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Research Articles: Targets

Differential roles of checkpoint kinase 1, checkpoint kinase 2, and mitogen-activated protein kinase–activated protein kinase 2 in mediating DNA damage–induced cell cycle arrest: implications for cancer therapy

Cancer Research, Abbott Laboratories, Abbott Park, Illinois

Requests for reprints: Haiying Zhang, Cancer Research, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-6101. Phone: 847-938-4857; Fax: 847-935-7551. E-mail: haiying.zhang{at}abbott.com

	Abstract

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Mammalian cells initiate cell cycle arrest at different phases of the cell cycle in response to various forms of genotoxic stress to allow time for DNA repair, and thus preserving their genomic integrity. The protein kinases checkpoint kinase 1 (Chk1), checkpoint kinase 2 (Chk2), and mitogen-activated protein kinase–activated protein kinase 2 (MK2) have all been shown to be involved in cell cycle checkpoint control. Recently, cell cycle checkpoint abrogation has been proposed as one way to sensitize cancer cells to DNA-damaging agents due to the expected induction of mitotic catastrophe. Due to their overlapping substrate spectra and redundant functions, it is still not clear which kinase is mainly responsible for the cell cycle arrests conferred by clinically relevant chemotherapeutics. Thus, the issue remains about which kinase is the most therapeutically relevant target and, more importantly, whether multiple kinases might need to be targeted to achieve the best efficacy in light of recent studies showing superior efficacy for pan-receptor tyrosine kinase inhibitors. To clarify this issue, we investigated the roles of the three kinases in response to different genotoxic stresses through small interfering RNA–mediated specific target knockdowns. Our result showed that only the down-regulation of Chk1, but not of Chk2 or MK2, abrogated camptothecin- or 5-fluorouracil–induced S-phase arrest or doxorubicin-induced G₂-phase arrest. This was followed by mitotic catastrophe and apoptosis. Moreover, double inhibition of Chk1 and Chk2 failed to achieve better efficacy than Chk1 inhibition alone; surprisingly, inhibition of MK2, in addition to Chk1 suppression, partially reversed the checkpoint abrogation and negated mitotic catastrophe. We further showed that this is due to the fact that in MK2-deficient cells, Cdc25A protein, which is critically required for the mitotic progression following checkpoint abrogation, becomes greatly depleted. In summary, our findings show that Chk1 is the only relevant checkpoint kinase as a cancer drug target and inhibition of other checkpoint kinases in addition to Chk1 would be nonproductive. [Mol Cancer Ther 2006;5(8):1935–43]

	Introduction

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DNA-targeted agents are among the most effective in clinical cancer therapy and constitute the cornerstones of modern cancer treatment. These agents can be divided into four main classes: alkylating agents, antimetabolites, topoisomerase inhibitors, and radiomimetics. Their exact mechanisms of action vary greatly; however, a common theme is that they all confer DNA damage, directly or indirectly, and induce cell cycle checkpoints. Despite their effectiveness at elevating the overall survival rates of cancer patients, they have serious intrinsic limitations such as widespread tumor-resistance and severe toxicity in normal tissues, resulting in a narrow therapeutic window (1).

Mammalian cells have established highly elaborate surveillance systems to detect DNA damages and other forms of genotoxic stress, which is essential to maintain the genomic integrity and, hence, cellular viability. When damage is detected, cells activate sophisticated pathways, called cell cycle checkpoints, to arrest cells in different phases of the cell cycle to allow sufficient time for DNA repair. In normal cells, checkpoint responses are a critical safeguard to prevent tumorigenesis promoted by genetic instability; however, in tumor cells, checkpoints constitute a major mechanism of resistance to chemotherapeutic drugs that damage DNA because they reduce the effects of these drugs (2, 3).

It has previously been postulated that targeting the cellular checkpoint pathway could be an attractive approach to circumvent the cancer resistance as discussed above. The rationale is that in normal cells, DNA damage would arrest the cells mostly in G₁ in a p53-dependent manner, whereas p53-deficient tumors, accounting for over half of all tumor-types, have to rely on the checkpoint mediators to arrest cells at S or G₂-M checkpoint. Therefore, checkpoint inhibition would only abrogate the cell cycle blocks in tumor cells to induce mitotic catastrophe and apoptosis, but mostly spare the normal cells. This may offer a potential feasible therapeutic window (4, 5).

