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Aurora-A interacts with Cyclin B1 and enhances its stability
Cancer Letters 275 (2009) 77–85
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Aurora-A interacts with Cyclin B1 and enhances its stability Lili Qin a,b, Tong Tong a, Yongmei Song a, Liyan Xue a, Feiyue Fan b, Qimin Zhan a,* a b
State Key Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300192, China
a r t i c l e
i n f o
Article history: Received 14 February 2008 Received in revised form 25 September 2008 Accepted 2 October 2008
Keywords: Cyclin B1 Aurora-A Cell cycle Protein stability Carcinogenesis
a b s t r a c t The mitotic regulator Aurora-A is an oncogenic protein that is over-expressed in many types of human tumors. However, the underlying mechanism through which Aurora-A promotes tumorigenesis remains unclear. Here, we show that overexpression of Aurora-A causes an elevation of Cyclin B1 expression. Cyclin B1 degradation is delayed in AuroraA over-expressing cells, which depends on Aurora-A kinase activity. In contrast, AuroraA RNAi enhances Cyclin B1 degradation. Furthermore, we found that Aurora-A interacts with Cyclin B1, and that Aurora-A overexpression reduces the interaction of Cyclin B1 with APC subunits. In human esophageal squamous cell carcinomas (ESCC), overexpression of Aurora-A was correlated with deregulated expression of Cyclin B1. Taken together, these findings suggest that overexpression of Aurora-A may stabilize Cyclin B1 through inhibiting its degradation. These results provide new insight into the mechanism of how deregulated Aurora-A contributes to genomic instability and carcinogenesis. Ó 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The eukaryotic cell cycle is a complex process tightly controlled by many regulators, and aberrations in the cell cycle may lead to chromosomal instability, the hallmark of most human malignancies. Several protein kinases control cell cycle progression, such as Aurora kinases and CDKs/Cyclins kinases. Abnormal regulation or expression of these kinases may result in genomic instability and subsequently carcinogenesis [1,2]. Aurora-A kinase belongs to a serine/threonine kinase family important for proper execution of various mitotic events and for maintaining genomic integrity [3]. AuroraA participates in several crucial mitotic events, including centrosome maturation and separation, bipolar spindle assembly, chromosome alignment and segregation, and cytokinesis. Overexpression of Aurora-A has been shown to lead to centrosome amplification and aneuploidy, which usually results from incomplete cytokinesis and can be a driving cause of genomic instability and tumorigenesis * Corresponding author. Tel.: +86 10 87788422; fax: +86 10 67715058. E-mail address: [email protected] (Q. Zhan). 0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.10.011
[4–7]. Aurora-A has also been implicated in regulation of cell cycle checkpoints. Overexpression of Aurora-A was shown to abrogate the G2/M checkpoint and spindle assembly checkpoint, allowing cells to inappropriately enter into mitosis and anaphase with damaged DNA and defective spindles. These checkpoint defects may ultimately contribute to genomic instability and carcinogenesis [8–10]. The human Aurora-A gene is located at chromosome 20q13.2, a region frequently amplified and over-expressed in a variety of human tumors and cancerderived cell lines [3,6,11]. Recent studies have shown that overexpression of Aurora-A in cultured cells induces several cancer-related phenotypes, including increased cell proliferation and colony formation and inhibition of UVor cisplatin-induced apoptosis [12]. Overexpression of Aurora-A can also transform rat-1 and NIH3T3 cells and form tumors in null mice [5,7]. Collectively, this evidence indicates that Aurora-A acts as an oncogene and plays an important role in cell cycle progression and carcinogenesis. Cyclin B was the first mitotic Cyclin to be discovered. It accumulates during interphase and is rapidly degraded as cells exit from mitosis [13]. Cyclin B1 functions mainly during late G2 phase and mitosis as the regulatory subunit
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of CDK1 [14]. Previous studies have shown that accumulation and nuclear translocation of Cyclin B1 at the G2/M transition is required for CDK1 activation and mitotic entry, and that Cyclin B1 degradation is essential to inactivation of CDK1 and exit from mitosis [15,16]. Cyclin B1 is degraded by the APC/C-mediated ubiquitination pathway [17], and both CDC20 and CDH1 are involved in its degradation [18,19]. Stable Cyclin B1 causes dosedependent mitotic arrest phenotypes, suggesting that different thresholds of Cyclin B1/Cdk1 activity are required for the metaphase-to-anaphase and the anaphaseto-telophase transition, and that gradual degradation of Cyclin B1 is indispensable for late mitotic events [20]. Several lines of evidence have demonstrated that elevated levels of Cyclin B1 result in decreased G2 checkpoint function and contribute to the immortalization of human cells [21]. Overexpression of Cyclin B1 can also impair the mitotic spindle checkpoint and induce chromosome instability and aneuploidy, caused by incomplete cytokinesis [22]. Cyclin B1 has been shown to be over-expressed in many types of human tumors [23–25]. Moreover, the level of Cyclin B1 expression has been positively correlated with malignancy and poor prognosis in ESCC [26–28]. Ubiquitin-mediated proteolysis plays a crucial role in cell cycle progression in eukaryotes. APC/C is a multi-subunit ubiquitin ligase that catalyzes the ubiquitination of specific substrates and targets them for degradation by the 26S proteasome [29]. In vertebrates, APC/C is composed of at least 13 subunits, including APC2, which is a structural component of the core of APC/C, and CDC27, which regulates activation of APC/C by association with CDC20 and CDH1[29]. APC/C-mediated proteolysis helps trigger the onset of anaphase and facilitates mitotic exit by targeting the anaphase inhibitor and mitotic cyclins for proteolysis. Also, APC/C helps establish and maintain the G1 state by degrading G1 proteins until conditions are appropriate to begin another round of cell division [30–32]. CDC20 and CDH1 act as activators of APC/C, and they help determine the substrate specificity of APC/C as well [33,34]. APC/C is also controlled by a regulatory mechanism called the spindle assembly checkpoint, which monitors kinetochore-spindle microtubules interactions. This checkpoint prevents APC/C activation until all chromosomes are properly attached, to ensure that all chromosomes segregate faithfully [35]. In addition, phosphorylation plays an important role in controlling the timing of APC activation [36]. In this study, we show that Aurora-A overexpression causes upregulation of Cyclin B1 and delays the degradation of Cyclin B1, in a manner dependent on Aurora-A kinase activity. Further, Aurora-A RNAi enhances Cyclin B1 degradation, and Aurora-A overexpression reduces the interaction between Cyclin B1 and APC/C. In ESCC we show that both Aurora-A and Cyclin B1 proteins are over-expressed, and that the expression pattern of these two proteins is correlated. Collectively, these results suggest that Aurora-A and Cyclin B1 cooperate to regulate the cell cycle, and defects in the regulation of these proteins contribute to genomic instability and tumorigenesis.
2. Materials and methods 2.1. Cell culture, synchronization and drug treatment Human ESCC KYSE150 and ESCC EC9706 cell lines (gifts from Dr. Shimada, Kyoto University) were cultured in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin, and 100 lg/ml streptomycin at 37 °C. pEGFP-Aurora-A transfected KYSE150 cells and Aurora-A siRNA transfected EC9706 cells were cultured in the presence of 400 lg/ml of G418 (Geneticin sulfate, GIBCO). For stability analysis of protein, 100 lg/ml CHX (cycloheximide) (Sigma–Aldrich Corp., St. Louis, MO) was added to cell culture, and then cells were harvested at the indicated time points. The Aurora-A kinase inhibitor Cyclopropanecarboxylic acid-(3-(4-(3-trifluoromethyl-phenylamino)pyrimidin-2-ylamino)-phenyl)-amide (Merck Calbiochem Inc. Darmstadt, Germany. 189405, dissolved in DMSO and stored at 50 mg/ml at 20 °C), was used at 1 lM for 2 h prior to treatment with CHX. 2.2. RNA isolation and RT-PCR analysis Total RNA was purified and reverse transcription was carried out at 42 °C for 90 min using SuperScript II (Invitrogen Technologies Inc.). The cDNA was used for PCR amplification. Aurora-A PCR was performed for 35 cycles, at an annealing temperature of 60 °C (30 s) using primers: 50 AAT GAT TGA AGG TCG GAT GC-30 (forward) and 50 -TTC TCT GAG CAT TGG CCT CT-30 (reverse). Cyclin B1 amplification was carried out for 31 cycles, at an annealing temperature of 58 °C for 30 s. Primers for Cyclin B1 were: 50 -TCC AAG CCC AAT GGA AAC AT-30 (forward); 50 -ATG CTC TCC GAA GGA AGT GC-30 (reverse). PCR products were analyzed on 1% agarose gels stained with ethidium bromide and visualized with a UV transilluminator. 2.3. Antibodies Antibody against Cyclin B1 was from BD Biotechnology (BD Pharmingen 554176). Anti-Aurora-A antibody was from Cell Signaling (Cell Signaling, Beverly, MA, 3092). Antibodies to APC2 (Santa Cruz Biotechnology sc-20984), CDC27 (Santa Cruz Biotechnology sc-5618), CDC20 (Santa Cruz Biotechnology sc-8358), Ubiquitin (Santa Cruz Biotechnology sc-9133), Plk1 (Santa Cruz Biotechnology sc5585), Rb (Santa Cruz Biotechnology sc-50), b-catenin (Santa Cruz Biotechnology sc-7963), E-cadherin (Santa Cruz Biotechnology sc-7870) and b-actin (Santa Cruz Biotechnology sc-8432) were obtained from Santa Cruz Technology. Antibodies to CDH1 (Sigma, St. Louis, MO c-7855) were purchased from Sigma. 2.4. Cell cycle analysis For flow cytometry analysis, cells were seeded and grown. Then cells were trypsinized, washed twice with pre-chilled PBS and fixed in 70% ice-cold ethanol. After incubation at 20 °C for at least 2 h, cells were washed with PBS twice and re-suspended in 1 ml of PBS containing
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40 lg/ml propidium iodide and 100 lg/ml RNase A. The suspension was then used for flow cytometry analysis.
