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ARID3B Induces Malignant Transformation of Mouse

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ARID3B Induces Malignant Transformation of Mouse Priority Report ARID3B Induces Malignant Transformation of Mouse Embryonic Fibroblasts and Is Strongly Associated with Malignant Neuroblastoma Kenichiro Kobayashi,1 Takumi Era,1 Atsushi Takebe,2 Lars Martin Jakt,1 and Shin-Ichi Nishikawa1 1Laboratory for Stem Cell Biology, RIKEN Center for Development Biology and 2Division of Gastroenterological Surgery, Graduate School of Medicine, Kobe University, Kobe, Hyogo, Japan Abstract Interestingly, the most similar ARID protein, ARID3A was shown to ARID3B, a member of the AT-rich interaction domain (ARID) induce malignant transformation of mouse embryonic fibroblasts family of proteins, plays an essential role in the survival of (MEF) in collaboration with activated oncogenes such as Ras (5). neural crest during embryogenesis.Here, we report evidence These two observations inspired us to examine the possibility that ARID3B is involved in the development of malignant that ARID3B is involved in the formation of neuroblastoma, which neuroblastoma, a childhood tumor derived from neural crest. is the archetypal neural crest tumor. Here, we provide strong (a) ARID3B is expressed by all five cell lines derived from evidence that ARID3B is involved in development of malignant neuroblastoma tested by us.( b) Analysis of published DNA neuroblastoma. microarray data of fresh neuroblastoma tumors showed that ARID3B is expressed in 80% of stage IV tumors, whereas only Materials and Methods in 9% of stage I-III+IVs tumors.( c) In vitro growth of several Cell culture, gene transfection, and tumor growth assay. Human neuroblastoma cell lines is suppressed significantly by neuroblastoma cells (SH-SY5Y, TGW, CHP-126, NBLS, and IMR-32) were d antisense as well as siRNA treatment.( ) An increase of the maintained in a 1:1 mixture of DMEM and Ham’s F-12 medium (Invitrogen, ARID3B expression level by transfection in the SY5Y neuro- Carlsbad, CA) supplemented with 10% FCS. Human hematopoieticcells, blastoma cell line enhances the malignancy in tumor growth K-562 (American Type Culture Collection, Manassas, VA), were maintained assays in nu/nu mice.( e) ARID3B by itself can immortalize in RPMI 1640 (Invitrogen) supplemented with 10% FCS. Primary MEFs were mouse embryonic fibroblasts (MEFs) in vitro and confers isolated from 14.5 dpcmouse embryos and cultured in DMEM supplemented malignancy to MEF when transfected together with MYCN, the with 10% FCS. Cells were maintained in a humidified 5% CO2 atmosphere. best characterized oncogene for neuroblastoma.Thus, Soft agar assays were done as previously described (6). The retroviral ARID3B seems to play a key role in the malignant transfor- expression vectors, MSCV-IRES-EGFP and MSCV-IRES-DsREDT4, were used for transfecting ARID3B fused with the flag epitope and MYCN fused with mation of neuroblastoma and may serve not only as a marker the hemagglutinin-epitope, respectively (7). Production and infection of of malignancy but also as a potential target for cancer therapy retroviruses were done as described previously (8). Following purification of of stage IV neuroblastoma for which there is currently no GFP+ or DsREDT4+ cells by fluorescence-activated cell sorting (FACS), the effective treatment available. (Cancer Res 2006; 66(17): 8331-6) expression of ARID3B and MYCN was confirmed by Western blot analysis with antibodies against each tag. Primary MEF (passage 2) were infected Introduction with a retrovirus carrying ARID3B or MYCN. In some experiments, MEF that forcibly expressed ARID3B were further infected with MYCN-carrying Neuroblastoma is the most common malignant infant tumor (1). virus at passage 18. For evaluating tumorigenicity, either 106 MEFs or 107 It affects the sympathetic nervous system derived from neural crest neuroblastoma cells in 100 AL PBS were s.c. transplanted into BALB/c-nu/ and has a remarkable variability in its clinical course (1, 2). Roughly nu mice. Tumor growth was monitored every 3 days. half are discovered as solitary tumors without metastasis, of which RNA quantification. Northern blot analysis was done as described a considerable proportion undergo spontaneous regression. In previously using a part of the hARID3B cDNA (positioned from 1,298 to contrast, the other half are discovered with metastasis and take a 3,480 bp, Genbank accession no. BC041792) as the probe (4). Primers used poor clinical course. Although the MYCN gene has been implicated for reverse transcription-PCR (RT-PCR) and quantitative PCR analyses are in the development of neuroblastoma, the molecular mechanisms listed in Supplementary Fig. S1A and B. underlying such a diversity remain unknown (1, 3). Antisense oligonucleotides