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The O6-methylguanine (O6MeG) DNA lesion is well known for its mutagenic, carcinogenic, and cytotoxic properties, and understanding how a cell processes such damage is of critical importance for improving current cancer therapy. Here we use human cells differing only in their O6MeG DNA methyltransferase (MGMT) or mismatch repair (MMR) status to explore the O6MeG/MMR-dependent molecular and cellular responses to treatment with the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We find that O6MeG triggers MMR-dependent cell cycle perturbations in both the first and second cell cycle post treatment. At lower levels of damage, we show that a transient arrest in the second S-phase precedes survival and progression into subsequent cell cycles. However, at higher levels of damage, arrest in the second S-phase coincides with a cessation of DNA replication followed by initiation of apoptotic cell death. Further, we show that entry into apoptotic cell death is specifically from S-phase of the second cell cycle. Finally, we demonstrate the key role of an O6MeG/MMR-dependent multi-pathway, multi-time-scale signaling network activation, led by early ATM, H2AX, CHK1, and p53 phosphorylation and followed by greatly amplified late phosphorylation of the early pathway nodes along with activation of the CHK2 kinase and the stress-activated JNK kinase.
Our DNA is constantly under attack by DNA damaging agents that are present not only in our environment but are also formed endogenously during normal cellular metabolism.1 If not repaired, DNA damage can lead to permanent genetic alterations such as mutations and chromosomal aberrations that ultimately increase our risk of cancer and other diseases.2 In order to maintain genomic integrity, cells activate a DNA damage response that serves to slow down cell cycle progression so that repair of DNA damage can take place before mutations and aberrations are formed.3–4 In cases where damage is gauged as too severe or irreparable, cells activate a programmed cell death response resulting in their elimination from the population. The accurate execution of the DNA damage response is not only important for preventing cancer, but also for treating cancer as many commonly used cancer chemotherapeutic agents function by generating DNA damage that preferentially induces death in fast growing cancer cells.5
Methylating agents are an important class of chemotherapeutic DNA damaging agents currently in use in the cancer clinic.6 The monofunctional SN1 type methylating agent MNNG, like the chemotherapeutic agent temozolomide, produces a spectrum of DNA damage that includes over a dozen different methylated base lesions.7 Although it makes up a small fraction of the overall DNA alkylation, the specific repair of O6MeG by O6MeG DNA methyltransferase (MGMT) prevents many of the detrimental effects of MNNG, pinpointing O6MeG as the major genotoxic, cytotoxic, and carcinogenic lesion in cells treated with agents like MNNG and temozolomide.8 MGMT directly repairs O6MeG by transferring the methyl group from the O6 position of guanine to an internal cysteine residue on the protein; such transfer causes a conformational change leading to MGMT’s ubiquitin-mediated degradation.9–11 If left unrepaired, O6MeG preferentially mispairs with T during DNA replication ultimately giving rise to G:C to A:T transition mutations.12–13 Importantly, O6MeG-induced cytotoxicity is dependent on DNA mismatch repair (MMR), as cells deficient in this pathway are resistant to killing by SN1 type methylating agents, especially in the absence of O6MeG repair by MGMT.14–15 With loss of MMR, cells become ‘methylation tolerant’, escaping cell death even though O6MeG remains unrepaired; however, the cost of such tolerance is vastly increased sensitivity to the mutagenic and carcinogenic effects of methylating agents that produce O6MeG lesions.16
The methylating agent MNNG is known to induce apoptotic cell death in an O6MeG- and MMR-dependent manner.17–21 Two models have been proposed for the mechanism of MMR-mediated sensitivity to this methylating agent, the direct signaling model and the futile repair model; it should be noted that the two models are not mutually exclusive. The first replication past an unrepaired O6MeG lesion gives rise to an O6MeG/T mismatch that can be recognized and bound by MutSα (MSH2/MSH6), followed by the recruitment of MutLα (MLH1/PMS2) to the mismatch.22–24 In the direct signaling model, MMR proteins bound to the O6MeG/T base pair are thought to directly activate a signaling kinase cascade that controls downstream events such as DNA repair, cell cycle arrest, and apoptosis. In support of this hypothesis, recruitment of the ATR protein kinase to the MutSα/MutLα-bound O6MeG /T mismatch, accompanied by ATR kinase activation, was shown to occur in vitro.23 In addition, it was demonstrated that a particular missense mutation in Msh2 or Msh6 uncouples the DNA repair function of MMR from its role in DNA damage-induced apoptosis, suggesting that excision of DNA opposite the O6MeG lesion may not be required for the DNA damage response function of MMR.25–26 Alternatively, it is thought that the futile rounds of excision and resynthesis of DNA at O6MeG /T mismatches (on the newly synthesized T-containing strand) provide the trigger for activation of the DNA damage response. Following recognition of the mismatch by MutSα and MutLα, MMR initiates the excision and reinsertion of the nucleotides in the newly-synthesized daughter strand. Given that O6MeG almost always directs the incorporation of T, iterative futile repair attempts by MMR would lead to secondary lesions in the DNA in the form of nicks and single strand gaps.27 It is hypothesized that these nicks and gaps formed in the first S-phase after damage are encountered by replication forks in the next S-phase resulting in the blockage or collapse of DNA replication forks with ensuing DNA double strand break (DSB) formation. These DSBs in turn trigger a signaling response that activates the cell cycle checkpoint and the apoptotic signaling pathways. Multiple lines of evidence support this model. In agreement with the idea that a cell requires two rounds of replication to induce a DNA damage response, O6MeG/MMR-dependent apoptosis and G2 cell cycle arrest have been shown to be activated only after the second S-phase following treatment with SN1 type methylating agents.28–30 Indeed, it was shown that MMR-dependent single-stranded gaps accumulate and persist in newly replicated DNA after MNNG treatment, providing in vivo evidence of iterative excision by MMR.31 In addition, the importance of the excision step in inducing apoptosis was demonstrated using a mouse model deficient in the major MMR exonuclease, exonuclease 1 (Exo1); the absence of Exo1 conferred significant resistance to O6MeG-induced cell death, although the extent of resistance was cell type dependent.32 Finally, the futile repair model is thought to lead to stalled replication forks at single strand breaks during the second S-phase. The stalled forks are ultimately rescued by recombination bypass or, in the event of fork collapse, give rise to DSBs that also require recombination in order to be resolved. Indeed, both sister chromatid exchanges (SCEs) and chromosome aberrations, two endpoints predicted by the futile repair model, are induced by the O6MeG lesion in a MMR-dependent manner.33 Importantly, the induction of SCEs and chromosome aberrations observed in these studies occurred almost exclusively in the second cell cycle post-treatment. In addition, the loss of homologous recombination grossly sensitizes cells to MNNG in an O6MeG and MMR-dependent manner, suggesting that stalled replication forks and DSB lesions formed at O6MeG/MMR-induced strand breaks are ordinarily resolved by homologous recombination.34–36 Finally, it is important to note that while the futile repair model requires two S-phases to begin signaling the DNA damage response, direct signaling can, in principle, begin during the first S-phase after the induction of O6MeG.
