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Inducible degradation of checkpoint kinase 2 links to cisplatin-induced resistance in ovarian cancer cells
BBRC Biochemical and Biophysical Research Communications 328 (2005) 567–572 www.elsevier.com/locate/ybbrc
Inducible degradation of checkpoint kinase 2 links to cisplatin-induced resistance in ovarian cancer cells Peilin Zhanga,b, Weiyi Gaoa, Hongli Lia, Eddie Reedb, Fei Chena,c,* a
Department of Pathology, West Virginia University, Morgantown, WV 26506, USA Mary Baab Randolph Cancer Center, West Virginia University, Morgantown, WV 26506, USA Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA b
c
Received 29 December 2004 Available online 13 January 2005
Abstract Checkpoint kinase 2 (Chk2) is one of the critical kinases governing the cell cycle checkpoint, DNA damage repair, and cell apoptosis in response to DNA damaging signals. In the present report, we demonstrate that Chk2 kinase is degraded at the protein level in response to cisplatin through ubiquitin–proteasome pathway. This degradation was independent of the Thr68 phosphorylation, ATM kinase, and BRCA1 tumor suppressor. Examination of Chk2 protein revealed a decreased expression of Chk2 protein in cisplatin-resistant ovarian cancer cell lines, suggesting that degradation or decreased expression of Chk2 is partially responsible for chemo-resistance. Site-directed mutation of the putative destruction box in the Chk2 protein did not affect the Chk2 degradation induced by cisplatin. Therefore, these results are the first to indicate a novel mechanism of regulating Chk2 in cisplatin-induced resistance of cancer cells. 2005 Elsevier Inc. All rights reserved. Keywords: Chk2 kinase; Cancer; Degradation; Ubiquitination; Cisplatin resistance
Platinum-based agents are the mainstream chemotherapeutic drugs for almost all advanced cancers in the Western world. The effects of cisplatin in treatment of advanced cancers are mediated through high levels of DNA damage, leading to the programmed cell death or cell cycle arrest [1]. The cellular response to the DNA damage induced by cisplatin and other agents is mediated through a central DNA damage-signaling pathway controlled by ataxia telangiectasia mutated kinase (ATM) as well as several other DNA-damaging responsive kinases [2,3]. The activated ATM kinase has been shown to phosphorylate many downstream targets including the p53 tumor suppressor [4], the Chk1 and Chk2 serine/threonine kinases, and other molecular targets [5,6]. Chk2 kinase is a newly defined tumor suppressor and the germline mutation of Chk2 gene has been *
Corresponding author. Fax: +1 304 285 5938. E-mail address: [email protected] (F. Chen).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.01.007
associated with a minority of patients with Li–Fraumeni syndrome [7,8]. Once activated by ATM, Chk2 kinase can phosphorylate and activate other downstream targets such as CDC25A, CDC25C, BRCA1, p53, and E2F [9–13]. These target molecules are important for cell cycle checkpoint regulation, DNA repair, and apoptosis. Activation of Chk2 by DNA damage is dependent on ATM-mediated phosphorylation of its Thr68 [14]. The Chk2 kinase is critical for DNA damage-induced apoptosis, since the Chk2 null cells from the knockout mice showed remarkable resistance to ionizing radiation [15]. It is thought that the resistance to DNA damage-induced apoptosis in the Chk2 null cells is mediated through down-regulation of p53-regulated genes such as Bax and Noxa although the detailed mechanism has yet to be fully elucidated through genetic and biochemical studies [16]. In the present report, we show that cisplatin can rapidly induce Chk2 protein degradation in some cell lines derived from ovarian cancer. This
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degradation is independent of ATM kinase and is mediated through ubiquitination and proteasome pathway. Moreover, in the cisplatin resistant ovarian cancer cells, down-regulation of Chk2 kinase expression by degradation is at least in part responsible for the resistance of the cancer cell to cisplatin-induced apoptosis. These results provide a novel mechanism by which cisplatin induces the acquired resistance of the tumors after initial response to the drug.