Two structurally unrelated but functionally similar protein serine/threonine kinases, checkpoint kinase 1 (Chk1) and checkpoint kinase 2 (Chk2), have emerged as the major mediators of cell cycle checkpoints in response to genotoxic stress. Homozygous Chk1 knockout is lethal in mouse embryonic stem cells. The mouse embryonic stem cells that conditionally lack the Chk1 gene cannot prevent mitotic entry in response to ionizing radiation, showing that Chk1 is required for the G₂-M checkpoint (6). In adult somatic cells, Chk1 deficiency does not result in lethality and cells are viable and display normal cell cycle profiles (7, 8). Complete deficiency of Chk1 in avian DT-40 lymphoma cells abolished DNA damage–induced G₂ arrest and undermined S-phase checkpoint in response to replication stress (8). Small interfering RNA (siRNA)–mediated knockdown of Chk1 in various human cancer cell lines also revealed an essential role of this kinase in both S and G₂-M DNA damage checkpoints (9, 10). Unlike Chk1, Chk2 knockout mice are viable and fertile (11). Although structurally distinct from Chk1, Chk2 shares overlapping substrate specificity with Chk1 and can phosphorylate critical Chk1 substrates, such as Cdc25A and Cdc25C, both in vitro and in vivo. Additionally, Chk2 is rapidly phosphorylated and activated following exposure to various DNA-damaging agents such as ionizing radiation or topoisomerase inhibitors. This indicated that Chk2 also plays a role in cell cycle checkpoints (12). Consistently, studies with dominant-negative Chk2, siRNA-mediated Chk2 ablation, or intrinsic cellular Chk2 deficiency have all confirmed a role of Chk2 in the S and G₂ checkpoints in response to double-strand breaks in various immortalized human cell types (13–15). In contrast to these reports showing that Chk2 is required for checkpoint induction in human cell lines, murine fibroblasts with Chk2 deletion showed no significant defects in the S-phase checkpoints, indicating that it is not a major checkpoint mediator in mice (11, 16, 17). This discrepancy was attributed to the differential requirements of Chk2 between human and murine systems or to the slow proliferation rate of murine fibroblasts versus the fast growth rate of immortalized human cell lines (12).

In the current paradigm of S or G₂ checkpoint pathway, genotoxic stress activates ataxia telangiectasia mutated or ataxia telangiectasia mutated and Rad3-related, which phosphorylate and activate both Chk1 and Chk2 (5, 12). Key substrates for these two checkpoint kinases are the Cdc25A, B, and C tyrosine phosphatases, which regulate the timely activation of cyclin-dependent kinases at the G₁-S and G₂-M transitions. For agents that induce S-phase arrest, Chk1/Chk2 target Cdc25A to proteolysis through the ubiquitin pathway, resulting in the suppression of cyclin-dependent kinase-2 activity and, hence, S-phase arrest. Agents that confer G₂-M arrest also activate Chk1/Chk2 to target Cdc25A to degradation. Additionally, they induce the cytoplasmic sequestration of Cdc25B and Cdc25C through the 14-3-3 protein, eventually leading to G₂ arrest due to the failure to activate the mitotic cyclin-dependent kinase 1/Cdc2 kinase (5). Therefore, it is reasonable to conclude that simultaneous inhibition of both Chk1 and Chk2 should be necessary to fully abrogate the checkpoint and, hence, confer maximal efficacy of potentiation (12, 18).