In this study, we made use of the TK6 human lymphoblastoid cell line and its isogenic derivatives TK6/MGMT+ and TK6/MMR− to explore the O6MeG/MMR-dependent signaling in more detail. It was previously shown that the O6MeG DNA base lesion triggers a MMR-dependent apoptotic response in TK6 cells, and that this response is mediated at least in part by the mitochondrial signaling pathway.37 Expression of the MGMT protein that specifically repairs the O6MeG lesion in the TK6/MGMT+ cells suppressed, in its entirety, the apoptotic response of TK6 cells upon treatment with the methylating agent MNNG. In addition, the MMR-deficient TK6-derivative cell line, TK6/MMR−, was used to demonstrate that the O6MeG-induced apoptotic response is entirely MMR dependent; the MMR-deficient phenotype of TK6/MMR− cells is caused by a mutation in MSH6 that disables the MutSα sliding clamp from translocating along the DNA, an important early event in the MMR process.38–39 TK6/MMR− cells are resistant to MNNG despite the persistence of O6MeG lesions in their DNA.15, 40 Here we systematically investigated the cellular response of TK6 and its derivatives to the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) to gain a better understanding of how cells coordinate cell cycle progression and cell death triggered by the critical DNA base lesion O6MeG. Quantitative, dynamic measurements of the DNA signaling response were simultaneously obtained to further our understanding of the signaling events that trigger such cellular responses.
To explore the cell death decision mechanism triggered by O6MeG, TK6 human lymphoblastoid cell line and its isogenic derivatives TK6/MGMT+ and TK6/MMR− were treated with varying doses of the methylating agent MNNG. The treatment conditions examined include no (0 µg/ml; 0 µM), low dose (0.01 µg/ml; 0.068 µM), and high dose (0.1 µg/ml; 0.68 µM) MNNG. Concentrations were chosen to provide different levels of induction of O6MeG- and MMR-dependent cell death. Current evidence suggests that cells require two rounds of replication to induce toxic lesions; therefore, phenotypic and signaling measurements were taken over a period of 48 hours post damage (TK6 cell doubling time 14–16 hours). Multiple molecular and cell behavioral endpoints were simultaneously examined to provide multi-variate insight into how DNA damage-induced cell behavior is regulated downstream of this chemotherapeutically-relevant DNA damage lesion.
Treatment of TK6 cells with MNNG resulted in a dose-dependent inhibition of cell growth. The viable cell density of TK6 cells was monitored over time following exposure to MNNG (Figure 1A). We observed a significant decrease in the viable cell density (compared with untreated control cells) as early as 12 and 24 hours after treatment with a high and low MNNG dose (0.1 and 0.01 µg/ml respectively) and this inhibition increased with time (Figure 1B). At 48 hours, TK6 control growth was 60 % and 19 % for low dose and high dose conditions, respectively (Figure 1B). The decreased cell growth was dependent on the persistence of O6MeG lesions and on functional MMR as no growth inhibition was observed in MNNG-treated TK6/MGMT+ or TK6/MMR− cells (Figures 1A and 1C). It is important to note that the viable cell density of untreated TK6 cells doubled within the first 16 hours of the time course and that this doubling was delayed following treatment with low dose (0.01 µg/ml) and high dose (0.1 µg/ml) MNNG (Figure 1A). This would suggest that the growth inhibition we observed at 12 hours is due to events occurring within the first cell cycle following treatment with MNNG (Figure 1B). Although delayed, the viable cell density of TK6 cells treated with low dose (0.01 µg/ml) MNNG doubled twice within the 48 hour time course experiment, once by 20 hours and again at 40 hours (Figure 1A), indicating cell division and progression into subsequent cell cycles. In contrast, when TK6 cells were treated with high dose (0.1 µg/ml) MNNG the viable cell density doubled only once. To gain more insight into the source of this DNA damage-induced growth inhibition we chose to look at the cellular responses of cell death and cell cycle progression in more detail.