Materials and methods Cell culture and transient transfection. The ovarian cancer cell lines A2780, CP70, the lung cancer cell line A549, oral cavity squamous carcinoma cell line SCC-15, ATM null lymphoblast cell line GM03182, BRCA1 null lymphoblast cell line GM14093, and human epithelial cell line 293T cells were cultured as described elsewhere [17]. The cells were cultured in 6-well plates and treated with cisplatin (33 lM) (Sigma– Aldrich, St. Louis, MO) for time periods as listed for each experiment. The cells were treated with cycloheximide (10 lg/ml) (Sigma–Aldrich, St. Louis, MO) for 1, 2, and 6 h in the presence or absence of cisplatin. When appropriate, actinomycin D (1 mg/ml) (Sigma–Aldrich, St. Louis, MO) was added to the cultured cells in the presence or absence of cisplatin (33 lM). Treatment of the cells with MG132 (Sigma–Aldrich, St. Louis, MO) was performed as follows: the cells were cultured overnight with or without 20 lM MG132 in DMSO. The cell lysates were prepared at the specific time point after the treatment. The HA-tagged wild type Chk2 and the T68A mutant expression plasmid were kindly provided by Dr. Junjie Chen, Mayo Clinic, Rochester, MN. The plasmids were transiently transfected into A2780 and CP70 cells using Lipofectamine 2000 (Invitrogen, San Diego, CA), and the total protein lysates were used for immunoblotting analyses. The site-directed mutagenesis of the R180DM (R180G/L183V) was performed using the Quickchange II sitedirected mutagenesis kit (from Stratagene, San Diego, CA) following the manufacturerÕs instruction. The mutant vector was sequenced using commercial sequencing services (Certigen, Lubbock, TX). Immunoblot analyses. Total cellular proteins were separated on 10% polyacrylamide gels under SDS denaturing conditions and electrophoretically transferred to nitrocellulose membranes (Bio-Rad, CA). Membranes were blocked with a 5% non-fat milk solution and incubated for 1 h with a 1:200 dilution of primary antibodies. Antibodies for Chk2, p53, and b-actin were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). After washing off the unbound antibodies three times with 1· TBS–Tween 20, the membranes were incubated for 1 h with a 1:2,000 dilution of sheep anti-rabbit horseradish peroxidase-conjugated IgG (Santa Cruz Biotechnologies, CA) and washed three times. Immunodetection was performed with a chemiluminescence Western blotting kit according to the supplierÕs instructions (Pierce Biotechnology, Rockford, IL). Anti-HA monoclonal antibody was purchased from Sigma–Aldrich (St. Louis, MO). Northern blot analyses. Northern blot analyses of the Chk2 mRNA were performed as described [17]. Total RNA was isolated from the cultured cells using TriZol reagent (Invitrogen, San Diego, CA) and 20 lg of RNA was used for each lane in Northern blot analyses. The RNA was loaded onto 1.2% formaldehyde agarose gel as described [17]. The RNA was blotted onto a nylon membrane (Hybond N, Amersham Biosciences), and the membrane was UV crosslinked according to standard procedures. Full length Chk2 cDNA was amplified by PCR based on the sequence from the expression vector kindly provided by Dr. Junjie Chen at Mayo Clinic (Rochester, MN). The cDNA probe was labeled by random priming using a Prime-aGene labeling kit as instructed by the manufacturer (Promega, WI). Hybridization overnight at 65 C was followed by washes (2 · 15 min
at room temperature in 2· SSC, 1% SDS and then 2 · 30 min at 68 C in 0.1· SSC, 1% SDS). The results were analyzed by using a Phosphoimager (Molecular Dynamics). Immunoprecipitation. Total cell lysates were prepared and directly used for immunoprecipitation using anti-Chk2 antibody (Santa Cruz Biotechnologies, CA). The protein A/G-conjugated agarose beads (20 ll) and 10 ll anti-Chk2 antibody were added to the 500 ll total cellular extracts from the cells grown in 6-well culture plates treated with or without the appropriate drugs. The antibody and the conjugated agarose beads were incubated at 4 C overnight, and the proteins bound to the beads were washed five times with RIPA buffer containing protease inhibitor cocktail (1·) (Invitrogen, CA). The immunoprecipitates were separated on 10% SDS–PAGE, blotted onto the nitrocellulose membranes, and incubated with the appropriate antibody for immunodetection. The signals were visualized using a chemiluminescence kit (Pierce Biotechnology, Rockford, IL).
Results Chk2 kinase expression is diminished in response to DNA damage We have examined the Chk2 protein levels in the oral cavity squamous carcinoma SCC-15 cells and the human epithelial cell line 293T cells in the presence or absence of DNA damage agent cisplatin (33 lM). Treatment of the cells with cisplatin for 6 h decreased the level of Chk2 protein substantially (Fig. 1A). We have also examined the A2780 ovarian cancer cells in response to cisplatin. After treatment of the cells with cisplatin (33 lM) for 1, 2, and 6 h, the expression level of Chk2 kinase was decreased progressively to a non-detectable level (Fig 1B). Treatment of the cells for 24 h or longer showed identical results to those seen after 6 h (data not shown). Unless stated otherwise, we used a 6-h treatment throughout the experiments using cisplatin concentration of 33 lM. Expression of Chk2 kinase was not observed in the A549 lung adenocarcinoma cells due to hypermethylation of the Chk2 gene promoter [17]. While the expression level of Chk1 kinase was not altered in the ovarian cancer A2780 cells, p53 expression was gradually increased, in close correlation with the decrease of Chk2 kinase expression. In contrast to A2780 cells, the increase of the p53 expression level in the lung cancer A549 cells in response to cisplatin was not seen until 6 h. Increase of the cisplatin concentrations resulted in an earlier degradation of Chk2. Fig. 1C shows that cisplatin at 256 lM can down-regulate the expression level of Chk2 kinase within a 1 h time period. These results indicate that the level of Chk2 kinase expression is regulated by cisplatin in both timeand dose-dependent manners. Chk2 kinase is degraded in response to cisplatin We determined if down-regulation of Chk2 expression in response to cisplatin is at the transcriptional or post-translational levels. We used the protein synthesis
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Fig. 2. Chk2 protein is degraded in response to cisplatin. (A) The stability of Chk2 in A2780 cells. Cells were treated with 10 lg/ml cycloheximide for 0, 1, 2, and 6 h in the absence or presence of 33 lM cisplatin. The protein level of Chk2 was assayed by Western blotting with anti-Chk2 antibody. As a control, the expression of b-actin after treatment of cycloheximide is also determined. (B) Northern blot analyses of Chk2 mRNA expressions in A2780 cells in the presence or absence of actinomycin D (1 mg/ml). The 28S and 18S rRNAs were used to normalize the loading between each lane (lower panel). Fig. 1. Chk2 kinase expression is diminished in response to cisplatin treatment. (A) The oral cavity squamous carcinoma SCC-15 and the 293T cells in the presence or absence of 33 lM cisplatin for 6 h. (B) The ovarian cancer A2780 cells and the lung cancer A549 cells were cultured and treated without (C, control) or with cisplatin for the time period listed above, and the total cell lysates were harvested and subjected to immunoblotting analyses using different antibodies as listed. b-Actin was used to normalize the loading. (C) The ovarian cancer A2780 cells were treated with increasing concentrations of cisplatin for 1 h, and the total cell lysates were used for immunoblotting analyses of Chk2 expression.
