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  • One of the hallmarks of the terminal stages of apoptosis

    2019-10-08

    One of the hallmarks of the terminal stages of apoptosis is the fragmentation of chromosomal DNA that proceeds in a two-step manner: the DNA is initially cleaved into 50–300kb fragments and eventually into oligonucleosomal pieces [12], [13]. The two major apoptotic nucleases are DNA fragmentation factor (DFF)/caspase-activated DNase (CAD) and endonuclease G (Endo G) [14]. Cleavage of DNA by these nucleases also triggers the chromatin condensation that is observed concurrently with DNA fragmentation [15]. Apoptotic chromatin condensation essentially involves the aggregation of chromatin fragments into large clumps of condensed chromatin; it is not yet clear if this aggregation is a non-specific phenomenon or an ordered event. While DNA fragmentation and chromatin condensation do not grossly affect cell death, these events are still very significant as they are necessary for the efficient clearance of genomic DNA by phagocytosis during PCD [13], [14]. Clearance of genomic DNA and nucleosomes is required to prevent auto-immunization of the organism: patients and animal models with defective apoptotic DNA processing are predisposed to auto-immune disease characterized by the appearance of anti-DNA and anti-nucleosomal GSK1016790A mass [16], [17], [18]. Moreover, clearance of genomic DNA of dying tumor cells or virus-infected cells precludes the potential transformation of healthy cells with oncogenes [19]. Finally, apoptotic DNA fragmentation may be reversible albeit at a low frequency and, this, in turn, may result in genomic instability and cellular transformation. For these reasons, even though DNA fragmentation may occur at a late stage in apoptosis, it is very important to understand the events that occur during this stage. The DNA-dependent protein kinase (DNA-PK) is a nuclear serine/threonine kinase whose activity is stimulated by double-stranded DNA ends [20]. DNA-PK is involved in diverse cellular processes including DNA repair, V(D)J recombination, innate immunity, telomere maintenance, and apoptosis. DNA-PK consists of an approximately 470-kDa catalytic subunit (DNA-PKcs) and a DNA end-binding component, the Ku70/80 heterodimer [21]. DNA-PKcs, by virtue of its C-terminal kinase domain, belongs to a family of phosphatidylinositol 3-kinase-like protein kinases (PIKKs) other members of which include ATM, ATR, ATX, mTOR, and TRRAP [22]. The activation of DNA-PK upon DNA damage involves its autophosphorylation at several serine/threonine residues [20], notably serine 2056 [23] and threonine 2609 [24]. While DNA-PK plays a critical role in DNA double-strand break (DSB) repair in mammalian cells, it also appears to play an important role in triggering apoptosis in response to excessive or unrepairable DNA damage. Ionizing radiation (IR)-induced apoptosis in mouse thymocytes and fibroblasts, a p53-dependent process, is significantly suppressed in the absence of DNA-PKcs [25], [26]. DNA-PK also appears to be involved in triggering apoptosis in response to critically shortened telomeres in mouse germ cells [27], [28]. Interestingly, DNA-PKcs itself is inactivated by cleavage into 240-, 150-, and 120-kDa fragments by a CPP32-like protease during the terminal stages of apoptosis [29], [30]. The fundamental unit of eukaryotic chromatin is the nucleosome consisting of 146 base pairs of DNA wrapped around a histone core (two each of histones H3, H4, H2A, and H2B) [31]. Adjacent nucleosomes are linked together by at least 20 base pairs of DNA in complex with histone H1. An unexpected role of histones in PCD has recently been revealed by several independent studies [32], [33]. DSBs induce translocation of nuclear H1.2 (a histone H1 isoform) to the cytoplasm where it induces apoptosis by promoting the release of cytochrome c from mitochondria [34]. Histone H2B is phosphorylated at Ser 14 by Mst1 kinase in cells undergoing PCD and it has been suggested that this modification could facilitate apoptotic chromatin condensation and cell death [35]. A third histone, histone H2AX (a histone H2A variant), is phosphorylated in its unique C-terminal tail (at serine 139) immediately upon the introduction of DSBs into eukaryotic cells [36]. Phosphorylated H2AX, also called γH2AX, forms discrete foci at the sites of DSBs, facilitates the recruitment of damage-responsive proteins and chromatin-remodeling complexes to the sites of DNA damage, and influences both the efficiency and fidelity of DSB repair [37], [38], [39], [40]. We have reported earlier that ATM is the primary kinase that phosphorylates H2AX in response to DNA damage induced by IR in mouse cells while DNA-PK plays a minor role in the process [41]. Other studies have implicated ATM, ATR, as well as DNA-PK in IR-induced H2AX phosphorylation in human cells [37], [42], [43], [44], [45]. H2AX is also phosphorylated during apoptosis concurrently with the initiation of DNA fragmentation [46]. As an understanding of the events occurring during apoptotic DNA fragmentation is of fundamental importance, we wanted to identify the kinase responsible for H2AX phosphorylation during PCD. In this study, we demonstrate that histone H2AX is phosphorylated during staurosporine-induced PCD in mouse fibroblasts, Chinese hamster ovary (CHO) cells, and human fibroblasts. The phosphorylation of H2AX is coincident with apoptotic DNA fragmentation and limited to nuclei with characteristic chromatin condensation. Using DNA-PKcs- and ATM-deficient cells and NU7026, a very specific inhibitor of DNA-PKcs, we show that DNA-PK is primarily responsible for H2AX phosphorylation during apoptosis while ATM is dispensable for the process. The differences in γH2AX induction in the various cell lines used is not due to differences in the extent of apoptosis. Moreover, the kinase activity of DNA-PKcs is specifically required for the induction of γH2AX. We further show that DNA-PKcs is activated in apoptotic cells, as evidenced by autophosphorylation at serine 2056, before it is inactivated by cleavage. Thus, at around the time when γH2AX induction is seen to occur there are sufficiently high levels of active uncleaved DNA-PKcs available for H2AX phosphorylation. In contrast, ATM is observed to be degraded well before DNA fragmentation and γH2AX induction resulting in the predominance of DNA-PK at this stage of apoptosis. Finally, we show that DNA-PKcs autophosphorylation and γH2AX induction occur only in apoptotic nuclei with characteristic chromatin condensation but not in non-apoptotic nuclei from the same culture establishing the most direct link between DNA fragmentation, DNA-PKcs activation, and H2AX phosphorylation.