ARID3B Induces Malignant Transformation of Mouse

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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

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 D, and propidium iodide stainings. siRNAs were transfected using LipofectAMINE 2000 (Invitrogen) according Analysis of DNA chip data. Expression data from McArdle et al. (10) to the protocol of the manufacturer. In AS experiments, the relative growth was obtained from ArrayExpress,3 accession code E-MEXP-83. Expression ratio against scramble-treated cells was calculated by the ATP assay, using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Madison, WI). Each assay consists of triplicate cultures. In some experiments, the 3 http://www.ebi.ac.uk/arrayexpress.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. ARID3B, a New Oncogene Associated with Neuroblastoma values, detection calls, and detection P values were extracted from the microarray data. We used the data set of McArdle et al. (10) as entire data set using a series of perl scripts. Additional data from human these data is available from ArrayExpress (11),3 has been described tissues and tumor samples was also obtained from ArrayExpress in detail and uses the highly standardized Affymetrix oligonucle- (accession nos. E-MEXP-66, E-MEXP-97, E-MEXP-101, E-MEXP-114, E- otide arrays, enabling us to extract the relevant data and to MEXP-120, E-MEXP-214, E-MEXP-167, E-MEXP-353, E-MEXP-149, E-MIMR- compare this across other data sets. Of 23 malignant neuroblas- 17, and E-AFMX-5). The data sets were selected to cover a wide range of different tumors and normal tissues and to avoid samples that had been toma samples, ARID3B was detected as present (++) or marginal subjected to experimental treatments. A full list of samples is available in (+) in 11 samples (Fig. 1C). Of these, two are neuroblastoma cell the Supplementary Data. The association between different types of lines (not shown in Fig. 1C), one is a stage II neuroblastoma, and neuroblastoma and the expression of various genes was calculated using the remaining eight represent stage IV neuroblastomas. Thus, the hypergeometric distribution. This calculates the probability of ARID3B expression was found in 9 of 21 (not counting the cell observing an overlap of a given size or larger between two groups of a lines) neuroblastoma cases (42.8%; Fig. 1C). Strikingly, this given population. For the purpose of statistical calculations, a call of expression is strongly associated with stage IV tumors (8 of 10 of marginal was considered as detected (i.e., present). The neuroblastoma stage IV versus 1 of 11 of stage I-II+IVs, P = 0.0018) and also with cell lines included in McArdle et al. (10) were considered as stage IV tumors with 11q deletion (9 of 11, P = 0.00019). ARID3A is neuroblastoma when association P values were calculated for all probe expressed in five of the ARID3B+ samples but in none of the sets (Fig. 1D) but were excluded from calculations for specific P values À quoted in the text. ARID3B tumors, suggesting a possible relationship (P = 0.06). MYCN was detected in the two cell lines and in 19 of the 21 neuroblastomas, including in all ARID3B+ tumors. Results and Discussion In mouse, the expression of ARID3B is detected in only a small To test ARID3B expression in neuroblastoma, we obtained number of embryonicand adult tissues (4). To determine whether five human neuroblastoma cell lines and examined the expres- the same is true in human, we obtained microarray data created sion of ARID3A, ARIDB, and MYCN. All obtained cell lines using the same array used by McArdle et al. (10) from a total of express significant although variable levels of ARID3B (Fig. 1A). 12 different experiments covering a wide range of normal tissues In contrast, ARID3A was not detected in any of the cell lines and cell types (f70) and tumors (f30) represented by a total of even by RT-PCR (Fig. 1B).Ofthefivecelllines,strongMYCN 483 samples (Supplementary Data). Of these 483 samples, ARID3B expression was detected in four cell lines, whereas one line was detected as expressed (present or marginal) in only 25 expresses a low level that is detectable by RT-PCR. In contrast, samples. Eleven of these are the previously described neuroblas- we detected ARID3B expression in only two (K-562 and SW480) toma samples. Hence, ARID3B can be said to be specific to stage of eight nonneuroblastoma cancer cell lines (Supplementary IV neuroblastoma, both in the narrow sense of not being Fig. S1C). expressed in earlier stage neuroblastoma, but also in the broader We next examined the expression of ARID3B, ARID3A, and sense of being expressed in only a few other cell types. If there MYCN in clinical samples of neuroblastoma using published are only a few molecules that are specific to neuroblastoma in

