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. 2016 Feb 18;15(5):730–739. doi: 10.1080/15384101.2016.1148841

Dissociation of gemcitabine chemosensitization by CHK1 inhibition from cell cycle checkpoint abrogation and aberrant mitotic entry

Leslie A Parsels a,b, Daria M Tanska b, Joshua D Parsels a,b, Sonya D Zabludoff c,d, Kyle C Cuneo a, Theodore S Lawrence a, Jonathan Maybaum a,b,#, Meredith A Morgan a,#,✉
PMCID: PMC4845922  PMID: 26890478

ABSTRACT

In order to determine the relative contribution of checkpoint abrogation and subsequent aberrant mitotic entry to gemcitabine chemosensitization by CHK1 inhibition, we established a model utilizing the CDK inhibitors roscovitine or purvalanol A to re-establish cell cycle arrest and prevent aberrant mitotic entry in pancreatic cancer cells treated with gemcitabine and the CHK inhibitor AZD7762. In this study, we report that the extent of aberrant mitotic entry, as determined by flow cytometry for the mitotic marker phospho-Histone H3 (Ser10), did not reflect the relative sensitivities of pancreatic cancer cell lines to gemcitabine chemosensitization by AZD7762. In addition, re-establishing gemcitabine-induced cell cycle arrest either pharmacologically, with roscovitine or purvalanol A, or genetically, with cyclin B1 siRNA, did not inhibit chemosensitization uniformly across the cell lines. Furthermore, we found that AZD7762 augmented high-intensity γH2AX signaling in gemcitabine-treated cells, suggesting the presence of replication stress when CHK1 is inhibited. Finally, the ability of roscovitine to prevent chemosensitization correlated with its ability to inhibit AZD7762-induced high-intensity γH2AX, but not aberrant pHH3, suggesting that the effects of AZD7762 on DNA replication or repair rather than aberrant mitotic entry determine gemcitabine chemosensitization in pancreatic cancer cells.

KEYWORDS: aberrant mitotic entry, AZD7762, checkpoint abrogation, CHK1, gemcitabine, pancreatic cancer

Introduction

Pancreatic cancer is a highly aggressive disease associated with both local and systemic disease progression. Although new chemotherapy regimens such as FOLFIRINOX or the addition of nab-paclitaxel to gemcitabine have improved survival compared to gemcitabine alone1,2 median survival for patients with metastatic disease is still less than one year. In addition, these new therapies, while improving quality of life in a majority of patients, are accompanied by increased toxicity.1 Thus, it would be logical to attempt to add novel targeted therapies that may improve response without substantially increasing toxicity.

One promising approach to improve therapy for metastatic disease is to employ cell cycle checkpoint inhibitors in combination with existing regimens of chemotherapy and/or radiation. This strategy is largely based on the idea that, because tumor cells are often deficient in one or more checkpoint activities as part of their neoplastic phenotype (especially the G1/S checkpoint enforced by P53), they should be more sensitive to ablation of additional checkpoint activities, compared to normal cells.3

The protein kinase CHK1 plays a central role in both the S-phase4 and G2 checkpoints5 and is activated in response to either replication stress or direct DNA damage induced by gemcitabine.6-8 In addition, CHK1 promotes HRR (homologous recombination repair)9 and functions in DNA replication to stabilize stalled replication forks,10,11 as well as to prevent excess origin firing and consequently promote recovery of normal replication fork progression.12 Thus, CHK1 inhibitors have been developed as potential targeted therapies predicted to enhance existing therapeutic treatments in part by magnifying chemotherapy-induced DNA damage, either by directly inhibiting DNA repair pathways, or indirectly, by permitting cell cycle progression in the presence of unrepaired lesions or exacerbating chemotherapy-induced replication stress. For example, we and others have shown synergy between CHK1 inhibition and gemcitabine,6,13,14 an antimetabolite with multiple activities that target DNA synthesis including both direct mis-incorporation into DNA and depletion of the deoxynucleotides required for DNA replication via inhibition of the enzyme ribonucleotide reductase.

