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Genes to Cells (2005) 10, 97-106. doi:10.1111/j.1365-2443.2005.00821.x
© 2005 Blackwell Publishing or its licensors
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Role of Chk1 and Chk2 in Ara-C-induced differentiation of human leukemia K562 cells

Kazuchika Takagaki*, Susumu Katsuma, Yoshinori Kaminishi, Tatsuya Horio, Teruo Tanaka, Tadaaki Ohgi and Junichi Yano

Research Laboratories, Nippon Shinyaku Co. Ltd, 3-14-1 Sakura, Tsukuba, Ibaraki 305-0003, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 Supplementary material
 References
 
Human chronic myelogenous leukemia K562 cells are relatively resistant to the anti-metabolite cytosine arabinoside (Ara-C) and, when treated with Ara-C, they differentiate into erythrocytes without undergoing apoptosis. In this study we investigated the mechanism by which Ara-C induces K562 cells to differentiate. We first observed that Ara-C-induced differentiation of these cells is completely inhibited by the radiosensitizing agent caffeine, an inhibitor of ATM and ATR protein kinases. We next found that Ara-C activates Chk1 and Chk2 in the cells, and that the activation of Chk1, but not of Chk2, was almost completely inhibited by caffeine. Proteasome-mediated degradation of Cdc25A and phosphorylation of Cdc25C were induced by Ara-C treatment, presumably due to the activation of Chk2 and Chk1, respectively. To directly observe the effects of checkpoint kinase activation in Ara-C-induced differentiation, we suppressed Chk1 or Chk2 with the Chk1-specific inhibitor Gö6976, by generating cell lines stably over-expressing dominant-negative forms of Chk2, or by siRNA-mediated knock-down of the Chk1 or the Chk2 gene. The results suggest that Ara-C-induced erythroid differentiation of K562 cells depends on both Chk1 and Chk2 pathways.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 Supplementary material
 References
 
The anti-metabolite cytosine arabinoside (Ara-C) is one of the oldest drugs in leukemia therapy and is still the most commonly used agent in the treatment of adult acute myeloid leukemia. Ara-C is a deoxycytidine analogue that is phosphorylated in the cell to its active form, cytosine arabinoside triphosphate (Ara-CTP), which competes with deoxycytidine triphosphate (dCTP) for incorporation into DNA. When incorporated, Ara-C disrupts further DNA synthesis, resulting in the initiation of the downstream cellular response to DNA damage (Huang & Plunkett 1995). In mammals, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function at the top of the signal-transduction cascade that is triggered by DNA damage (Banin et al. 1998; Canman et al. 1998; Khanna et al. 1998; Tibbetts et al. 1999). The checkpoint functions of ATR and ATM are mediated in part by a pair of checkpoint effector kinases termed checkpoint kinase 1 (Chk1) and checkpoint kinase 2 (Chk2) (Rotman & Shiloh 1999; Zhou & Elledge 2000). Although they have distinct regulatory domains, Chk1 and Chk2 are functionally related kinases that phosphorylate overlapping pools of cellular substrates (Shieh et al. 2000).

The chronic myelogenous leukemia cell line K562 is induced in vitro to undergo erythroid differentiation by a variety of compounds, among them Ara-C (Fang et al. 2000; Huang et al. 2002). We have previously analysed Ara-C-treated K562 cells by cDNA microarray, and we found that a variety of hemoglobin transcripts were up-regulated and that several chaperone transcripts were down-regulated in treated cells (Takagaki et al. 2003). However, we do not know the signal-transduction pathways leading to Ara-C-induced K562-cell differentiation. In this study, we examined signaling pathways to Ara-C-induced DNA damage in K562 cells, and we found that Ara-C-induced K562 erythroid differentiation depends on both the Chk1 and the Chk2 pathway.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 Supplementary material
 References
 
Ara-C-induced differentiation of K562 cells is mediated by a caffeine-sensitive pathway