Figure 2. Suppression of ARID3B expression impairs the proliferation of neuroblastoma cell lines. A to D, growth inhibition of neuroblastoma cells by siRNA treatment. Neuroblastoma cell lines were transfected with either double-stranded scrambled RNA oligonucleotide or short double-stranded RNA molecules against ARID3B, siRNA1 (A and B) and siRNA2 (C and D). Four days later, viable cell numbers were calculated by staining with trypan blue dye. Data are ratios of cell numbers observed with specific siRNA treatment to numbers observed with the scrambled siRNA. Columns, mean from triplicate assays. siRNA treatments suppressed the growth of neuroblastoma cells (A and C). B and D, suppression of ARID3B expression by siRNA treatment. Total RNA was purified from siRNA- transfected cells as well as control cells. The graphs showthe ratio of expression of ARID3B expression observed after treatment with specific siRNA molecules compared with the scrambled control. Expression levels of ARID3B were normalized by the expression level of glyceraldehyde-3-phosphate dehydrogenase (ARID3B/GAPDH ratio) before calculating the expression ratios. Bars, SD. The statistical significance was calculated by the Student’s t test. Significant suppression was induced in four of five cell lines.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Cancer Research this manner, then this strengthens our contention that ARID3B is a key molecule in the progression of neuroblastoma. To ascertain this, we calculated P values (Supplementary Data) for all probe sets for the specific association with stage IV neuroblastoma and a general association (compared against the data for the 30 tumor types in our data sets). Although there are five probe sets that are more strongly associated with stage IV versus other stages of neuroblastoma than ARID3B, these are also expressed in many other tumor types. Surprisingly, ARID3B is easily distinguished from all other probe sets as being the only gene that is specific to stage IV neuroblastoma in both a specific and a broader sense (Fig. 1D). To investigate the role of ARID3B in the growth of neuroblastoma cell lines, we did suppression and overexpression of ARID3B. First, we suppressed ARID3B expression using two types of siRNAs (Fig. 2A-D). Although the SY5Y cell line was insensitive to this treatment, siRNA treatment suppressed the growth of other neuroblastoma cell lines to a variable extent (85-10%; Fig. 2A and C). We also treated these cell lines with AS molecules to ARID3B and obtained similar results to the siRNA experiments (Supple- mentary Fig. S1D). These results clearly indicate the involvement of ARID3B in the growth of some, although perhaps not all, neuroblastoma cell lines. Interestingly, expression of additional ARID3B by transfection showed no significant effect on the cell lines whose growth was strongly affected by siRNA or AS (Supplementary Fig. S1D). However, both anchorage-dependent and anchorage-independent growth of the SY5Y cell line was markedly enhanced by transfection of the ARID3B gene (Fig. 3A and B; Supplementary Fig. S2A). Moreover, malignancy, as assessed by the mean survival times of tumor transplanted nu/nu mice, was accelerated from 47 to 33 days (Fig. 3C). Taken together, these data suggest that ARID3B is a key molecule supporting the growth of neuroblastoma. Finally, we analyzed the transforming activity of ARID3B by exploiting the MEF transformation assay (6). Primary MEF cells were transfected with a retrovirus vector containing ARID3B (Supplementary Fig. S2B). In the control groups (untransfected or vector alone), the proliferative ability of MEF decreased over passage (Fig. 4A), whereas transfection by ARID3B immortalized MEF (Fig. 4A). Thus, ARID3B by itself can transform MEF, which is similar to the activity of ARID3A (5). We next assessed whether ARID3B can collaborate with MYCN to alter the growth of MEF. Additional transfection of the MYCN gene to ARID3B+ MEF indeed enhanced their growth (Fig. 4B). More importantly, coexpression of ARID3B and MYCN in MEF conferred both anchorage-independent growth (Fig. 4C) and tumorigenicability in nu/nu mice(Fig. 4 D; Supplementary Fig. S2C). Thus, ARID3B is a transforming gene and Figure 3. ARID3B overexpression enhances the oncogenic potential of the SY5Y neuroblastoma cell line. A, overexpression of ARID3B accelerates the is likely to be involved in the malignant transformation of proliferation of the SY5Y neuroblastoma cell line. The cells were infected with neuroblastoma. retrovirus carrying Flagtag-fused ARID3B and GFP markers. The proliferation of Although ARID family molecules, such as JARID1B (12, 13) and cells overexpressing ARID3B is higher than cells infected with the control vector. B, colony formation assay in soft agar. Culture medium containing 2.5 Â 104cells ARID4B (12, 14), have been detected in specific types of and 0.4% low-melting agarose was poured on a layer of 1% agar. The number of carcinoma, there is only one report directly demonstrating the colonies (>2 mm in diameter) was counted after 2 weeks. Overexpression of ARID3B enhances the potential for colony formation. C, survival curves of BALB/ transforming activity of ARID family molecules (5). Peeper et al. c-nu/nu mice implanted with SY5Y cells that were transfected with ARID3B. (5) showed that expression of ARID3A by itself immortalizes MEF SY5Y neuroblastoma cells (1 Â 107) with or without ARID3B transfection were and induces malignant transformation when expressed together s.c. injected into BALB/c nu/nu mice. The mean survival periods were 47.3 and 32.5 days for untransfected and transfected SY5Y cells, respectively. with oncogenic RAS. Despite such a clear oncogenic activity in vitro, its involvement in actual tumors has not been docu- mented. Our present study shows that ARID3B satisfies the two transformation of MEF and that it is expressed preferentially in main criteria for tumorigenic molecules, the ability to transform stage IV tumors is consistent with the idea that ARID3B plays a MEF and the specific association with the given cancer. The key role in the development of malignant neuroblastoma. Our fact that ARID3B collaborates with MYCN to induce malignant results suggest that MYCN expression is required for the

