In this study we take a systems-level approach towards achieving a more complete understanding of the cellular response to a chemotherapeutically-relevant class of DNA damaging agents, the SN1 type methylating agents. Although SN1 type methylating agents cause damage to numerous sites within DNA, as well as other macromolecules within the cell (e.g. RNA, lipids, proteins), it is well documented that the O6MeG lesion, in the presence of a functional MMR pathway, is responsible for the toxic effects of these agents. However, the precise mechanism by which the MMR pathway triggers downstream events like cell cycle arrest and apoptosis is less clear. Two models have been proposed that link the MMR pathway to SN1 methylating agent sensitivity, a direct signaling model and an indirect futile repair model. Our results indicate that both may be working simultaneously to control cellular outcomes in response to the classical SN1 type methylating agent MNNG.
In the direct signaling model, the recognition of an O6
MeG/T mismatch by MMR proteins is thought to provoke a signaling response through direct interaction between MMR proteins and key DNA damage signaling kinases. In this event, we would expect to observe a cellular response soon after MNNG treatment due to misincorporation of T opposite O6
MeG in the first S-phase. In support of this, we observed O6
MeG/MMR-dependent inhibition of cell growth and dynamic cell cycle changes in TK6 cells within the first cell cycle post MNNG treatment. Specifically, we detected an increase in the proportion of cells in S- and G2-phase of the cell cycle alongside a decrease in the proportion of cells in mitosis as early as eight hours following MNNG treatment, suggesting induction of a checkpoint response. It is important to keep in mind that this early checkpoint response is transient as cells are ultimately permitted to progress into subsequent cell cycles with unresolved DNA damage. Based on previous studies showing that a permanent G2 arrest is only sustained above a certain threshold of damage, it is possible that the MNNG damage signal is too low to induce a robust G2-M arrest in the first cell cycle post treatment.42
Finally, we point out that the damage signal produced during the first round of replication is not sufficient to invoke cell death, as apoptosis is observed only after cells progress into their second round of replication.
Within the first cell cycle, we additionally detected a low persistent level of phosphorylation of a number of proteins involved in the DNA damage response, namely ATM, H2AX, CHK1, and p53, and this phosphorylation proved to be O6
MeG and MMR dependent. Previous studies have provided evidence that components of the MMR pathway, MutSα (MSH2/MSH6) and MutLα (MLH1/PMS2), physically interact with several of these signaling molecules in the presence and/or absence of DNA damage.43–45
In a comprehensive study, MutSα and MutLα were found to directly interact with multiple components of the ATR-CHK1 signaling pathway, namely ATR, TOPBP1, and CHK1, and moreover the recruitment of these proteins to chromatin was enriched following treatment with MNNG.46
In addition, it was demonstrated that the ATR protein kinase is recruited in vitro
MeG/T mismatches in the presence of MMR proteins, resulting in ATR’s activation and phosphorylation of its downstream target kinase CHK1.23
As the ATR-CHK1 pathway is known for mediating cell cycle transitions in response to DNA lesions that give rise to replication protein A (RPA)-coated single-stranded DNA, including repair excision intermediates, we do not rule out the possibility that the CHK1 phosphorylation observed could originate from single-stranded DNA (ssDNA) gaps formed during MMR-mediated futile repair processing.47
Along these lines, a study by Mojas et al.
demonstrated that ssDNA gaps accumulate in cellular DNA within the first S-phase following treatment with MNNG in an MMR-dependent manner; but whether this is sufficient to invoke a signaling response is not clear.31
While substantial evidence supporting direct activation of the ATR-CHK1 pathway by MMR exists, a more unexpected result was the O6
MeG/MMR-dependent induction of phosphorylated ATM(S1981) within one hour after treatment with MNNG. ATM responds primarily to DSBs, an event that leads to its autophosphorylation at serine-1981 and activation.48
However, the formation of DSBs is not expected to occur at such early time points following induction of the O6
MeG lesion. It is therefore important to point out that while ATM activation is often associated with DSB formation, it was shown that other forms of stress can activate ATM in a DSB-independent manner, in particular oxidative stress.49
Additionally, ATM phosphorylation at serine-1981 was shown to be mediated by ATR, and not by ATM autophosphorylation, following UV treatment or replication fork stalling.50
Moreover, ATM has been shown to interact directly with the MMR protein MLH1, providing a mechanism for ATM localization to the site of damage indepenent of DSBs.43
While the phosphorylation of ATM(S1981) is associated with ATM kinase activation and subsequent CHK2 phosphorylation, we did not detect CHK2 phosphorylation at threonine-68, nor did we detect CHK2 kinase activation, during this initial period of DDR signaling following MNNG treatment. From this study, we are unable to say whether the observed phosphorylation of ATM at serine-1981 is sufficent to trigger full kinase activation. Indeed, a number of post-translational modifications in addition to the phosphorylation at serine-1981 are required for the the complete activation of ATM.51
