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  • br Materials and methods br Results br Discussion

    2020-01-09


    Materials and methods
    Results
    Discussion The phosphatidylinositol 3-kinase-like protein kinases, ATM, ATR, and DNA-PK, have all been implicated in H2AX phosphorylation in response to different stimuli [37], [41], [42], [43], [44], [45]. We show here that H2AX is phosphorylated in rodent and human CD 2665 treated with staurosporine to trigger apoptosis. Apoptotic H2AX phosphorylation occurs late in the process and can be correlated with the DNA fragmentation/chromatin condensation stage. While H2AX has been reported before to be phosphorylated during apoptosis [46], the kinase responsible is yet to be identified. We report here that DNA-PK is essential for H2AX phosphorylation in apoptotic mammalian cells while ATM is dispensable for the process and that this difference is not due to differences in the extent of apoptosis induced. Clearly, an apoptotic cell responds to DNA breaks differently from a cell in normal physiological condition. The differential requirements for DNA-PK and ATM may reflect differences in their mode of activation and their roles within the cell. DNA-PK is activated by directly binding to DNA ends [21] and its activation by DNA damage (as measured by in vivo autophosphorylation) increases proportionately with the extent of damage [23]. The massive DNA fragmentation during apoptosis could thus result in very high levels of activated DNA-PK making it the predominant H2AX kinase in these cells. In fact, our observations of robust DNA-PKcs autophosphorylation at Ser2056 [23] in apoptotic cells is the first clear indicator of DNA-PK activation during PCD. We also show that though DNA-PKcs is cleaved during apoptosis, there are sufficiently high levels of uncleaved and active DNA-PKcs around the time when γH2AX induction occurs. ATM, on the other hand, is postulated to be activated both by DNA breaks [67], [68] as well as by changes in chromatin structure induced by breaks [66], [69] and its activation plateaus with low levels of DNA damage [66]. Perhaps, the chromosomal changes during late apoptosis precludes ATM activation making it dispensable for H2AX phosphorylation. Indeed, we fail to observe any activation of ATM upon Western blotting with anti-phospho-ser1981 antibody [66]. However, we cannot rule out that low levels of ATM activation may have been undetected due to the low affinity of the anti-phospho-ser1981 antibody used. ATM is also reported to be cleaved and inactivated during PCD before the onset of DNA fragmentation [65], which might make it unavailable for H2AX phosphorylation. We, too, find that ATM is largely degraded well before the onset of H2AX phosphorylation. While DNA-PKcs is also cleaved during apoptosis [29], [30], our observations indicate that DNA-PKcs is active as a kinase before it is degraded. The time between DNA-PK activation and its inactivation by cleavage might provide this kinase with a window of opportunity in which to successfully phosphorylate H2AX and, perhaps, other substrates. We also find that immunoprecipitated DNA-PK can phosphorylate recombinant H2AX in vitro (Supplementary Fig. 1); thus, it is very likely that DNA-PK directly phosphorylates H2AX within the cell. The most striking correlation between apoptotic DNA fragmentation, DNA-PK activation, and H2AX phosphorylation comes from our observation of DNA-PKcs autophosphorylation and γH2AX induction in apoptotic nuclei with chromatin condensation but not in non-apoptotic nuclei from the same culture. In contrast to our observations of DNA-PK-dependent H2AX phosphorylation during apoptosis, we have previously shown that ATM is primarily responsible for γH2AX induction in murine cells in response to IR [41]. However, other reports have implicated both ATM and DNA-PK in H2AX phosphoryaltion in irradiated human cells [37], [42], [44], [45]. We, too, have shown previously that the residual levels of H2AX phosphorylation in Atm−/− cells is dependent on DNA-PK [41]. In this study we find that though IR-induced H2AX phosphorylation is largely reduced in ATM-deficient or ATM-inhibited human cells as assayed by Western blotting, H2AX foci with reduced intensity are still discernible. We surmise, therefore, that though IR-induced H2AX phosphorylation is largely reduced in the absence of ATM, back-up phosphorylation by DNA-PK is sufficient for the development of IR-induced H2AX foci though of lower intensity.