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Cells were harvested, rinsed with PBS twice, and lysed in ice-cold lysis buffer on ice for 40 min. Lysate was collected by centrifugation at 12,000g at 4 °C for 20 min. 80 lg of protein was subjected to 10% SDS–PAGE. Immunoblotting was done as described before [37]. For immunoprecipitation, the total lysate containing 1 mg of protein was incubated with the indicated antibodies at room temperature for 2 h. Protein A/G-agarose beads (Santa Cruz Technology Inc.) were then added and incubated overnight at 4 °C. After washed six times with lysis buffer, the immuno-complex was boiled and subjected to SDS–PAGE, followed by immunoblotting assay.
transfected with an empty pEGFP vector (Fig. 1A). This result demonstrates that when over-expressed, Aurora-A can induce upregulation of Cyclin B1 protein. In addition to Cyclin B1, the levels of Plk1 and b-catenin were also upregulated by Aurora-A overexpression. Conversely, the level of Rb appeared to decrease, and E-cadherin level was not affected by Aurora-A overexpression. These results suggest that Aurora-A overexpression may affect the levels of other oncogenes or tumor suppressor genes. To determine whether the high level of Cyclin B1 in Aurora-A over-expressing cells is a result of G2/M arrest, we performed flow cytometry to examine cell cycle distributions. As shown in Fig. 1B, Aurora-A over-expressing cells did not appear to accumulate in G2 phase in greater numbers than control cells, which is consistent with previous results [41]. This result demonstrates that the increased level of Cyclin B1 protein in Aurora-A over-expressing cells is not caused by a delay in the G2/M transition. Additionally, the level of Cyclin B1 mRNA was examined via semi-quantitative RT-PCR, and there was no evidence of an increase in Cyclin B1 mRNA level following Aurora-A overexpression (data not shown). This result suggests that accumulation of cyclin B1 in Aurora-A over-expressing cells is not due to regulation at the level of transcription but may occur at the post-transcriptional level.
2.6. Pull-down assays
3.2. Overexpression of Aurora-A delays the degradation of Cyclin B1
Expression and purification of recombinant GST fusion proteins was carried out as previously described [38]. For GST-pull-down assay, the total lysate containing 2 mg of protein was incubated with GST, GST-Aurora-A and GSTCyclin B1 bound to glutathione Sepharose overnight at 4 °C. After washed six times with lysis buffer, samples were boiled and then subjected to SDS–PAGE and immunoblotting analysis.
We next investigated whether upregulation of Cyclin B1 protein occurred as a result of decreased protein degradation. We examined the stability of Cyclin B1 protein in both Aurora-A over-expressing and control cells treated with CHX. In control cells, the amount of Cyclin B1 protein dramatically decreased by 50% at 0.5 h, and disappeared at about 2 h after treatment with CHX. However, in Aurora-A over-expressing cells, the level of Cyclin B1 was reduced by about 50% at 1.5 h, and disappeared at about 6 h after treatment with CHX. This result suggests that Aurora-A overexpression might inhibit the degradation of Cyclin B1 (Fig. 2A). Additionally, we found that Aurora-A degradation occurs later and more slowly than Cyclin B1 degradation, although both proteins are degraded through the APC/C-mediated pathway. To investigate whether the delay in Cyclin B1 degradation in Aurora-A over-expressing cells depends on Aurora-A kinase activity, we treated cells with an Aurora-A kinase inhibitor for 2 h prior to CHX treatment. Western blot analysis demonstrated that when Aurora-A kinase activity is inhibited, Cyclin B1 degradation is not delayed, suggesting that Aurora-A kinase activity is needed to regulate Cyclin B1 degradation (Fig. 2C).
2.5. Cell lysis, immunoblotting and immunoprecipitation
2.7. Patients, clinical tissue sample collection and tissue microarray immunohistochemical analysis Fresh tumor tissues (173) were obtained from patients with pathologically and clinically confirmed ESCC. The institutional Review Board approved use of the tumor specimens in this study and clinical consultation reports were available for all tumor samples. TMAs were prepared as described [39] with slight modifications. Core cylinders with a diameter of 0.6 mm were punched and deposited into a recipient paraffin block. Four-lm sections of the resulting microarray block were transferred to glass slides. Then, antibodies against Aurora-A and Cyclin B1 were used for IHC analysis. Immunohistochemical staining and semiquantitative evaluation of staining was performed as previously described [40]. Aurora-A and Cyclin B1 expression was analyzed using the v2-Test with SPSS software. Statistical significance was considered at the value of P < 0.05. 3. Results 3.1. Overexpression of Aurora-A induces upregulation of Cyclin B1 expression Our previous studies indicated that Aurora-A is over-expressed in human ESCC, and that the overexpression of Aurora-A is associated with increased malignancy and poor prognosis of patients [40]. Expression of exogenous Aurora-A promotes cell proliferation, stimulates colony formation, and inhibits apoptosis in ESCC KYSE150 cells [12]. To investigate the underlying mechanism through which Aurora-A promotes tumorigenesis, we first examined the expression levels of different oncogenic proteins by Western analysis. We found that Cyclin B1 was upregulated in KYSE150 cells that had been stably transfected with pEGFP-Aurora-A, but was expressed at relatively lower levels in untransfected KYSE150 cells (which express low levels of endogenous Aurora-A kinase), and in KYSE150 cells
3.3. Aurora-A RNAi enhances the degradation of Cyclin B1 To further study the role of Aurora-A in Cyclin B1 degradation, we examined whether downregulation of Aurora-A results in an increased amount of Cyclin B1 degradation in EC9706 cells. EC9706 cells have relatively higher levels of endogenous Aurora-A expression than KYSE150 cells, and in EC9706 ESCC cells, the half-life of Cyclin B1 is longer than in KYSE150 cells. As shown in Fig. 3A, Aurora-A protein levels were dramatically reduced in EC9706 cells following treatment with siRNA targeting Aurora-A. We observed that downregulation of Aurora-A resulted in increased degradation of Cyclin B1 (Fig. 3B). Taken together with the result described above, these data show that Aurora-A helps regulate the stability of Cyclin B1 protein. 3.4. Cyclin B1 ubiquitination and interactions with APC/C subunits are affected by Aurora-A overexpression It has been reported previously that Cyclin B1 is degraded by the APC/C-mediated ubiquitin pathway, and that CDC20 and CDH1 are involved in this process. Therefore, we hypothesized that Aurora-A may inhibit Cyclin B1 degradation by disrupting interactions between Cyclin B1 and proteins involved in its degradation. We performed immunoprecipitation experiments using Cyclin B1 antibody to investigate this hypothesis. As expected, the level of ubiquitinated Cyclin B1 was lower in Aurora-A over-expressing cells than that in control cells (shown in Fig. 4A). Additionally, reduced amounts of APC2, CDC27, CDC20 and CDH1 were detected in Cyclin B1 immuno-complexes from Aurora-A over-expressing cells compared to control cells. However, the levels of these proteins in total lysate from Aurora-A transfected cells were higher than in control cells (shown in Fig. 4B). These results suggest
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Fig. 1. Aurora-A overexpression induces upregulation of Cyclin B1 in ESCC KYSE150 cells. (A) Protein expression was analyzed by western blot with indicated antibodies. Genes that were upregulated, down-regulated or not affected by Aurora-A overexpression are shown. (B) Flow cytometry analysis of cell cycle distributions. The results are expressed as the percentage of cells at G1/S or G2/M phase. The data is representative of three independent experiments. that the interactions between Cyclin B1 and degradation-associated proteins are weakened by increased levels of Aurora-A, which results in an inhibition of ubiquitin-mediated degradation of Cyclin B1. 3.5. Aurora-A interacts with Cyclin B1 Previous studies have shown that Aurora-A and Cyclin B1 are both centrosome-associated proteins, and that centrosomal localization of Aurora-A is required for Cyclin B1 localization and for activation of the Cyclin B1/CDK1 complex [42–44]. Therefore, it is possible that both proteins are present in the same complex. To investigate this, immunoprecipitation assays were carried out using antibodies against Aurora-A and Cyclin B1. As shown in Fig. 5A, both Aurora-A and Cyclin B1 were detected in Aurora-A-immunoprecipitated complex. Similarly, these two proteins were observed in Cyclin B1-immunoprecipitated complex. In contrast, neither Aurora-A nor Cyclin B1 was detected in precipitates obtained with b-actin antibody. To further confirm the interaction between Aurora-A and Cyclin B1, we performed an in vitro binding assay using purified GST-tagged proteins. We expressed and purified GST, GST-Aurora-A, and GST-Cyclin B1 fusion proteins in Escherichia coli, and performed pull-down assays. As shown in Fig. 5B, both Aurora-A and Cyclin B1 were found in protein complexes obtained from GST-Cyclin B1 and GST-Aurora-A pull-down assays, but not from GST pull-down assays. Taken together, these results indicate that Aurora-A interacts with Cyclin B1 directly or indirectly. 3.6. Expression relevance of Aurora-A and Cyclin B1 in clinical ESCC Previous studies have shown that Aurora-A and Cyclin B1 proteins are over-expressed in many types of human tumors. We used immunohistochemical assays to examine the expression of these two proteins simultaneously in clinical tumor specimens from 173 ESCC patients, to further assess the relevance of this overexpression. The immunohistochemical staining results are summarized in Table 1. Consistent with previous findings [23,40], we found that both Aurora-A and Cyclin B1 are over-expressed in ESCC (in Fig. 6). Aurora-A overexpression was detected in 119 of 173 samples (68.8%), and Cyclin B1 overexpression was detected in 124 of 173 samples (71.7%). v2-Test analysis using SPSS software was performed, and we found there was a positive correlation between Aurora-A expression and Cyclin B1 expression in human ESCC (P < 0.05).
