VKGILS-NH2 br Materials and methods br Results br Discussion

Materials and methods
Results
Discussion 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 VKGILS-NH2 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. 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 cells 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.