Mitogen-activated protein kinase–activated protein kinase-2 (MK2) was discovered as a protein kinase activated by extracellular signal–regulated kinase 1/2 from rabbit skeletal muscle. Extracellular signal–regulated kinase 1/2 and p38 were first reported to activate MK2 and MK3 in vitro, but it was later found that extracellular signal–regulated kinase 1 and 2 are not physiologic kinases for MK2. Rather, MK2 activity is potently stimulated by various activators of p38 and p38ß (19, 20). The targeted deletion of the MK2 gene in mice provided the unexpected result that although p38 mediates the activation of many similar kinases, MK2 seems to be the key kinase responsible for p38-dependent biological processes involving cytokine synthesis (21). Loss of MK2 leads to a defect in lipopolysaccharide-induced synthesis of cytokines such as TNF- and -IFN. Consistent with a role for MK2 in inflammatory responses, MK2-deficient mice show increased susceptibility to infection (22). A recent finding has added new complexities to the above paradigm. Manke et al. showed that MK2 recognizes the same phosphorylation sites on Cdc25B/C as Chk1/Chk2. More importantly, MK2 is directly responsible for Cdc25B/C phosphorylation and their subsequent 14-3-3 binding in response to UV-induced DNA damage in mammalian cells. Down-regulation of MK2 eliminates DNA damage–induced G₂-M and intra-S phase checkpoints. Therefore, it was proposed that MK2 is a new member of the DNA damage checkpoint kinase family that functions in parallel with Chk1 and Chk2 to integrate DNA damage signaling responses and cell cycle arrest in mammalian cells (23, 24).

Judging from these results, it is still far from clear which checkpoint kinase is the major mediator of cell cycle arrest in response to various DNA-damaging agents, especially the clinically relevant ones. It is possible that different forms of DNA damage require different checkpoint kinases to enforce the arrest. This issue will be critical for the development of small-molecule inhibitors targeting the checkpoint kinase(s). Additionally, it remains unknown whether inhibition of multiple checkpoint kinases may confer better efficacy in potentiation of chemotherapeutics. In comparison, it has been well established that, targeting multiple receptor tyrosine kinases, a pan-receptor tyrosine kinase inhibitor confers better efficacy in both antiangiogenesis and overall tumor regression (25–28). Here we attempted to clarify these pressing issues with three frequently used chemotherapeutics including the topoisomerase-I inhibitor camptothecin, the topoisomerase-II inhibitor doxorubicin, or the antimetabolite 5-fluorouracil (5-FU). Using siRNA technology, we selectively down-regulated each of the checkpoint kinases singly or in various combinations and examined the effects on checkpoint abrogation, mitotic progression, and cell survival. Our result show that only Chk1 inhibition is required for significant efficacy whereas knockdown of other kinases in addition to Chk1 not only fails to deliver better efficacy but also, in some cases, attenuates or eliminates the potentiation effect seen with Chk1 inhibition alone.

	Materials and Methods

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Cell Culture
Human cervical cancer cell line HeLa and human non–small-cell lung cancer cell line H1299 were obtained from American Type Culture Collection (Manassas, VA). H1299 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum, 1 mmol/L sodium pyruvate, 1% penicillin-streptomycin, and 0.45% glucose at 37°C in a 5% CO₂ incubator. HeLa cells were grown in DMEM supplemented with 10% fetal bovine serum, 1 mmol/L sodium pyruvate, 1% penicillin-streptomycin, and 0.45% glucose at 37°C in a 5% CO₂ incubator. Before transfection, the cells were switched to medium without any penicillin-streptomycin.

Antibodies
Antibodies against Chk1, Chk2, and Cdc2 Y15P were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against MK2 was purchased from Cell Signaling Technology (Beverly, MA). Antibodies for phospho-histone H3 (P-H3) and phospho-histone H2AX (P-H2AX) were obtained from Upstate Technology (Waltham, MA).

Transfection
Human Chk1 siRNA and control luciferase siRNA were as previously described and obtained from Dharmacon Technology (Lafayette, CO; ref. 29). siRNA transfection protocols were also as previously described (29). HeLa cells were transfected with Oligofectamine whereas H1299 cells were transfected with Lipofectamine-2000 (Invitrogen, Carlsbad, CA). The final concentration of each siRNA was 50 nmol/L.