Several CHK1 inhibitors have been advanced to clinical trial15-18 and like other targeted agents their development requires pharmacodynamic biomarkers to verify target inhibition and subsequent modulation of appropriate molecular pathways in vivo. One of the most widely used markers to confirm the biological activity of CHK1 inhibitors in experimental systems has been phosphorylation of histone H3 at Ser10, which normally occurs in cells entering mitosis and is therefore indicative of either passage through the G2 checkpoint or direct entry of S-phase arrested cells into mitosis (termed premature mitosis).6,7,19 Markers of DNA damage response pathways, such as pCHK1(Ser345) and γH2AX have also been used to confirm the downstream effects of CHK1 inhibition.13,20-22 In particular, the pattern and intensity of γH2AX staining may be informative in terms of discriminating DSBs from the replication stress that results from CHK1 inhibition.10,23-25

In previous studies, we found that sensitization to gemcitabine by the CHK1 inhibitors AZD7762 and PD321852 was substantial, but highly variable, among several pancreatic cancer cell lines.13,20 While investigating the basis for the divergent responses of these cell lines, we observed (contrary to initial expectations) that cell cycle checkpoint abrogation could be clearly dissociated from potentiation of gemcitabine cytotoxicity. Those findings led us to conduct the current study, in which we established a model to evaluate the relative contribution of cell cycle checkpoint abrogation resulting in aberrant mitotic entry to gemcitabine chemosensitization in each of these cell lines. We found that aberrant mitotic entry was not required for chemosensitization by AZD7762, and that the persistent DNA damage (as indicated by high-intensity γH2AX staining) that results from CHK1 inhibition in the presence of gemcitabine better correlates with chemosensitization.

Results

Differences in the extent and kinetics of mitotic entry induced by gemcitabine + AZD7762 among human pancreatic tumor cell lines

In a previous report of gemcitabine chemosensitization by the CHK1 inhibitor AZD7762, we found that CHK1 inhibition 24 h after gemcitabine treatment was optimal for chemosensitization.20 We therefore began the current study by assessing mitotic entry induced by AZD7762 using that schedule (Fig. 1A). Consistent with our previous data,13 while cells treated with gemcitabine alone remained arrested in early S-phase 30 h post-treatment (Fig. S1), over time, MiaPaCa2 and Panc1 cells treated with gemcitabine and AZD7762 exhibited significant levels of aberrant mitotic entry, consisting of both mitotic cells with a normal 4N DNA content and premature mitotic cells with a sub-4N DNA content cells (Fig. 1B and E). The small percentage of MiaPaCa2 cells treated with gemcitabine + AZD7762 entering mitosis with a 4N DNA content at t = 30 h (1.6 ± 0.3%) may reflect the first cells to begin to recover from gemcitabine-induced replication stress. In contrast, gemcitabine-treated BxPC3 cells did not undergo aberrant mitotic entry in the presence of AZD7762 (Fig. 1C) and AsPC1 cells showed only a small level of 4N mitotic entry with very little premature mitotic entry (Fig. 1D). In addition to these variations in the extent of mitotic entry among the cell lines, we noted that the kinetics of mitotic entry also differed: In MiaPaCa2 cells, the greatest number of pHH3-positive cells was detected at t = 30 h (6 h after addition of CHK1 inhibitor), while in Panc1 cells the increase in mitotic cell number was undetectable at t = 30 h, and a longer incubation with AZD7762 was required to force aberrant mitotic entry in gemcitabine-treated cells (16 to 24 h).

Figure 1.

Figure 1.

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Time-dependence of AZD7762-mediated mitotic entry in pancreatic cancer cell lines. Cells treated with gemcitabine ± AZD7762 were collected 26, 30, 40 or 48 h post-gemcitabine (2, 6, 16 or 24 h AZD7762, respectively; (A) and assayed for pHH3 staining by flow cytometry (B-E). Data presented are the mean ± SEM of the percentage of pHH3-positive cells with either a 4N (normal mitosis – shaded bars) or sub-4N (premature mitosis – open bars) DNA content (n = 3–6; p < 0.05 as indicated for *normal or °premature mitosis, one-way ANOVA)

Detection of latent G2 checkpoint abrogation by nocodazole trapping

Given the lack of or minimal aberrant mitotic entry observed in BxPC3 and AsPC1 cells, we considered the possibility that in some cell lines, G2 checkpoint abrogation may be masked by AZD7762-induced abrogation of the mitotic exit checkpoint, which also depends upon CHK1,26 allowing cells to quickly slip through mitosis rather than accumulating in mitosis. This hypothesis is consistent with previously published data from our group demonstrating that AZD7762 accelerates the transit of cells from S-phase, through G2/M and into G1.27