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. ARID3B, a New Oncogene Associated with Neuroblastoma tumorigenicactivityof ARID3B. Although our data analysis express MYCN. Indeed, it is actually difficult to establish whether suggests that ARID3B expression is correlated with 11q deletion ARID3B expression is associated with 11q deletion or advanced- rather than with MYCN amplification, there is no inconsistency stage neuroblastoma because almost all (8 of 10) stage IV samples with our experimental findings since all ARID3B+ tumors also have 11q deletions. However, because two of three non–stage IV

Figure 4. Oncogenic potential of ARID3B in primary MEFs. A, ARID3B overexpression can immortalize primary MEFs. MEF at passage 2 were infected with retrovirus carrying both ARID3B and GFP. GFP+ cells were purified by FACS and evaluated for proliferation. MEF carrying the control vector could not survive beyond eight passages (5). In contrast, overexpression of ARID3B can bypass the spontaneous senescence in MEF resulting in immortalization (n). B, the effect of ARID3B and MYCN on the growth of MEF. MYCN was transfected to passage 18 ARID3B-overexpressing MEF (o). Cells expressing both ARID3B and MYCN (n) growat a faster rate than those expressing only one of the two genes. The experiments were independently done thrice and the representative results were shown in (A and B). C, colony-forming potential of MEF transfected either with ARID3B alone, MYCN alone, or both ARID3B and MYCN. Culture medium containing 2.5 Â 104 cells and 0.4% low-melting agarose was poured on a layer of 1% agar. The number of colonies (>1 mm in diameter) was counted after 2 weeks. Overexpression of both molecules (right) but not ARID3B alone (left)orMYCNalone(middle) confers the potential of anchorage-independent growth in MEF. D, ARID3B in collaboration with MYCN induces malignant transformation of MEF. MEFs transfected with ARID3B, MYCN, or ARID3B + MYCN were s.c. injected into BALB/c nu/nu mice and the formation of tumors was assessed 7 days after transplantation. No tumors were generated from MEF that were transfected with either ARID3B (left in the picture)orMYCN(center in the picture) alone over 2 months of observation. In contrast, tumors were detected within 7 days in all mice implanted with MEF that were transfected with both ARID3B and MYCN,andallmicediedwithin 3weeks(right in the picture). Each group consisted of five recipient mice injected in both flanks (total of 10 sites). Bars (A-C), results from three independent cultures initiated from a single viral infection. Cells used in (C and D) were derived from a single infection event but represent heterogeneous populations. Cells expressing both MYCN and ARID3B were used at four passages after infection with MYCN, whereas cells expressing only one of the genes were taken at equivalent number of passages after the primary infection. The experiments of (C and D) were independently done twice and the representative results are shown in the pictures. www.aacrjournals.org 8335 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 tumors carrying 11q deletions do not express ARID3B, ARID3B Although ARID3B has not been recognized by previous extensive expression may be more coupled to tumor progression than 11q searches for molecules involved in neuroblastoma formation (10, deletion. Interestingly, ARID3A, whose AT-rich interaction domain 16–18), our analysis of previously published data suggests that it has 84% homology with that of