and short interfering RNA. The ARID3B antisense (AS) oligonucleotides used for transfection, were designed and Recently, we have reported that neural crest development synthesized by Gene Design, Inc. (Osaka, Japan), and contained 2¶-O,4¶-C- is severely suppressed in embryos that bear null mutations in methylene bridged nucleic acid (9). AS1 (5¶-CCATTTTTgcCTCaagcttC-3¶)and the ARID3B gene (4). This phenotype suggests that the role of AS2 (5¶-TGCccAgGTTcagagtCATG-3¶) were targeted at starting locations 141 ARID3B is to maintain the survival of proliferating neural crest. and 108 of human ARID3B mRNA (Genbank accession no. BC041792), respectively. Scrambled oligonucleotides (5¶-gCTTCcCtTcTtgCAaCaTT-3¶ to AS1 and 5¶-GccACaTgGGcTggTaCAtT-3¶ to AS2) have the same base com- position as hARID3B AS, but in a random order. Capital letters indicate Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). bridged nucleic acid residues. Requests for reprints: Takumi Era, Laboratory for Stem Cell Biology, RIKEN Short interfering RNA (siRNA) sequences targeting ARID3B correspond Center for Development Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, to the coding regions 944 to 964 (siRNA1) and 1,037 to 1,057 (siRNA2) Hyogo 650-0047, Japan. Phone: 81-78-306-1893; Fax: 81-78-306-1895; E-mail: relative to the first nucleotide of the start codon. Scrambled siRNAs [email protected]. ¶ ¶ ¶ I2006 American Association for Cancer Research. (5 -GAGCGAUGCCUAGAAUAAU-3 to siRNA1 and 5 -GCUCGCAAUAUGUA- doi:10.1158/0008-5472.CAN-06-0756 ACAGU-3¶ to siRNA2) were designed and used as a control. All sequences www.aacrjournals.org 8331 Cancer Res 2006; 66: (17).September 1, 2006 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Cancer Research Figure 1. ARID3B expression in neuroblastoma. A, Northern blot analysis of ARID3B and MYCN in neuroblastoma cell lines. Total RNA was purified from neuroblastoma cell lines and hybridized with either 32P-labeled ARID3B–, MYCN-, or h-actin–specific cDNA probes. Although the expression level is variable, a 4.2 kb band of ARID3B signal was detected in all neuroblastoma cell lines. Strong MYCN expression was observed in four of five lines. B, RT-PCR analysis of ARID3A, ARID3B, and MYCN in neuroblastoma and hematopoietic cell lines. Although both ARID3B and MYCN signals were detected in all neuroblastoma cell lines, that of ARID3A was undetected. K562, a chronic myelogenous leukemia cell line, in contrast, expresses both ARID3A and 3B molecules. SH-SY5Y (SY5Y, ATCC), TGW (19), CHP-126 (20), NBLS, and IMR-32 (ATCC) are all neuroblastoma cell lines. K562 (ATCC) is hematopoietic cell line. C, ARID3B expression in stage IV neuroblastoma tumors. The results of case analyses, including stages, MYCN amplification, and 11q deletion are taken from McArdle et al. (10). Expression of ARID3B, MYCN, and ARID3A were obtained from published microarray data. ARID3B expression is associated with advanced-stage IV tumors. D, association of ARID3B with stage IV neuroblastoma. The plot shows the probability of observing the number of stage IV samples (including the two neuroblastoma cell lines and 10 clinical samples) expressing the given gene within two different populations of samples for all probe sets present on the HGU133A array. The probabilities within the neuroblastoma only sample group of McArdle et al. (10) is shown along the X axis (24 samples in total, 12 stage IV samples and 12 non–stage IV neuroblastoma samples). The probabilities within a larger population of samples containing data from a range of tumor samples (290 samples in total, 12 of which are stage IV neuroblastoma, 12 non–stage IV neuroblastoma, and the remainder which are from around 30 types of nonneuroblastoma tumor). X-axis, specific association with stage IV versus non–stage IV neuroblastoma; Y-axis, extent of specificity of expression in stage IV neuroblastoma within the larger group of samples (since the total number of samples is much larger than the number of neuroblastoma samples, genes that are expressed in all neuroblastoma will also score highly in this test). The probe sets for MYCN, ARID3B, and ARID3A have been plotted with special markers as indicated in the figure. Only ARID3B scores highly in both tests and can be clearly distinguished from the other probe sets on the array. P values were calculated using the hypergeometric distribution and are shown as Àlog values on the X and Y axes. The specific samples used in this test, as well as the values of the probabilities, are given in the Supplementary Data. were compared with the human genome (using Basic Local Alignment proportion of apoptotic and dead cells was measured by flowcytometer Search Tool) to ensure their specificities. Antisense oligonucleotides and using Annexin, 7-amino-actinomycin
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