To test this hypothesis, the microtubule inhibitor nocodazole was added concurrently with AZD7762 in the 2 cell lines that showed little or no mitotic entry after gemcitabine + AZD7762 to “trap” any cells escaping from G2 into mitosis (Fig. 2 and Fig. S2). In BxPC3 cells, nocodazole did indeed reveal a small (2.1 ± 0.5%) but significant accumulation of pHH3-positive cells following exposure to gemcitabine + AZD7762. This result suggests that if CHK1 inhibition abrogates a mitotic cell cycle arrest, in addition to the G2 checkpoint, pHH3 may not be an adequate marker of CHK1 inhibition and G2 checkpoint abrogation. In contrast, nocodazole had little effect on pHH3-positive cell accumulation after treatment with gemcitabine + AZD7762 in AsPC1 cells. As these 2 cell lines are similarly sensitized to gemcitabine by AZD7762,20 the data in Figs. 1 and 2 suggest that the extent of checkpoint abrogation leading to aberrant mitotic entry, as determined by pHH3 staining, does not reflect the extent of gemcitabine chemosensitization by AZD7762.

Figure 2.

Figure 2.

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Nocodazole-mediated mitotic arrest allows detection of G2 checkpoint abrogation. BxPC3 and AsPC1 cells treated with gemcitabine ± AZD7762 ± roscovitine (Ros) as illustrated in Fig. 1A were collected 30 h post-gemcitabine (6 h AZD7762 ± 100 ng/mL nocodazole ± Ros) and assayed for the mitotic marker pHH3 by flow cytometry. Data presented are the mean ± SEM of the percentage of pHH3-positive cells with either a 4N (normal mitosis – shaded bars) or sub-4N (premature mitosis – open bars) DNA content (n = 3–4; *p < 0.05 as indicated for normal mitosis relative to the gemcitabine + AZD7762 condition, one-way ANOVA). Representative histograms are presented in Fig. S2.

Isolation of the effects of AZD7762 on cell cycle progression by use of CDK inhibitors

Although we have established that aberrant mitotic entry, as determined by pHH3 flow cytometry, is not proportional to gemcitabine chemosensitization, the data presented above do not address whether the aberrant mitotic entry that does result from CHK1 inhibition is required for chemosensitization. We previously suggested that potentiation of gemcitabine cytotoxicity by CHK1 inhibitors may instead depend upon inhibition of CHK1-mediated DNA repair and persistent DNA damage.13,28 One approach to separating the effects of CHK1 inhibition on cell cycle progression from its effects on DNA repair is to use inhibitors of the cyclin-dependent kinases CDK2 and CDK1, which mediate CHK1-regulated S-phase and G2 checkpoints, respectively. We hypothesized that short-term chemical inhibition of these CDKs, which act downstream of CHK1 to prevent cell cycle progression, would re-establish or preserve cell cycle checkpoints that are abrogated by AZD7762 and thereby prevent chemosensitization resulting from checkpoint abrogation and aberrant mitotic entry. Consistent with this model, we found that either of the CDK inhibitors roscovitine or purvalanol A effectively prevented the appearance of pHH3 in cells treated with gemcitabine + AZD7762 (Fig. 3). Roscovitine also prevented the appearance of pHH3 in nocodazole-treated BxPC3 cells (Fig. 2). Similar results from experiments with an alternative mitotic marker, MPM2, support the conclusion that the loss of pHH3-positive cells in the presence of CDK inhibitors is a direct consequence of preventing aberrant mitotic entry (vs. non-specific inhibition of pHH3 kinases; Fig. S3).

Figure 3.

Figure 3.

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CDK inhibition with roscovitine or purvalanol A prevents AZD7762-mediated checkpoint abrogation and aberrant mitotic entry. Cells treated with gemcitabine ± AZD7762 ± roscovitine (Ros) or purvalanol A (Pur A) as illustrated in Figure 1A were collected 30 or 48 h post-gemcitabine (6 or 24 h AZD7762 ± Ros or Pur A) and assayed for the mitotic marker pHH3 by flow cytometry. Premature mitotic entry (sub-4N; PM) was defined by the left gate and normal mitosis (4N; NM) by the right. A representative experiment in MiaPaCa2 cells with the percentage of cells in each gate is shown (A; t = 30 h). Data presented in (B) are the mean ± SEM of the percentage of pHH3-positive cells with either a 4N (normal mitosis – shaded bars) or sub-4N (premature mitosis – open bars) DNA content (n = 3–6; p < 0.05 as indicated for *normal or °premature mitosis, one-way ANOVA). Note that by the 48 h time point, Panc1 cells treated with gemcitabine alone had recovered from the drug-induced S-phase arrest.