Distinct from the cell cycle effects observed at early time points, TK6 cells treated with MNNG undergo an O6
MeG/MMR-dependent intra-S-phase arrest in the second cell cycle that is marked by a dramatic accumulation of S-phase cells as well as a reduction in S-phase DNA replication. In addition, apoptotic cell death is triggered directly out of this second S-phase. That cells initiate such a response only after entering S-phase of the second cell cycle supports the futile repair model for MMR-induced O6
MeG cytotoxicity. According to this model, the futile repair of O6
MeG/T mismatches by MMR generates gapped DNA that interferes with DNA replication in the second S-phase, leading to fork collapse and double strand break formation, both of which are well known activators of the DDR network. Our results indicate a clear intra-S-phase arrest in the second cell cycle following both a low and high dose of MNNG. Further, we note that cells with low levels of damage, upon S-phase completion, continue through the second G2 without a prolonged cell cycle arrest. This result differs from previous studies that have demonstrated a dramatic accumulation of G2/M cells in the second cell cycle following treatment with both toxic and nontoxic doses of MNNG.28, 30
We note that such differences could be attributed to differences in cell type. While our experiments were performed with TK6 human lymphoblastoid cells, reports indicating a G2 arrest after MNNG treatment utilized chinese hamster ovary CHO-9 and human embryonic kidney 293T cell lines, both of which have defective p53 functions.28, 30
Most importantly, we were able to show for the first time a direct connection between cells in S-phase of the second cell cycle, where the toxic damage is presumed to form, and the initiation of cell death via apoptosis.
As described above, it is hypothesized that the ultimate trigger for cell death in response to MNNG is replication forks stalling, ultimately causing fork collapse and the formation of DSBs. In general, the ATM-CHK2 pathway is primarily activated in response to DSB lesions whereas the ATR-CHK1 pathway is activated by various lesions that give rise to ssDNA, like stalled replication forks and DSB repair intermediates. Based on the described model, all of these events (i.e. fork stalling, DSB formation, DSB repair intermediates) are presumed to take place in an MMR-dependent manner in the second S-phase following MNNG exposure. In agreement with this, our data show O6
MeG/MMR-dependent activation of both the ATM-CHK2 pathway and the ATR-CHK1 pathway that coincides perfectly with the accumulation of cells in the second S-phase. At this time, we detect the phosphorylation of ATM(S1981), H2AX(S139), CHK1(S317), and p53(S15 and S20), plus a dramatic increase in p53 levels, and CHK2 and JNK kinase activation. While many of the molecular players overlap between the first and second cell cycle response there are several key differences. For one, the damage signals triggered in the second S-phase are of a much greater magnitude than the damage signals triggered in the first cell cycle. Particularly with regard to the p53 tumor suppressor protein, the extent of DNA damage is an important factor in cellular outcome; low/transient stress induces a p53 transcriptional response that promotes ‘survival’ (i.e. genes involved in cell cycle arrest, DNA repair, metabolic homeostasis, etc.) while high/sustained levels of stress induces a p53 transcriptional response that promotes cell death or senescence.52
Another difference between the early and late signaling response is the activation of CHK2, which occurs only in the second cell cycle. While both CHK1 and CHK2 have roles in regulating cell cycle checkpoints in response to DNA damage, CHK2 plays a prominent role in the regulation of p53 and apoptosis in response to DSBs.53
Therefore, we might speculate that with regard to MNNG, CHK2 mediates cell death while CHK1 serves a protective role by regulating cell cycle progression and DNA replication in the presence of persistent damage. In regards to this, Bartek and Lukas53
coined CHK1 as a “workhorse” and CHK2 as an “amplifier” of the DDR. Interestingly, like CHK2, the stress-activated protein kinase JNK was activated by MNNG in an O6
MeG/MMR-dependent manner around the time that we also observed the accumulation of cells in S phase of the second cell cycle and accumulation of apoptotic cells. JNK has been implicated in the apoptotic response to a variety of stresses and therefore may be important for the induction of apoptosis following exposure to MNNG.54
depicts our model for O6MeG/MMR-dependent cell cycle arrest and cell death according to the results discussed. Briefly, O6MeG lesions are converted to O6MeG/T mispairs in the first replication cycle. Subsequently, members of the MMR pathway, MutSα and MutLα, bind to the O6MeG/T mismatch leading to direct activation of the DDR network that delays cell cycle progression through S- and G2- phase of the first cell cycle. In parallel, ssDNA gaps arise due to futile repair attempts by the MMR pathway. These gaps persist into the second cell cycle where they are encountered by replication forks in the second S-phase. Ultimately, this gives rise to stalled replication forks, fork collapse, and DSBs. Such events in turn trigger activation of the ATR-CHK1, ATM-CHK2, and JNK signaling pathways that together coordinate various cellular responses including an intra-S-phase arrest and, depending on the dose, either repair and survival, or apoptotic cell death.
Proposed model for O6MeG/MMR-dependent cell cycle arrest and cell death