Ara-C exerts its cytotoxic activity against cancer cells mainly by incorporation into DNA and disruption of further DNA synthesis, resulting in the initiation of the cellular response to DNA damage (Huang & Plunkett 1995). The mammalian DNA damage-response pathway includes members of several families of conserved protein kinases; and ATM and ATR, members of the phosphoinositide 3-kinase superfamily, are at the top of this signal-transduction cascade (Banin et al. 1998; Canman et al. 1998; Khanna et al. 1998; Tibbetts et al. 1999). To assess whether Ara-C-induced differentiation of K562 cells is mediated by ATM or ATR, we examined Ara-C-induced hemoglobin expression in the presence or absence of the radiosensitizing agent caffeine, a well-known inhibitor of ATM and ATR but not an inhibitor of DNA-dependent protein kinase (DNA-PK) (Sarkaria et al. 1999). Caffeine completely inhibited the Ara-C-induced up-regulation of the hemoglobin {gamma} G gene (Fig. 1A) as well as that of the gene for porphobilinogen deaminase (PBGD), the third enzyme of the heme biosynthetic pathway (see Fig. S1 at http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC821/GTC821sm.htm). Furthermore, the accumulation of hemoglobin protein in cells treated with Ara-C was almost completely inhibited by caffeine three and six days after treatment with Ara-C (Fig. 1B). These results show that the Ara-C-induced differentiation of K562 cells is mediated by a caffeine-sensitive pathway. Another pluripotent erythroleukemic cell line, HEL, showed similar responses to Ara-C and caffeine (see Fig. S2A at http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC821/GTC821sm.htm).



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  		Figure 1  Effect of caffeine on expression of Ara-C-induced fetal hemoglobin in K562 cells. Cells were preincubated with or without caffeine (2 mM) for 30 min followed by incubation with Ara-C (20 µM). (A) Hemoglobin {gamma} G mRNA levels. At the indicated times total RNA was prepared, reverse-transcribed, and used as a PCR template for the hemoglobin {gamma} G gene. (B) Production of hemoglobin. Cells were stained with benzidine after 3 or 6 days’ incubation with Ara-C.

 
Both Chk1 and Chk2 are activated by Ara-C

In response to DNA damage, ATM and ATR phosphorylate and activate the protein kinases Chk1 and Chk2 (Rotman & Shiloh 1999; Shieh et al. 2000; Zhou & Elledge 2000). To examine the involvement of checkpoint kinases in Ara-C-induced K562-cell differentiation, we investigated Chk1 and Chk2 phosphorylation by immunoblotting with anti-phospho-Chk1 and anti-phospho-Chk2 antibodies, which recognize activated Chk1 and activated Chk2, respectively. When K562 cells were treated with Ara-C for 8 or 24 h, Chk1 was phosphorylated at Ser345 and Chk2 at Thr68 (Fig. 2A). The phosphorylation of Chk1 was almost completely abolished by pretreatment with caffeine, whereas the phosphorylation of Chk2 was dramatically enhanced by caffeine pretreatment (Fig. 2A). These results show that both Chk1 and Chk2 can be activated through Ara-C, but that the effects of caffeine on their activation are very different. In HEL cells, as in K562 cells, both Chk1 and Chk2 were activated through Ara-C, and phosphorylation of Chk2 was enhanced by caffeine pretreatment (see Fig. S2B at http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC821/GTC821sm.htm). (Since the growth rate and the total amount of Chk1 protein in HEL cells were decreased by caffeine treatment, the abolishment of the phosphorylation of Chk1 could not be confirmed.) Among the reagents that induce K562-cell differentiation, the genotoxic agents hydroxyurea and 5-fluorouracil promoted the activation of Chk1 and Chk2 (data not shown) as well as inducing hemoglobin expression. In addition, this activation of Chk1 and Chk2 was inhibited by caffeine. Correspondingly, the non-genotoxic agents STI-571 (a tyrosine kinase inhibitor) and sodium butyrate (a histone deacetylase inhibitor) induced hemoglobin expression without activating Chk1 or Chk2 (data not shown). Furthermore, the mitosis inhibitor vinblastine, which affects the cell cycle distribution, did not activate Chk1 or Chk2 and did not induce hemoglobin expression (see Fig. S3 at http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC821/GTC821sm.htm).



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  		Figure 2  Effect of Ara-C on phosphorylation status of checkpoint kinases in K562 cells. (A) Effect of caffeine on activation of Ara-C-induced checkpoint kinases. Cells were incubated with or without caffeine (2 mM) for 30 min, treated with Ara-C (20 µM) for 8 or 24 h, lysed and immunoblotted with antibodies against Chk1, phospho-Chk1, Chk2, phospho-Chk2, or actin. (B) Effect of wortmannin on activation of Ara-C-induced checkpoint kinases. Cells were preincubated with or without caffeine, then incubated with or without Ara-C and/or wortmannin (10 µM) for 8 h, lysed and immunoblotted. (C) Effect of wortmannin on up-regulation of Ara-C-induced fetal hemoglobin. Cells were preincubated with or without caffeine, and then incubated with or without Ara-C and/or wortmannin. After 36 h, total RNA was prepared, reverse-transcribed, and used as a PCR template for the hemoglobin {gamma} G gene.

