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Dipartimenti di Biologia e Patologia Cellulare e Molecolare (R.S., S.Lib., P.L.P., D.S.-C., A.F., G.P.), Scienze Biomorfologiche e Funzionali-Istituto di Anatomia Patologica (G.T., L.P.), and Chimica Biologica (P.L.), Università Federico II, 80131 Naples, Italy; Cancer Research U.K. Centre for Cancer Therapeutics (V.B., S.Lin.), Institute of Cancer Research 15, Belmont, Sutton, Surrey SM2 5NG, United Kingdom; The Breakthrough Breast Cancer Research Centre (S.Lin.), Institute of Cancer Research, London SW3 6JB, United Kingdom; and Dipartimento di Medicina Sperimentale (P.C.), II Università di Napoli, 80138 Naples, Italy
Address all correspondence and requests for reprints to: Giuseppe Portella, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facoltà di Medicina e Chirurgia di Napoli, Università Federico II, via S. Pansini 5, 80131 Napoli, Italy. E-mail: portella{at}unina.it
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Abstract |
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We analyzed Aurora B expression in human thyroid carcinomas. Cell lines originating from different histotypes showed an increase in Aurora B expression. Immunohistochemical analysis of archive samples showed a high expression of Aurora B in anaplastic thyroid carcinomas; conversely, Aurora B expression was not detectable in normal thyroid tissue. Real-time PCR analysis confirmed a strong expression of Aurora B in anaplastic thyroid carcinomas.
The block of Aurora B expression induced by RNA interference or by using an inhibitor of Aurora kinase activity significantly reduced the growth of thyroid anaplastic carcinoma cells.
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Introduction |
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Materials and Methods |
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The human anaplastic thyroid carcinoma cell lines used in the study are ARO, FRO, 8550-C, BHT 101, KAT-18, Cal 62, and KAT-4.
ARO and FRO human thyroid anaplastic carcinoma cell lines were obtained by Dr. G. F. Juillard (University of California, Los Angeles). ARO and FRO cell lines were kindly provided by Prof. J. A. Fagin (University of Cincinnati College of Medicine, Cincinnati, OH).
KAT-4 and KAT-18 cell lines were established and obtained from Dr. K. B. Ain (University of Kentucky, Lexington, KY). 8505-C anaplastic thyroid cell line was established by Dr. M. Akiyama (Radiation Effects Research Foundation, Hiroshima, Japan), BHT 101 anaplastic thyroid carcinoma cell line was established by Dr. I. Palyi (National Institute of Oncology, Budapest, Hungary), and Cal 62 anaplastic thyroid carcinoma cell line was established by Dr. J. Gioanni (Centre A. Lacassagne, Nice, France). 8505-C, BHT 101, and Cal 62 cell lines were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).
The NPA line derived from poorly differentiated papillary carcinoma and was obtained by Dr. Juillard, the TPC 1 cell line derived from a papillary thyroid carcinoma (31), and the WRO and FB1 cell lines derived from a follicular carcinoma (32, 33).
Cells were grown in DMEM medium supplemented with 10% fetal calf serum and ampicillin/streptomycin.
Cell proliferation was evaluated by using a colorimetric assay for determining the number of viable cells, Cell Titer 96 Aqueous cell proliferation assay (Promega, Madison, WI) according to the manufacturer’s suggestions.
The pCMV-AIM1 construct, containing the human Aurora B gene cDNA (accession no. NM 004217), was obtained as follows: mRNA from human testis (Clontech, Palo Alto, CA) was reverse transcribed using random hexamers as primers and Muloney leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA) to yield cDNA. The forward (5'-CTCTGGATCCATGGCCCAGAAGGAGAAC-3') and reverse (5'-ACAGGGATCCTCAGGCGACAGATTGAAG-3') primers were used to amplify the cDNA. The PCR product was cloned into the BamH1 site of the pcDNA His-tagged (Invitrogen Life Technologies, Carlsbad, CA).
The pCMV AIM-1 sense and antisense constructs were obtained by inserting the Aurora B cDNA in both sense and antisense orientation in the pCMV vector under the transcriptional control of the cytomegalovirus promoter. The vector also carries the gene for G418 resistance. The transfection was performed according to the calcium phosphate transfection protocol (32). After 36 h the cells were trypsinized and expanded, and stable transfectants were generated after selection with 400 µg/ml geneticin G418 (Life Technologies, Italy). Six independent clones were isolated from pCMV AIM-1 sense, pCMV AIM-1 antisense, and control transfections. The clones carrying sense or antisense plasmid were then selected for further experiments.
