The primary objective of these studies was to determine if we could detect the formation of γ-H2AX under conditions that did not induce overt changes in chromatin structure associated with DNA strand breaks. Here we present evidence that γ-H2AX can be formed in significant yield under minimally toxic conditions of altered tonicity. Cells subjected to hypotonic conditions rapidly form γ-H2AX in a manner dependent on the time of exposure (). This effect appears to be general, at least, for neural precursors and fibroblasts, cells that routinely showed elevated γ-H2AX levels following hypotonic treatments.
Cells retain the requisite kinase activity necessary to phosphorylate histone H2AX under hypotonic conditions (). While not the focus of this investigation, the capability of wortmannin to inhibit the formation of γ-H2AX suggests that the PI3 kinases (e.g. ataxia telangiectasia mutated, ataxia telangiectasia and Rad3 related and/or DNA-dependent protein kinase) are likely to be involved in mediating H2AX phosphorylation after hypotonic exposure (18
). This idea is also supported by a number of past studies analyzing the substrate specificities of this kinase family after different types of cellular stress (25
). The formation of γ-H2AX in response to hypotonic treatment persists well after return to isotonic medium (), suggesting that certain disruptions to chromatin structure persist, and/or the ability of phosphatases to return H2AX to the unphosphorylated state are compromised. It may also be possible that hypotonic conditions compromise cellular metabolism and energy pools, potentially impacting H2AX phosphorylation by altering the response of DNA damage and chromatin sensing pathways. While different salt levels are likely to have many effects in cells, the impact of the resultant structural alterations to chromatin induced under hypotonic conditions are still likely to play a contributory if not causal role in eliciting γ-H2AX.
Past studies have tried to quantify the cellular response to DNA damage through immunohistochemical approaches aimed at analyzing the nuclear staining of γ-H2AX (4
). Such efforts have clearly demonstrated that agents and/or conditions capable of causing DNA DSBs directly (e.g. ionizing radiation) or indirectly (e.g. via replication fork breakdown) lead to the formation of brightly staining γ-H2AX foci of widely varying size. However, efforts to quantify fluorescent γ-H2AX foci within nuclei are often hampered by the marked heterogeneity in the level of background γ-H2AX staining. If, as we suggest, γ-H2AX levels are sensitive to chromatin change, then much of the heterogeneity in nuclear γ-H2AX staining profiles may simply reflect the multiple structural conformations of DNA actively undergoing replication and/or repair. In support of this possibility, past studies from us and others have found that background H2AX staining is highest during S-phase (4
For the reasons alluded to above, we chose to focus on the quantification of γ-H2AX levels via FACS analysis. Immunohistochemical analyses undertaken for comparative purposes demonstrated unequivocally the capability of hypotonic treatments to elicit γ-H2AX nuclear staining. The different nuclear staining profiles evident after various treatments did, however, reveal marked variations in γ-H2AX signal intensity and distribution in the nucleus. Overall, hypotonic treatments led to more uniform pan-nuclear staining while γ-irradiated cells all showed very discrete punctate patterns of nuclear staining (). The number of weakly staining nuclei increased from 30 to 50% as hypotonic incubations increased from 30 to 120 min (, ). Similar hypotonic treatments had a smaller impact on the fraction of cells staining more intensely, where the yield of positive cells scored as strong or moderate fluctuated around mean values of 6.9 ± 2% and 14 ± 2%, respectively (, ).
Clearly, there are caveats associated with this type of subjective immunohistochemical analyses, which limits the types of conclusions that can be drawn. We reiterate that these types of studies were included with the intent of highlighting qualitative differences between γ-H2AX staining patterns found after irradiation and those found after hypotonic salt. It is possible that the most brightly staining cells were those destined to die (possibly via apoptosis), but at the time at which our H2AX measurements were performed, annexin V staining was minimal (). If in fact pan-nuclear staining was a marker for early apoptosis, then it also did not impact clonogenic kill. Based on the increased H2AX phosphorylation observed after hypotonic treatments, it is unlikely that apoptosis could account for the majority of the increased H2AX phosphorylation we report in this study. Despite inherent differences between the quantification of γ-H2AX-positive cells by FACS or immunohistochemistry, each technique did show that exposure to hypotonic conditions increased the number of cells positive for γ-H2AX.
Further support that γ-H2AX is a response sensitive to chromatin disruptions comes in a recent report showing that UV light induces significant increases in γ-H2AX levels (30
), a response that was muted in cells deficient for nucleotide excision repair (NER). UVC light does not produce DNA DSBs, but at the fluences used, hundreds of thousands of thymine dimers and 6–4 photoproducts are formed throughout the genome (33
). These UV-induced lesions are excised during NER, a repair process that involves the formation of intermediate D-loop structures and gap-filling synthesis in the DNA. If γ-H2AX acts to respond to structural change in chromatin, then activation of NER would be predicted to increase γ-H2AX levels. Repair competent cells exposed to UVC light not only exhibited marked increases in γ-H2AX but also showed pan-nuclear staining patterns for γ-H2AX (i.e. similar to that shown in ) (30
). These results lend support to the idea that more subtle changes in chromatin structure can elicit γ-H2AX formation.
One of the critical issues these investigations sought to clarify was if γ-H2AX formation was not entirely (if at all) dependent on DNA strand breakage. Because measuring low-level yields of DNA DSBs in mammalian cells (i.e. 1 Gy equivalent, ~40/cell) presents a considerable technical challenge (35
), it becomes difficult to conclusively determine whether a cell at any given time contains only a few or no DSBs. Consequently, to unequivocally substantiate that the hypotonic treatments used here do not create any DNA DSBs would be difficult. However, the survival curves shown () do provide an unambiguous assessment of survival and allow one to estimate relative DSB yields. Thus, at a dose of 5 Gy, ~200 DSBs elicit 80% cell kill and a 1.8-fold increase in γ-H2AX levels () in neural precursor cells. In comparison, a 60-min hypotonic treatment elicits equivalent γ-H2AX levels (~1.7-fold over background) but only results in 13% cell kill, survival levels typically found at doses <1 Gy (i.e. <40 DSBs). Therefore, based on the survival data shown, the level of γ-H2AX induced under hypotonic conditions cannot be explained solely by the induction of DNA DSBs, even if such treatments were in fact found to induce low yields (≤1 Gy) of these lesions.
In summary, our investigation was initiated not to dispute the role of the DSB-dependent formation of γ-H2AX but rather to provide evidence that the phosphorylation of this histone is responsive to alterations in chromatin structure that are not strictly dependent upon strand break formation. Changes in tonicity elicit predictable changes governing the interactions between macromolecules in cells, and based on these expectations, it is difficult to reconcile how the hypotonic conditions used here would not elicit at least minimal changes in chromatin structure. While we did not attempt to measure such structural alterations, salt-induced changes were in fact sufficient to elicit the phosphorylation of histone H2AX. We maintain that γ-H2AX formation is principally responsive to changes in chromatin structure and that such changes likely provided the selective pressure for the adaptation of γ-H2AX into a DNA damage-responsive niche.