inhibitor cycloheximide (CHX) to block new protein synthesis and determined the natural half-life of the existing Chk2 protein in the presence or absence of cisplatin. We treated the A2780 cells with CHX alone or in combination with cisplatin, and determined the Chk2 expression levels by immunoblot (Fig. 2A). In the presence of CHX alone, the Chk2 expression level was not significantly altered within 6 h, whereas cisplatin can induce down-regulation of Chk2 expression in the presence of CHX. This result suggests that cisplatin shortens the half-life of Chk2 kinase and induces accelerated Chk2 protein degradation. We also used the transcription inhibitor actinomycin D (Act D) to block the new mRNA synthesis and determined the half-life of the Chk2 mRNA by Northern blot analysis (Fig. 2B). Cisplatin appeared to have no significant effect on the half-life of the Chk2 mRNA. These results suggest to us that down-regulation of Chk2 expression in response to cisplatin in the ovarian cancer A2780 cells is mediated through protein degradation, rather than the decrease of
transcription rate of the Chk2 gene or decrease of mRNA half-life. Chk2 degradation is mediated by a ubiquitin–proteasome pathway The ubiquitin–proteasome system is the most common protein degradation pathway in mammalian cells. To determine whether the degradation of Chk2 kinase in response to cisplatin is mediated through this system, we performed immunoprecipitation using anti-Chk2 antibody and immunoblot by using either anti-ubiquitin antibody or anti-Chk2 antibody. Fig. 3A indicates that Chk2 is ubiquitinated under the basal condition. Treatment of the cells with cisplatin enhanced the ubiquitination of Chk2 kinase appreciably. We also used the proteasome inhibitor MG132 to treat the A2780 cells in combination with cisplatin to see if MG132 can inhibit the Chk2 degradation in the presence of cisplatin (Fig. 3B). The Chk2 degradation induced by cisplatin can be inhibited by MG132, indicating that the degradation of Chk2 kinase in response to cisplatin is mediated through the ubiquitin–proteasome pathway. Chk2 degradation is independent of ATM kinase activation ATM kinase is activated in response to cisplatininduced DNA damage, and the activated ATM
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Fig. 3. Chk2 degradation is mediated through ubiquitin–proteasome pathway. (A) The ovarian cancer A2780 cells were treated with cisplatin (33 lM) for the time period listed above, and the total cell lysates were prepared for immunoprecipitation using Chk2 antibody. The immunoprecipitates were separated on the 10% SDS–PAGE gel, and the proteins were immunoblotted by using anti-ubiquitin antibody (upper panel) and anti-Chk2 antibody. (B) Chk2 degradation in response to cisplatin treatment can be inhibited by the presence of MG132 (20 lM), a wide spectrum ubiquitin–proteasome inhibitor.
phosphorylates Thr68 of Chk2 kinase to transduce the DNA damaging signals [8]. Therefore, we next investigated the possible involvement of ATM-mediated Thr68 phosphorylation in cisplatin-induced Chk2 degradation. We transfected the CP70 cells, in which the endogenous Chk2 kinase is minimal to absent by immunoblot analysis (see Fig. 5, below) [17], with the wild type HA-Chk2 and the HA-T68A expression constructs (kindly provided by Dr. Junjie Chen, Mayo Clinic, Rochester, MN), and analyzed the expression of the wild type Chk2 and the T68A mutant by immunoblot with both anti-HA antibody and anti-Chk2 antibody (Fig. 4A). Treatment of the transfected cells with cisplatin induced degradation of the HA-tagged wild type Chk2 protein as well as the T68A mutant. Moreover, Chk2 degradation was also seen in the ATM null lymphoblasts (GM03182, Coriell Cell Repositories, NJ), indicating that degradation of Chk2 kinase in response to cisplatin is independent of ATM kinase (Fig. 4B). Since BRCA1 is one of the Chk2 downstream targets, and BRCA1 possesses ubiquitin E3 ligase activity at the N-terminus as shown in in vitro study [18], we sought to see if BRCA1 is required for Chk2 degradation. Cisplatin can indeed induce Chk2 degradation in the BRCA1 null lymphoblasts (GM14093, Coriell Cell Repositories, NJ), suggesting that BRCA1 is not required for the degradation of Chk2 induced by cisplatin. Chk2 degradation mediates cisplatin resistance We have obtained a pair of the ovarian cancer cell lines, A2780 and CP70. The CP70 cells were developed
Fig. 4. Chk2 kinase degradation in response to cisplatin is independent of ATM kinase and BRCA1 tumor suppressor. (A) HA-tagged wild type Chk2 expression vector and the T68A mutant were transfected into the ovarian cancer CP70 cells and treated with cisplatin(33 lM) for the time period listed above. The total cell lysates were prepared and used for immunoblotting using anti-HA antibody (upper panel) and anti-Chk2 antibody (lower panel) for the detection of endogenous Chk2 protein. (B) The ATM / and BRCA1 / lymphoblasts were obtained from Coreill Cell Repository (New Jersey) and cultured under the condition described. The cells were treated with cisplatin (33 lM) for the time listed above, and the total proteins were used for immunoblotting analyses for Chk2 expression.