 
Since preincubation of K562 cells with a combination of caffeine and wortmannin (a phosphatidylinositol 3-kinase (PI3K) inhibitor) completely blocks pathways mediated by ATM/ATR and DNA-PK (Sarkaria et al. 1999; Roos-Mattjus et al. 2002), we examined the effects of wortmannin and caffeine on Ara-C-induced checkpoint kinase activation in K562 cells. The activation of both Chk1 and Chk2 by Ara-C was only slightly inhibited by wortmannin (Fig. 2B), even though wortmannin completely inhibited the Ara-C-induced up-regulation of hemoglobin (Fig. 2C). This indicates that wortmannin inhibited Ara-C-induced K562-cell differentiation through a part of the signaling pathway that is downstream from checkpoint kinase activation. The activation of Chk2 by Ara-C was not inhibited at all even in cells preincubated with a combination of caffeine and wortmannin (Fig. 2B).

Proteasome-mediated degradation of Cdc25A is induced by Ara-C treatment

Chk2 has been proposed to regulate the G1/S checkpoint by phosphorylation of the Cdc25A protein phosphatase at Ser123, which leads to the ubiquitin-proteasome-mediated degradation of Cdc25A (Bernardi et al. 2000; Falck et al. 2002). To investigate the signaling pathway downstream from Chk2 activation, we determined the abundance of Cdc25A in Ara-C-induced K562 cells. Immunoblot analysis showed that in Ara-C-treated cells Cdc25A was drastically down-regulated, while the levels of slower-migrating forms of Cdc25A increased (Fig. 3A). The Ara-C-induced down-regulation of Cdc25A was slightly accentuated by pretreatment with caffeine (Fig. 3A). When a cell lysate of Ara-C-treated cells was immunoprecipitated with anti-Cdc25A antibody and then immunoblotted with anti-ubiquitin antibody, Cdc25A-ubiquitin conjugates were observed, and caffeine pretreatment slightly increased the accumulation of these conjugates (Fig. 3B). The proteasome inhibitor MG-132 abolished the Ara-C-induced down-regulation of Cdc25A (Fig. 3C), consistent with a scheme in which Ara-C treatment leads to the ubiquitin-proteasome-mediated degradation of Cdc25A. Furthermore, a lysate of Ara-C-induced K562 cells treated with anti-Chk2 antibody yielded an immunoprecipitate that could phosphorylate recombinant Cdc25A, and the activity was significantly enhanced by caffeine (Fig. 3D).



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  		Figure 3  Ara-C induces Chk2-mediated phosphorylation and proteasome-mediated degradation of Cdc25A. (A) Degradation of Cdc25A in Ara-C-treated K562 cells. Cells were preincubated with or without caffeine (2 mM) for 30 min, incubated with or without Ara-C (20 µM) for 8 h, lysed and immunoblotted with antibodies against Cdc25A or actin. (B) Ubiquitination of Cdc25A. Cells were incubated with or without Ara-C for 8 h, lysed, immunoprecipitated with anti-cdc25A antibody, subjected to SDS-PAGE and immunoblotted with anti-ubiquitin antibody. (C) Effect of MG132 on Cdc25A degradation. Cells were incubated with Ara-C after preincubation with or without caffeine (2 mM) and/or MG132 (5 µM). Cells were lysed 8 h after Ara-C treatment and immunoblotted with antibodies against Cdc25A or actin. (D) Chk2 kinase assay. Cells were preincubated with or without caffeine (2 mM), incubated with or without Ara-C for 8 h, lysed, immunoprecipitated with anti-Chk2 antibody, and assayed for kinase activity with recombinant Cdc25A protein as the substrate.

 
Phosphorylation of Cdc25C is induced by Ara-C treatment

Chk1 has been proposed to regulate the G2/M checkpoint by phosphorylating the Cdc25C protein phosphatase on residues that facilitate the binding of 14-3-3 proteins (Sanchez et al. 1997; Yarden et al. 2002). To determine the signaling pathway downstream from Chk1 activation in Ara-C-induced K562 cells, we examined the phosphorylation of Cdc25C at Ser216, which leads to the accumulation of Cdc2 phosphorylated on Thr14 and Tyr15 and thence to G2/M cell-cycle arrest (McGowan & Russell 1993; Galaktionov et al. 1995; Hunter 1995; Watanabe et al. 1995). When K562 cells were treated with Ara-C, the phosphorylation of Cdc25C at Ser216 was considerably enhanced, and this phosphorylation was prevented by pretreatment with caffeine (Fig. 4A). The phosphorylation of Cdc2 at Tyr15 was also considerably enhanced by Ara-C, and the phosphorylation was partially prevented by caffeine (Fig. 4B). Furthermore, a lysate of Ara-C-treated K562 cells treated with anti-Chk1 antibody yielded an immunoprecipitate that could phosphorylate recombinant Cdc25C (Fig. 4C). These results, taken together, suggest that Ara-C promotes Chk1 activation, resulting in Cdc25C phosphorylation and the accumulation of phosphorylated Cdc2.