Western Blot Analysis
Adherent cells in the well were rinsed with PBS and directly lysed in Laemmli sample buffer. Floating cells or cell fragments in the same well were collected, lysed, and combined with the above lysates. Samples were heated at 95°C for 5 minutes and resolved on the Novex mini-gel system (Invitrogen) under denaturing conditions and blotted to polyvinylidene difluoride membrane using a semidry transfer device (Amersham Biosciences, Piscataway, NJ). The membrane was blocked with 5% nonfat dry milk and probed with various antibodies. Enhanced chemiluminescent detection was done with enhanced chemiluminescence reagents according to the protocols of the vendor (Santa Cruz Biotechnology).

Cell Proliferation Assay [3-(4,5-Dimethyl-Thiazol-2yl)-5-(3-Carboxymethoxyphenyl)-2-(4-Sulfophenyl)-2H-Tetrazolium Assay]
HeLa cells were seeded in 96-well plates and transfected with the indicated siRNA. Eight hours after transfection, the cells were treated with the indicated DNA-damaging agents for 48 hours. After treatment, 3-(4,5-dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent, which measures the amount of live cells (Promega, Madison, WI), was added to the cells and allowed to develop for 20 minutes to 2 hours. Colorimetric measurement was taken at 490 nm on Spectra MAX 190 from Molecular Devices (Sunnyvale, CA).

Colony Formation Assay
H1299 cells in six-well plates were transfected with the indicated siRNA (or combinations of siRNAs). Twenty-four hours posttransfection, the cells were trypsinized and split into new six-well plates at 500 per well. After attachment overnight, the cells were treated with either DMSO (control) or low doses of camptothecin for 9 days. The formed colonies were then fixed and stained with methylene blue. Total colony formation (considering both surface area and staining intensity) was scanned as pixel counts and calculated with Image-Pro program.

Caspase Assay
Fluorometric assay for caspase-3 was done essentially according to the recommendations of the manufacturer (Roche Applied Sciences, Nutley, NJ) in a 96-well plate. HeLa cells transfected with the indicated siRNAs were treated or not with 150 nmol/L of camptothecin for 32 hours and assayed for caspase activity. Excitation was at 400 nm and fluorescence emission was detected at 505 nm. Caspase-3 activity was calculated as the differential emission at 505 nm between treated sample and blank buffer only.

Cell Cycle Analysis
Cells undergoing the indicated treatments were washed once in PBS and fixed in 70% ethanol. The fixed cells were washed again twice with PBS and treated with RNase A at 37°C for 30 minutes. Finally, the cells were stained with propidium iodide and incubated in the dark for 60 minutes or overnight before analysis. The samples were analyzed by flow cytometry with a fluorescence-activated cell sorter manufactured by BD Bioscience (San Jose, CA) using the CellQuest program.

	Results

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To clarify the unresolved issue of which checkpoint kinase represents the best cancer target for sensitization of tumor cells to various clinically relevant DNA-damaging agents, and additionally, whether inhibition of multiple kinases confers superior efficacy than inhibition of a single kinase, we transfected HeLa cells with siRNAs that specifically targeted Chk1, Chk2 or MK2, either singly or in various combinations. We first investigated the morphologic profiles of HeLa cells undergoing the various knockdowns, treated with the topoisomerase-I inhibitor camptothecin at 150 nmol/L or with the topoisomerase II inhibitor doxorubicin at 150 nmol/L for 1 day (Fig. 1 ). The doses correspond to 25% of the cytotoxic EC₅₀ values of the drugs in cell proliferation assay so that they would not confer extensive cell death by themselves, making it easier to detect the potentiation effect of the siRNAs.

View larger version (34K): [in this window] [in a new window]   Figure 1. Morphologic study of HeLa cells transfected with Chk1, Chk2, and MK2 siRNAs, either singly or in combination, and treated with topoisomerase inhibitors. HeLa cells were transfected in six-well plates with siRNA(s) targeting either control luciferase, Chk1, Chk2, or MK2, either singly or in combination as indicated. Sixteen hours posttransfection, cells were treated or not with 150 nmol/L camptothecin (CPT) or 150 nmol/L doxorubicin (Dox) for 24 h. Cells were examined under phase-contrast microscope to analyze the sensitization effect of the differential siRNA-treatments (A). Cells under each condition were also harvested for Western blot analysis to confirm the specific knockdown of the target of each siRNA (B).