A study by Quiros et al. showed that MNNG-induced apoptosis in CHO-9 cells is a late event occurring only after cells pass through two replication cycles and enter their second G2/M-phase post MNNG treatment.30 To examine the timing of the cell death response in TK6 cells, we monitored several distinct markers of apoptosis including caspase-3 and PARP cleavage, phosphatidylserine exposure, membrane permeability, and DNA fragmentation. An early event in apoptosis is the flipping of the phospholipid phosphatidylserine (PS) to the outer surface of the cell membrane. This translocation of PS was analyzed by flow cytometry using the phospholipid-binding protein annexin V as a probe to detect PS exposure in cells undergoing apoptosis. Staining with annexin V in combination with the vital dye 7AAD allowed us to distinguish between cells that were early apoptotic (annexin+7AAD−), mid apoptotic or necrotic (annexin+7AAD+), and late apoptotic or necrotic (annexin−7AAD+). Both mid to late apoptotic and necrotic cells lose their membrane integrity and stain positive for 7AAD. We observed a small but significant increase in the number of cells staining positive for annexin V and/or 7AAD (total cell death) as early as 24 hours after treatment with low dose (0.01 µg/ml) MNNG and 16 hours after treatment with high dose (0.1 µg/ml) MNNG (Figure 2A). Cell death increased steadily with time in a dose-dependent manner, with 11%, 30%, and 76% cell death at 48 hours for untreated, low dose MNNG, and high dose MNNG respectively (Figure 2A). Representative flow cytometry plots of untreated TK6 or TK6 treated with high dose MNNG are shown in Figure 2B. Although differentiation between necrosis and mid- to late-apoptosis could not be determined directly, detection of PS by annexin V preceded the loss of membrane integrity, providing evidence that the cell death observed in these studies is apoptotic rather than necrotic (illustrated in Figure 2C).
Caspases play a central role in the execution of apoptotic cell death, and we used flow cytometry to monitor the active form of the effector caspase, caspase-3, along with cleavage of its substrate, PARP. We observed a small yet significant increase in apoptosis in TK6 cells at 16 hours in response to both low (0.01 µg/ml) and high (0.1 µg/ml) doses of MNNG that increased with time in a dose-dependent manner (Figure 3A); in other words the increase in apoptosis with time was steeper at the high dose versus the low dose of MNNG. At 48 hours, cleaved (active) caspase-3 and/or cleaved (inactive) PARP levels (total apoptosis) was detected at 4%, 13%, and 68% for untreated, low dose MNNG, and high dose MNNG respectively (Figure 3A). Figure 3B shows the representative flow cytometry plots obtained for untreated TK6 and TK6 treated with high dose MNNG. Importantly, the induction of apoptosis observed in TK6 cells was O6MeG- and MMR-dependent, as TK6/MGMT+ cells and TK6/MMR− cells did not display any significant increase in apoptosis following treatment with high dose (0.1 µg/ml) MNNG (Figure 3C). Another commonly used marker and hallmark of apoptotic cell death is the fragmentation of DNA. In agreement with the caspase-3 and PARP results, flow cytometry analysis of cells stained with propidium iodide (PI) showed a dose-dependent accumulation of subG1 DNA content beginning 16 hours after treatment with MNNG (Figure 4G); these results are discussed later in the context of other events in the cell cycle. In summary, using multiple methods of detection, we have demonstrated that O6MeG and MMR-dependent apoptosis was triggered as cells entered the second cell cycle post MNNG treatment, supporting an indirect futile cycling model for mismatch repair-mediated signaling for cytotoxicity. With that being said, we observed a significant MNNG-induced reduction in viable cell density (compared to untreated cells) as early as 12 hours after high dose treatment (Figure 1B), several hours prior to the induction of apoptosis, leading us to hypothesize that additional behaviors besides cell death are influencing cell growth after MNNG treatment. Cells exposed to DNA damage undergo cell cycle arrest leading to a delay in cell cycle progression. To examine whether the growth inhibition we observed was brought about by a delay in cell cycle progression we monitored the dynamics of the cell cycle response in cells exposed to MNNG.
To characterize the cell cycle response to DNA alkylation damage we monitored DNA content in combination with BrdU incorporation as a marker of active DNA replication following MNNG exposure. At various time points after the addition of MNNG, cells were pulse labeled with BrdU for one hour and subsequently analyzed both for the incorporation of BrdU into replicating DNA and for total DNA content. Coupling a measure of active replication with DNA content allowed us not only to visualize and quantitate cell cycle stage, but also to monitor cellular events during S-phase in more detail. Figure S1 depicts BrdU versus DNA content flow cytometry plots for a subset of time points analyzed in order to demonstrate the gating used to quantify the fraction of cells in each phase of the cell cycle. As shown, G1 and G2/M cells have 2N and 4N DNA content respectively. Using this method we could detect very early S-phase replicating cells even though they stain as G1 cells with PI, and we could detect very late S-phase replicating cells with apparent G2/M DNA content. Moreover, in mid S-phase it was possible to distinguish replicating and non-replicating cells by whether or not they had incorporated BrdU during the one hour pulse labeling time. As mentioned previously, subG1 DNA content was used as a measure of apoptotic cell death.