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  		Figure 4  Ara-C induces phosphorylation of Cdc25C by Chk1 and phosphorylation of Cdc2. (A) Phosphorylation of Cdc25C in Ara-C-treated K562 cells. Cells were preincubated with or without caffeine (2 mM) for 30 min, incubated with or without Ara-C for 8 h, lysed, immunoprecipitated with anti-Cdc25C antibody, subjected to SDS-PAGE, and immunoblotted with anti-Cdc25C and anti-phospho-Cdc25C antibodies. (B) Phosphorylation of Cdc2 in Ara-C-treated K562 cells. Cells were preincubated with or without caffeine, incubated with or without Ara-C for 8 h, lysed, and immunoblotted with anti-Cdc2, anti-phospho-Cdc2, or anti-actin antibodies. (C) Chk1 kinase assay. Cells were preincubated with or without caffeine, incubated with or without Ara-C for 8 h, lysed, immunoprecipitated with anti-Chk1 antibody, and assayed for kinase activity with recombinant Cdc25C protein as the substrate.

 
Both Chk1 and Chk2 mediate Ara-C-induced differentiation of K562 cells

To directly investigate the role of checkpoint kinases in Ara-C-induced K562-cell differentiation, we attempted to isolate cell lines stably over-expressing wild-type (wt) or dominant negative (dn) forms of checkpoint kinases. In spite of our efforts, we could not generate cell lines showing more than 2.1-fold induction of Chk1 compared to cells transfected with empty vector (data not shown). However, we did succeed in isolating cell lines over-expressing wt or dn Chk2 at up to 13 times the levels of endogenous Chk2 (see Fig. S4A at http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC821/GTC821sm.htm). In these cell lines, the phosphorylation of Chk2 at Thr68 was enhanced both in the presence and in the absence of Ara-C (see Fig. S4B at http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC821/GTC821sm.htm). Cell lines with exogenous expression of wt Chk2 showed enhanced hemoglobin expression without Ara-C stimulation; and over-expression of dn Chk2 resulted in a reduction in the Ara-C-induced up-regulation of hemoglobin (Fig. 5A).



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  		Figure 5  Exogenous over-expression of dn Chk2 or the Chk1-specific inhibitor Gö6976 strongly reduces Ara-C-induced expression of hemoglobin {gamma} G gene in K562-cells. (A) Expression of hemoglobin {gamma} G gene. Cells stably expressing wt or dn Chk2 were incubated with or without Ara-C and after 36 h total RNA was prepared, reverse-transcribed, and used as a PCR template for the hemoglobin {gamma} G gene. Data are shown as the mean ± SEM of three independent experiments. (B, C) Expression of the hemoglobin {gamma} G gene in the presence of inhibitors. Cells were preincubated with up to 100 nM Gö6976 or bisindolylmaleimide I and after incubation in the presence or absence of Ara-C for 36 h total RNA was prepared, reverse-transcribed, and used as a PCR template for the hemoglobin {gamma} G gene. The expression of GAPDH mRNA was also determined as an internal control and was found to be almost constant. Data are shown as the mean ± SEM of three independent experiments.

 
To clarify the role played by Chk1 kinase in hemoglobin expression in K562 cells treated with Ara-C, Gö6976, an inhibitor of Chk1 kinase but not of Chk2 kinase (Kohn et al. 2003; Ishimi et al. 2003), was added to the medium. Gö6976 inhibited hemoglobin and PBGD expression in K562 cells in a dose-dependent manner (Fig. 5B, and see Fig. S5A at http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC821/GTC821sm.htm). Because the drug is also known as an inhibitor of protein kinase C (PKC), it was necessary to demonstrate that inhibition of hemoglobin expression had not been caused by inhibition of PKC. When bisindolylmaleimide I, an inhibitor of PKC but not of Chk1, was added to K562 cells, it did not inhibit hemoglobin expression (Fig. 5C).