Western blot confirmed efficient and specific knockdowns of the various kinase targets by only their respective siRNAs but not the other siRNAs. Additionally, double siRNA transfections achieved comparable extents of target knockdown as single siRNA (Fig. 1B).

We further probed the underlying mechanisms of the above finding by molecular marker analysis. HeLa cells were transfected with the various siRNAs and treated with 150 nmol/L camptothecin. Twenty-four hours later, the cells were harvested for immunoblot analysis (Fig. 2A ). We first investigated the profile of P-H3, the major M-phase marker. Camptothecin treatment in control cells (transfected with Luciferase siRNA) suppressed the P-H3 signal, in line with the expected S-G₂ phase arrest and a corresponding depletion of mitotic cells (lanes 1 and 2). Chk1 siRNA rescued the P-H3 signal, indicating a successful abrogation of cell cycle block and renewed mitotic progression (lanes 3 and 4). In contrast, Chk2 or MK2 siRNA failed to rescue P-H3 (lanes 5–8), suggesting a failure to abrogate the camptothecin-induced checkpoint. Chk1 and Chk2 combination knockdown showed similar efficacy as Chk1 knockdown alone (lanes 9 and 10) whereas Chk2 and MK2 siRNA combination did not lead to a recovery of the P-H3 signal (lanes 13 and 14). In contrast, the combination of Chk1 and MK2 siRNAs failed to abrogate the checkpoint and induce mitotic progression (lanes 11 and 12), suggesting that MK2 knockdown not only failed to enhance the efficacy of Chk1 siRNA but actually blocked its ability to abrogate cell cycle checkpoint. This is consistent with the surprising finding in Fig. 1 showing a failure of the double Chk1/MK2 knockout to potentiate the cytotoxicity of camptothecin or doxorubicin.

View larger version (22K): [in this window] [in a new window]   Figure 2. Cell cycle marker analysis of HeLa cells transfected with siRNAs targeting various checkpoint kinases and treated with camptothecin (A) or 5-FU (B). HeLa cells were treated similarly as in Fig. 1. Cells were harvested for protein analysis for P-H3 and P-H2AX to ascertain mitotic progression and double-strand break induction. Chk1, Chk2, and MK2 immunoblots were done to confirm target knockdowns by the corresponding siRNAs. Actin was probed as loading control. Representative of three separate tests. C, HeLa cells transfected with the indicated siRNAs were treated or not with 150 nmol/L camptothecin for 32 h and assayed for caspase activity according to the suggestions of the manufacturer. Excitation was at 400 nm and fluorescence emission was detected at 505 nm.

Because we and others have previously shown that besides DNA-damaging agents such as topoisomerase inhibitors, Chk1 inhibition could also greatly potentiate the efficacy of antimetabolites and other replication inhibitors such as 5-FU, hydroxyurea, and gemcitabine (29–32), we tested whether we could extend the above observations to 5-FU. HeLa cells were similarly transfected with various siRNAs and subjected to treatment with 5-FU at 50 µmol/L for 24 hours. This dose corresponds to 20% of the EC₅₀ value of 5-FU in suppressing HeLa cell growth in a cell proliferation assay. We used the same two markers, P-H3 and P-H2AX, to examine the effect of each siRNA on the checkpoint abrogation and potentiation of DNA damages (Fig. 2B). As expected, in control cells, 5-FU successfully depleted the mitotic cell population as shown by the elimination of the P-H3 signal, consistent with the expected G₁-S arrest. Chk1 siRNA, but not Chk2 or MK2 siRNA, recovered the P-H3 signal, indicating a successful checkpoint abrogation followed by mitotic progression, which led to enhanced double-strand breaks as denoted by increased P-H2AX signal. Again, additional MK2 knockdown blocked the efficacy of Chk1 siRNA. However, distinct from Fig. 2A, in which Chk1 and Chk2 double knockdown induced similar effect as Chk1 siRNA alone in potentiation of camptothecin, Chk1 and Chk2 double knockdown failed to achieve the full effect of Chk1 inhibition alone in potentiation of 5-FU both in terms of checkpoint abrogation (P-H3 signal recovery) and potentiation of DNA damage (P-H2AX signal elevation), indicating that to sensitize tumor cells to antimetabolites, only Chk1 inhibition is necessary and additional Chk2 or MK2 inhibition may be counterproductive.

