CS is a complex chemical mixture of gases and suspended particulate matter that contains a wide range of carcinogens, mutagens, and free radicals that can induce an array of DNA lesions (e.g., adducts, deletions, insertions, point mutations, structural chromosomal aberrations, aberrant sister chromatid exchanges, micronuclei, and single strand DNA breaks) that have been strongly implicated in the development and progression of human cancers by epidemiological studies [39–48]. Our previous studies showed that CS and cigarette smoke condensate (i.e., 'tar') can also directly induce DSBs in both normal and malignant lung cells [24, 35]. DSBs are a major type of DNA damage that can lead to translocations and chromosomal instability, two important mechanisms in the generation of malignant tumors . Consequently, detection of DSBs and the various proteins that sense and orchestrate repair of these lesions may ultimately provide a more precise measurement of the potential cancer risk in individuals exposed to CS.
In the current study, we show that exposure of A549 cells to CS led to concurrent activation of ATM and phosphorylation of H2AX. Moreover, there was a close relationship between ATM activation and H2AX phosphorylation both in timing (dose) and in the degree of response to CS. Thus, both events were triggered after 10 min exposure to CS (Fig. 3), and the degree of response, when cells were treated with CS from cigarettes with different tobacco and filter combinations, was strongly correlated (Figs. 4 and 5). In addition, both ATM activation and H2AX phosphorylation were partially suppressed by caffeine but not by NU7026, implying that CS-induced H2AX phosphorylation was predominantly mediated by ATM. DNA damage that primarily results in formation of DSBs is believed to trigger H2AX phosphorylation mediated by ATM rather than by ATR or DNA-PKcs [2, 4, 6–9, 50]. This observation is in contrast to other genotoxic agents such as hydroxyurea, aphidicolin, and UVR that induce H2AX phosphorylation as a function of replication stress, but which is mediated by ATR and not by ATM [28, 29]. However, despite the apparent causal relationship between ATM activation and H2AX phosphorylation in CS-treated A549 cells, a definitive study in which ATM expression is suppressed by interfering RNAs (RNAi) is currently being conducted in order to prove conclusively that ATM is the primary responsive kinase.
How might CS induce DSBs? The most common mechanism by which DSBs are thought to be generated is as a result of stalled DNA replication forks at the sites of single strand DNA lesions such as single-strand breaks (SSBs) during DNA replication . Unrepaired DSBs generated by such mechanisms may result in chromosome breakage and mironucleation leading to genetic instability and malignant transformation [52, 53]. However, although it is clear that CS can cause numerous SSBs that may induce replication fork stalling [47, 54], some data suggest that even large amounts of SSBs are incapable of provoking ATM activation . Whether this is a general phenomenon or restricted to a specific cell type or cell context remains to be determined, but it may suggest that the induction of DSBs by CS occurs via a mechanism other than SSB formation. An important observation in this regard is that if stalled replication forks at SSB lesions were the dominant mechanism of DSB formation, it would be expected to reflect a characteristic pattern of H2AX phosphorylation that is distinctly maximal in S-phase cells [27, 55]. While we observed somewhat higher levels of H2AX phosphorylation in S-phase cells compared to G1 cells upon exposure to CS [see Fig 2 and [24, 35]], the difference was rather small. These data imply that the contribution of this mechanism to the overall level of DSB damage, as reflected by the increased expression of γH2AX, was not predominant. One conjecture to mechanistically explain this observation is the possible activation of SMC1, a critical downstream event in the ATM-NBS1-BRCA1 pathway that engages the S-phase checkpoint and prevents movement of the replication forks and thus their collisions with ssDNA lesions [17, 56, 57]. However, whether SMC1 is activated in CS-exposed cells remains to be determined.
CS also contains a range of carcinogens known to result in covalently linked DNA adducts either directly or indirectly after metabolic activation . Upon cell proliferation, these type of adducts can induce DNA replication errors that may result in carcinogenic events [59, 60]. Moreover, as these DNA-carcinogen adducts are removed by base excision repair mechanisms, defects in one or more of the genes involved in these processes can directly result in DSBs . Another potentially unique capability of CS may be that it can induce DSBs directly due to its large concentration of highly reactive organic and inorganic substances that either are free radicals or can generate free radicals within the cell that can subsequently attack DNA [62, 63]. It has been speculated that multiple hydroxyl radicals produced in close proximity could directly cause DSBs and lead to ATM activation . It is well documented that increased oxidative stress is a major mechanism by which CS causes airway damage that can lead to a host of pathogenic conditions including lung cancer [64, 65], possible as a result of the induction of oxidatively derived DNA damage such as altered or mismatched bases, deletions and insertions, intra and inter-strand cross-links, or possibly DSBs. Additional research will be required to determine the primary mechanism(s) by which DSBs are formed in cells exposed to CS. Regardless of the mechanism by which CS induces DSBs, repair of this defect occurs through two major pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ). In vertebrate cells, NHEJ is the dominant DSB repair mechanism, particularly in G1-phase cells . However, reliance on NHEJ for accurate repair of CS-induced DSBs may be problematic for the damaged cell since this process is error-prone, often resulting in deletion of base pairs that can lead to the accumulation of defective DNA with each cell cycle following exposure to CS [67, 68].
Assessing the sites of ATM activation and H2AX phosphorylation by UV fluorescence microscopy revealed different patterns of localization in CS-exposed cells. The sites of γH2AX localization had a distinct punctate pattern (Fig. 8C). This was evident despite the close proximity of individual foci with each other and the frequent overlap of their images. The presence of distinct foci of γH2AX IF is considered to be a reliable marker of DSBs [18–20]. In contrast to γH2AX, no distinct ATM-S1981P IF foci were observed, and activated ATM was spread rather uniformly over the whole nucleoplasm (Fig. 7C). ATM activation in response to DSB formation, similar as H2AX phosphorylation, is purported to present as distinct ATM-S1981P IF foci [3, 17]. It is likely, therefore, that the observed pattern of ATM-S1981P IF in A549 cells reflects, not only ATM activation in response to formation of DSBs, but also to other types of CS-induced DNA lesions that may have more global effects such as altering chromatin structure [3, 17]. Such diverse spatial patterns of response in terms of nuclear ATM activation and H2AX phosphorylation are consistent with a variety of different DNA lesions generated by CS [39–48]. It has been proposed by Kitagawa et al.  that ATM phosphorylation may be triggered by "structural" changes in chromatin as a result of formation of DSBs or other types of DNA damage that leads to altered topological stress on the DNA double helix in the proximity of the damage. In support of this mechanism are the observations that chromatin condensation during mitosis [69, 70] or premature chromosomes condensation  is accompanied by ATM activation. The altered topological stress on DNA in mitosis is reflected by DNA's increased propensity to undergo denaturation  and its sensitivity to single-strand specific endonucleases such as S1 or mung bean nuclease . The observed CS-induced H2AX phosphorylation may also be mediated by the mechanism associated with NER and linked with the excision of DNA adducts generated by CS. This mechanism would be consistent with the recent findings of Marti et al  who reported H2AX phosphorylation in G1 cells upon exposure to UV-C light, which was abrogated in the NER mutant cells.