4. Discussion Proper cell cycle progression is essential to maintain genomic integrity. Aurora-A and Cyclin B1 are two crucial regulators involved in cell cycle regulation, and they function mainly in mitosis. Multiple lines of evidence indicate that Aurora-A and Cyclin B1 are over-expressed in a variety of human tumors, and increased expression of Aurora-A and Cyclin B1 has been correlated with malignant behavior of tumors [23,40]. We previously reported that Aurora-A overexpression may induce malignant transformation of a human ESCC cell line [12]. However, the exact mechanism(s) through which Aurora-A overexpression promotes the development and progression of cancer remains unclear. Here we provided new insight into the molecular mechanism through which Aurora-A may promote carcinogenesis. First, we demonstrated that the protein level of Cyclin B1 is increased when Aurora-A is over-expressed in ESCC KYSE150 cells. Flow cytometry analysis revealed that upregulation of Cyclin B1 is not caused by a delay in the G2/M transition in Aurora-A over-expressing cells, and we found no evidence that Aurora-A regulates Cyclin B1 at the level of transcription. Based on this evidence, we hypothesized that upregulation of Cyclin B1 in Aurora-A over-expressing cells is probably due to regulation at the post-transcriptional level. Additionally, we demonstrated that the expression of some other cancer-associated proteins, such as PLK1 and b-catenin, is induced in Aurora-A over-expressing cells, suggesting that Aurora-A plays an oncogenic role through regulation of these proteins as well. Interestingly, we found that when Aurora-A is over-expressed, the half-life of Cyclin B1 is increased, and the degradation of Cyclin B1 is delayed. The delay of Cyclin B1
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Fig. 2. (A) Overexpression of Aurora-A delays the degradation of Cyclin B1. Cells were treated with CHX and then harvested at the indicated time points. Cyclin B1 protein level at each time point was determined by Western Blot. (B) Effects of Cyclopropanecarboxylic acid-(3-(4-(3-trifluoromethylphenylamino)-pyrimidin-2-ylamino)-phenyl)-amide on Aurora kinase biomarkers. Cells were exposed to the indicated concentration of Aurora-A kinase inhibitor for 2 h. Western Blotting was performed with antibodies recognizing pThr288 on Aurora-A, or total Aurora-A. (C) The effect of Aurora-A overexpression on Cyclin B1 degradation depends on the kinase activity of Aurora-A. Cells were exposed to Aurora-A kinase inhibitor for 2 h prior to treatment with CHX. Cells were harvested and analyzed by Western blotting. (D) To quantitatively evaluate the effect of Aurora-A on Cyclin B1 degradation, Cyclin B1 protein levels were quantified using QuantityOne software and normalized to b-actin level.
degradation in Aurora-A over-expressing cells was found to be dependent on the kinase activity of Aurora-A. In line with these results, Aurora-A RNAi enhanced the degradation of Cyclin B1. Furthermore, Aurora-A over-expressing cells had lower level of ubiquitinated Cyclin B1 compared with control cells, and the interactions between Cyclin B1 and proteins involved in Cyclin B1 ubiquitin-mediated destruction (such as APC2, CDC27, CDC20 and CDH1) were much weaker. In addition, we demonstrated an interaction between Aurora-A and Cyclin B1 in vivo and in vitro. Taken together, these results suggest that overexpression of Aurora-A delays Cyclin B1 degradation through disrupting the interactions between Cyclin B1 and the APC/C complex.
Aurora-A may interfere with the ubiquitin-mediated degradation of Cyclin B1 in two different ways. First, the physical interaction of Aurora-A and Cyclin B1 may alter the conformation of Cyclin B1, which could inhibit the ability of APC/CCDC20 and APC/CCDH1 to recognize and bind to Cyclin B1. Second, as a protein kinase, Aurora-A could regulate Cyclin B1 degradation through phosphorylation. Previous work has shown that phosphorylation may regulate proteolysis; for example, Aurora-A degradation can be inhibited by phosphorylation of serine 51 in the A box [45]. In addition to observations that Aurora-A interacts with and stabilizes Cyclin B1, findings from clinical ESCC
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Fig. 3. Aurora-A siRNA transfection enhances the degradation of Cyclin B1. (A) Aurora-A RNAi down-regulates Aurora-A and Cyclin B1 expression. (B) The degradation of Cyclin B1 is enhanced by Aurora-A RNAi.
Fig. 4. Effects of Aurora-A overexpression on ubiquitination of Cyclin B1. Cells were grown, harvested and lysed. Cell lysate (1 mg) was immunoprecipitated with anti-Cyclin B1 antibody, followed by immunoblotting using antibodies against ubiquitin, APC2, CDC27, CDC20 and CDH1, Cyclin B1 and b-actin. Immunoprecipitation with anti-b-actin antibody was used as a negative control. (A) The level of ubiquitinated-Cyclin B1 is decreased in Aurora-A overexpressing cells. (B) The interaction between Cyclin B1 and APC/C complexes is weaker in Aurora-A over-expressing cells. The asterisk marks the immunoglobin heavy chains, and the upper bands shown in the cyclin B1 IP correspond to cyclin B1. Ubi: ubiquitin.
samples indicate that expression of these two proteins may be correlated. Although the expression of both Aurora-A and Cyclin B1 is deregulated in human ESCC, we
demonstrated a positive correlation in expression profile between them. Thus, Aurora-A may cooperate with Cyclin B1 to induce cell transformation and carcinogenesis.
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Fig. 5. Interaction between Aurora-A and Cyclin B1. (A) In vivo interaction of Aurora-A and Cyclin B1. Immunoprecipitation of Aurora-A and Cyclin B1 from KYSE150/GFP-Aur-A cell lysates was carried out as described in Section 2 using antibodies against Aurora-A and Cyclin B1. Immunoblotting was performed, using b-actin as a negative control. (B) In vitro interaction between Aurora-A and Cyclin B1. GST, GST-Aurora-A and GST-Cyclin B1 fusion proteins were expressed in E. coli and purified using glutathione agarose beads. Pull-down assays were performed as described in Section 2 using whole-cell extracts of KYSE150/GFP-Aur-A cells. Samples were analyzed by Western blot. GST was used as a negative control. Table 1 The expression correlation of Aurora-A and Cyclin B1. CyclinB1 expression
Aurora-A expression () or (±) (+) (++) *
P value
() or (±)
(+)
(++)
16 16 7
24 10 19
14 28 29
0.038*
3 3 Pearson v2-Test.
Multiple lines of evidence have demonstrated a connection between aneuploidy and genomic instability, cell malignant transformation and tumorigenesis. Aneuploidy
is thought to be caused by abortive cytokinesis, due to deregulation of mitotic regulator proteins. Previous experiments have shown that Aurora-A overexpression leads to premature anaphase entry, which subsequently results in centrosome amplification and aneuploidy due to incomplete cytokinesis [10]. However, up to now, no evidence has demonstrated a direct role for Aurora-A in cytokinesis. As the regulatory subunit of CDK1, Cyclin B1’s scheduled degradation plays a crucial role in late mitotic events, such as mitotic exit and completion of cytokinesis [20,22]. Although CyclinB1/Cdk1 kinase activity is required for inhibiting separase activity before cells enter anaphase, CyclinB1 degradation is not thought to be necessary for the
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Fig. 6. The expression of Aurora-A and Cyclin B1 in ESCC and normal adjacent tissues studied with IHC on tissue microarrays. 173 ESCC samples and normal adjacent tissues were collected, arrayed and subjected to immunohistochemical staining with antibodies to either Aurora-A or Cyclin B1 (see Section 2). In esophageal tumor samples (c–f), Aurora-A and Cyclin B1 exhibit strong cytoplasmic staining, but in normal adjacent esophageal tissues (a and b), Aurora-A and Cyclin B1 show no positive staining. 100, 200: original magnification.
metaphase-to-anaphase transition; however, it is required for late mitotic events [20,46]. Non-degradable Cyclin B1 causes dose-dependent mitotic arrest phenotypes, including incomplete cytokinesis and aneuploidy [20]. Considering this evidence along with our present findings, we speculate that when over-expressed, Aurora-A might lead to abnormal cytokinesis through delaying (but not blocking) Cyclin B1 degradation and CDK1 inactivation. Future research is needed to define the precise mechanism through which Aurora-A delays the degradation of Cyclin B1 and disrupts cytokinesis. 5. Conflict of interest None declared.
Acknowledgements We thank Dr. Shemada of Kyoto University for providing us with KYSE esophageal carcinoma cell lines. This work is supported by funds from the 973 National Key Fundamental Research Program of China (2002 CB513101) and the National Natural Science Foundation of China (30730046 and 30721001). References [1] S. Ferrari, Protein kinases controlling the onset of mitosis, Cell. Mol. Life Sci. 63 (2006) 781–795. [2] E.A. Nigg, Mitotic kinases as regulators of cell division and its checkpoints, Nat. Rev. Mol. Cell Biol. 2 (2001) 21–32. [3] T. Marumoto, D. Zhang, H. Saya, Aurora-A—a guardian of poles, Nat. Rev. Cancer 5 (2005) 42–50.