Fig. 5. Diminished Chk2 expression in the cisplatin-resistant CP70 ovarian cancer cells in comparison with the parental A2780 cells. Ovarian cancer A2780 cells and CP70 cells were treated with cisplatin for the time period listed above, and total proteins were used for immunoblotting analyses for Chk2 expression. b-Actin was used to normalize the equal loading of the proteins in each lane.
by exposure of A2780 cells to low dose cisplatin (1– 2 lM) for long period of time over a total of 70 cell passages. A2780 cells are sensitive to cisplatin (IC50 = 3 lM), and CP70 cells are resistant to cisplatin (IC50 = 45 lM) [19]. We have shown that cisplatin induced Chk2 degradation in A2780 cells, and we reasoned that development of resistance to cisplatin of CP70 cells may be the direct result of down-regulation of Chk2 by degradation. CP70 cells may have much lower or diminished Chk2 kinase expression. To test this, we measured Chk2 protein by immunoblot (Fig. 5). Chk2 expression in the CP70 cells was almost non-detectable and represents approximately 5–10% of that seen in the parental A2780 cells. This result indicates that diminished expression of Chk2 kinase is at least in part responsible for cisplatin resistance of CP70 cells. Thus, Chk2 degradation may be one of the primary mechanisms by which a large number of clinically relevant tumors develop the acquired resistance to DNA damage agent.
P. Zhang et al. / Biochemical and Biophysical Research Communications 328 (2005) 567–572
Fig. 6. Chk2 degradation is not affected by the D-box sequence. (A) Diagrammatic representation of the Chk2 sequences and the mutational design at the D-box sequence. (B) Wild type Chk2 (WT-HA), T68A-mutated Chk2 (T68A), and R180G/L183V-mutated Chk2 (R180DM) are degraded in response to cisplatin treatment.
Chk2 degradation is independent of a putative D-box sequence To further investigate the molecular mechanism of Chk2 degradation in response to cisplatin, we examined the Chk2 amino acid sequences. At the position 180, there appears to be a putative consensus sequence of destruction box (D-box motif), defined by yeast genetics from degradation of cyclin B [20] (Fig. 6A). D-box sequence is the known interaction domain with the anaphase-promoting complex (APC), an E3 ubiquitin ligase complex and it is associated WD-domain-containing activator CDC20 or CDH1 [20,21]. To determine the possible involvement of D-box in cisplatin-induced Chk2 degradation, we constructed a D-box-mutated Chk2 expression vector, R180G/L183V (referred to as R180DM) by site-directed mutation. The expression plasmids were transfected into the CP70 cells and treated with cisplatin (Fig. 6B). The wild type Chk2 kinase, T68A mutant, and R180DM were all degraded in response to cisplatin. These results suggested that Chk2 degradation is mediated through a D-box independent mechanism.
Discussion In response to DNA damage, Chk2 kinase is rapidly activated by ATM kinase through T68 phosphorylation in response to DNA damaging signals [10]. The importance of Chk2 kinase function in DNA damage response is exemplified by the Chk2 gene knockout study in
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which deficiencies in DNA-damage-induced apoptosis and G1/S checkpoint were observed [16,22]. Clinically, most ovarian cancers are sensitive to cisplatin-based chemotherapy [23–25]. However, cisplatin-induced resistance to treatment is of clinical significance [25]. We felt that down-regulation of Chk2 kinase expression in these cancer cells, at least in part, mediated the cancer cell resistance to cisplatin. Furthermore, the CP70 ovarian cancer cells were derived from the parental A2780 cells by exposure to low dose cisplatin over total of 70 passages [19]. The level of Chk2 kinase expression in the CP70 cells was remarkably lower compared with that in the parental A2780 cells, while the resistance of CP70 cells to cisplatin was found to be 15-fold higher [19]. Although other proteins may have played roles in mediating the resistance of the CP70 cells to cisplatin, down-regulation of Chk2 by degradation in these cells may be the predominant event in development of cisplatin resistance. Chk2 degradation in response to cisplatin treatment in cancer cells is a novel observation. The degradation of Chk2 kinase appears to be independent of ATM kinase or BRCA1 tumor suppressor (Fig. 4). Examination of the Chk2 amino acid sequence revealed a consensus destruction box (D-box) domain at the amino acid 180–188 that is known to serve as a recognition box by anaphase-promoting complex (APC/CDC20 and/or APC/CDH1), leading to protein ubiquitination and proteolysis through proteasome pathway [21,26,27]. Mutation of this D-box sequence of Chk2 kinase has no effect on the cisplatin-induced degradation of Chk2 (Fig. 6). These data, however, do not exclude the potential role that the APC/CDC20 or APC/CDH1 complex may play in mediating Chk2 degradation. Other potential sites within the Chk2 sequence may be targeted by APC complex. In addition to APC complex, other ubiquitin ligases may also contribute to the ubiquitination and degradation of Chk2. Identification of the E3 ligases that mediates Chk2 degradation will significantly help in the understanding of how Chk2 degradation is regulated in the DNA damage signaling pathway. Acknowledgments The authors thank Dr. Junjie Chen, at the Mayo Clinic for providing the Chk2 expression plasmids. This study is supported in part by the Sara Crile Allen and James F. Allen endowment fund for lung cancer research, and the West Virginia University Senate Research Grant. References [1] E. Reed, Cancer Treat. Rev. 24 (1998) 331–344. [2] W.C. Lin, F.T. Lin, J.R. Nevins, Genes Dev. 15 (2001) 1833– 1844.