To correlate the observed checkpoint abrogation with induction of apoptosis, we further tested the caspase activation of the differentially transfected HeLa cells with or without camptothecin treatment for 32 hours (Fig. 2C). Due to the relative short duration of the treatment and the low dose of camptothecin used, control cells did not show a dramatic induction of caspase activity in response to camptothecin treatment. Although not activating caspase by itself, Chk1 siRNA greatly potentiated the camptothecin-induced caspase activity (>5-fold increase). Again, Chk2 or MK2 siRNAs were not effective in this assay. Combination knockdowns additionally confirmed that Chk1/Chk2 knockdown was modestly lower than Chk1 knockdown alone, and MK2 knockdown significantly diminished the induction of caspase activity conferred by Chk1 siRNA.

To ascertain whether the above conclusions can be applied to other cancer cell lines besides HeLa cells, we carried out similar siRNA transfections in H1299 cells, a non–small-cell lung cancer cell line (Fig. 3 ). In addition to P-H3, we also characterized the profile of another cell cycle marker, phospho-Cdc2 Y15P. Cdc2 kinases undergo phosphorylation at Y15 position in response to DNA damage, resulting in inhibition of the kinase activity and, hence, S or G₂ phase arrest. Therefore, this marker is commonly used as an indicator of cell cycle arrest. H1299 cells with various siRNA transfections were treated with camptothecin or doxorubicin and cell lysates were probed for Cdc2 Y15P (Y15P) and P-H3. As expected, doxorubicin and camptothecin conferred G₂ or S phase arrest in control cells by inducing the Y15P signal, and correspondingly, the depletion of the mitotic marker, P-H3 (lanes 1–3, Fig. 3A). Chk1 knockout completely eliminated the increases in Y15P level and rescued the P-H3 signal (lanes 4–6), indicating an efficient checkpoint abrogation followed by mitotic progression. Interestingly, Chk2 knockdown also significantly diminished the elevation of the Y15P signal, albeit to a lesser extent than Chk1 siRNA. This suggests that Chk2 knockdown may have partially activated Cdc2. However, the P-H3 profile showed no recovery, indicating that partially activated Cdc2 was insufficient for complete checkpoint abrogation or mitotic progression. MK2 siRNA did not abolish the doxorubicin-induced increase in Y15P level but abrogated the camptothecin-induced increase, suggesting that MK2 knockdown may partially relieve camptothecin-induced checkpoint (lanes 10–12). However, the P-H3 profile again showed that this partial effect failed to translate into productive mitotic progression.

View larger version (25K): [in this window] [in a new window]   Figure 3. Cell cycle marker analysis of H1299 cells transfected with siRNAs targeting Chk1, Chk2, and MK2 and treated with doxorubicin or camptothecin. H1299 cells were transfected in six-well plates with the indicated siRNAs either singly (A) or in various combinations (B) and then treated with 200 nmol/L doxorubicin or 200 nmol/L camptothecin for 24 h. Protein lysates were harvested for immunoblot analysis for the checkpoint marker Cdc2 Y15P and the mitotic marker P-H3.