ARID3B, is also able to collaborate might have been possible to identify ARID3B from existing data if with MYCC to alter the proliferation of MEF (4, 5). However, different questions had been asked. Our experience suggests that it the combination of MYCC and ARID3A cannot confer the ability would have been necessary to analyze a range of data sets from a to undergo anchorage-independent proliferation, nor to form variety of sources to identify ARID3B as involved in neuroblastoma tumors in nu/nu mice (5). It is thus likely that ARID3A is less from a pure data mining exercise. The ability to combine data sets tumorigenic in combination with MYC-induced transformation from a wide range of sources is a major advantage of standardized than ARID3B. array analyses that is often overlooked but has great potential for Our data strongly implicates dysregulated ARID3B expression in future research. the development of malignant neuroblastoma, but addresses In conclusion, this study shows that ARID3B is a tumorigenic neither the function of ARID3B in the development of neuroblas- molecule that can collaborate with MYCN to induce malignancy toma nor the causes underlying its expression. However, previous and implies its role in the progression of malignant neuroblastoma. findings suggest that ARID3B is involved in avoiding oncogene- The strong association of ARID3B expression with stage IV induced apoptosis: (a) ARID3A, the closest relative of ARID3B, has neuroblastoma suggests that ARID3B may serve as a good tumor been shown to be able to bypass Ras or MYCC-induced apoptosis marker for neuroblastoma. As siRNA and AS treatments can (5); (b) AS treatment of the CHP126 cell line resulted in accelerated suppress growth of some neuroblastoma cell lines, ARID3B may apoptosis (Supplementary Fig. S2D); and (c) the function of also serve as a target for the therapy of stage IV neuroblastoma for ARID3B during embryogenesis is to protect neural crest cells from which development of effective treatments have been lacking. apoptosis at their proliferative phase (4). As ARID3B was initially isolated as a RB1-binding protein (15), it seems likely that the Rb pathway is involved in apoptoticavoidancein neuroblastoma. Acknowledgments In normal mouse, the ARID3B gene is expressed under strict Received 2/27/2006; revised 6/19/2006; accepted 7/11/2006. spatiotemporal control, with expression limited to nascent Grant support: Ministry of Education and Science grants 16606005 and 17045039 (T. Era), Knowledge Cluster Initiative grant (T. Era), and Leading Project for mesoderm and neural crest (4). Our data suggests that ARID3B Realization of Regenerative Medicine grant (S.I. Nishikawa). expression in neuroblastoma is aberrant, as it is expressed only in a The costs of publication of this article were defrayed in part by the payment of page specific type or stage of neuroblastoma. Thus, the trigger for the charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. aberrant expression of ARID3B seems likely to be an important We thank Dr. H. Enomoto and Dr. M. Royle for a critical reading of the manuscript event in the development of malignant neuroblastoma. and a helpful discussion.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. ARID3B Induces Malignant Transformation of Mouse Embryonic Fibroblasts and Is Strongly Associated with Malignant Neuroblastoma

Kenichiro Kobayashi, Takumi Era, Atsushi Takebe, et al.

Cancer Res 2006;66:8331-8336.

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