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Inducible degradation of checkpoint kinase 2 links to cisplatin-induced resistance in ovarian cancer cells Peilin Zhanga,b, Weiyi Gaoa, Hongli Lia, Eddie Reedb, Fei Chena,c,* a
Department of Pathology, West Virginia University, Morgantown, WV 26506, USA Mary Baab Randolph Cancer Center, West Virginia University, Morgantown, WV 26506, USA Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA b
c
Received 29 December 2004 Available online 13 January 2005
Abstract Checkpoint kinase 2 (Chk2) is one of the critical kinases governing the cell cycle checkpoint, DNA damage repair, and cell apoptosis in response to DNA damaging signals. In the present report, we demonstrate that Chk2 kinase is degraded at the protein level in response to cisplatin through ubiquitin–proteasome pathway. This degradation was independent of the Thr68 phosphorylation, ATM kinase, and BRCA1 tumor suppressor. Examination of Chk2 protein revealed a decreased expression of Chk2 protein in cisplatin-resistant ovarian cancer cell lines, suggesting that degradation or decreased expression of Chk2 is partially responsible for chemo-resistance. Site-directed mutation of the putative destruction box in the Chk2 protein did not affect the Chk2 degradation induced by cisplatin. Therefore, these results are the first to indicate a novel mechanism of regulating Chk2 in cisplatin-induced resistance of cancer cells. 2005 Elsevier Inc. All rights reserved. Keywords: Chk2 kinase; Cancer; Degradation; Ubiquitination; Cisplatin resistance
Platinum-based agents are the mainstream chemotherapeutic drugs for almost all advanced cancers in the Western world. The effects of cisplatin in treatment of advanced cancers are mediated through high levels of DNA damage, leading to the programmed cell death or cell cycle arrest [1]. The cellular response to the DNA damage induced by cisplatin and other agents is mediated through a central DNA damage-signaling pathway controlled by ataxia telangiectasia mutated kinase (ATM) as well as several other DNA-damaging responsive kinases [2,3]. The activated ATM kinase has been shown to phosphorylate many downstream targets including the p53 tumor suppressor [4], the Chk1 and Chk2 serine/threonine kinases, and other molecular targets [5,6]. Chk2 kinase is a newly defined tumor suppressor and the germline mutation of Chk2 gene has been *
Corresponding author. Fax: +1 304 285 5938. E-mail address: [email protected] (F. Chen).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.01.007
associated with a minority of patients with Li–Fraumeni syndrome [7,8]. Once activated by ATM, Chk2 kinase can phosphorylate and activate other downstream targets such as CDC25A, CDC25C, BRCA1, p53, and E2F [9–13]. These target molecules are important for cell cycle checkpoint regulation, DNA repair, and apoptosis. Activation of Chk2 by DNA damage is dependent on ATM-mediated phosphorylation of its Thr68 [14]. The Chk2 kinase is critical for DNA damage-induced apoptosis, since the Chk2 null cells from the knockout mice showed remarkable resistance to ionizing radiation [15]. It is thought that the resistance to DNA damage-induced apoptosis in the Chk2 null cells is mediated through down-regulation of p53-regulated genes such as Bax and Noxa although the detailed mechanism has yet to be fully elucidated through genetic and biochemical studies [16]. In the present report, we show that cisplatin can rapidly induce Chk2 protein degradation in some cell lines derived from ovarian cancer. This
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degradation is independent of ATM kinase and is mediated through ubiquitination and proteasome pathway. Moreover, in the cisplatin resistant ovarian cancer cells, down-regulation of Chk2 kinase expression by degradation is at least in part responsible for the resistance of the cancer cell to cisplatin-induced apoptosis. These results provide a novel mechanism by which cisplatin induces the acquired resistance of the tumors after initial response to the drug.