Fluorescence-activated cell sorting analysis was carried out to corroborate the above molecular marker analysis (Fig. 4 ). As expected, control cells displayed prominent G₂-M arrest in response to doxorubicin and a hybrid S-G₂ arrest in response to camptothecin. Under our experimental condition (150 nmol/L doxorubicin or camptothecin for 1 day), no significant cell death population (sub-G₁ peak) was observed. Consistent with previous reports (7, 29), Chk1 down-regulation abrogated the doxorubicin-induced G₂-phase arrest by lowering the G₂ peak, resulting in apoptosis (G₂-population decreased from 60% to 33% whereas cells with a sub-G₁ DNA content increased from 1.5% to 13.3%; Fig. 4B). Similarly, Chk1 siRNA also abrogated the camptothecin-induced hybrid S-G₂ peak and drove the cells into sub-G₁ phase (total S-G₂ population decreased from 72% to 57% whereas cells with a sub-G₁ DNA content increased from 3.6% to 15.7%). Chk2 siRNA only showed a modest abrogation effect compared with Chk1 siRNA (decreasing doxorubicin-induced G₂-population from 60% to 52% and camptothecin-induced S-G₂ population from 72% to 64% but conferring no significant change to the cells with a sub-G₁ DNA content). This is fully consistent with the Chk2 siRNA immunoblot result in Fig. 3A showing a modest abrogation of the Cdc2 Y15P signal but no rescue of the mitotic marker P-H3. Similarly, MK2 siRNA also only showed a very limited abrogation profile. Chk1 and Chk2 double knockdown decreased doxorubicin-induced G₂-phase arrest from 60% to 39% and decreased camptothecin-induced S-G₂ arrest from 72% to 58%. Concomitantly, it increased the cells with a sub-G₁ DNA content from 1.5% to 12.4% for doxorubicin and from 3.6% to 12.8% for camptothecin. These figures are virtually identical to those achieved by Chk1 siRNA alone, again indicating no additional benefits from Chk2 knockdown. In contrast, double Chk1 and MK2 knockdown only decreased doxorubicin-induced G₂-phase arrest from 60% to 52% (compared with 33% achieved by Chk1 siRNA alone) and only increased apoptotic cells in sub-G₁ phase from 1.5% to 6.4% (compared with 13% achieved by Chk1 siRNA alone). For camptothecin, the MK2/Chk1 double siRNA increased the cells with a sub-G₁ DNA content from 3.6% to 5.5%, much less than the 15% accomplished with Chk1 siRNA alone. Therefore, MK2 siRNA significantly abolished the ability of Chk1 siRNA to abrogate the G₂ or S phase arrest, conferring apoptosis. In summary, these results confirmed the above molecular marker analysis and further established that Chk1 is the dominant checkpoint kinase.

View larger version (22K): [in this window] [in a new window]   Figure 4. Cell cycle analysis of H1299 cells transfected with siRNAs targeting various checkpoint kinases and treated with doxorubicin or camptothecin. A, H1299 cells were treated similarly as in Fig. 2 and subjected to fluorescence-activated cell sorting analysis. All profiles were uniformly scaled. B, cells in different phases were quantitated with the CellQuest program and the percentages of cells in sub-G1, G1, S, and G2-M phases were plotted to display the extent of checkpoint abrogation and induction of apoptosis. Representative of three independent trials.

View larger version (25K): [in this window] [in a new window]   Figure 5. Knockdown of Chk1, but not of Chk2 or MK2, potentiates chemotherapeutics in both short-term cell proliferation assay and long-term colony formation assay. A, HeLa cells were transfected with the indicated siRNAs (single or combination) and treated or not with 150 nmol/L camptothecin or 50 µmol/L 5-FU for 2 d. Cell proliferation assay (MTS assay) was done to assess cell growth under each condition. Luciferase siRNA–transfected cells with no drug treatment was used as 0% growth inhibition. B, H1299 cells transfected with the indicated siRNAs were trypsinized and replated into six-well plates at 500 per well. Cells were treated with vehicle or 0.3 nmol/L camptothecin for 9 d to allow colony formation. Colonies were then stained with methylene blue and scanned. Total colony formation was calculated with Image-Pro program. Luciferase siRNA was used as control (100% colony formation). Representative of three separate trials.