Having demonstrated that roscovitine and purvalanol A re-established cell cycle checkpoints and prevented aberrant mitotic entry, we assessed the impact of these treatments on clonogenic survival, with the expectation that if AZD7762-mediated sensitization were dependent on checkpoint abrogation, then it should be antagonized by the presence of the CDK inhibitors. In two of the 4 cell lines (Panc1 and AsPC1), roscovitine significantly inhibited AZD7762-mediated gemcitabine chemosensitization (Fig. 4C and D). In contrast, roscovitine provided only minimal protection of MiaPaCa2 and BxPC3 cells, and purvalanol A conferred minimal protection in all 4 cells lines, effects which did not reach statistical significance. Taken together, these results demonstrate that while CDK1/2 inhibition may be sufficient to prevent aberrant mitotic entry in response to gemcitabine + AZD7762, it is not sufficient to rescue chemosensitization in all cell types.

Figure 4.

Figure 4.

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Quantification of clonogenic survival assays for pancreatic cancer cell lines treated with gemcitabine or gemcitabine + AZD7762 ± roscovitine or purvalanol A. Assays were performed in triplicate for each condition and normalized to the corresponding non-gemcitabine plating efficiency. Data are presented as the mean surviving fraction ± SEM from 3–6 independent experiments; p < 0.05 compared to cells treated with *gemcitabine alone or °gemcitabine + AZD7762, one-way ANOVA

Direct assessment of the contribution of checkpoint abrogation to gemcitabine chemosensitization using siRNA directed against cyclin B1

Because CDK inhibitors impinge on multiple cell cycle checkpoints, we developed a second model to more selectively determine the importance of aberrant mitotic entry on AZD7762-mediated chemosensitization, namely, siRNA-mediated depletion of cyclin B1, the primary cyclin responsible for mitotic entry.29 Using either of 2 different siRNAs, we were able to deplete cyclin B1 protein and prevent aberrant mitotic entry, as indicated by a reduction in the number of pHH3-positive MiaPaCa2 or Panc1 cells after treatment with gemcitabine + AZD7762 (Fig. 5A and C). Despite the ability of cyclin B1 siRNA to prevent aberrant mitotic entry, it did not rescue chemosensitization in any cell line (Fig. 5D–E). These data further dissociate checkpoint abrogation from AZD7762-mediated chemosensitization.

Figure 5.

Figure 5.

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Cyclin B1 depletion with siRNA prevents AZD7762-mediated checkpoint abrogation and aberrant mitotic entry but does not protect cells from AZD7762-mediated chemosensitization. MiaPaCa2, AsPC1 and Panc1 cells were transfected with cyclin B1 siRNA. Forty-eight h post-transfection, cells were treated with gemcitabine for 2 h, followed 24 h later by AZD7762 for either 6 (MiaPaCa2 and AsPC1) or 24 h (Panc1) as illustrated in Figure 1A. Cyclin B1 depletion was verified by western blot 72 h post-transfection (insets). At the end of treatment, cells were collected and assayed for the mitotic marker pHH3 by flow cytometry. Data presented (A – C) are the mean ± SEM of the percentage of pHH3-positive cells with either a 4N (normal mitosis – shaded bars) or sub-4N (premature mitosis – open bars) DNA content (n = 3–4; p < 0.05 as indicated for *normal or °premature mitosis, one-way ANOVA). Cells were also assayed for drug-induced loss of clonogenicity following depletion of cyclin B1 (D – F). Data presented are the mean surviving fraction ± SEM from 3 independent experiments; p < 0.05 relative to control cells transfected with non-specific (N.S.) siRNA, one-way ANOVA.