Materials and methods Cell culture and transient transfection. The ovarian cancer cell lines A2780, CP70, the lung cancer cell line A549, oral cavity squamous carcinoma cell line SCC-15, ATM null lymphoblast cell line GM03182, BRCA1 null lymphoblast cell line GM14093, and human epithelial cell line 293T cells were cultured as described elsewhere [17]. The cells were cultured in 6-well plates and treated with cisplatin (33 lM) (Sigma– Aldrich, St. Louis, MO) for time periods as listed for each experiment. The cells were treated with cycloheximide (10 lg/ml) (Sigma–Aldrich, St. Louis, MO) for 1, 2, and 6 h in the presence or absence of cisplatin. When appropriate, actinomycin D (1 mg/ml) (Sigma–Aldrich, St. Louis, MO) was added to the cultured cells in the presence or absence of cisplatin (33 lM). Treatment of the cells with MG132 (Sigma–Aldrich, St. Louis, MO) was performed as follows: the cells were cultured overnight with or without 20 lM MG132 in DMSO. The cell lysates were prepared at the specific time point after the treatment. The HA-tagged wild type Chk2 and the T68A mutant expression plasmid were kindly provided by Dr. Junjie Chen, Mayo Clinic, Rochester, MN. The plasmids were transiently transfected into A2780 and CP70 cells using Lipofectamine 2000 (Invitrogen, San Diego, CA), and the total protein lysates were used for immunoblotting analyses. The site-directed mutagenesis of the R180DM (R180G/L183V) was performed using the Quickchange II sitedirected mutagenesis kit (from Stratagene, San Diego, CA) following the manufacturerÕs instruction. The mutant vector was sequenced using commercial sequencing services (Certigen, Lubbock, TX). Immunoblot analyses. Total cellular proteins were separated on 10% polyacrylamide gels under SDS denaturing conditions and electrophoretically transferred to nitrocellulose membranes (Bio-Rad, CA). Membranes were blocked with a 5% non-fat milk solution and incubated for 1 h with a 1:200 dilution of primary antibodies. Antibodies for Chk2, p53, and b-actin were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). After washing off the unbound antibodies three times with 1· TBS–Tween 20, the membranes were incubated for 1 h with a 1:2,000 dilution of sheep anti-rabbit horseradish peroxidase-conjugated IgG (Santa Cruz Biotechnologies, CA) and washed three times. Immunodetection was performed with a chemiluminescence Western blotting kit according to the supplierÕs instructions (Pierce Biotechnology, Rockford, IL). Anti-HA monoclonal antibody was purchased from Sigma–Aldrich (St. Louis, MO). Northern blot analyses. Northern blot analyses of the Chk2 mRNA were performed as described [17]. Total RNA was isolated from the cultured cells using TriZol reagent (Invitrogen, San Diego, CA) and 20 lg of RNA was used for each lane in Northern blot analyses. The RNA was loaded onto 1.2% formaldehyde agarose gel as described [17]. The RNA was blotted onto a nylon membrane (Hybond N, Amersham Biosciences), and the membrane was UV crosslinked according to standard procedures. Full length Chk2 cDNA was amplified by PCR based on the sequence from the expression vector kindly provided by Dr. Junjie Chen at Mayo Clinic (Rochester, MN). The cDNA probe was labeled by random priming using a Prime-aGene labeling kit as instructed by the manufacturer (Promega, WI). Hybridization overnight at 65 C was followed by washes (2 · 15 min
at room temperature in 2· SSC, 1% SDS and then 2 · 30 min at 68 C in 0.1· SSC, 1% SDS). The results were analyzed by using a Phosphoimager (Molecular Dynamics). Immunoprecipitation. Total cell lysates were prepared and directly used for immunoprecipitation using anti-Chk2 antibody (Santa Cruz Biotechnologies, CA). The protein A/G-conjugated agarose beads (20 ll) and 10 ll anti-Chk2 antibody were added to the 500 ll total cellular extracts from the cells grown in 6-well culture plates treated with or without the appropriate drugs. The antibody and the conjugated agarose beads were incubated at 4 C overnight, and the proteins bound to the beads were washed five times with RIPA buffer containing protease inhibitor cocktail (1·) (Invitrogen, CA). The immunoprecipitates were separated on 10% SDS–PAGE, blotted onto the nitrocellulose membranes, and incubated with the appropriate antibody for immunodetection. The signals were visualized using a chemiluminescence kit (Pierce Biotechnology, Rockford, IL).
Results Chk2 kinase expression is diminished in response to DNA damage We have examined the Chk2 protein levels in the oral cavity squamous carcinoma SCC-15 cells and the human epithelial cell line 293T cells in the presence or absence of DNA damage agent cisplatin (33 lM). Treatment of the cells with cisplatin for 6 h decreased the level of Chk2 protein substantially (Fig. 1A). We have also examined the A2780 ovarian cancer cells in response to cisplatin. After treatment of the cells with cisplatin (33 lM) for 1, 2, and 6 h, the expression level of Chk2 kinase was decreased progressively to a non-detectable level (Fig 1B). Treatment of the cells for 24 h or longer showed identical results to those seen after 6 h (data not shown). Unless stated otherwise, we used a 6-h treatment throughout the experiments using cisplatin concentration of 33 lM. Expression of Chk2 kinase was not observed in the A549 lung adenocarcinoma cells due to hypermethylation of the Chk2 gene promoter [17]. While the expression level of Chk1 kinase was not altered in the ovarian cancer A2780 cells, p53 expression was gradually increased, in close correlation with the decrease of Chk2 kinase expression. In contrast to A2780 cells, the increase of the p53 expression level in the lung cancer A549 cells in response to cisplatin was not seen until 6 h. Increase of the cisplatin concentrations resulted in an earlier degradation of Chk2. Fig. 1C shows that cisplatin at 256 lM can down-regulate the expression level of Chk2 kinase within a 1 h time period. These results indicate that the level of Chk2 kinase expression is regulated by cisplatin in both timeand dose-dependent manners. Chk2 kinase is degraded in response to cisplatin We determined if down-regulation of Chk2 expression in response to cisplatin is at the transcriptional or post-translational levels. We used the protein synthesis
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Fig. 2. Chk2 protein is degraded in response to cisplatin. (A) The stability of Chk2 in A2780 cells. Cells were treated with 10 lg/ml cycloheximide for 0, 1, 2, and 6 h in the absence or presence of 33 lM cisplatin. The protein level of Chk2 was assayed by Western blotting with anti-Chk2 antibody. As a control, the expression of b-actin after treatment of cycloheximide is also determined. (B) Northern blot analyses of Chk2 mRNA expressions in A2780 cells in the presence or absence of actinomycin D (1 mg/ml). The 28S and 18S rRNAs were used to normalize the loading between each lane (lower panel). Fig. 1. Chk2 kinase expression is diminished in response to cisplatin treatment. (A) The oral cavity squamous carcinoma SCC-15 and the 293T cells in the presence or absence of 33 lM cisplatin for 6 h. (B) The ovarian cancer A2780 cells and the lung cancer A549 cells were cultured and treated without (C, control) or with cisplatin for the time period listed above, and the total cell lysates were harvested and subjected to immunoblotting analyses using different antibodies as listed. b-Actin was used to normalize the loading. (C) The ovarian cancer A2780 cells were treated with increasing concentrations of cisplatin for 1 h, and the total cell lysates were used for immunoblotting analyses of Chk2 expression.