L. Qin et al. / Cancer Letters 275 (2009) 77–85 [4] H. Zhou, J. Kuang, L. Zhong, W.L. Kuo, J.W. Gray, A. Sahin, B.R. Brinkley, S. Sen, Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation, Nat. Genet. 20 (1998) 189–193. [5] J.R. Bischoff, L. Anderson, Y. Zhu, K. Mossie, L. Ng, B. Souza, B. Schryver, P. Flanagan, F. Clairvoyant, C. Ginther, C.S. Chan, M. Novotny, D.J. Slamon, G.D. Plowman, A homologue of Drosophila aurora kinase is oncogenic and amplified in human colorectal cancers, EMBO J. 17 (1998) 3052–3065. [6] P. Meraldi, R. Honda, E.A. Nigg, Aurora-A overexpression reveals tetraploidization as a major route to centrosome amplification in p53/ cells, EMBO J. 21 (2002) 483–492. [7] X. Wang, Y.X. Zhou, W. Qiao, Y. Tominaga, M. Ouchi, T. Ouchi, C.X. Deng, Overexpression of aurora kinase A in mouse mammary epithelium induces genetic instability preceding mammary tumor formation, Oncogene 25 (2006) 7148–7158. [8] T. Marumoto, T. Hirota, T. Morisaki, N. Kunitoku, D. Zhang, Y. Ichikawa, T. Sasayama, S. Kuninaka, T. Mimori, N. Tamaki, M. Kimura, Y. Okano, H. Saya, Roles of aurora-A kinase in mitotic entry and G2 checkpoint in mammalian cells, Genes Cells 7 (2002) 1173–1182. [9] Y. Jiang, Y. Zhang, E. Lees, W. Seghezzi, AuroraA overexpression overrides the mitotic spindle checkpoint triggered by nocodazole, a microtubule destabilizer, Oncogene 22 (2003) 8293–8301. [10] S. Anand, S. Penrhyn-Lowe, A.R. Venkitaraman, AURORA-A amplification overrides the mitotic spindle assembly checkpoint, inducing resistance to Taxol, Cancer Cell 3 (2003) 51–62. [11] S.L. Warner, D.J. Bearss, H. Han, D.D. Von Hoff, Targeting Aurora-2 kinase in cancer, Mol. Cancer Ther. 2 (2003) 589–595. [12] X.X. Wang, R. Liu, S.Q. Jin, F.Y. Fan, Q.M. Zhan, Overexpression of Aurora-A kinase promotes tumor cell proliferation and inhibits apoptosis in esophageal squamous cell carcinoma cell line, Cell Res. 16 (2006) 356–366. [13] T. Evans, E.T. Rosenthal, J. Youngblom, D. Distel, T. Hunt, Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division, Cell 33 (1983) 389–396. [14] A.W. Murray, M.J. Solomon, M.W. Kirschner, The role of cyclin synthesis and degradation in the control of maturation promoting factor activity, Nature 339 (1989) 280–286. [15] P. Nurse, Universal control mechanism regulating onset of M-phase, Nature 344 (1990) 503–508. [16] R.W. King, R.J. Deshaies, J.M. Peters, M.W. Kirschner, How proteolysis drives the cell cycle, Science 274 (1996) 1652–1659. [17] M. Glotzer, A.W. Murray, M.W. Kirschner, Cyclin is degraded by the ubiquitin pathway, Nature 349 (1991) 132–138. [18] P. Clute, J. Pines, Temporal and spatial control of cyclin B1 destruction in metaphase, Nat. Cell Biol. 1 (1999) 82–87. [19] W. Zachariae, K. Nasmyth, Whose end is destruction: cell division and the anaphase-promoting complex, Genes Dev. 13 (1999) 2039– 2058. [20] F. Wolf, C. Wandke, N. Isenberg, S. Geley, Dose-dependent effects of stable cyclin B1 on progression through mitosis in human cells, EMBO J. 25 (2006) 2802–2813. [21] W.K. Kaufmann, E.N. Levedakou, H.L. Grady, R.S. Paules, G.H. Stein, Attenuation of G2 checkpoint function precedes human cell immortalization, Cancer Res. 55 (1995) 7–11. [22] M. Roh, C. Song, J. Kim, S.A. Abdulkadir, Chromosomal instability induced by Pim-1 is passage-dependent and associated with dysregulation of cyclin B1, J. Biol. Chem. 280 (2005) 40568–40577. [23] Y. Song, C. Zhao, L. Dong, M. Fu, L. Xue, Z. Huang, T. Tong, Z. Zhou, A. Chen, Z. Yang, N. Lu, Q. Zhan, Overexpression of cyclin B1 in human esophageal squamous cell carcinoma cells induces tumor cell invasive growth and metastasis, Carcinogenesis 29 (2008) 307–315. [24] J.C. Soria, S.J. Jang, F.R. Khuri, K. Hassan, D. Liu, W.K. Hong, L. Mao, Overexpression of cyclin B1 in early-stage non-small cell lung cancer and its clinical implication, Cancer Res. 60 (2000) 4000–4004. [25] M. Yu, Q. Zhan, O.J. Finn, Immune recognition of cyclin B1 as a tumor antigen is a result of its overexpression in human tumors that is caused by non-functional p53, Mol. Immunol. 38 (2002) 981–987. [26] S. Takeno, T. Noguchi, R. Kikuchi, Y. Uchida, S. Yokoyama, W. Muller, Prognostic value of cyclin B1 in patients with esophageal squamous cell carcinoma, Cancer 94 (2002) 2874–2881.
85
[27] H. Murakami, M. Furihata, Y. Ohtsuki, S. Ogoshi, Determination of the prognostic significance of cyclin B1 overexpression in patients with esophageal squamous cell carcinoma, Virchows Arch. 434 (1999) 153–158. [28] T. Nozoe, D. Korenaga, A. Kabashima, T. Ohga, H. Saeki, K. Sugimachi, Significance of cyclin B1 expression as an independent prognostic indicator of patients with squamous cell carcinoma of the esophagus, Clin. Cancer Res. 8 (2002) 817–822. [29] A. Castro, C. Bernis, S. Vigneron, J.C. Labbe, T. Lorca, The anaphasepromoting complex: a key factor in the regulation of cell cycle, Oncogene 24 (2005) 314–325. [30] J.M. Peters, The anaphase-promoting complex: proteolysis in mitosis and beyond, Mol. Cell 9 (2002) 931–943. [31] A.M. Fry, H. Yamano, APC/C-mediated degradation in early mitosis: how to avoid spindle assembly checkpoint inhibition, Cell Cycle 5 (2006) 1487–1491. [32] J.W. Harper, J.L. Burton, M.J. Solomon, The anaphase-promoting complex: it’s not just for mitosis any more, Genes Dev. 16 (2002) 2179–2206. [33] E. Eytan, Y. Moshe, I. Braunstein, A. Hershko, Roles of the anaphasepromoting complex/cyclosome and of its activator Cdc20 in functional substrate binding, Proc. Natl. Acad. Sci. USA 103 (2006) 2081–2086. [34] M. Rape, S.K. Reddy, M.W. Kirschner, The processivity of multiubiquitination by the APC determines the order of substrate degradation, Cell 124 (2006) 89–103. [35] A. de Gramont, O. Ganier, O. Cohen-Fix, Before and after the spindle assembly checkpoint—an APC/C point of view, Cell Cycle 5 (2006) 2168–2171. [36] E.R. Kramer, N. Scheuringer, A.V. Podtelejnikov, M. Mann, J.M. Peters, Mitotic regulation of the APC activator proteins CDC20 and CDH1, Mol. Biol. Cell 11 (2000) 1555–1569. [37] S. Shao, Y. Wang, S. Jin, Y. Song, X. Wang, W. Fan, Z. Zhao, M. Fu, T. Tong, L. Dong, F. Fan, N. Xu, Q. Zhan, Gadd45a interacts with auroraA and inhibits its kinase activity, J. Biol. Chem. 281 (2006) 28943– 28950. [38] Y. Wang, Q. Zhan, Cell cycle-dependent expression of centrosomal ninein-like protein in human cells is regulated by the anaphasepromoting complex, J. Biol. Chem. 282 (2007) 17712–17719. [39] C. Ginestier, E. Charafe-Jauffret, F. Bertucci, F. Eisinger, J. Geneix, D. Bechlian, N. Conte, J. Adelaide, Y. Toiron, C. Nguyen, P. Viens, M.J. Mozziconacci, R. Houlgatte, D. Birnbaum, J. Jacquemier, Distinct and complementary information provided by use of tissue and DNA microarrays in the study of breast tumor markers, Am. J. Pathol. 161 (2002) 1223–1233. [40] T. Tong, Y. Zhong, J. Kong, L. Dong, Y. Song, M. Fu, Z. Liu, M. Wang, L. Guo, S. Lu, M. Wu, Q. Zhan, Overexpression of Aurora-A contributes to malignant development of human esophageal squamous cell carcinoma, Clin. Cancer Res. 10 (2004) 7304–7310. [41] M. Cazales, E. Schmitt, E. Montembault, C. Dozier, C. Prigent, B. Ducommun, CDC25B phosphorylation by Aurora-A occurs at the G2/ M transition and is inhibited by DNA damage, Cell Cycle 4 (2005) 1233–1238. [42] C. Roghi, R. Giet, R. Uzbekov, N. Morin, I. Chartrain, R. Le Guellec, A. Couturier, M. Doree, M. Philippe, C. Prigent, The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycledependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly, J. Cell Sci. 111 (Pt. 5) (1998) 557–572. [43] M. Jackman, C. Lindon, E.A. Nigg, J. Pines, Active cyclin B1-Cdk1 first appears on centrosomes in prophase, Nat. Cell Biol. 5 (2003) 143–148. [44] T. Hirota, N. Kunitoku, T. Sasayama, T. Marumoto, D. Zhang, M. Nitta, K. Hatakeyama, H. Saya, Aurora-A and an interacting activator, the LIM protein Ajuba, are required for mitotic commitment in human cells, Cell 114 (2003) 585–598. [45] V. Horn, J. Thelu, A. Garcia, C. Albiges-Rizo, M.R. Block, J. Viallet, Functional interaction of Aurora-A and PP2A during mitosis, Mol. Biol. Cell 18 (2007) 1233–1241. [46] F.M. Yeong, H.H. Lim, C.G. Padmashree, U. Surana, Exit from mitosis in budding yeast: biphasic inactivation of the Cdc28-Clb2 mitotic kinase and the role of Cdc20, Mol. Cell 5 (2000) 501–511.