The effects of AZD7762 on gemcitabine-induced DNA damage signaling

As we and others have shown, CHK1 inhibitors also have a role in the cellular response to gemcitabine-induced DNA damage, either through inhibition of DNA repair pathways9,13,28 or via inhibition of the cell's ability to recover from gemcitabine-induced replication stress.11 To begin to determine the relative contribution of replication stress to gemcitabine chemosensitization by AZD7762, we next assayed cells treated with gemcitabine + AZD7762 for the DNA damage marker γH2AX. In particular, we wished to look for a high-intensity γH2AX staining pattern which has been documented as a response to either gemcitabine8 or CHK1 inhibition alone10 and may represent persistent replication stress.24,25 We found that AZD7762 did not consistently increase the number of γH2AX positive cells after gemcitabine treatment, in part because in some cell lines, even moderately toxic concentrations of gemcitabine resulted in >80% of cells staining positive for γH2AX (Fig. S4). AZD7762 did, however, increase the number of early S-phase cells with a high-intensity γH2AX signal (Fig. 6). In two of the cell lines, MiaPaCa2 and Panc1, the combination of gemcitabine and AZD7762 was required to induce significant levels of high-intensity γH2AX staining, while in BxPC3 and AsPC1 cells, treatment with gemcitabine alone was sufficient to cause this staining pattern. Importantly, roscovitine significantly attenuated induction of the high-intensity γH2AX staining after CHK1 inhibition in gemcitabine-treated AsPC1 and Panc1 cells, but not in MiaPaCa2 or BxPC3 cells. Furthermore, the ability of roscovitine to inhibit high-intensity γH2AX staining corresponded to the effects of roscovitine on chemosensitization (Fig. 4), where roscovitine significantly protected AsPC1 and Panc1 cells but only weakly protected MiaPaCa2 or BxPC3 cells. These findings suggest that AZD7762-mediated high-intensity γH2AX staining may be a better marker for chemosensitization by CHK1 inhibition than simple γH2AX positivity (or aberrant mitotic entry).

Figure 6.

Figure 6

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The effects of roscovitine on AZD7762-mediated high-intensity γH2AX-staining in gemcitabine-treated pancreatic cancer cells. Cells treated as illustrated in Figure 1A were collected 30 or 48 h post-gemcitabine (6 or 24 h AZD7762 ± Ros) and assayed for γH2AX by flow cytometry (A – D). In each representative dot plot, the lower number is the total percentage of cells in the population considered γH2AX-positive, as defined by the larger gate, while the upper number is the percentage of cells with a high-intensity γH2AX-staining pattern, as defined by the upper gate. Cells treated with gemcitabine ± AZD7762 ± Ros were collected 26, 30, 40 or 48 h post-gemcitabine (2, 6, 16 or 24 h AZD7762, respectively) and assayed for γH2AX by flow cytometry (E – H). Data presented are the mean ± SEM of the percentage of cells with a high-intensity γH2AX staining pattern (n = 3–4)

Discussion

In this paper, we present data which clearly dissociate the ability of the CHK1/CHK2 inhibitor AZD7762 to abrogate cell cycle checkpoints from its ability to sensitize pancreatic cancer cells to the antimetabolite gemcitabine. While we have previously reported that CHK1 inhibition does not uniformly cause checkpoint abrogation and premature mitosis under chemosensitizing conditions,13 to our knowledge this is the only study to show that preventing CHK1-mediated checkpoint abrogation, either pharmacologically or with siRNA, does not necessarily prevent chemosensitization as determined by clonogenic survival. Given the multiple roles of CHK1 in DNA replication10,12,30 and HRR,9,13,27 we also measured the DNA damage response in cells treated with gemcitabine + AZD7762. We found that a high-intensity γH2AX staining pattern previously associated with replication stress and collapsed replication forks best correlated with chemosensitization. These results suggest that DNA replication or cellular responses to DNA replication stress may be a better pharmacological target for sensitizing cancer cells to gemcitabine, and possibly other antimetabolites, than cell cycle checkpoints.

The development of CHK1 inhibitors as chemosensitizers was originally based on the hypothesis that abrogation of CHK1-dependent cell cycle checkpoints, whether intra-S or G2, would sensitize cancer cells to DNA damaging agents due to the lost opportunity to repair DNA prior to cell division.3,31 Furthermore, both premature mitotic entry and G2 checkpoint abrogation are established markers for verifying CHK1 inhibition and chemosensitization.13,32,33 However, in a previous investigation into the ability of the CHK1 inhibitor PD321852 to chemosensitize pancreatic cancer cells to gemcitabine, we found that CHK1 inhibition can promote premature mitotic entry without chemosensitization.13 In our current study, we present data which systematically demonstrate that aberrant mitotic entry (either premature or that resulting from G2 checkpoint abrogation) is not required for gemcitabine chemosensitization by the CHK inhibitor AZD7762 (Figs. 1 and 4). In addition, we show that preservation of cell cycle checkpoints and inhibition of aberrant mitotic entry with either roscovitine, purvalanol A or cyclin B1 siRNA was unable to completely rescue cells from AZD7762-mediated chemosensitization, further disassociating the ability of AZD7762 to abrogate cell cycle checkpoints and force aberrant mitotic entry from its ability to chemosensitize pancreatic cancer cells to gemcitabine (Figs. 3, 4 and 5). These results suggest that the loss of other CHK1-mediated functions, such as stabilization of stalled replication forks8 or regulation of origin firing,10,12 play a significant role in chemosensitization to gemcitabine.

