Rad51 foci (Fig. 6C) but rather to formation of longer stretches of resected DNA at the damage sites. To determine the role of cell cycle checkpoints in our results, we carried out detailed analysis of proportions of cell cycle phases after 2Gy γ-rays and high LET 56Fe and 28Si radiation. The results reveal a potent G1 phase arrest along with a stronger G2 block in cells irradiated with high LET particles as compared to γ-rays, which show only a G2 block. Although ATM signaling is expected across all phases of cell cycle, the persistence of pATM signal in 56Fe and 28Si treated samples may be due to the observed stronger G1 arrest, thus higher proportion of cells in G1 phase, even 48h post exposure (Fig. 5 and suppl. Fig. 2). The differences in pATR signal (S and G2 phases) between γ-rays and high LET radiation treated samples indicate that ATM-to-ATR switch is prolonged in high LET radiation-induced complex DNA damage. The kinetics of Cyclin B1, a G2/M phase marker, also supported the kinetics of pATR signal for γ-rays and 56Fe particle irradiation (suppl. Fig. 3). Normalizing our Western blot density data to residual DSBs (γ-H2AX foci) for γ-rays and 56Fe irradiated samples at each corresponding time point (suppl. Fig. 4) reveals that resection reaches a peak at about 8h and then steadily declines in case of γ-rays, whereas 56Fe treated samples show extended resection with steady increase in values until 24h post exposure. Cyclin A (S/G2 marker) stained 82-6 cells were examined for Rad51 foci after 1Gy of γ-rays and 56Fe particle irradiation. Although a higher number of Rad51 foci were observed at all time points with 56Fe irradiation (Fig. 6B), normalizing the data to number of DSBs (γ-H2AX foci) for each corresponding time point reveals similar number of Rad51 foci after γ-rays and 56Fe particles (Fig. 6C), suggesting that Rad51 foci kinetics is probably independent of type of radiation damage. The initial resection/end processing is similar for both low and high LET induced DNA damage, however the increase in pATR levels observed only with high LET radiation until 24h, suggests sustained resection at these damage sites due to breaks remaining unrepaired owing to the complexity of the damage induced. This probably leads to transition from ATM to ATR signaling at DSB sites for extended periods of time post damage induction. Building up on our phospho-kinase activation results, we speculate that this prolonged transition from ATM to ATR kinases at damage sites may partly contribute to the increased biological effectiveness of high LET radiation.