The quinazoline derivative: N-[4-(6,7-dimethoxy-quinazolin-4-ylamino)-phenyl]-benzamide (34) was dissolved in dimethylsulfoxide at a concentration of 10 mM. Then 1 x 103 ARO cells were plated in a 96-well plate, and after 24 h the cells were treated with 5, 10, or 15 µM of the inhibitor. Cell proliferation was evaluated after 24 and 48 h by using the Cell Titer 96 aqueous cell proliferation assay (Promega). Soft agar colony assays were performed according to a previously described technique (35).
Synthesis of Aurora kinase inhibitor
For the synthesis of Aurora kinase inhibitor, the methodology described elsewhere (34) was followed.
RNAi and transfection of RNA oligonucleotides
For design of small interfering RNA (siRNA) oligos targeting Aurora B, the sequence AAG GAGAACUCCUACCCCUGG (RNAi) was selected, corresponding to the nucleotides located 67–87 bp of the human Aurora B gene. As a control of siRNA oligo, the following aspecific sequence was used AAG GUC CCC AUC CUC AAG AGG (RNAc). The siRNAs were purchased from Dharmacon Research (Lafayette, CO).
Approximately 6 x 105 cells were plated per 6-well plate in media containing 10% fetal bovine serum to give 30–50% confluence. Transfection of the RNA oligonucleotides was performed after 24 h to result in a final RNA concentration of 200 nM. Transfections were performed using Oligofectamine (Invitrogen, Carlsbad, CA).
Human thyroid tissue samples, semiquantitative RT-PCR, real-time PCR, and immunohistochemistry
Normal and neoplastic human thyroid tissue was obtained from surgical specimens and immediately frozen in liquid nitrogen. Thyroid tumors were collected at the Laboratoire d’Histologie et de Cytologie, Centre Hospitalier Lyon Sud, France, and the Laboratoire d’Anatomie Pathologique, Hospital de L’Antiquaille Lyon, France. Total RNA was isolated and DNase digested using the RNeasy minikit (Qiagen, Valencia, CA) according to the manufacturer’s recommendations. Three micrograms of total RNA from each sample were reverse transcribed using random hexamers as primers and Moloney leukemia virus reverse transcriptase (Applied Biosystems) to yield cDNA. Semiquantitative RT-PCR was carried out on cDNA using the GeneAmp PCR System 9600 (Applied Biosystems). A RNA PCR core kit (Applied Biosystems) was used to perform PCR. The human ß-actin gene primers, amplifying a 109-bp cDNA fragment, were used as a control with the following program: 95 C for 10 min; 25 cycles at 95 C for 30 sec, 60 C for 30 sec, and 72 C for 30 sec; and 72 C for 5 min for a final extension.
To amplify the Aurora B mRNA, RT-PCR was performed using two primers that amplified a 128-bp nucleotide cDNA fragment encompassing exons 5 and 6 with the following program: 95 C for 10 min; 29 cycles of 95 C for 30 sec, 59.5 C for 30 sec, and 72 C for 30 sec; and a final extension of 72 C for 5 min.
Quantitative PCR was performed in triplicate using iCycler (Bio-Rad Laboratories, München, Germany) with SYBR Green PCR master mix (Applied Biosystems) as follows: 95 C 10 min and 40 cycles (95 C 15 sec and 60 C 1 min). Fold mRNA overexpression was calculated according to the formula 2(Rt–Et)/2(Rn–En) as described previously (36), where Rt is the threshold cycle number for the reference gene (ß-actin) in the tumor, Et for the experimental gene in the tumor, Rn for the reference gene in the normal sample, and En for the experimental gene in the normal sample. Specific primers used are as follows. Aurora B forward, 5'-CTGGAATATGCACCACTTGGA, reverse, 5'CGAATGACAGTAAG-ACAGGG; and ß-actin-forward, 5'-TCGTGCGTGACATTAAGGAG; ß-actin-reverse, 5'-GTCAGGCAGCTCGTAGCTCT.
The cellular distribution of Aurora B protein was assessed by immunohistochemical analysis. Aurora B expression was evaluated in both nonneoplastic and neoplastic thyroid tissues. Hematoxylin and eosin-stained slides were retrieved from the files of the Department of Bio-Morphological Sciences at the University Federico II of Naples; in all cases the histological diagnosis was confirmed on review. Paraffin-embedded sections were obtained from these samples, and Aurora B expression was evaluated. Xylene-dewaxed and alcohol-rehydrated paraffin sections were placed in Coplin jars filled with a 0.01 M trisodium citrate solution and heated for 3 min in a conventional pressure cooker. After heating, slides were thoroughly rinsed in cool running water for 5 min and then washed in Tris-buffered saline (pH 7.4).