The visualization and detailed quantitation of cell cycle stage with time following treatment of TK6 cells with low and high doses of MNNG revealed interesting cell cycle dynamics (Figures 4A–H and S2). As early as eight hours after MNNG treatment TK6 cells showed a small but significant increase (p-value<0.01, two sample t-test comparing treated to untreated control) in the proportion of cells in mid S-phase (high dose) and G2/M-phase (low and high dose) of the cell cycle with a simultaneous dose-dependent decrease in the G1 population (Figures 4A–C). These data indicate a delay in cell cycle progression within the first cell cycle following treatment with MNNG and explains the observed delay in growth at early time points (Figure 1A). This initial checkpoint response appeared to dissolve by 16 hours as cells were observed to progress from S to G2/M and from G2/M to G1, indicated by a drop in mid S-phase (total) cells in parallel with an increase in the G2/M and G1 population at this time (Figures 4A–C; high dose). At 24 hours post treatment we observed a distinct accumulation of mid S-phase (total) cells under both low (0.01 µg/ml) and high (0.1 µg/ml) dose conditions compared with untreated cells (Figure 4B). Notably, at this time the number of viable cells under both treatment conditions had doubled suggesting that these cells are entering into the second S-phase post treatment (Figure 1A). Under low dose conditions this accumulation was transient and was followed by an increase in the G2/M and G1 fraction of cells at 32 and 40 hours respectively, indicating resumed progression through the cell cycle (Figures 4A and 4C). In support of cell cycle progression, the density of these cells reached a second doubling around 40 hours (Figure 1A). Following treatment with the high dose, cells showed an increased delay in S-phase compared to the low dose, with accumulation of total mid S-phase cells peaking at 32 hours (Figure 4B). In contrast to the low dose, the subsequent decrease of mid S-phase cells at late time points was not accompanied by an increase in the G2/M or G1 populations (Figures 4A and 4C); rather we observed a corresponding increase in cells with subG1 DNA content (Figure 4G). An increase in subG1 was also observed in cells treated with the low dose of MNNG, albeit to a lesser extent. It is important to note that the cell cycle perturbations that we observed in TK6 cells were O6MeG- and MMR-dependent. Equivalent cell cycle analysis was carried out using TK6/MGMT+ and TK6/MMR− cell lines following high dose treatment with MNNG (Figures S3–S6). Although a significant change (p-value<0.05, two sample t-test comparing treated to untreated control) was observed at eight hours in the G2/M- and early S-phase populations after high dose treatment in TK6/MGMT+ cells, the perturbation was minimal and much smaller than that seen for TK6 cells (Figure S4). At all the subsequent time points, no significant differences were observed with high dose treatment compared to untreated cells in TK6/MGMT+ cells (Figure S4). TK6/MMR− cells showed no significant differences between the untreated and treated conditions at any of the time points measured (Figure S6). From this analysis we have demonstrated that MNNG induces dynamic changes in all phases of the cell cycle and that all of these perturbations are O6MeG- and MMR-dependent. Delays in cell cycle progression in response to DNA damage are brought about by the activation of cell cycle checkpoints that arrest cells in a particular cell cycle stage allowing time for DNA repair. Here we look more closely at several of these cell cycle checkpoints.
Previous studies have demonstrated a G2 arrest in the second, but not the first, cell cycle following MNNG treatment.28, 30 To test whether the early cell cycle perturbations we observed in TK6 cells involved a bona fide G2 arrest, cells were stained with an antibody against the mitotic marker, phospho-histone H3, and mitotic cells were detected by flow cytometry (Figure 4I). As discussed previously, a small but significant increase (p-value<0.01, two sample t-test comparing treated to untreated control) in the percent of G2/M-phase cells was seen at eight hours following treatment with both low and high doses of MNNG (Figure 4C). At this time, we also detect a significant decrease (p-value<0.05, two sample t-test comparing treated to untreated control) in the proportion of mitotic cells staining positive for phospho-histone H3 (Figure 4I). This data suggests that shortly after MNNG treatment TK6 cells activate a G2 checkpoint preventing G2 cells from entering mitosis. However, this checkpoint appeared to be short-lived as the fraction of mitotic cells returned to untreated levels at 16 hours, accompanied by a corresponding movement of cells into G1 (compare high dose curves in Figures 4A and 4I). Cell division and the entrance of cells into G1 from G2 requires movement through mitosis; indeed the changes in phospho-histone H3 positive cells (mitotic cells) and G1-phase cells mirror each other throughout the time course experiment (compare Figures 4A and 4I). Although a transient G2 arrest cannot be eliminated in the second cell cycle, a strong delay in the progression of cells into G1 from G2/M at late time points was not evident following low dose treatment. Following the movement of cells into G2/M-phase from S-phase, we detected an increase in the fraction of phospho-histone H3 positive cells between 32 and 40 hours in parallel with as an increase in the fraction of G1-phase cells at this time (compare low dose curves in Figures 4A and 4I). In addition, cell division and cell cycle progression after low dose treatment was supported by a second cell density doubling around 40 hours as discussed previously (Figure 1A). Under high dose conditions, cells did not appear to progress into G2/M from the second cell cycle S-phase but rather we observed an increase in the fraction of cells with subG1 DNA content at this time. In agreement with this, phospho-histone H3 levels remained low at late time points (Figure 4I). The observation that TK6 cells do not arrest in G2 of the second cell cycle in response to MNNG is in contrast to previous studies that show a dramatic accumulation of cells in this phase of the cell cycle.28, 30 Here we observed that TK6 cells accumulate in mid S-phase of both the first and second cell cycle, and under high doses do not appear to escape the second S-phase indicating activation of an intra-S phase checkpoint that we chose to look into further.