To test for a more direct involvement of Chk1 or Chk2 in the Ara-C-induced expression of hemoglobin, we synthesized siRNAs that specifically eliminated Chk1 or Chk2 expression as described by Ahn et al. (2003). Chk1 siRNA, Chk2 siRNA, or control RNA (GL3, a firefly luciferase sequence which has no target genes in mammalian cells), were transfected into K562 cells. After 24 h of incubation, the mRNA levels of Chk1 or Chk2 in a portion of the cells was measured by RT-PCR. The remaining cells were incubated with or without Ara-C, and after 36 h the mRNA levels of the hemoglobin {gamma} G gene were measured. Suppression of Chk1 and Chk2 mRNA levels at 24 h was observed in the siRNA-transfected cells (Fig. 6A,B), and after each siRNA transfection, the protein levels of Chk1 and Chk2 were also found to have been down-regulated (Fig. 6C). Under these conditions, the Ara-C-induced expression of the hemoglobin {gamma} G gene was suppressed by transfection with either Chk1 siRNA or Chk2 siRNA (Fig. 6D,E). The Ara-C-induced expression of the PBGD gene was also suppressed by Chk1 or Chk2 siRNA, but the basal level of PBGD expression was increased by transfection with either siRNA (see Fig. S5B at http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC821/GTC821sm.htm). The results shown in Figs 5 and 6 suggest that the activation of both Chk1 and Chk2 is indispensable for Ara-C-induced K562 erythroid differentiation.



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  		Figure 6  siRNA-mediated knock-down of Chk1 or Chk2 gene leads to reduced expression of hemoglobin {gamma} G gene. (A, B) Chk1/2 mRNA levels of siRNA-transfected K562 cells. K562 cells were transfected with Chk1- or Chk2-specific siRNA, and 24 h later total RNA was prepared, reverse-transcribed, and used as a PCR template. (C) Protein levels of Chk1, Chk2, and their phosphorylated forms in siRNA-transfected K562 cells. K562 cells were transfected with Chk1- or Chk2-specific siRNA, and 24 h later the cells were incubated with or without Ara-C. After 36 h cells were harvested for immunoblotting. (D, E) Hemoglobin {gamma} G mRNA levels of siRNA-transfected K562 cells. K562 cells were transfected with Chk1- or Chk2-specific siRNA, and 24 h later the cells were incubated with or without Ara-C. After 36 h cells were harvested for RT-PCR. Data are shown as the mean ± S.E. of two independent experiments (A, B, D, E).

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 Supplementary material
 References
 
Human chronic myelogenous leukemia K562 cells, which are relatively resistant to Ara-C, can be induced to undergo erythroid differentiation in vitro by Ara-C as well as a variety of other structurally diverse compounds. When Ara-C is incorporated into DNA, it aborts further DNA synthesis, resulting in the initiation of the cellular response to DNA damage and finally leading to apoptosis or differentiation (Huang & Plunkett 1995). However, the precise mechanism by which Ara-C induces K562-cell differentiation has not been elucidated. In the present study, we investigated the signaling cascade involved in the erythroid differentiation of K562 cells exposed to Ara-C.

We have previously found by cDNA microarray analysis that various chaperones are down-regulated after Ara-C treatment of K562 cells (Takagaki et al. 2003). Chaperones promote cell growth by binding to and stabilizing a variety of growth-related kinases (Nimmanapalli et al. 2003). In the present work, we tested the possibility that this down-regulation of chaperones could lead to the down-regulation of Bcr-Abl, which could in turn cause the K562 cells to undergo erythroid differentiation. However, Ara-C treatment did not affect the mRNA levels, protein levels, or phosphorylation status of Bcr-Abl (data not shown). We then tested whether the Ara-C-induced response to DNA damage was related to Ara-C-induced differentiation. Our experiments clearly show that caffeine, an inhibitor of ATM/ATR, inhibited Ara-C-induced K562-cell differentiation as well as Chk1 phosphorylation at Ser345 (Fig. 2A,B). We also observed that both the phosphorylation of Cdc25C at Ser216 and the phosphorylation of Cdc2 at Tyr15 were reduced by pretreatment with caffeine (Fig. 4A,B). In vitro kinase assays directly demonstrated that Chk1 activated by Ara-C can phosphorylate Cdc25C (Fig. 4C). These results suggest that Ara-C activates Chk1, resulting in the phosphorylation of Cdc25C and Cdc2 and finally leading to G2/M cell-cycle arrest. This signaling pathway was inhibited by caffeine and therefore seems to be important in Ara-C-induced K562-cell differentiation. In contrast, Ara-C-induced phosphorylation of Chk2, as well as the kinase activity of Chk2, was enhanced by caffeine pretreatment (Fig. 2A,B). Furthermore, treatment with caffeine and Ara-C slightly increased the accumulation of Cdc25A-ubiquitin conjugates (Fig. 3A,B) (Cdc25A is an important regulator of the G1/S transition in mammalian cells (Bartek & Lukas 2001)). These results suggest that the activation of Chk2 and the downstream activity of the pathway were not abolished by caffeine, but in fact slightly enhanced by it, giving the appearance that this signaling pathway is not important in K562-cell differentiation. However, over-expression of a dn form of the Chk2 gene, specific inhibition of Chk1 kinase activity by Gö6976, and siRNA-mediated knock-down of the Chk1 and Chk2 genes demonstrated that pathways downstream of Chk1 and Chk2 were indeed both required for Ara-C-induced K562-cell differentiation.