Aurora B protein was detected by using the polyclonal antibody raised in rabbit (no. 611082; BD Transduction Laboratories, San Diego, CA). The incubation with the primary antibody was followed by incubation with biotinylated antimouse immunoglobulins and by peroxidase-labeled streptavidin (LSAB-Dako, Carpinteria, CA). The signal was developed by using diaminobenzidine chromogen as substrate. Negative controls were run with normal rabbit serum instead of the primary antibody, or the antibody was preadsorbed with the cognate peptide (10–6 M). Cases were scored by assessing the percentage of labeled cells, and percentage of positive cells was evaluated by analyzing 1000 neoplastic cells in five different high power fields. The study was approved by the Ethics Committee of the Centre Hospitalier Lyon Sud and the University Federico II Napoli. In all cases written informed consent was obtained.
Protein extraction and Western blot analysis
Cells were homogenized directly into lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1%Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 0.5 mM sodium orthovanadate, and 20 mM sodium pyrophosphate). The lysates were clarified by centrifugation at 14,000 x 10 min. Protein concentrations were estimated by an assay (Bio-Rad) and boiled in Laemmli buffer [0.125 M Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.002% bromophenol blue] for 5 min before electrophoresis. Proteins were subjected to SDS-PAGE (10% polyacrylamide) under reducing conditions. After electrophoresis, proteins were transferred to nitrocellulose membranes (Immobilon, Millipore Corp., Bedford, MA); complete transfer was assessed using prestained protein standards (Bio-Rad, Hercules, CA). After blocking with Tris-buffered saline-BSA [25 mM Tris (pH7.4), 200 mM NaCl, and 5% BSA], the membrane was incubated with the primary antibody against Aurora B for 1 h (at room temperature). Membranes were then incubated with the horseradish peroxidase-conjugated secondary antibody (1:10,000) for 45 min (at room temperature), and the reaction was detected with an enhanced chemiluminescence system (Amersham Life Science, Buckinghamshire, UK).
Tumorigenicity assay
Experiments were performed in 6-wk-old male athymic mice (Charles River, Italy). Untransfected ARO cells (1 x 106) or respectively transfected with a plasmid-containing Aurora B in the sense or antisense orientation were injected into the right flank of the mice. The animals were monitored for the appearance of tumors and tumor latency evaluated. The mean tumor latency is the time needed for tumors to reach 10 mm in diameter. All mice were maintained at the Dipartimento di Biologia e Patologia Animal Facility. Animal experimentations described in the present paper have been conducted in accordance with accepted standards of animal care, in accordance with the Italian regulations for the welfare of animals used in studies of experimental neoplasia, and the study was approved by Ethics Committee on Animal Care of the University Federico II Napoli.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionEffects of the suppression...References |
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Thyroid carcinoma cell lines derived from papillary carcinomas, follicular carcinomas, and anaplastic thyroid carcinomas were analyzed for Aurora B expression by Western blot. An Aurora B-specific band of 41 kDa was detected in all the samples. As shown in Fig. 1
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To confirm that Aurora B gene expression is increased in highly malignant thyroid carcinomas, Aurora B expression in thyroid glands obtained from animal models of thyroid carcinogenesis was evaluated by Western blot. We used transgenic animals lines expressing RET/PTC3, HPV 16 E7, and LT SV40 oncogenes under the transcriptional control of the thyroglobulin promoter.
RET/PTC3 is an oncogene frequently activated in human thyroid papillary carcinomas, and it has been shown to induce thyroid papillary carcinomas when expressed in the thyroid cells of transgenic animals (37). Transgenic mice carrying HPV 16 E7 oncogene under the transcriptional control of the thyroglobulin gene promoter develop follicular carcinomas of the thyroid (38), whereas thyroid anaplastic carcinomas are obtained in animals with thyroid-targeted expression of LT SV40 oncogene (39). Thyroid glands of animals treated with the goitrogenic agent propylthiouracil (PTU) for 2 wk and 1 month (40) were used as a control for nonneoplastic proliferation. Furthermore, the thyroid gland of a 6-month-old wild-type mouse was used as a control (Fig. 1C
). The results shown are representative of three different experiments.
Elevated levels of Aurora B protein expression were observed in the experimental thyroid anaplastic carcinomas. A weaker signal was observed in the papillary and follicular carcinomas. Conversely, very low levels of Aurora B were observed in normal thyroid tissue as in thyroid glands stimulated by PTU.
Aurora B expression in the normal human thyroid and thyroid neoplastic lesions
To evaluate the expression of Aurora B in human thyroid carcinoma tissues, a panel of matched tumor/normal tissues was analyzed by RT-PCR. No significant differences in Aurora B gene expression were observed in papillary carcinomas matched with normal thyroid tissue (Fig. 2
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionEffects of the suppression...References |
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The Aurora protein kinases play a crucial role in the regulation of the cell cycle processes. Three Aurora-related kinases have been described in mammals, Aurora A (STK15), Aurora B (AIM 1), and Aurora C (STK13) (19, 20, 21). All three mammalian members of this family are overexpressed in human cancer cells. Overexpression of Aurora A is thought to relate to multiple centrosome production, and its role has been evaluated in human mammary (45), pancreatic (46), and other neoplastic lesions (47, 48).