In an effort to protect the genome, cells that encounter damage to their DNA during DNA replication can activate an intra-S-phase checkpoint. This checkpoint slows the progression of S-phase, allowing time for repair, both by suppressing the firing of late replication origins and by stabilizing stalled replication forks. As discussed previously, we observed an O6MeG/MMR-dependent accumulation of cells in mid S-phase in both the first and second cell cycle following treatment with MNNG, suggesting activation of an intra-S-phase checkpoint (Figure 4B). To look more closely at the replication status of these cells, we divided the mid S-phase population into replicating and non-replicating populations defined by whether or not they incorporated BrdU during a one hour period prior to the time point indicated. Figures 4E and 4H break down the data in Figure 4B into replicating (4E) and non-replicating (4H) cells; note that the data look dramatically different in Figure 4E versus 4H, while 4B represents a composite. At late time points, we observed an increase in mid S-phase cells with impaired DNA replication following MNNG treatment (Figure 4H). To account for changes in the number of mid S-phase cells specifically, loss of replication potential is more clearly represented in Figure 5 that shows mid S BrdU− cells as a proportion of total mid S-phase cells as opposed to the proportion of total cells (as displayed in Figure 4). Beginning 16 and 24 hours after high dose and low dose treatment, respectively, we observed an increase in the fraction of TK6 cells in mid S-phase with grossly reduced BrdU incorporation, and the proportion of cells with such low replication activity increased with time in a dose-dependent manner (Figure 5). At 48 hours the majority (80%) of cells remaining in mid-S phase following treatment with high dose MNNG were not engaging in DNA replication (Figure 5). From this data it is clear that the DNA lesion O6MeG provokes an intra-S phase checkpoint that, through either stalled replication forks or a reduction in the firing of replication origins (or both) results in a dose-dependent decrease in DNA replication, accompanied by the accumulation or trapping of cells in S-phase.
Cell cycle analysis of TK6 cells treated with MNNG indicated an intra-S-phase arrest in both the first and second cell cycle post treatment (Figure 4). The second intra-S-phase arrest was marked by an increase in the overall S-phase population as well as a decrease in the replication status of those S-phase cells. Importantly, the second intra-S-phase arrest coincided with the induction of cell death via apoptosis. We hypothesized that the MNNG-induced intra-S-phase arrest in the second cell cycle, is accompanied by an inability to recover, and that this prompts cells to trigger apoptosis. To test the connection between triggering apoptosis and cell cycle stage we treated TK6 cells with MNNG and measured cleaved (active) caspase-3 in combination with DNA content. MNNG induced an increase in cleaved caspase-3 as cells entered the second cell cycle post treatment (>16 hours) and the majority of cleaved caspase-3 positive cells had S-phase or subG1 DNA content across all time points (Figure 6A, Figure S7). At 40 hours, 92% of caspase-3 positive cells had either S phase or subG1 DNA content following treatment with high dose (0.1 µg/ml) MNNG (Figure 6A). The specificity for cell death out of S-phase is evident in Figure 6B, which depicts the cell population 32 hours after treatment with MNNG according to their cleaved caspase-3 and DNA content status. Under low dose conditions, a fraction of cells in S-phase showed an induction of apoptosis, illustrated by their positive cleaved caspase-3 status on the flow cytometry plot, while cells that had progressed through S into G2/M were negative for cleaved caspase-3 and therefore not undergoing caspase-dependent apoptosis (Figure 6B). Under high dose conditions, a similar but more pronounced S-phase-dependent activation of caspase-3 was observed (Figure 6B). It is important to note that a significant fraction of the subG1 population is negative for cleaved caspase-3 indicating a caspase-independent mode of apoptosis whose cell cycle origin cannot be accounted for using this assay (Figure S7, see 48 hour time point). Similar results were obtained for cleaved PARP (Figure S8). Overall, our results suggest that following cell cycle arrest, cells that have repairable or tolerable amounts of damage are able to avoid triggering apoptosis and are permitted to proceed through the cell cycle whereas if severe damage is uncovered during the S-phase of the second cell cycle post treatment with MNNG, cells are shunted directly into apoptosis without entering G2.
We have demonstrated that MNNG induces O6MeG/MMR-dependent cell cycle perturbations in TK6 cells not only in the second cell cycle following treatment, as expected, but also in the first cell cycle (Figure 4). In addition, while low levels of damage ultimately give rise to survival and progression into subsequent cell cycles, high levels of damage induce apoptotic cell death out of the second S-phase post treatment. To enhance our understanding of the O6MeG/MMR-dependent cell cycle checkpoint and apoptotic cell death response induced by MNNG, we chose to monitor the activation or phosphorylation of a number of key signaling proteins thought to mediate such outcomes in response to DNA damage and replication stress. As described previously, TK6 cells were treated with various doses of MNNG and collected at specific time points post treatment for subsequent analysis. Quantitative western blotting was used to monitor dynamic changes in total- and phospho-protein levels while protein kinase activation was monitored directly using an IP kinase activity assay. The results presented below are based on the average of three independent time course experiments. Figure 7 provides an illustration of typical western blot results used for the quantitation shown in subsequent figures.