Furthermore, flow-cytometric analysis showed that treatment with Ara-C decreased the percentage of K562 cells in the S phase (data not shown). This suggests that activation of Chk2 promotes G1/S cell-cycle arrest. Pretreatment with caffeine followed by treatment with Ara-C abolished the G2/M phase, which suggests that the suppression of Ara-C-induced Chk1 activation leads to release of G2/M cell-cycle arrest. These flow-cytometric results correspond to the phosphorylation states of Chk1 and Chk2 observed under the respective experimental conditions. Treatment with Ara-C induced DNA damage, which could arrest the cell cycle in a manner dependent on Chk1 and Chk2. These results suggest that Chk1 and Chk2 are required only for cell-cycle arrest, and that cell-cycle arrest leads to differentiation. However, the fact that the mitosis inhibitor vinblastine, which causes cells to accumulate in the G2/M phase, did not activate Chk1 or Chk2 and did not induce hemoglobin expression demonstrates that a perturbation of the cell-cycle distribution by itself does not necessarily lead to differentiation. The simultaneous activation of Chk1 and Chk2 is required for Ara-C-induced K562-cell differentiation. The fact that another erythroleukemic cell line, HEL, showed similar DNA-damage-dependent activation of Chk1 and Chk2 and erythroid differentiation supports our conclusion that the activation of Chk1 and Chk2 are both important in DNA-damage-dependent differentiation. (Only about 4% of the HEL cells were benzidine positive after three days’ incubation with Ara-C (see Fig. S2A at http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC821/GTC821sm.htm), a much lower percentage than observed in K562 cells. Previous work reports similar results (e.g. Clausen et al. 1997).)

Caffeine enhanced the Ara-C-induced activation of Chk2 even though it almost completely inhibited the Ara-C-induced activation of Chk1 (Fig. 2A). Ara-C presumably brought about the phosphorylation, ubiquitination, and degradation of Cdc25A through the activation of Chk2 (Fig. 3A–C). To our knowledge, this is the first report of the inactivation of Chk1 and the simultaneous activation of Chk2. Since caffeine is often used to inhibit ATM and ATR, we find it somewhat surprising that these opposite effects of caffeine on Chk1 and Chk2 have never been reported. The mechanism of the selective activation of Chk2, however, is not understood. Caffeine is a relatively non-selective agent that has many effects in cells. Illustrating the diverse effects of caffeine in different experimental systems, a recent study suggests that both Chk1 and Chk2 are hyperphosphorylated in HCT116 human colon cancer cells treated with concentrations of caffeine twofold or fourfold higher than those used in our experiments (Cortez 2003). Nevertheless, caffeine was still found to inhibit signaling through ATM and ATR, although the mechanism of inhibition is unclear. We also observed activation of Chk2 when the expression of Chk1 was suppressed by siRNA transfection (Fig. 6C). So the activation of Chk2 seems to be an unexpected effect of caffeine, and indeed a loss of feedback control downstream from Chk1 may activate the substrates of ATM and/or ATR, resulting in the hyperactivation of Chk2. In addition, the PI3K inhibitor wortmannin only slightly inhibited the activation of Chk1 and Chk2 (Fig. 2B), even though it completely inhibited the Ara-C-induced up-regulation of hemoglobin (Fig. 2C). This suggests that wortmannin inhibits Ara-C-induced K562-cell differentiation through a part of the signal-transduction pathway downstream from the checkpoint-kinase-activation step. Erythropoietin-induced differentiation of K562 cells is mediated by PI3K (Kubota et al. 2001), and our data suggest that Ara-C-induced K562-cell differentiation may also depend on PI3K activation, perhaps downstream from cell-cycle arrest. Wortmannin preferentially inhibits DNA-PK and ATM, although it also (albeit less potently) inhibits ATR (Hartley et al. 1995; Sarkaria et al. 1998). In contrast, caffeine preferentially inhibits ATM and ATR but does not inhibit DNA-PK (Blasina et al. 1999; Hall-Jackson et al. 1999; Falck et al. 2002). Taken together with these results, our findings suggest that Ara-C-induced Chk1 activation is mainly mediated by ATR, as is the case with other genotoxic agents, and that Ara-C-induced Chk2 activation may be mediated by species other than ATM, ATR and DNA-PK. During Ara-C-induced K562-cell differentiation, the incorporated Ara-CTP may halt DNA replication; and the DNA replication intermediate would then trigger ATR-mediated cell-cycle arrest. It has been reported that Ara-C might also act by trapping topoisomerase I cleavage complexes after its incorporation into DNA (Pourquier et al. 2000; Chrencik et al. 2003). It remains to be seen which effect is dominant in the activation of Chk1 or Chk2.