Exploiting replication stress as a mechanism for chemosensitization by CHK1 inhibition was first suggested by work from Feijoo and colleagues34 who reported that CHK1 blocks activation of late replication origins when replication forks have stalled. Subsequent studies10 found that loss of CHK1 activity led to aberrant rates of DNA initiation, accumulation of single strand DNA and a high-intensity γH2AX-staining pattern similar to that observed in cells treated with gemcitabine + AZD7762 (Fig. 6), and more recent studies35,36 have confirmed the potential importance of CHK1 inhibitor–mediated replication stress as a primary mechanism of chemosensitization (via a process termed ‘replication catastrophe’).37 As inhibiting CHK1 during periods of replication stress may ultimately result in MUS81-mediated DNA double strand breaks,21 and CHK1 directly regulates RAD51 during DNA damage-induced HRR,9 it remains to be determined whether the γH2AX signaling in pancreatic cancer cells treated with gemcitabine + AZD7762 reflects persistent replication stress, subsequent DNA double strand breaks, loss of HRR activity or a combination of these effects.

We began these studies with the assumption that we would be able to selectively discern the contributions of cell cycle checkpoint abrogation to AZD7762-mediated chemosensitization with CDK inhibitors such as roscovitine or purvalanol A. In many instances, however, the replication stress associated with CHK1 inhibition is also CDK2-dependent and thus attenuated by these drugs.38 It would be difficult, therefor, to evaluate CDK-dependent checkpoint abrogation as a mechanism of chemosensitization from these data alone. The inability of checkpoint preservation conferred by cyclin B1 depletion with siRNA, however, supports the conclusion that restoration of cell cycle checkpoints and prevention of aberrant mitotic entry does not necessarily prevent gemcitabine chemosensitization by CHK1 inhibition (Fig. 5).

Prior studies have attributed chemosensitization by CHK1 inhibitors to one of several mechanisms including premature mitotic entry or abrogation of the G2 checkpoint,39 inhibition of HRR,13 or faulty DNA replication.35 While individual studies tend to focus on single mechanisms, it is unlikely that one mechanism is solely responsible for chemosensitization. Through the ‘rescue’ types of experiments presented in this study, for the first time the relative contribution of the cell cycle checkpoints preventing aberrant mitotic entry to chemosensitization was discerned. This work clearly shows that in some pancreatic cancer cells checkpoint abrogation resulting in aberrant mitotic entry is not required for gemcitabine chemosensitization and further suggests that replication stress is a contributing mechanism. It should be noted however, that in 2 of the 4 models presented in this study, preventing aberrant mitotic entry caused significant, although partial, protection from chemosensitization (Fig. 4), suggesting that the relative contribution of these mechanisms is model-dependent.

One question that often arises in studies with cell cycle checkpoint inhibitors is whether or not P53 status plays a role in efficacy. For example, Aarts et al reported that breast cancer cells arrested in S-phase by gemcitabine were driven directly into mitosis by the WEE1 kinase inhibitor MK1775 (now AZD1775) without passing through G2,32 similar to the response in MiaPaCa2 and Panc1 cells treated with gemcitabine + AZD7762. Furthermore, this forced mitotic entry was found to be a characteristic of P53 defective cells and correlated with gemcitabine chemosensitization. The data from our panel of pancreatic cell lines, however, do not precisely fit this model. As none of the cell lines in this study have wild-type P53 function, P53 status cannot account for the lack of premature mitotic entry in BxPC3 and AsCP1 cells treated with gemcitabine + AZD7762. Furthermore, direct entry into mitosis from S-phase was not a prerequisite for gemcitabine chemosensitization in these cells, and preventing premature mitotic entry with cyclin B1 siRNA did not prevent chemosensitization. While it is likely that the defective P53 phenotype in these cells contributes to their ability to be chemosensitized by AZD7762, this sensitization is not dependent upon aberrant mitotic entry.