Following TK6 cell treatment with MNNG, we detected the phosphorylation of ATM(S1981), CHK1(S317), p53(S15), and p53(S20), as well as CHK2 kinase activation (Figure 8A–F). While evidence points to ATR playing a prominent role in the DNA damage response to MNNG, we do not have a good measure for its direct activation and therefore infer activity based on the phosphorylation of its downstream target CHK1 on the ATR-dependent residue serine-317.41 In addition to the protein modifications mentioned, the stability of p53 appeared to be enhanced as we detected an MNNG-induced accumulation of total p53 with kinetics similar to its phosphorylation on serine-15 and serine-20 (Figure 9A). To address p53’s primary role as a transcriptional regulator of genes we additionally assessed the levels of several well studied p53-regulated transcripts involved in apoptosis, cell cycle regulation, and DNA repair and found a similar pattern of induction between their expression and p53 protein levels with MNNG treatment (Figure 9B). Taken together these results suggest that p53 may play a transcriptional role in the regulation of cell behavior following MNNG treatment in TK6 cells though further studies are needed to determing p53’s function in this context. In addition to the canonical DNA damage signaling proteins just mentioned, the activation of several members of the MAPK pathway known to regulate various cellular processes in response to extracellular stimuli were monitored. While no or little activation of ERK or MK2 was observed, respectively, we did observe a dramatic induction of JNK in TK6 cells after treatment with MNNG (Figure 8G–I). To address whether the observed signaling events originated from MNNG-induced lesions other than O6MeG, we repeated these experiments in TK6/MGMT+ and TK6/MMR− cell lines. To our surprise, time course experiments in TK6/MGMT+ and TK6/MMR− demonstrated that MNNG-induced activation of all of the signaling events tested, namely H2AX(S139), CHK1(S317), p53(S15 and S20), total p53, CHK2 kinase activation, and JNK kinase activation, were indeed triggered solely by the O6MeG lesion in a MMR-dependent fashion across all time points monitored (Figures S9 and S10).
Though such signaling responses have been noted in previous studies in relation to the methylating agent MNNG, the quantitative and temporal approach taken here allows us to examine the timing of such events in further detail. For example, we observed a significant increase in the phosphorylation of ATM at serine-1981 above untreated levels beginning 16 hours after treatment with low dose (0.01 µg/ml) MNNG that, while minimal, remained upregulated up to 36 hours (Figure 8A). Interestingly, ATM (S1981) phosphorylation kinetics under high dose (0.1 µg/ml) conditions appeared biphasic with a detectable early response (0–16 hours) and more dramatic late response (16–36 hours) (Figure 8A). Of note, transition into the second phase occurred as cells began to enter their second cell cycle post treatment, at which time we observed the accumulation of cells in S phase, loss of DNA replication in S phase cells, and accumulation of apoptotic cells (Figure 4B, Figures 4H and and5,5, and Figure 4G respectively). We detected a pattern of phosphorylation similar to ATM for H2AX(S139), CHK1(S317), p53(S15) and p53(S20). That is, for each of these proteins, a modest yet significant increase in phosphorylation levels were observed at early (0–16 hours) time points followed by a more pronounced increase in phosphorylation levels at late (16–36 hours) time points (Figure 8B). In contrast, the induction of CHK2 and JNK kinase activation was significantly delayed. An increase in CHK2 activity above untreated levels was not detected until 20 and 28 hours after exposure to high dose (0.1 µg/ml) and low dose (0.01 µg/ml) MNNG, respectively, after which time CHK2 activation continued to rise (Figure 8D). Since we observed ATM phosphorylation immediately after cell exposure to MNNG, the lack of activation of CHK2, a downstream target of the ATM kinase, at early time points was unexpected. To confirm that the unexpected observation of delayed CHK2 activation was not due to differences in assay sensitivity (western blot versus kinase activity assay) we measured the phosphorylation of CHK2 at threonine-68 by western blot. Indeed, the phosphorylation of CHK2 at threonine-68 by western blot was detected in MNNG treated cells with the same kinetics to those seen with kinase activity measurements, confirming lack of CHK2 activation at early time points (Figure S11). Further, as with CHK2, our results demonstrated a delayed induction of JNK activation following MNNG treatment (Figure 8I); JNK activity increased beginning 12 hours and 20 hours after treatment with 0.1 µg/ml MNNG and 0.01 µg/ml MNNG, respectively, in the late phase after MNNG exposure.
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 O6MeG/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 O6MeG in the first S-phase. In support of this, we observed O6MeG/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 O6MeG 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 to O6MeG/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 O6MeG/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 O6MeG 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 O6MeG/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 O6MeG cytotoxicity. According to this model, the futile repair of O6MeG/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 O6MeG/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 O6MeG/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
Figure 10 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.
The TK6 B-lymphoblastoid cell line and its derivatives, TK6/MGMT+ and MT1 (here referred to as TK6/MMR−) have been described previously.37 Cells were maintained in RPMI medium 1640 (Gibco #11875) supplemented with 10% equine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Due to the large number of cells needed, time course experiments were performed in 1 L or 3 L spinner flasks. For drug treatment, logarithmically growing cells were split to a density of 3×105 cells/ml and MNNG, in 0.1 M sodium acetate (pH 5.0) was added directly to the culture medium. Cells were collected at the indicated time points and prepared as described for further analysis. Viable and total cell density were monitored using a Vi-cell XR cell viability analyzer (Beckman-Coulter) which determines cellular viability using a trypan blue exclusion test. Percent control growth was quantified as the percent viable cell density of treated cells compared to untreated control cells at the indicated time points post treatment.
Cells were washed once with annexin binding buffer (ABB: 10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) and resuspended in 50 µl ABB containing PE-conjugated annexin V (1:25 dilution) and 7AAD (15 µg/ml). Cells were incubated for 15 minutes at room temperature. Following incubation, 250 µl of ABB was added to the cell suspension and analyzed immediately on a BD FACScan flow cytometer. 7-aminoactinomycin D (7AAD) (#A1310) and PE-conjugated annexin V (#A35111) were obtained from Molecular Probes.