To identify the underlying mechanisms of this interesting but puzzling finding, we focused on Cdc25A, the critical downstream effector molecule of Chk1 in the checkpoint pathway. It has been shown that Chk1 mediates cell cycle checkpoint by targeting Cdc25A, which is required for both S and G₂-M transitions, to proteasome-mediated degradation. Therefore, Chk1 down-regulation rescues Cdc25A from proteolysis, which then drives cells into premature mitosis despite unrepaired DNA damage or unfinished replication (5, 9). Because the endogenous Cdc25A level in H1299 cells was very low, we used H1299 cells expressing low levels of ectopic Cdc25A for this study. The cells were transfected with siRNAs targeting either luciferase, MK2, or Chk1 and then were harvested 24 hours later for immunoblot analysis of Cdc25A expression profile (Fig. 6 ). In comparison with luciferase siRNA transfection, Chk1 siRNA increased the Cdc25A protein level, consistent with previous studies showing that Chk1 constitutively targets Cdc25A to degradation even in the absence of DNA damage (10, 13). In contrast, MK2 down-regulation abolished the Cdc25A signal, indicating that loss of MK2 destabilized the Cdc25A protein. On the other hand, Cdc25C, another member of the Cdc25 phosphatase family, did not show significant variation among the different knockdowns. Hence, we speculated that MK2 siRNA may have prevented Chk1 siRNA from abrogating the cell cycle checkpoint due to its ability to destabilize the Cdc25A protein, which is required for the checkpoint abrogation and cell cycle progression. We are in the process of further characterizing this interesting discovery. We also carried out Chk2 siRNA knockdown in a similar assay, which showed that Chk2 depletion does not affect Cdc25A stability (data not shown).

View larger version (22K): [in this window] [in a new window]   Figure 6. MK2 knockdown destabilizes Cdc25A. H1299 cells were transfected with very low level of Cdc25A expression vector (0.03 µg/well in a 12-well plate) and then additionally transfected with either luciferase, MK2, or Chk1 siRNA at 50 nmol/L. Protein lysates were analyzed 24 h later for Cdc25A or Cdc25C expression. Chk1 and MK2 were also probed to confirm the siRNA efficacy.

	Discussions

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Our cell cycle marker analysis reveals that partial Cdc2/cyclin-dependent kinase activation is not sufficient to mount an effective checkpoint abrogation. In H1299 cells (Fig. 3A), DNA-damaging agents (doxorubicin or camptothecin) confer cell cycle arrest by increasing the inhibitory Cdc2 Y15P (G₂-phase arrest indicator) level and suppressing the P-H3 signal (mitotic indicator). Chk1 siRNA efficiently abrogates the increase in Y15P and rescues the P-H3 signal, indicating successful mitotic progression following checkpoint abrogation. Chk2 knockdown also depressed the Y15P level, albeit to a lesser extent. However, unlike Chk1 siRNA, the critical difference is that this abrogation does not result in any recovery of the P-H3 signal, indicating that partial increase of the activity Cdc2 did not result in productive mitotic progression and checkpoint abrogation. In comparison, MK2 knockdown eliminated the camptothecin-induced increase of Cdc2 Y15P, but not the doxorubicin-induced increase, and failed to rescue the P-H3 signal in both cases. Therefore, it seems that whereas all three siRNAs were able to induce different degrees of cyclin-dependent kinase activation, only Chk1 siRNA induced sufficient extent of cyclin-dependent kinase activation, which is required for the checkpoint abrogation and mitotic transition.

In summary, our study clarified the differential roles of the three checkpoint kinases in cell cycle control by convincingly showing that inhibition of Chk1 is both necessary and sufficient for complete checkpoint abrogation, and furthermore, suppression of Chk2 or MK2 not only fails to enhance the efficacy of Chk1 inhibition but may actually antagonize it. Therefore, Chk1 is the only relevant cancer target among the checkpoint kinases, and efforts should be made during the compound screening process to ensure that the obtained Chk1 inhibitor does not target Chk2 or MK2.

	Acknowledgments

 
We thank Dr. Saul Rosenberg for his critical reading of the manuscript and valuable inputs.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2/ 9/06; revised 4/11/06; accepted 5/31/06.

	References

Top Abstract Introduction Materials and Methods Results Discussions References

 

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