inhibitor cycloheximide (CHX) to block new protein synthesis and determined the natural half-life of the existing Chk2 protein in the presence or absence of cisplatin. We treated the A2780 cells with CHX alone or in combination with cisplatin, and determined the Chk2 expression levels by immunoblot (Fig. 2A). In the presence of CHX alone, the Chk2 expression level was not significantly altered within 6 h, whereas cisplatin can induce down-regulation of Chk2 expression in the presence of CHX. This result suggests that cisplatin shortens the half-life of Chk2 kinase and induces accelerated Chk2 protein degradation. We also used the transcription inhibitor actinomycin D (Act D) to block the new mRNA synthesis and determined the half-life of the Chk2 mRNA by Northern blot analysis (Fig. 2B). Cisplatin appeared to have no significant effect on the half-life of the Chk2 mRNA. These results suggest to us that down-regulation of Chk2 expression in response to cisplatin in the ovarian cancer A2780 cells is mediated through protein degradation, rather than the decrease of
transcription rate of the Chk2 gene or decrease of mRNA half-life. Chk2 degradation is mediated by a ubiquitin–proteasome pathway The ubiquitin–proteasome system is the most common protein degradation pathway in mammalian cells. To determine whether the degradation of Chk2 kinase in response to cisplatin is mediated through this system, we performed immunoprecipitation using anti-Chk2 antibody and immunoblot by using either anti-ubiquitin antibody or anti-Chk2 antibody. Fig. 3A indicates that Chk2 is ubiquitinated under the basal condition. Treatment of the cells with cisplatin enhanced the ubiquitination of Chk2 kinase appreciably. We also used the proteasome inhibitor MG132 to treat the A2780 cells in combination with cisplatin to see if MG132 can inhibit the Chk2 degradation in the presence of cisplatin (Fig. 3B). The Chk2 degradation induced by cisplatin can be inhibited by MG132, indicating that the degradation of Chk2 kinase in response to cisplatin is mediated through the ubiquitin–proteasome pathway. Chk2 degradation is independent of ATM kinase activation ATM kinase is activated in response to cisplatininduced DNA damage, and the activated ATM
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Fig. 3. Chk2 degradation is mediated through ubiquitin–proteasome pathway. (A) The ovarian cancer A2780 cells were treated with cisplatin (33 lM) for the time period listed above, and the total cell lysates were prepared for immunoprecipitation using Chk2 antibody. The immunoprecipitates were separated on the 10% SDS–PAGE gel, and the proteins were immunoblotted by using anti-ubiquitin antibody (upper panel) and anti-Chk2 antibody. (B) Chk2 degradation in response to cisplatin treatment can be inhibited by the presence of MG132 (20 lM), a wide spectrum ubiquitin–proteasome inhibitor.
phosphorylates Thr68 of Chk2 kinase to transduce the DNA damaging signals [8]. Therefore, we next investigated the possible involvement of ATM-mediated Thr68 phosphorylation in cisplatin-induced Chk2 degradation. We transfected the CP70 cells, in which the endogenous Chk2 kinase is minimal to absent by immunoblot analysis (see Fig. 5, below) [17], with the wild type HA-Chk2 and the HA-T68A expression constructs (kindly provided by Dr. Junjie Chen, Mayo Clinic, Rochester, MN), and analyzed the expression of the wild type Chk2 and the T68A mutant by immunoblot with both anti-HA antibody and anti-Chk2 antibody (Fig. 4A). Treatment of the transfected cells with cisplatin induced degradation of the HA-tagged wild type Chk2 protein as well as the T68A mutant. Moreover, Chk2 degradation was also seen in the ATM null lymphoblasts (GM03182, Coriell Cell Repositories, NJ), indicating that degradation of Chk2 kinase in response to cisplatin is independent of ATM kinase (Fig. 4B). Since BRCA1 is one of the Chk2 downstream targets, and BRCA1 possesses ubiquitin E3 ligase activity at the N-terminus as shown in in vitro study [18], we sought to see if BRCA1 is required for Chk2 degradation. Cisplatin can indeed induce Chk2 degradation in the BRCA1 null lymphoblasts (GM14093, Coriell Cell Repositories, NJ), suggesting that BRCA1 is not required for the degradation of Chk2 induced by cisplatin. Chk2 degradation mediates cisplatin resistance We have obtained a pair of the ovarian cancer cell lines, A2780 and CP70. The CP70 cells were developed
Fig. 4. Chk2 kinase degradation in response to cisplatin is independent of ATM kinase and BRCA1 tumor suppressor. (A) HA-tagged wild type Chk2 expression vector and the T68A mutant were transfected into the ovarian cancer CP70 cells and treated with cisplatin(33 lM) for the time period listed above. The total cell lysates were prepared and used for immunoblotting using anti-HA antibody (upper panel) and anti-Chk2 antibody (lower panel) for the detection of endogenous Chk2 protein. (B) The ATM / and BRCA1 / lymphoblasts were obtained from Coreill Cell Repository (New Jersey) and cultured under the condition described. The cells were treated with cisplatin (33 lM) for the time listed above, and the total proteins were used for immunoblotting analyses for Chk2 expression.