Cells were washed once in cold PBS and resuspended in 500 µl 4% paraformaldehyde in PBS (Electron Microscopy Sciences #15700). Cells were fixed for 15 minutes at room temperature. After fixation, cells were washed with PBS and resuspended in 500 µl 100% methanol. Methanol suspensions were stored at −20°C. All antibody staining steps was performed in 96-well v-bottom plates. Fixed cells were resuspended in 200 µl PBS-B (PBS + 1% BSA) and blocked at room temperature for 30 minutes. Following incubation, cells were incubated in 100 µl of primary antibody diluted at 1:200 in PBS-TB (PBS-B + 0.1% Tween 20) for 1 hour at room temperature. Anti-cleaved (active) caspase-3 antibody (#559565) and anti-cleaved (inactive) PARP antibody (#552597) were purchased from BD Biosciences. Following primary antibody incubation, cells were washed twice with PBS-T (PBS + 0.1% Tween 20). Cells were then resuspended in 100 µl PBS-TB containing secondary antibody diluted at 1:600 and incubated for 1 hour at room temperature. The secondary antibodies used, PE-conjugated goat anti-rabbit IgG (#P2771) and Alexa Fluor 647-conjugated goat anti-mouse IgG (#A21235), were purchased from Molecular Probes. Finally, cells were washed twice with PBS-T, resuspended in 100 µl PBS-TB, and analyzed on a BD FACSCalibur flow cytometer.
For the simultaneous detection of DNA content and apoptosis, cells were fixed and stained for cleaved (active) casapase-3 and cleaved (inactive) PARP as described above. Following secondary antibody incubation, cells were washed once with PBS-T (PBS + 0.1% Tween 20) and resuspended in 200 µl Hoechst 33258 (10 mg/ml stock solution, Invitrogen) at 1:10,000 dilution in PBS. Cells were incubated in Hoechst 33258 for 10 minutes at room temperature in the dark. Finally, cells were washed with PBS-T and resuspended in 120 µl PBS-TB (PBS-T + 1% BSA) for analysis on a FACS LSR II flow cytometer.
Cells were washed once in cold PBS and resuspended in 500 µl 4% paraformaldehyde in PBS (Electron Microscopy Sciences #15700). Cells were fixed for 15 minutes at room temperature. After fixation, cells were washed with PBS and resuspended in 500 µl 100% methanol. Methanol suspensions were stored at −20°C. Fixed cells were washed once with PBS followed by permeabilization with Triton X-100. For permeabilization, cells were suspended in 400 µl of PBS + 0.25% Triton X-100 for 15 minutes at room temperature. Afterwards, cells were washed with PBS-B (PBS + 1% BSA) and resuspended in 100 µl PBS-B containing primary antibody against phosphorylated Histone H3 (0.75 µg). Anti-phospho-Histone H3 (Ser 10) antibody was purchased from Upstate (#06-570). Cells were incubated with primary antibody for three hours at room temperature. Following antibody incubation, cells were washed twice with PBS-B and incubated with Alexa Flour 647-conjugated goat anti-rabbit IgG antibody (Molecular Probes #A21244) diluted in 100 µl PBS-B (1:30) for 30 minutes at room temperature. Finally, cells were washed twice with PBS and resuspended in 1 ml PBS containing 50 µg propidium iodide (Molecular Probes # P21493) and 500 µg RNase A (Sigma #R6513). Cells were analyzed on a BD FACSCalibur flow cytometer for phospho-Histone H3 detection and DNA content.
One hour before cell collection, BrdU was added to the culture medium at a concentration of 10 µM. At the corresponding time point, cells were collected, washed with cold PBS, and resuspended in 500 µl cold PBS. Cells were fixed by adding 5 ml ethanol (-20°C) while vortexing to prevent clumping and stored at 4°C overnight. In order to provide access to the incorporated BrdU, partial DNA denaturation was needed prior to staining with anti-BrdU antibody. Fixed cells were washed with PBS-B (PBS + 1% BSA) and incubated in 1 ml 2 M HCl + 0.5% Triton X-100 for 30 minutes at room temperature, followed by incubation in 1 ml 0.1 M Na2B4O7 for 2 minutes at room temperature. Next, cells were washed in PBS-TB (PBS-B + 0.5% Tween 20) and resuspended with anti-BrdU antibody (BD Biosciences #555627) diluted in 60 µl PBS-TB (5 µg antibody per approx 5×106 cells) and incubated at room temperature for 2 hours. Cells were washed twice with PBS-T (PBS + 0.5% Tween 20) and resuspended in 55 µl PBS-T containing 5 µl Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (1:11 dilution, 10 µg of antibody per approx 5×106 cells). Cells were incubated with secondary antibody for 1 hour at room temperature (Molecular Probes #A21235). Cells were then washed twice with PBS-T and resuspended in 1 ml PBS containing 50 µg propidium iodide (Molecular Probes # P21493) and 500 µg RNase A (Sigma #R6513). Cells were analyzed on a BD FACSCalibur flow cytometer for BrdU detection and DNA content.
Cells were washed with cold PBS and lysed in 1% Triton X-100, 50 mM Tris-HCl (pH 7.5), 150 mM sodium chloride, 50 mM β-glycerophosphate (pH 7.3), 10 mM sodium pyrophosphate, 30 mM sodium fluoride, 1 mM benzamidine, 2 mM EGTA, 100 µM sodium orthovanadate (NaVO4), 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml microcystin-LR. Lysates were incubated on ice for 30 minutes, and clarified by centrifugation at max speed (16110×g) for 10 minutes at 4°C. Protein concentrations of clarified extracts were determined using the BCA protein assay (Pierce). Lysates prepared for western blotting were sonicated (Branson Model 450 Digital Sonifier: Amplitude 20%, Pulse Time 2 seconds, Repeat 3×) prior to centrifugation.