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Aurora-A interacts with Cyclin B1 and enhances its stability Lili Qin a,b, Tong Tong a, Yongmei Song a, Liyan Xue a, Feiyue Fan b, Qimin Zhan a,* a b
State Key Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300192, China
a r t i c l e
i n f o
Article history: Received 14 February 2008 Received in revised form 25 September 2008 Accepted 2 October 2008
Keywords: Cyclin B1 Aurora-A Cell cycle Protein stability Carcinogenesis
a b s t r a c t The mitotic regulator Aurora-A is an oncogenic protein that is over-expressed in many types of human tumors. However, the underlying mechanism through which Aurora-A promotes tumorigenesis remains unclear. Here, we show that overexpression of Aurora-A causes an elevation of Cyclin B1 expression. Cyclin B1 degradation is delayed in AuroraA over-expressing cells, which depends on Aurora-A kinase activity. In contrast, AuroraA RNAi enhances Cyclin B1 degradation. Furthermore, we found that Aurora-A interacts with Cyclin B1, and that Aurora-A overexpression reduces the interaction of Cyclin B1 with APC subunits. In human esophageal squamous cell carcinomas (ESCC), overexpression of Aurora-A was correlated with deregulated expression of Cyclin B1. Taken together, these findings suggest that overexpression of Aurora-A may stabilize Cyclin B1 through inhibiting its degradation. These results provide new insight into the mechanism of how deregulated Aurora-A contributes to genomic instability and carcinogenesis. Ó 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The eukaryotic cell cycle is a complex process tightly controlled by many regulators, and aberrations in the cell cycle may lead to chromosomal instability, the hallmark of most human malignancies. Several protein kinases control cell cycle progression, such as Aurora kinases and CDKs/Cyclins kinases. Abnormal regulation or expression of these kinases may result in genomic instability and subsequently carcinogenesis [1,2]. Aurora-A kinase belongs to a serine/threonine kinase family important for proper execution of various mitotic events and for maintaining genomic integrity [3]. AuroraA participates in several crucial mitotic events, including centrosome maturation and separation, bipolar spindle assembly, chromosome alignment and segregation, and cytokinesis. Overexpression of Aurora-A has been shown to lead to centrosome amplification and aneuploidy, which usually results from incomplete cytokinesis and can be a driving cause of genomic instability and tumorigenesis * Corresponding author. Tel.: +86 10 87788422; fax: +86 10 67715058. E-mail address: [email protected] (Q. Zhan). 0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.10.011
[4–7]. Aurora-A has also been implicated in regulation of cell cycle checkpoints. Overexpression of Aurora-A was shown to abrogate the G2/M checkpoint and spindle assembly checkpoint, allowing cells to inappropriately enter into mitosis and anaphase with damaged DNA and defective spindles. These checkpoint defects may ultimately contribute to genomic instability and carcinogenesis [8–10]. The human Aurora-A gene is located at chromosome 20q13.2, a region frequently amplified and over-expressed in a variety of human tumors and cancerderived cell lines [3,6,11]. Recent studies have shown that overexpression of Aurora-A in cultured cells induces several cancer-related phenotypes, including increased cell proliferation and colony formation and inhibition of UVor cisplatin-induced apoptosis [12]. Overexpression of Aurora-A can also transform rat-1 and NIH3T3 cells and form tumors in null mice [5,7]. Collectively, this evidence indicates that Aurora-A acts as an oncogene and plays an important role in cell cycle progression and carcinogenesis. Cyclin B was the first mitotic Cyclin to be discovered. It accumulates during interphase and is rapidly degraded as cells exit from mitosis [13]. Cyclin B1 functions mainly during late G2 phase and mitosis as the regulatory subunit
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of CDK1 [14]. Previous studies have shown that accumulation and nuclear translocation of Cyclin B1 at the G2/M transition is required for CDK1 activation and mitotic entry, and that Cyclin B1 degradation is essential to inactivation of CDK1 and exit from mitosis [15,16]. Cyclin B1 is degraded by the APC/C-mediated ubiquitination pathway [17], and both CDC20 and CDH1 are involved in its degradation [18,19]. Stable Cyclin B1 causes dosedependent mitotic arrest phenotypes, suggesting that different thresholds of Cyclin B1/Cdk1 activity are required for the metaphase-to-anaphase and the anaphaseto-telophase transition, and that gradual degradation of Cyclin B1 is indispensable for late mitotic events [20]. Several lines of evidence have demonstrated that elevated levels of Cyclin B1 result in decreased G2 checkpoint function and contribute to the immortalization of human cells [21]. Overexpression of Cyclin B1 can also impair the mitotic spindle checkpoint and induce chromosome instability and aneuploidy, caused by incomplete cytokinesis [22]. Cyclin B1 has been shown to be over-expressed in many types of human tumors [23–25]. Moreover, the level of Cyclin B1 expression has been positively correlated with malignancy and poor prognosis in ESCC [26–28]. Ubiquitin-mediated proteolysis plays a crucial role in cell cycle progression in eukaryotes. APC/C is a multi-subunit ubiquitin ligase that catalyzes the ubiquitination of specific substrates and targets them for degradation by the 26S proteasome [29]. In vertebrates, APC/C is composed of at least 13 subunits, including APC2, which is a structural component of the core of APC/C, and CDC27, which regulates activation of APC/C by association with CDC20 and CDH1[29]. APC/C-mediated proteolysis helps trigger the onset of anaphase and facilitates mitotic exit by targeting the anaphase inhibitor and mitotic cyclins for proteolysis. Also, APC/C helps establish and maintain the G1 state by degrading G1 proteins until conditions are appropriate to begin another round of cell division [30–32]. CDC20 and CDH1 act as activators of APC/C, and they help determine the substrate specificity of APC/C as well [33,34]. APC/C is also controlled by a regulatory mechanism called the spindle assembly checkpoint, which monitors kinetochore-spindle microtubules interactions. This checkpoint prevents APC/C activation until all chromosomes are properly attached, to ensure that all chromosomes segregate faithfully [35]. In addition, phosphorylation plays an important role in controlling the timing of APC activation [36]. In this study, we show that Aurora-A overexpression causes upregulation of Cyclin B1 and delays the degradation of Cyclin B1, in a manner dependent on Aurora-A kinase activity. Further, Aurora-A RNAi enhances Cyclin B1 degradation, and Aurora-A overexpression reduces the interaction between Cyclin B1 and APC/C. In ESCC we show that both Aurora-A and Cyclin B1 proteins are over-expressed, and that the expression pattern of these two proteins is correlated. Collectively, these results suggest that Aurora-A and Cyclin B1 cooperate to regulate the cell cycle, and defects in the regulation of these proteins contribute to genomic instability and tumorigenesis.
2. Materials and methods 2.1. Cell culture, synchronization and drug treatment Human ESCC KYSE150 and ESCC EC9706 cell lines (gifts from Dr. Shimada, Kyoto University) were cultured in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin, and 100 lg/ml streptomycin at 37 °C. pEGFP-Aurora-A transfected KYSE150 cells and Aurora-A siRNA transfected EC9706 cells were cultured in the presence of 400 lg/ml of G418 (Geneticin sulfate, GIBCO). For stability analysis of protein, 100 lg/ml CHX (cycloheximide) (Sigma–Aldrich Corp., St. Louis, MO) was added to cell culture, and then cells were harvested at the indicated time points. The Aurora-A kinase inhibitor Cyclopropanecarboxylic acid-(3-(4-(3-trifluoromethyl-phenylamino)pyrimidin-2-ylamino)-phenyl)-amide (Merck Calbiochem Inc. Darmstadt, Germany. 189405, dissolved in DMSO and stored at 50 mg/ml at 20 °C), was used at 1 lM for 2 h prior to treatment with CHX. 2.2. RNA isolation and RT-PCR analysis Total RNA was purified and reverse transcription was carried out at 42 °C for 90 min using SuperScript II (Invitrogen Technologies Inc.). The cDNA was used for PCR amplification. Aurora-A PCR was performed for 35 cycles, at an annealing temperature of 60 °C (30 s) using primers: 50 AAT GAT TGA AGG TCG GAT GC-30 (forward) and 50 -TTC TCT GAG CAT TGG CCT CT-30 (reverse). Cyclin B1 amplification was carried out for 31 cycles, at an annealing temperature of 58 °C for 30 s. Primers for Cyclin B1 were: 50 -TCC AAG CCC AAT GGA AAC AT-30 (forward); 50 -ATG CTC TCC GAA GGA AGT GC-30 (reverse). PCR products were analyzed on 1% agarose gels stained with ethidium bromide and visualized with a UV transilluminator. 2.3. Antibodies Antibody against Cyclin B1 was from BD Biotechnology (BD Pharmingen 554176). Anti-Aurora-A antibody was from Cell Signaling (Cell Signaling, Beverly, MA, 3092). Antibodies to APC2 (Santa Cruz Biotechnology sc-20984), CDC27 (Santa Cruz Biotechnology sc-5618), CDC20 (Santa Cruz Biotechnology sc-8358), Ubiquitin (Santa Cruz Biotechnology sc-9133), Plk1 (Santa Cruz Biotechnology sc5585), Rb (Santa Cruz Biotechnology sc-50), b-catenin (Santa Cruz Biotechnology sc-7963), E-cadherin (Santa Cruz Biotechnology sc-7870) and b-actin (Santa Cruz Biotechnology sc-8432) were obtained from Santa Cruz Technology. Antibodies to CDH1 (Sigma, St. Louis, MO c-7855) were purchased from Sigma. 2.4. Cell cycle analysis For flow cytometry analysis, cells were seeded and grown. Then cells were trypsinized, washed twice with pre-chilled PBS and fixed in 70% ice-cold ethanol. After incubation at 20 °C for at least 2 h, cells were washed with PBS twice and re-suspended in 1 ml of PBS containing
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40 lg/ml propidium iodide and 100 lg/ml RNase A. The suspension was then used for flow cytometry analysis.
