What could be the possible significance of H AX

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 ML-291 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. The other scenario pertaining to the significance of H2AX phosphorylation is based on the fact that the initiation of DNA fragmentation by CAD facilitates the chromatin condensation that occurs concurrently [15]. Apoptotic DNA fragmentation and chromatin condensation are important for the efficient clearing of genomic DNA and nucleosomes. This step in apoptosis, though not absolutely required for cell death, is very important in protecting the organism from auto-immmunization and oncogenic transformation [13], [14]. However, the molecular mechanisms underlying apoptotic chromatin condensation are still rather obscure. It has recently become increasingly clear that γH2AX plays a very important role in recruiting chromatin remodeling complexes and damage-responsive factors to the sites of DSBs [37], [38], [39], [40]. Therefore, it is quite plausible that H2AX phosphorylation may also facilitate apoptotic chromatin condensation to some extent by recruiting chromatin-modifying activities, i.e., factors involved in cleavage or condensation. Or else, it may simply promote the aggregation of nucleosomal fragments. The observed masking of the H2AX epitope in regions of chromatin condensation within the nucleus does indicate that γH2AX may be tightly complexed with other proteins. A similar function in apoptosis has been proposed for the phosphorylated version of histone H2B [35].