A microtiter-based assay was used in these studies to monitor protein kinase activity following DNA damage. The assay was adapted from Janes et al.55 who monitored various kinase activities to identify critical nodes within the signaling network regulating cytokine-induced apoptosis.56 The assay is divided into three parts: immunoseparation of the kinase in microtiter wells; in vitro kinase reaction; and measurement of 32P incorporation into the substrate. A detailed description of the experimental procedure was published previously55 and will be described here briefly along with any adjustments made to the original protocol. Protein A or G coated microtiter wells were incubated overnight at 4°C with 10 µg/ml antibody against the kinase of interest. The following antibodies were used to perform the immunoprecipitation step: Anti-MAP Kinase 1/2 (Erk1/2), CT (Upstate #06-182), Anti-JNK1 (C-17) (Santa Cruz Biotechnology #sc-474), Anti-MAPKAPK-2 (Stressgen #KAP-MA015), Anti-Chk2 (Bethyl Laboratories #BL1432). The next day, wells were washed and incubated with cell lysate (150 µg for CHK2, 37.5 µg for ERK, 150 µg for JNK, and 150 µg for MK2) for three hours at 4°C. After being washed, the wells were filled with kinase buffer containing cold ATP and [32P]ATP along with the kinase-specific substrate. The substrate along with the concentration of cold ATP and [32P]ATP used in each kinase reaction can be found in Table S1. The substrates include myelin basic protein (purchased from Sigma #M1891), MK2tide (described in 57), and GST-ATF2 (1–109) (described in 58). Following incubation at 37°C for one hour, the reaction was quenched with 20 mM EDTA or 75 mM phosphoric acid depending on the kinase being tested (see Table S1). The contents were then applied to a 96-well phosphocellulose filter to capture the phosphorylated substrate. After repeated washings, the filter was punched into scintillation vials and the [32P]substrate was measured by liquid scintillation counting. The linearity and range of the assay, as well as its sensitivity to external stimuli, was tested in our model system for each of the protein kinases monitored in this study. All measurements were normalized to a positive control in order to control for day to day assay variability; results are reported as fold activation above untreated background levels (activity at 0 hours).
Regulatory phosphorylation events and total protein levels of several key DNA damage signaling proteins were monitored by quantitative western blotting. Whole cell lysates were prepared at a concentration of 2.5 mg/ml in sample buffer, containing 1X LDS (Lithium Dodecyl Sulfate) Sample Buffer (NuPage, Invitrogen) and 2.5% β-mercaptoethanol, and heated at 80°C for 10 minutes. Protein samples were separated by SDS-PAGE using the NuPage Electrophoresis System (Invitrogen), and transferred to a PVDF or nitrocellulose membrane in transfer buffer (49.1 mM TrisBase, 38.6 mM Glycine, 0.04% SDS, 20% Methanol) for one hour at 100V using a Criterion Blotter (BioRad). After the transfer, membranes were blocked at room temperature for one hour with Odyssey Blocking Buffer (OBB, Licor Biosciences) and then probed with primary antibodies. Primary antibodies were diluted in a 1:1 mixture of OBB:PBS-T (PBS + 0.1% Tween 20) and incubated with the membrane overnight at 4°C. Following repeated washes with PBS-T, IRDye secondary antibodies (Licor Biosciences) were diluted in a 1:1 mixture of OBB:PBS-T (1:5000-1:10000 dilution) and incubated with the membrane for one hour at room temperature covered from light. The membrane was washed again in PBS-T and stored in PBS at 4°C in the dark until detection using the Odyssey Infrared Imaging System (Licor Biosciences). Using the Odyssey’s two-color detection system we were able to detect both phopho- and total- antigen targets simultaneously by probing with secondary antibodies that could be visualized in different fluorescence channels. Following protein detection, membranes were stained with actin antibody which was used as a loading control. The specific immunoblot conditions for each of the proteins detected are described in Table S2. Antibodies were tested for linearity and sensitivity using a positive control. Positive controls were run on every gel and signal intensities were normalized to the intensity of the reference sample to correct for differences in transfer and probing efficiencies. Each time course is reported as fold change above untreated background levels (signal at 0 hours).
TK6 cells were treated with 0 µg/ml, 0.01 µg/ml, or 0.1 µg/ml MNNG as described and collected at various time points following exposure to the drug. Total RNA was extracted from cells with TRIZOL reagent (Invitrogen) and purified using Qiagen’s RNeasy Mini Kit. Evaluation of sample quality was performed on the Agilent 2100 BioAnalyzer (BioMicro Center, MIT). Sample labeling, array hybridization, and data extraction were performed by the BioMicro Center (MIT). RNA was hybridized to HGU133 Plus 2.0 full human genome arrays (Affymetrix). Data normalization was performed in GenePattern.59 Affymetrix CEL files were uploaded to GenePattern (ExpressionFileCreator module) which was then used to carry out quantile normalization using the Robust Multichip Average (RMA) algorithm.
The authors would like to thank Gerard Ostheimer for help in the initial phases of this project. The authors have been supported by US National Institutes of Health grants CA112967, CA055042, CA 014051 and ES002109. L.D.S. is an American Cancer Society Research Professor.