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Cells were harvested, rinsed with PBS twice, and lysed in ice-cold lysis buffer on ice for 40 min. Lysate was collected by centrifugation at 12,000g at 4 °C for 20 min. 80 lg of protein was subjected to 10% SDS–PAGE. Immunoblotting was done as described before [37]. For immunoprecipitation, the total lysate containing 1 mg of protein was incubated with the indicated antibodies at room temperature for 2 h. Protein A/G-agarose beads (Santa Cruz Technology Inc.) were then added and incubated overnight at 4 °C. After washed six times with lysis buffer, the immuno-complex was boiled and subjected to SDS–PAGE, followed by immunoblotting assay.
transfected with an empty pEGFP vector (Fig. 1A). This result demonstrates that when over-expressed, Aurora-A can induce upregulation of Cyclin B1 protein. In addition to Cyclin B1, the levels of Plk1 and b-catenin were also upregulated by Aurora-A overexpression. Conversely, the level of Rb appeared to decrease, and E-cadherin level was not affected by Aurora-A overexpression. These results suggest that Aurora-A overexpression may affect the levels of other oncogenes or tumor suppressor genes. To determine whether the high level of Cyclin B1 in Aurora-A over-expressing cells is a result of G2/M arrest, we performed flow cytometry to examine cell cycle distributions. As shown in Fig. 1B, Aurora-A over-expressing cells did not appear to accumulate in G2 phase in greater numbers than control cells, which is consistent with previous results [41]. This result demonstrates that the increased level of Cyclin B1 protein in Aurora-A over-expressing cells is not caused by a delay in the G2/M transition. Additionally, the level of Cyclin B1 mRNA was examined via semi-quantitative RT-PCR, and there was no evidence of an increase in Cyclin B1 mRNA level following Aurora-A overexpression (data not shown). This result suggests that accumulation of cyclin B1 in Aurora-A over-expressing cells is not due to regulation at the level of transcription but may occur at the post-transcriptional level.
2.6. Pull-down assays
3.2. Overexpression of Aurora-A delays the degradation of Cyclin B1
Expression and purification of recombinant GST fusion proteins was carried out as previously described [38]. For GST-pull-down assay, the total lysate containing 2 mg of protein was incubated with GST, GST-Aurora-A and GSTCyclin B1 bound to glutathione Sepharose overnight at 4 °C. After washed six times with lysis buffer, samples were boiled and then subjected to SDS–PAGE and immunoblotting analysis.
We next investigated whether upregulation of Cyclin B1 protein occurred as a result of decreased protein degradation. We examined the stability of Cyclin B1 protein in both Aurora-A over-expressing and control cells treated with CHX. In control cells, the amount of Cyclin B1 protein dramatically decreased by 50% at 0.5 h, and disappeared at about 2 h after treatment with CHX. However, in Aurora-A over-expressing cells, the level of Cyclin B1 was reduced by about 50% at 1.5 h, and disappeared at about 6 h after treatment with CHX. This result suggests that Aurora-A overexpression might inhibit the degradation of Cyclin B1 (Fig. 2A). Additionally, we found that Aurora-A degradation occurs later and more slowly than Cyclin B1 degradation, although both proteins are degraded through the APC/C-mediated pathway. To investigate whether the delay in Cyclin B1 degradation in Aurora-A over-expressing cells depends on Aurora-A kinase activity, we treated cells with an Aurora-A kinase inhibitor for 2 h prior to CHX treatment. Western blot analysis demonstrated that when Aurora-A kinase activity is inhibited, Cyclin B1 degradation is not delayed, suggesting that Aurora-A kinase activity is needed to regulate Cyclin B1 degradation (Fig. 2C).
2.5. Cell lysis, immunoblotting and immunoprecipitation
2.7. Patients, clinical tissue sample collection and tissue microarray immunohistochemical analysis Fresh tumor tissues (173) were obtained from patients with pathologically and clinically confirmed ESCC. The institutional Review Board approved use of the tumor specimens in this study and clinical consultation reports were available for all tumor samples. TMAs were prepared as described [39] with slight modifications. Core cylinders with a diameter of 0.6 mm were punched and deposited into a recipient paraffin block. Four-lm sections of the resulting microarray block were transferred to glass slides. Then, antibodies against Aurora-A and Cyclin B1 were used for IHC analysis. Immunohistochemical staining and semiquantitative evaluation of staining was performed as previously described [40]. Aurora-A and Cyclin B1 expression was analyzed using the v2-Test with SPSS software. Statistical significance was considered at the value of P < 0.05. 3. Results 3.1. Overexpression of Aurora-A induces upregulation of Cyclin B1 expression Our previous studies indicated that Aurora-A is over-expressed in human ESCC, and that the overexpression of Aurora-A is associated with increased malignancy and poor prognosis of patients [40]. Expression of exogenous Aurora-A promotes cell proliferation, stimulates colony formation, and inhibits apoptosis in ESCC KYSE150 cells [12]. To investigate the underlying mechanism through which Aurora-A promotes tumorigenesis, we first examined the expression levels of different oncogenic proteins by Western analysis. We found that Cyclin B1 was upregulated in KYSE150 cells that had been stably transfected with pEGFP-Aurora-A, but was expressed at relatively lower levels in untransfected KYSE150 cells (which express low levels of endogenous Aurora-A kinase), and in KYSE150 cells
3.3. Aurora-A RNAi enhances the degradation of Cyclin B1 To further study the role of Aurora-A in Cyclin B1 degradation, we examined whether downregulation of Aurora-A results in an increased amount of Cyclin B1 degradation in EC9706 cells. EC9706 cells have relatively higher levels of endogenous Aurora-A expression than KYSE150 cells, and in EC9706 ESCC cells, the half-life of Cyclin B1 is longer than in KYSE150 cells. As shown in Fig. 3A, Aurora-A protein levels were dramatically reduced in EC9706 cells following treatment with siRNA targeting Aurora-A. We observed that downregulation of Aurora-A resulted in increased degradation of Cyclin B1 (Fig. 3B). Taken together with the result described above, these data show that Aurora-A helps regulate the stability of Cyclin B1 protein. 3.4. Cyclin B1 ubiquitination and interactions with APC/C subunits are affected by Aurora-A overexpression It has been reported previously that Cyclin B1 is degraded by the APC/C-mediated ubiquitin pathway, and that CDC20 and CDH1 are involved in this process. Therefore, we hypothesized that Aurora-A may inhibit Cyclin B1 degradation by disrupting interactions between Cyclin B1 and proteins involved in its degradation. We performed immunoprecipitation experiments using Cyclin B1 antibody to investigate this hypothesis. As expected, the level of ubiquitinated Cyclin B1 was lower in Aurora-A over-expressing cells than that in control cells (shown in Fig. 4A). Additionally, reduced amounts of APC2, CDC27, CDC20 and CDH1 were detected in Cyclin B1 immuno-complexes from Aurora-A over-expressing cells compared to control cells. However, the levels of these proteins in total lysate from Aurora-A transfected cells were higher than in control cells (shown in Fig. 4B). These results suggest
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Fig. 1. Aurora-A overexpression induces upregulation of Cyclin B1 in ESCC KYSE150 cells. (A) Protein expression was analyzed by western blot with indicated antibodies. Genes that were upregulated, down-regulated or not affected by Aurora-A overexpression are shown. (B) Flow cytometry analysis of cell cycle distributions. The results are expressed as the percentage of cells at G1/S or G2/M phase. The data is representative of three independent experiments. that the interactions between Cyclin B1 and degradation-associated proteins are weakened by increased levels of Aurora-A, which results in an inhibition of ubiquitin-mediated degradation of Cyclin B1. 3.5. Aurora-A interacts with Cyclin B1 Previous studies have shown that Aurora-A and Cyclin B1 are both centrosome-associated proteins, and that centrosomal localization of Aurora-A is required for Cyclin B1 localization and for activation of the Cyclin B1/CDK1 complex [42–44]. Therefore, it is possible that both proteins are present in the same complex. To investigate this, immunoprecipitation assays were carried out using antibodies against Aurora-A and Cyclin B1. As shown in Fig. 5A, both Aurora-A and Cyclin B1 were detected in Aurora-A-immunoprecipitated complex. Similarly, these two proteins were observed in Cyclin B1-immunoprecipitated complex. In contrast, neither Aurora-A nor Cyclin B1 was detected in precipitates obtained with b-actin antibody. To further confirm the interaction between Aurora-A and Cyclin B1, we performed an in vitro binding assay using purified GST-tagged proteins. We expressed and purified GST, GST-Aurora-A, and GST-Cyclin B1 fusion proteins in Escherichia coli, and performed pull-down assays. As shown in Fig. 5B, both Aurora-A and Cyclin B1 were found in protein complexes obtained from GST-Cyclin B1 and GST-Aurora-A pull-down assays, but not from GST pull-down assays. Taken together, these results indicate that Aurora-A interacts with Cyclin B1 directly or indirectly. 3.6. Expression relevance of Aurora-A and Cyclin B1 in clinical ESCC Previous studies have shown that Aurora-A and Cyclin B1 proteins are over-expressed in many types of human tumors. We used immunohistochemical assays to examine the expression of these two proteins simultaneously in clinical tumor specimens from 173 ESCC patients, to further assess the relevance of this overexpression. The immunohistochemical staining results are summarized in Table 1. Consistent with previous findings [23,40], we found that both Aurora-A and Cyclin B1 are over-expressed in ESCC (in Fig. 6). Aurora-A overexpression was detected in 119 of 173 samples (68.8%), and Cyclin B1 overexpression was detected in 124 of 173 samples (71.7%). v2-Test analysis using SPSS software was performed, and we found there was a positive correlation between Aurora-A expression and Cyclin B1 expression in human ESCC (P < 0.05).
