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  • The other interesting observation made during this study rel

    2020-01-29

    The other interesting observation made during this study relates to the differences between the discrete IR-induced γH2AX foci and the robust H2AX phosphorylation observed during apoptotic DNA fragmentation. The pattern of H2AX phosphorylation observed during DNA fragmentation possibly reflects the massive chromatin fragmentation and condensation during this stage and is similar to the pattern of histone H2B phosphorylation reported by Cheung et al [35]. Interestingly, areas in the apoptotic nucleus with chromatin condensation stain poorly with anti-γH2AX antibody indicating that the γH2AX epitope may be masked due to tight interactions with other proteins that might facilitate chromatin compaction. DNA-PKcs is involved in the induction of apoptosis in response to IR-induced DNA damage in mouse thymocytes and fibroblasts [25], [26]. DNA-PKcs is also responsible for triggering apoptosis in Ethyl 3-Aminobenzoate methanesulfonate with critically shortened telomeres [27], [28]. We find though that the extent of apoptosis induced by staurosporine in DNA-PKcs-deficient cells is the same as that in wild type cells. This is not surprising because the induction of apoptosis by staurosporine does not involve DNA damage [70]. Thus, while DNA-PKcs is required for signaling the presence of excessive DNA damage to the apoptotic machinery, probably via p53 [25], [26], it may be dispensable for the induction of PCD by agents that are not overtly genotoxic. Staurosporine is a powerful and commonly used inducer of apoptosis [9], [10], [11]. The induction of apoptosis by staurosporine apparently does not involve DNA damage as even enucleated cells can undergo staurosporine-induced cell death [70]. However, it was recently reported that a short pulse of staurosporine in high doses (3μM) induces DSBs and γH2AX foci within 3h [71]. The early induction of DSBs was proposed to be a byproduct of the effect of staurosporine on multiple kinases and is not critical for the subsequent induction of apoptosis. We do not observe staurosporine-induced H2AX phosphorylation at early time points probably because of the relatively lower doses of staurosporine used by us to induce apoptosis. The H2AX phosphorylation that is the subject of our study is distinct from the early phosphorylation reported by Andreau et al. [71] as it is observed at a late stage and is coincident with nuclear fragmentation. It is interesting though that the early H2AX phosphorylation induced by staurosporine, similar to that induced by IR, is ATM dependent [71] while the apoptotic H2AX phosphorylation observed by us is DNA-PK dependent. What could be the possible significance of H2AX phosphorylation during DNA fragmentation, a rather late event in apoptosis? H2AX phosphorylation, or the lack thereof, may not grossly affect the eventual outcome, i.e., cell death (case in point, equal levels of unviable cells in both wild type and DNA-PKcs−/− cultures treated with staurosporine). However, we envisage two scenarios in which H2AX phosphorylation may not simply be a “knee-jerk reflex” to DNA fragmentation and may be of some significance. The first scenario is based on the thesis that DNA fragmentation during PCD may be reversible, albeit at a low frequency [14], [72]. It is postulated that NHEJ-mediated end joining of DNA fragments may reverse apoptosis but lead to the development of chromosomal translocations and cancer [73], [74]. A very good example of this is therapy-related leukemia where treatment of leukemias with pro-apoptotic drugs leads to the development of secondary tumors with new chromosomal translocations [75]. DNA-PK plays a central role in the mammalian cell in the NHEJ pathway for DSB repair [20] and the phosphorylation of H2AX at the sites of DNA damage influences the efficiency and fidelity of DSB repair [37]. Therefore, the observed activation of DNA-PK during the late stages of PCD may reflect a final effort by the cell to repair DNA cleaved during the apoptotic process. Thus, the phosphorylation of H2AX might represent the first step in a counter-apoptotic repair process that is rapidly derailed by the degradation of DNA-PKcs during the terminal stages of PCD [29], [30]. It is well established that DNA-PK is proteolysed during apoptosis in order to forestall the repair of cleaved DNA [29], [30]. Our results demonstrate, for the first time, that DNA-PK is actually activated and is able to phosphorylate H2AX, an early step in the DNA-damage response, before it is inactivated by cleavage. In this context, it is interesting to note that DNA-PK-proficient CHO cells are more resistant to staurosporine as compared to DNA-PK-deficient CHOs in a colony forming assay (Supplementary Fig. 2). We do not know if the better long-term survival of cells with DNA-PK may reflect some reversal of apoptosis as proposed by Betti et al. [73] but it may be enlightening to see if the surviving wild type CHO cells harbor higher levels of chromosome translocations.