The tumor suppressor p53 mediates DNA-damage-induced apoptosis, and p53 is phosphorylated by Chk2 in normal cells; but more than 70% of cancer cell lines, including K562 and HEL, express a mutant p53 protein (Schultz et al. 2000; Jia et al. 1997). Because our purpose was to examine the mechanism of differentiation with a view to identifying a potential target for anti-cancer therapy, the present study focused on the differentiation induced by an anti-cancer drug in a p53-negative cancer cell line. We felt that such a study would be more clinically relevant than a study on p53-positive cells.

In conclusion, we have shown that Ara-C-induced erythroid differentiation of K562 cells is mediated by both Chk1 and Chk2 pathways, and we propose a mechanism for Ara-C-induced K562-cell differentiation (Fig. 7). Ara-C-induced signaling leading to erythroid differentiation is mediated by the activation of both Chk1 and Chk2. Phosphorylated Chk2 inactivates Cdc25A, which leads to cell-cycle arrest at G1/S; and phosphorylated Chk1 inactivates Cdc25C, which leads to cell-cycle arrest at G2/M. Ara-C-induced erythroid differentiation thus depends on both Chk1- and Chk2-mediated signaling pathways. We also carried out separate siRNA-mediated knock-down of Chk1 and Chk2 genes and confirmed that the corresponding proteins are required for complete erythroid differentiation, a result which emphasizes the general importance of these pathways in the chemically induced differentiation of leukemia cells. Further studies are required to clarify downstream events in Chk1- and Chk2-mediated signaling pathways during the Ara-C-induced erythroid differentiation of K562 cells.



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  		Figure 7  Proposed pathways of Ara-C-induced erythroid differentiation in K562 cells.

 

   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
Supplementary material
 References
 
Materials

Antibodies against actin (sc-1615), Chk1 (sc-8408 or sc-7898), Chk2 (sc-5278), Cdc25A (sc-7389), Cdc25C (sc-13138), phosphotyrosine (sc-7020), c-abl (sc-23) and ubiquitin (sc-8017) were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against phospho-Chk1 (Ser345; #2341), phospho-Chk2 (Thr68; #2661), phospho-Cdc25C (Ser216; #9528), Cdc2 (#9112), and phospho-Cdc2 (Tyr15; #9111) were from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase-conjugated secondary antibodies were from Zymed Laboratories (South San Francisco, CA, USA) and anti-phosphoserine antibody was from Calbiochem (San Diego, CA, USA). Ara-C was used as marketed by Nippon Shinyaku under the trade name Cylocide. MG-132, Gö6976, bisindolylmaleimide I, and wortmannin were from Calbiochem, and caffeine was from Nacalai Tesque (Kyoto, Japan). All other materials were of the highest available commercial grade.

Cell culture

Human leukemia K562 cells were from the American Type Culture Collection (Manassas, VA, USA), and human leukemia HEL cells were from the Health Science Research Resources Bank (Japan Health Science Foundation, Tokyo). Cells were grown in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and maintained at 37 °C in an atmosphere containing 5% CO2.

Assessment of erythroid differentiation of K562 cells

Erythroid differentiation was assessed by measuring hemoglobin production by benzidine staining as described by Huang et al. (2002), and benzidine-positive cells were counted in a hemocytometer.

Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)

Total RNA was reverse-transcribed for real-time PCR analysis with a LightCycler‘ Instrument (Roche Diagnostics, Basel, Switzerland). The PCR primers were 5'-TGCATGTGGATCCTGAGAAC-3' (sense) and 5'-CTTGAAGCTCTGCATCATGG-3' (anti-sense) for hemoglobin {gamma} G, 5'-ATGCTCGCTGGAGAATTGC-3' (sense) and 5'-ATAAGGAAAGACCTGTGCGG-3' (anti-sense) for Chk1, 5'-GCGCCTGAAGTTCTTGTTTC-3' (sense) and 5' -GCCTTTGGATCCACTACCAA-3' (anti-sense) for Chk2, and 5'-CTGACTGGAGGAGTCTGGA-3' (sense) and 5'-AAACCAGTTAATGGGCATCG-3' (anti-sense) for PBGD.