Although the first generation CHK1 inhibitors have largely been abandoned as clinical agents, the reasons behind their demise are due to the agents themselves, rather than CHK1 as a therapeutic target. Indeed, CHK1 inhibitors have played a critical role in establishing the potential of the general strategy of targeting DNA damage response pathways for cancer therapy and newer CHK1 inhibitors are in development.16 This field has grown considerably in the last decade with the introduction of many agents designed to target other proteins in the DNA replication, DNA repair, and cell cycle checkpoint pathways such as ATR,40 RPA41 and WEE1.32,42 Given the lack of effective strategies to treat pancreatic cancer, and the demonstrated efficacy of CHK1 inhibitors in sensitizing pancreatic cancers to gemcitabine or gemcitabine-based chemoradiation,13,20,27,28 second generation CHK1 inhibitors or other agents which target these pathways should continue to be explored as potential therapeutic options.

Materials and methods

Cell culture, transfections and drug solutions

AsPC1, BxPC3, MiaPaCa2 and Panc1 cells were obtained from the American Type Culture Collection and grown in either RPMI (AsPC1 and BxPC3; Invitrogen) or DMEM media (MiaPaCa2 and Panc1; Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 2 mM L-glutamine (Sigma). Specific knock-down of cyclin B1 was performed with X-tremeGENE siRNA Transfection Reagent (Roche) per the manufacturer's protocol using one of 2 different individual siRNAs purchased from Thermo Scientific. Gemcitabine (Gemzar, Eli Lilly) was dissolved in PBS and stored in aliquots at −20°C. AZD7762 (AstraZeneca), roscovitine (Cell Signaling), purvalanol A and nocodazole (Sigma) were each dissolved in DMSO and stored in aliquots at −20°C. All experiments were carried out as illustrated in Fig. 1A: cells were treated with gemcitabine for 2 h, followed 24 h later by 100 nM AZD7762 ± 20 μM roscovitine for 2, 6, 16 or 24 h. In experiments with a single timepoint, MiaPaCa2, BxPC3 and AsPC1 cells were assayed 30 h post-gemcitabine (6 h AZD7762) and Panc1 cells were assayed 48 h post-gemcitabine (24 h AZD7762).

Detection of pSer10 histone H3 or γH2AX by flow cytometry

Treated cells were trypsinized, washed with ice-cold PBS, and fixed at a concentration of 2 × 106 cells/mL in ice-cold 70% ethanol. For pSer10 histone H3 (pHH3) analysis, samples were first incubated with a rabbit anti-pHH3 antibody (#06–570, EMD Millipore) diluted 1:133 in PBS buffer containing 5% FBS and 0.5% Tween-20 (Sigma) overnight at 4°C, followed by incubation with a FITC-conjugated secondary antibody (Sigma Biochemical) as previously described.19 For γH2AX analysis, samples were incubated with a mouse monoclonal anti-γH2AX antibody (JBW301, EMD Millipore) diluted 1:50 in PBS buffer containing 1% FBS and 0.2% Triton X-100 (Sigma), followed by incubation with a FITC-conjugated anti-mouse secondary antibody as previously described.43 Samples were then stained with propidium iodide to assess total DNA content and analyzed on a FACScan flow cytometer (Becton Dickinsson) with FlowJo software (Tree Star).

Clonogenic survival assay

Cells were processed for clonogenic survival as previously described.44 Surviving fractions represent the plating efficiency for a given drug-treated sample divided by the plating efficiency for the corresponding non-gemcitabine condition. Sensitization or protection was defined by statistically significant differences in normalized surviving fractions between drug-treated groups.

Immunoblotting

Whole cell lysates were prepared in cold SDS lysis buffer (10 mM Tris, 2% SDS) supplemented with both phosphatase inhibitor and Complete protease inhibitor cocktails (Roche) as previously described.45 The following antibodies were used: Cyclin B1 (GNS1; Santa Cruz) and GAPDH (Cell Signaling).

Supplementary Material

2015CC6880R-f07-z-4c.pptx

Abbreviations

FBS

fetal bovine serum

γH2AX

phospho-Histone H2A.X (Ser139)

Gem

gemcitabine

Noc

nocodazole

pHH3

phospho-Histone H3 (Ser10)

Pur A

purvalanol A

Ros

roscovitine

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was funded by National Institute of Health grants R01CA163895, R01CA138723, P50CA130810 and an AstraZeneca research grant.

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