4. Discussion Proper cell cycle progression is essential to maintain genomic integrity. Aurora-A and Cyclin B1 are two crucial regulators involved in cell cycle regulation, and they function mainly in mitosis. Multiple lines of evidence indicate that Aurora-A and Cyclin B1 are over-expressed in a variety of human tumors, and increased expression of Aurora-A and Cyclin B1 has been correlated with malignant behavior of tumors [23,40]. We previously reported that Aurora-A overexpression may induce malignant transformation of a human ESCC cell line [12]. However, the exact mechanism(s) through which Aurora-A overexpression promotes the development and progression of cancer remains unclear. Here we provided new insight into the molecular mechanism through which Aurora-A may promote carcinogenesis. First, we demonstrated that the protein level of Cyclin B1 is increased when Aurora-A is over-expressed in ESCC KYSE150 cells. Flow cytometry analysis revealed that upregulation of Cyclin B1 is not caused by a delay in the G2/M transition in Aurora-A over-expressing cells, and we found no evidence that Aurora-A regulates Cyclin B1 at the level of transcription. Based on this evidence, we hypothesized that upregulation of Cyclin B1 in Aurora-A over-expressing cells is probably due to regulation at the post-transcriptional level. Additionally, we demonstrated that the expression of some other cancer-associated proteins, such as PLK1 and b-catenin, is induced in Aurora-A over-expressing cells, suggesting that Aurora-A plays an oncogenic role through regulation of these proteins as well. Interestingly, we found that when Aurora-A is over-expressed, the half-life of Cyclin B1 is increased, and the degradation of Cyclin B1 is delayed. The delay of Cyclin B1
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Fig. 2. (A) Overexpression of Aurora-A delays the degradation of Cyclin B1. Cells were treated with CHX and then harvested at the indicated time points. Cyclin B1 protein level at each time point was determined by Western Blot. (B) Effects of Cyclopropanecarboxylic acid-(3-(4-(3-trifluoromethylphenylamino)-pyrimidin-2-ylamino)-phenyl)-amide on Aurora kinase biomarkers. Cells were exposed to the indicated concentration of Aurora-A kinase inhibitor for 2 h. Western Blotting was performed with antibodies recognizing pThr288 on Aurora-A, or total Aurora-A. (C) The effect of Aurora-A overexpression on Cyclin B1 degradation depends on the kinase activity of Aurora-A. Cells were exposed to Aurora-A kinase inhibitor for 2 h prior to treatment with CHX. Cells were harvested and analyzed by Western blotting. (D) To quantitatively evaluate the effect of Aurora-A on Cyclin B1 degradation, Cyclin B1 protein levels were quantified using QuantityOne software and normalized to b-actin level.
degradation in Aurora-A over-expressing cells was found to be dependent on the kinase activity of Aurora-A. In line with these results, Aurora-A RNAi enhanced the degradation of Cyclin B1. Furthermore, Aurora-A over-expressing cells had lower level of ubiquitinated Cyclin B1 compared with control cells, and the interactions between Cyclin B1 and proteins involved in Cyclin B1 ubiquitin-mediated destruction (such as APC2, CDC27, CDC20 and CDH1) were much weaker. In addition, we demonstrated an interaction between Aurora-A and Cyclin B1 in vivo and in vitro. Taken together, these results suggest that overexpression of Aurora-A delays Cyclin B1 degradation through disrupting the interactions between Cyclin B1 and the APC/C complex.
Aurora-A may interfere with the ubiquitin-mediated degradation of Cyclin B1 in two different ways. First, the physical interaction of Aurora-A and Cyclin B1 may alter the conformation of Cyclin B1, which could inhibit the ability of APC/CCDC20 and APC/CCDH1 to recognize and bind to Cyclin B1. Second, as a protein kinase, Aurora-A could regulate Cyclin B1 degradation through phosphorylation. Previous work has shown that phosphorylation may regulate proteolysis; for example, Aurora-A degradation can be inhibited by phosphorylation of serine 51 in the A box [45]. In addition to observations that Aurora-A interacts with and stabilizes Cyclin B1, findings from clinical ESCC
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Fig. 3. Aurora-A siRNA transfection enhances the degradation of Cyclin B1. (A) Aurora-A RNAi down-regulates Aurora-A and Cyclin B1 expression. (B) The degradation of Cyclin B1 is enhanced by Aurora-A RNAi.
Fig. 4. Effects of Aurora-A overexpression on ubiquitination of Cyclin B1. Cells were grown, harvested and lysed. Cell lysate (1 mg) was immunoprecipitated with anti-Cyclin B1 antibody, followed by immunoblotting using antibodies against ubiquitin, APC2, CDC27, CDC20 and CDH1, Cyclin B1 and b-actin. Immunoprecipitation with anti-b-actin antibody was used as a negative control. (A) The level of ubiquitinated-Cyclin B1 is decreased in Aurora-A overexpressing cells. (B) The interaction between Cyclin B1 and APC/C complexes is weaker in Aurora-A over-expressing cells. The asterisk marks the immunoglobin heavy chains, and the upper bands shown in the cyclin B1 IP correspond to cyclin B1. Ubi: ubiquitin.
samples indicate that expression of these two proteins may be correlated. Although the expression of both Aurora-A and Cyclin B1 is deregulated in human ESCC, we
demonstrated a positive correlation in expression profile between them. Thus, Aurora-A may cooperate with Cyclin B1 to induce cell transformation and carcinogenesis.
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Fig. 5. Interaction between Aurora-A and Cyclin B1. (A) In vivo interaction of Aurora-A and Cyclin B1. Immunoprecipitation of Aurora-A and Cyclin B1 from KYSE150/GFP-Aur-A cell lysates was carried out as described in Section 2 using antibodies against Aurora-A and Cyclin B1. Immunoblotting was performed, using b-actin as a negative control. (B) In vitro interaction between Aurora-A and Cyclin B1. GST, GST-Aurora-A and GST-Cyclin B1 fusion proteins were expressed in E. coli and purified using glutathione agarose beads. Pull-down assays were performed as described in Section 2 using whole-cell extracts of KYSE150/GFP-Aur-A cells. Samples were analyzed by Western blot. GST was used as a negative control. Table 1 The expression correlation of Aurora-A and Cyclin B1. CyclinB1 expression
Aurora-A expression () or (±) (+) (++) *
P value
() or (±)
(+)
(++)
16 16 7
24 10 19
14 28 29
0.038*
3 3 Pearson v2-Test.
Multiple lines of evidence have demonstrated a connection between aneuploidy and genomic instability, cell malignant transformation and tumorigenesis. Aneuploidy
is thought to be caused by abortive cytokinesis, due to deregulation of mitotic regulator proteins. Previous experiments have shown that Aurora-A overexpression leads to premature anaphase entry, which subsequently results in centrosome amplification and aneuploidy due to incomplete cytokinesis [10]. However, up to now, no evidence has demonstrated a direct role for Aurora-A in cytokinesis. As the regulatory subunit of CDK1, Cyclin B1’s scheduled degradation plays a crucial role in late mitotic events, such as mitotic exit and completion of cytokinesis [20,22]. Although CyclinB1/Cdk1 kinase activity is required for inhibiting separase activity before cells enter anaphase, CyclinB1 degradation is not thought to be necessary for the
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Fig. 6. The expression of Aurora-A and Cyclin B1 in ESCC and normal adjacent tissues studied with IHC on tissue microarrays. 173 ESCC samples and normal adjacent tissues were collected, arrayed and subjected to immunohistochemical staining with antibodies to either Aurora-A or Cyclin B1 (see Section 2). In esophageal tumor samples (c–f), Aurora-A and Cyclin B1 exhibit strong cytoplasmic staining, but in normal adjacent esophageal tissues (a and b), Aurora-A and Cyclin B1 show no positive staining. 100, 200: original magnification.
metaphase-to-anaphase transition; however, it is required for late mitotic events [20,46]. Non-degradable Cyclin B1 causes dose-dependent mitotic arrest phenotypes, including incomplete cytokinesis and aneuploidy [20]. Considering this evidence along with our present findings, we speculate that when over-expressed, Aurora-A might lead to abnormal cytokinesis through delaying (but not blocking) Cyclin B1 degradation and CDK1 inactivation. Future research is needed to define the precise mechanism through which Aurora-A delays the degradation of Cyclin B1 and disrupts cytokinesis. 5. Conflict of interest None declared.
Acknowledgements We thank Dr. Shemada of Kyoto University for providing us with KYSE esophageal carcinoma cell lines. This work is supported by funds from the 973 National Key Fundamental Research Program of China (2002 CB513101) and the National Natural Science Foundation of China (30730046 and 30721001). References [1] S. Ferrari, Protein kinases controlling the onset of mitosis, Cell. Mol. Life Sci. 63 (2006) 781–795. [2] E.A. Nigg, Mitotic kinases as regulators of cell division and its checkpoints, Nat. Rev. Mol. Cell Biol. 2 (2001) 21–32. [3] T. Marumoto, D. Zhang, H. Saya, Aurora-A—a guardian of poles, Nat. Rev. Cancer 5 (2005) 42–50.