Immunoprecipitation and immunoblotting

K562 cells were incubated in the presence or absence of 20 µM Ara-C for 8 h. Cells were lysed and incubated overnight at 4 °C with 2–5 µg of antibody coupled to protein A/G agarose (Amersham). Precipitates were washed three times with lysis buffer and immunoblotted as described by Katsuma et al. (2002). Since the specificity of the anti-phospho-Cdc25C antibody by itself was insufficient, we first immunoprecipitated the cell lysate with anti-Cdc25C antibody, and then immunoblotted the precipitate with anti-phospho-Cdc25C antibody.

Kinase assay

To monitor the activity of endogenous Chk1 or Chk2, K562 cells were incubated with 20 µM Ara-C for 8 h. Cells were lysed and incubated overnight at 4 °C with 5 µg of antibody coupled to protein A/G agarose. Precipitates were washed twice with lysis buffer and twice with incomplete kinase buffer (50 mM Tris-HCl, pH 7.5, containing 10 mM MgCl2 and 1 mM dithiothreitol). Kinase reactions were carried out in the presence of complete kinase buffer (incomplete kinase buffer containing 100 µM ATP) and recombinant Cdc25A or Cdc25C (Upstate Technology, Charlottesville, VA, USA). Reaction mixtures were incubated at 30 °C for 30 min and analysed by SDS-PAGE. Anti-phosphoserine antibody was used to detect phospho-Cdc25 proteins.

Generation of stable transfectants

Genes encoding human Chk1 and Chk2 were cloned from K562 cells by RT-PCR and subcloned into the EcoRI/XhoI sites of the eukaryotic expression vector pBK-CMV (Stratagene, La Jolla, CA, USA) as described by Shieh et al. (2000). Dominant negative (dn) forms of Chk1 (D130A) and Chk2 (D347A) were constructed with a QuickChange‘ multi site-directed-mutagenesis kit from Stratagene (Shieh et al. 2000). Plasmids were transfected into K562 cells with DMRIE-C transfection reagent (Invitrogen, Carlsbad, CA). Three days after transfection, cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 800 µg/mL Geneticin disulphate (G418; Invitrogen). Stably transfected cells were cloned by the limiting-dilution method, expanded, and screened for Chk1 and Chk2 mRNA by RT-PCR and for Chk1 and Chk2 protein by immunoblotting.

RNA interference

Knock-down of Chk1 and Chk2 gene expression in K562 cells was achieved by the siRNA gene-silencing technique that has been used to study gene function in mammalian cells (Elbashir et al. 2001). Duplexes of 21-nucleotide siRNA with 3'-overhanging TT were designed according to Ahn et al. (2003) and synthesized by Qiagen (Hilden, Germany). The sense strand of the siRNA used to silence the Chk1 gene was UCGAAGUACUCAGCGUAAG, corresponding to positions 97–115 of the Chk1 mRNA relative to the start codon. The sense strand of the siRNA used to silence the Chk2 gene was GAACCUGAGGACCAAGAAC, corresponding to positions 235–253 of the Chk2 mRNA relative to the start codon. A control siRNA oligonucleotide, GL3, designed to silence the luciferase gene of the firefly, has no target gene in mammalian cells (Elbashir et al. 2001) and was used as a negative control for transfection. It had the sequence CUUACGCUGAGUACUUCGA (positions 155–173 relative to the start codon) of the luciferase gene.

Transfection was carried out by electroporation using the Nucleofection‘ system (Amaxa, Köln, Germany) according to the manufacturer's instructions. Briefly, 106 cells were resuspended in 100 µL of Nucleofector solution V (Cell line Nucleofector kit V) containing 100 pmol of double-stranded siRNAs. After electroporation, 400 µL of prewarmed culture medium was added to the electroporation cuvette and the cells were transferred on to culture plates containing prewarmed culture medium. After a 24-h rest, a small sample of cells was taken for RT-PCR analysis. The remaining cells were counted, 106 cells per 10 mL of culture medium were incubated with or without Ara-C, and after 36 h cells were harvested for RT-PCR analysis and immunoblotting.


   Supplementary material
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 Supplementary material
References
 
The following material is available from:

http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC821/GTC821sm.htm


   Acknowledgements
 
We thank Dr G. E. Smyth (Research Laboratories, Nippon Shinyaku Co.) for critical reading of the manuscript.


   Footnotes
 
Communicated by: Masayuki M. Yamamoto

* Correspondence: E-mail: k.takagaki{at}nippon-shinyaku.co.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 Supplementary material
 References
 
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Received: 16 July 2004
Accepted: 10 November 2004




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