Fig. 5. Diminished Chk2 expression in the cisplatin-resistant CP70 ovarian cancer cells in comparison with the parental A2780 cells. Ovarian cancer A2780 cells and CP70 cells were treated with cisplatin for the time period listed above, and total proteins were used for immunoblotting analyses for Chk2 expression. b-Actin was used to normalize the equal loading of the proteins in each lane.
by exposure of A2780 cells to low dose cisplatin (1– 2 lM) for long period of time over a total of 70 cell passages. A2780 cells are sensitive to cisplatin (IC50 = 3 lM), and CP70 cells are resistant to cisplatin (IC50 = 45 lM) [19]. We have shown that cisplatin induced Chk2 degradation in A2780 cells, and we reasoned that development of resistance to cisplatin of CP70 cells may be the direct result of down-regulation of Chk2 by degradation. CP70 cells may have much lower or diminished Chk2 kinase expression. To test this, we measured Chk2 protein by immunoblot (Fig. 5). Chk2 expression in the CP70 cells was almost non-detectable and represents approximately 5–10% of that seen in the parental A2780 cells. This result indicates that diminished expression of Chk2 kinase is at least in part responsible for cisplatin resistance of CP70 cells. Thus, Chk2 degradation may be one of the primary mechanisms by which a large number of clinically relevant tumors develop the acquired resistance to DNA damage agent.
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Fig. 6. Chk2 degradation is not affected by the D-box sequence. (A) Diagrammatic representation of the Chk2 sequences and the mutational design at the D-box sequence. (B) Wild type Chk2 (WT-HA), T68A-mutated Chk2 (T68A), and R180G/L183V-mutated Chk2 (R180DM) are degraded in response to cisplatin treatment.
Chk2 degradation is independent of a putative D-box sequence To further investigate the molecular mechanism of Chk2 degradation in response to cisplatin, we examined the Chk2 amino acid sequences. At the position 180, there appears to be a putative consensus sequence of destruction box (D-box motif), defined by yeast genetics from degradation of cyclin B [20] (Fig. 6A). D-box sequence is the known interaction domain with the anaphase-promoting complex (APC), an E3 ubiquitin ligase complex and it is associated WD-domain-containing activator CDC20 or CDH1 [20,21]. To determine the possible involvement of D-box in cisplatin-induced Chk2 degradation, we constructed a D-box-mutated Chk2 expression vector, R180G/L183V (referred to as R180DM) by site-directed mutation. The expression plasmids were transfected into the CP70 cells and treated with cisplatin (Fig. 6B). The wild type Chk2 kinase, T68A mutant, and R180DM were all degraded in response to cisplatin. These results suggested that Chk2 degradation is mediated through a D-box independent mechanism.
Discussion In response to DNA damage, Chk2 kinase is rapidly activated by ATM kinase through T68 phosphorylation in response to DNA damaging signals [10]. The importance of Chk2 kinase function in DNA damage response is exemplified by the Chk2 gene knockout study in
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which deficiencies in DNA-damage-induced apoptosis and G1/S checkpoint were observed [16,22]. Clinically, most ovarian cancers are sensitive to cisplatin-based chemotherapy [23–25]. However, cisplatin-induced resistance to treatment is of clinical significance [25]. We felt that down-regulation of Chk2 kinase expression in these cancer cells, at least in part, mediated the cancer cell resistance to cisplatin. Furthermore, the CP70 ovarian cancer cells were derived from the parental A2780 cells by exposure to low dose cisplatin over total of 70 passages [19]. The level of Chk2 kinase expression in the CP70 cells was remarkably lower compared with that in the parental A2780 cells, while the resistance of CP70 cells to cisplatin was found to be 15-fold higher [19]. Although other proteins may have played roles in mediating the resistance of the CP70 cells to cisplatin, down-regulation of Chk2 by degradation in these cells may be the predominant event in development of cisplatin resistance. Chk2 degradation in response to cisplatin treatment in cancer cells is a novel observation. The degradation of Chk2 kinase appears to be independent of ATM kinase or BRCA1 tumor suppressor (Fig. 4). Examination of the Chk2 amino acid sequence revealed a consensus destruction box (D-box) domain at the amino acid 180–188 that is known to serve as a recognition box by anaphase-promoting complex (APC/CDC20 and/or APC/CDH1), leading to protein ubiquitination and proteolysis through proteasome pathway [21,26,27]. Mutation of this D-box sequence of Chk2 kinase has no effect on the cisplatin-induced degradation of Chk2 (Fig. 6). These data, however, do not exclude the potential role that the APC/CDC20 or APC/CDH1 complex may play in mediating Chk2 degradation. Other potential sites within the Chk2 sequence may be targeted by APC complex. In addition to APC complex, other ubiquitin ligases may also contribute to the ubiquitination and degradation of Chk2. Identification of the E3 ligases that mediates Chk2 degradation will significantly help in the understanding of how Chk2 degradation is regulated in the DNA damage signaling pathway. Acknowledgments The authors thank Dr. Junjie Chen, at the Mayo Clinic for providing the Chk2 expression plasmids. This study is supported in part by the Sara Crile Allen and James F. Allen endowment fund for lung cancer research, and the West Virginia University Senate Research Grant. References [1] E. Reed, Cancer Treat. Rev. 24 (1998) 331–344. [2] W.C. Lin, F.T. Lin, J.R. Nevins, Genes Dev. 15 (2001) 1833– 1844.
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