L. Qin et al. / Cancer Letters 275 (2009) 77–85 [4] H. Zhou, J. Kuang, L. Zhong, W.L. Kuo, J.W. Gray, A. Sahin, B.R. Brinkley, S. Sen, Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation, Nat. Genet. 20 (1998) 189–193. [5] J.R. Bischoff, L. Anderson, Y. Zhu, K. Mossie, L. Ng, B. Souza, B. Schryver, P. Flanagan, F. Clairvoyant, C. Ginther, C.S. Chan, M. Novotny, D.J. Slamon, G.D. Plowman, A homologue of Drosophila aurora kinase is oncogenic and amplified in human colorectal cancers, EMBO J. 17 (1998) 3052–3065. [6] P. Meraldi, R. Honda, E.A. Nigg, Aurora-A overexpression reveals tetraploidization as a major route to centrosome amplification in p53/ cells, EMBO J. 21 (2002) 483–492. [7] X. Wang, Y.X. Zhou, W. Qiao, Y. Tominaga, M. Ouchi, T. Ouchi, C.X. Deng, Overexpression of aurora kinase A in mouse mammary epithelium induces genetic instability preceding mammary tumor formation, Oncogene 25 (2006) 7148–7158. [8] T. Marumoto, T. Hirota, T. Morisaki, N. Kunitoku, D. Zhang, Y. Ichikawa, T. Sasayama, S. Kuninaka, T. Mimori, N. Tamaki, M. Kimura, Y. Okano, H. Saya, Roles of aurora-A kinase in mitotic entry and G2 checkpoint in mammalian cells, Genes Cells 7 (2002) 1173–1182. [9] Y. Jiang, Y. Zhang, E. Lees, W. Seghezzi, AuroraA overexpression overrides the mitotic spindle checkpoint triggered by nocodazole, a microtubule destabilizer, Oncogene 22 (2003) 8293–8301. [10] S. Anand, S. Penrhyn-Lowe, A.R. Venkitaraman, AURORA-A amplification overrides the mitotic spindle assembly checkpoint, inducing resistance to Taxol, Cancer Cell 3 (2003) 51–62. [11] S.L. Warner, D.J. Bearss, H. Han, D.D. Von Hoff, Targeting Aurora-2 kinase in cancer, Mol. Cancer Ther. 2 (2003) 589–595. [12] X.X. Wang, R. Liu, S.Q. Jin, F.Y. Fan, Q.M. Zhan, Overexpression of Aurora-A kinase promotes tumor cell proliferation and inhibits apoptosis in esophageal squamous cell carcinoma cell line, Cell Res. 16 (2006) 356–366. [13] T. Evans, E.T. Rosenthal, J. Youngblom, D. Distel, T. Hunt, Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division, Cell 33 (1983) 389–396. [14] A.W. Murray, M.J. Solomon, M.W. Kirschner, The role of cyclin synthesis and degradation in the control of maturation promoting factor activity, Nature 339 (1989) 280–286. [15] P. Nurse, Universal control mechanism regulating onset of M-phase, Nature 344 (1990) 503–508. [16] R.W. King, R.J. Deshaies, J.M. Peters, M.W. Kirschner, How proteolysis drives the cell cycle, Science 274 (1996) 1652–1659. [17] M. Glotzer, A.W. Murray, M.W. Kirschner, Cyclin is degraded by the ubiquitin pathway, Nature 349 (1991) 132–138. [18] P. Clute, J. Pines, Temporal and spatial control of cyclin B1 destruction in metaphase, Nat. Cell Biol. 1 (1999) 82–87. [19] W. Zachariae, K. Nasmyth, Whose end is destruction: cell division and the anaphase-promoting complex, Genes Dev. 13 (1999) 2039– 2058. [20] F. Wolf, C. Wandke, N. Isenberg, S. Geley, Dose-dependent effects of stable cyclin B1 on progression through mitosis in human cells, EMBO J. 25 (2006) 2802–2813. [21] W.K. Kaufmann, E.N. Levedakou, H.L. Grady, R.S. Paules, G.H. Stein, Attenuation of G2 checkpoint function precedes human cell immortalization, Cancer Res. 55 (1995) 7–11. [22] M. Roh, C. Song, J. Kim, S.A. Abdulkadir, Chromosomal instability induced by Pim-1 is passage-dependent and associated with dysregulation of cyclin B1, J. Biol. Chem. 280 (2005) 40568–40577. [23] Y. Song, C. Zhao, L. Dong, M. Fu, L. Xue, Z. Huang, T. Tong, Z. Zhou, A. Chen, Z. Yang, N. Lu, Q. Zhan, Overexpression of cyclin B1 in human esophageal squamous cell carcinoma cells induces tumor cell invasive growth and metastasis, Carcinogenesis 29 (2008) 307–315. [24] J.C. Soria, S.J. Jang, F.R. Khuri, K. Hassan, D. Liu, W.K. Hong, L. Mao, Overexpression of cyclin B1 in early-stage non-small cell lung cancer and its clinical implication, Cancer Res. 60 (2000) 4000–4004. [25] M. Yu, Q. Zhan, O.J. Finn, Immune recognition of cyclin B1 as a tumor antigen is a result of its overexpression in human tumors that is caused by non-functional p53, Mol. Immunol. 38 (2002) 981–987. [26] S. Takeno, T. Noguchi, R. Kikuchi, Y. Uchida, S. Yokoyama, W. Muller, Prognostic value of cyclin B1 in patients with esophageal squamous cell carcinoma, Cancer 94 (2002) 2874–2881.
85
[27] H. Murakami, M. Furihata, Y. Ohtsuki, S. Ogoshi, Determination of the prognostic significance of cyclin B1 overexpression in patients with esophageal squamous cell carcinoma, Virchows Arch. 434 (1999) 153–158. [28] T. Nozoe, D. Korenaga, A. Kabashima, T. Ohga, H. Saeki, K. Sugimachi, Significance of cyclin B1 expression as an independent prognostic indicator of patients with squamous cell carcinoma of the esophagus, Clin. Cancer Res. 8 (2002) 817–822. [29] A. Castro, C. Bernis, S. Vigneron, J.C. Labbe, T. Lorca, The anaphasepromoting complex: a key factor in the regulation of cell cycle, Oncogene 24 (2005) 314–325. [30] J.M. Peters, The anaphase-promoting complex: proteolysis in mitosis and beyond, Mol. Cell 9 (2002) 931–943. [31] A.M. Fry, H. Yamano, APC/C-mediated degradation in early mitosis: how to avoid spindle assembly checkpoint inhibition, Cell Cycle 5 (2006) 1487–1491. [32] J.W. Harper, J.L. Burton, M.J. Solomon, The anaphase-promoting complex: it’s not just for mitosis any more, Genes Dev. 16 (2002) 2179–2206. [33] E. Eytan, Y. Moshe, I. Braunstein, A. Hershko, Roles of the anaphasepromoting complex/cyclosome and of its activator Cdc20 in functional substrate binding, Proc. Natl. Acad. Sci. USA 103 (2006) 2081–2086. [34] M. Rape, S.K. Reddy, M.W. Kirschner, The processivity of multiubiquitination by the APC determines the order of substrate degradation, Cell 124 (2006) 89–103. [35] A. de Gramont, O. Ganier, O. Cohen-Fix, Before and after the spindle assembly checkpoint—an APC/C point of view, Cell Cycle 5 (2006) 2168–2171. [36] E.R. Kramer, N. Scheuringer, A.V. Podtelejnikov, M. Mann, J.M. Peters, Mitotic regulation of the APC activator proteins CDC20 and CDH1, Mol. Biol. Cell 11 (2000) 1555–1569. [37] S. Shao, Y. Wang, S. Jin, Y. Song, X. Wang, W. Fan, Z. Zhao, M. Fu, T. Tong, L. Dong, F. Fan, N. Xu, Q. Zhan, Gadd45a interacts with auroraA and inhibits its kinase activity, J. Biol. Chem. 281 (2006) 28943– 28950. [38] Y. Wang, Q. Zhan, Cell cycle-dependent expression of centrosomal ninein-like protein in human cells is regulated by the anaphasepromoting complex, J. Biol. Chem. 282 (2007) 17712–17719. [39] C. Ginestier, E. Charafe-Jauffret, F. Bertucci, F. Eisinger, J. Geneix, D. Bechlian, N. Conte, J. Adelaide, Y. Toiron, C. Nguyen, P. Viens, M.J. Mozziconacci, R. Houlgatte, D. Birnbaum, J. Jacquemier, Distinct and complementary information provided by use of tissue and DNA microarrays in the study of breast tumor markers, Am. J. Pathol. 161 (2002) 1223–1233. [40] T. Tong, Y. Zhong, J. Kong, L. Dong, Y. Song, M. Fu, Z. Liu, M. Wang, L. Guo, S. Lu, M. Wu, Q. Zhan, Overexpression of Aurora-A contributes to malignant development of human esophageal squamous cell carcinoma, Clin. Cancer Res. 10 (2004) 7304–7310. [41] M. Cazales, E. Schmitt, E. Montembault, C. Dozier, C. Prigent, B. Ducommun, CDC25B phosphorylation by Aurora-A occurs at the G2/ M transition and is inhibited by DNA damage, Cell Cycle 4 (2005) 1233–1238. [42] C. Roghi, R. Giet, R. Uzbekov, N. Morin, I. Chartrain, R. Le Guellec, A. Couturier, M. Doree, M. Philippe, C. Prigent, The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycledependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly, J. Cell Sci. 111 (Pt. 5) (1998) 557–572. [43] M. Jackman, C. Lindon, E.A. Nigg, J. Pines, Active cyclin B1-Cdk1 first appears on centrosomes in prophase, Nat. Cell Biol. 5 (2003) 143–148. [44] T. Hirota, N. Kunitoku, T. Sasayama, T. Marumoto, D. Zhang, M. Nitta, K. Hatakeyama, H. Saya, Aurora-A and an interacting activator, the LIM protein Ajuba, are required for mitotic commitment in human cells, Cell 114 (2003) 585–598. [45] V. Horn, J. Thelu, A. Garcia, C. Albiges-Rizo, M.R. Block, J. Viallet, Functional interaction of Aurora-A and PP2A during mitosis, Mol. Biol. Cell 18 (2007) 1233–1241. [46] F.M. Yeong, H.H. Lim, C.G. Padmashree, U. Surana, Exit from mitosis in budding yeast: biphasic inactivation of the Cdc28-Clb2 mitotic kinase and the role of Cdc20, Mol. Cell 5 (2000) 501–511.