A significant overexpression of Aurora B has been observed in human cancer cell lines and colorectal tumors, and a correlation between Aurora B expression levels and Duke’s grade in colorectal tumors has been described (30, 49). Furthermore, it has been observed that the forced expression of Aurora B produced aneuploid cells with a malignant and aggressive phenotype, indicating that Aurora B is relevant in aneuploidy development during carcinogenesis (29). Recently it has been shown that high expression levels of Aurora B are a consistent feature of human seminomas (50).
In the present study, we evaluated the expression of Aurora B in thyroid carcinoma cell lines and experimental and human thyroid tumors, and we observed that the expression of Aurora B is increased in cell lines derived from thyroid anaplastic carcinomas. An increase of Aurora B expression was observed in experimental anaplastic thyroid tumors, whereas in differentiated experimental thyroid tumors, a lower expression of the Aurora B gene was observed. It is important to note that a very low expression of Aurora B was observed in the glands of animals treated with PTU, despite the intense mitotic activity of the follicular cells (40). These data indicate that Aurora B-increased expression is associated with the late stages of thyroid tumor progression and suggest that the Aurora B overexpression could contribute to thyroid tumor progression.
The immunohistochemical analysis of neoplastic or nonneoplastic thyroid lesions paralleled the results obtained using cell lines and experimental tumors, showing that the expression is abundant in anaplastic carcinomas. Conversely, no or low expression was observed in benign adenomas, nonneoplastic thyroid lesions such as Hashimoto disease, and normal thyroid tissue. Our data on thyroid carcinomas extend to this type of tumor the observation that the expression of Aurora B displays a tendency to group in higher grade of malignancy as previously observed in colon carcinomas (49). This observation suggests that during tumor progression, in particular during the transition from differentiated thyroid carcinomas to anaplastic thyroid carcinomas, Aurora B overexpression takes place, and Aurora B overexpression might confer a growth advantage to the neoplastic cells. Furthermore, our data indicate that Aurora B expression may serve as a prognostic marker in thyroid tumors.
In our study, the functional consequences of RNAi-mediated decrease of Aurora B expression in anaplastic thyroid cancer cells were determined using assays of gene expression and cellular proliferation. Our results demonstrate that the Aurora B siRNA treatment induces a significant reduction in cell proliferation. No cells with apoptotic figures were observed, and the analysis of the apoptotic cascade indicated that the pathway was not activated by the Aurora B RNAi treatment (data not shown). It is possible to conclude that the block of Aurora B protein synthesis does not induce apoptosis in ARO cell line; moreover, an accumulation of Aurora B RNAi-treated cells in G2/M phase was observed by flow cytometry (data not shown). The inhibition ARO cells proliferation was about 40% on RNAi treatment; these data indicate that cell proliferation is not completely dependent on Aurora B activity.
Aurora B cDNA antisense transfection decreased colony formation in soft agar and resulted in increased tumor latency. By using a selective Aurora B kinase inhibitor, we confirmed that the block of Aurora B activity dramatically reduces the proliferation of anaplastic thyroid carcinoma cells.
Anaplastic thyroid carcinoma is resistant to currently available therapeutic agents. The observation that the block of Aurora B expression or activity reduces the proliferation of anaplastic thyroid carcinoma cells could contribute to the development of novel therapeutic strategies.
In conclusion, our data indicate that in thyroid carcinomas Aurora B gene expression levels are correlated to the stage of malignant progression, and the block of Aurora B expression or its kinase activity significantly reduces the growth of thyroid anaplastic carcinoma cells, suggesting that Aurora B is a potential therapeutic target.
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Effects of the suppression of the Aurora B protein synthesis in anaplastic thyroid carcinoma cells |
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Acknowledgments |
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Footnotes |
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Abbreviations: PTU, Propylthiouracil; RNAi, RNA interference; siRNA, small interfering RNA.
This work was supported by the Italian Ministry of Instruction, University and Research, PRIN 2002, and the Associazione Italiana per la Ricerca sul Cancro.
Received July 31, 2004.
Accepted November 11, 2004.
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PH3) was observed at 48 h. Aurora A and H3 levels were not affected by the treatment. B, Time-dependent inhibition of ARO cell proliferation by Aurora B RNAi. ARO cells were transfected with 200 nM annealed sense (RNAi) and control (RNAc) 21-mer RNA oligonucleotides directed against Aurora B or mock-transfected (control). The data are expressed as percentages of the control cell proliferation, and the SD was calculated (bar). The data are the mean of three different experiments.
