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[Cancer Research 65, 4587-4597, June 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

LMO3 Interacts with Neuronal Transcription Factor, HEN2, and Acts as an Oncogene in Neuroblastoma

Mineyoshi Aoyama1, Toshinori Ozaki1, Hiroyuki Inuzuka1, Daihachiro Tomotsune3, Junko Hirato4, Yoshiaki Okamoto1, Hisashi Tokita2, Miki Ohira1 and Akira Nakagawara1

Divisions of 1 Biochemistry and 2 Animal Science, Chiba Cancer Center Research Institute; 3 Center for Functional Genomics, Hisamitsu Pharmaceutical Co., Inc., Chiba, Japan and 4 Department of Pathology, Gunma University School of Medicine, Gunma, Japan

Requests for reprints: Akira Nakagawara, Division of Biochemistry, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuoh-ku, Chiba 260-8717, Japan. Phone: 81-43-264-5431; Fax: 81-43-265-4459; E-mail: akiranak{at}chiba-cc.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
LIM-only proteins (LMO), which consist of LMO1, LMO2, LMO3, and LMO4, are involved in cell fate determination and differentiation during embryonic development. Accumulating evidence suggests that LMO1 and LMO2 act as oncogenic proteins in T-cell acute lymphoblastic leukemia, whereas LMO4 has recently been implicated in the genesis of breast cancer. However, little is known about the role of LMO3 in either tumorigenesis or development. In the present study, we have identified LMO3 and HEN2, which encodes a neuronal basic helix-loop-helix protein, as genes whose expression levels were higher in unfavorable neuroblastomas compared with those of favorable tumors. Immunoprecipitation and immunostaining experiments showed that LMO3 was associated with HEN2 in mammalian cell nucleus. Human neuroblastoma SH-SY5Y cells stably overexpressing LMO3 showed a marked increase in cell growth, a promotion of colony formation in soft agar medium, and a rapid tumor growth in nude mice compared with the control transfectants. More importantly, the increased expression of LMO3 and HEN2 was significantly associated with a poor prognosis in 87 primary neuroblastomas. These results suggest that the deregulated expression of neuronal-specific LMO3 and HEN2 contributes to the genesis and progression of human neuroblastoma in a lineage-specific manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The LIM domain–containing proteins are important regulators in determining cell fate and controlling cell growth and differentiation during embryonic development (1). The LIM domain is a highly conserved cysteine-rich zinc finger–like motif found in a variety of nuclear and cytoplasmic proteins and acts as a docking site for the assembly of multiprotein complexes (2–4). However, the precise role of the LIM domain is still unclear. Several distinct subgroups of the LIM domain–containing proteins are defined and some of them also possess a functionally divergent domain, including a DNA-binding homeodomain or a protein kinase domain (1, 2).

The LIM-only proteins (LMO) are one of the families of the LIM domain–containing proteins and possess only two tandem LIM domains. They consist of four members, including LMO1, LMO2, LMO3, and LMO4 (2, 4). LMO1 and LMO2 have been identified as the genes that are activated in human acute T-cell leukemia (T-cell ALL) by tumor-specific chromosomal translocations (4). Transgenic mice overexpressing LMO1 or LMO2 developed immature and aggressive T-cell leukemia, suggesting that these proteins act as T-cell oncoproteins (5–7). On the other hand, LMO4 has been identified as a nuclear protein that interacts with the adaptor protein Ldb1 (8). It has been shown recently that LMO4 is highly expressed in primary human breast cancers, and overexpression of LMO4 inhibits differentiation of mammary epithelial cells, suggesting that deregulated expression of LMO4 contributes to the breast carcinogenesis (9). LMO4 has also been reported to be associated with BRCA1 to repress its transcriptional activity (10). Thus, LMO1, LMO2, and LMO4 have been implicated in tumorigenesis. However, to date, little is known about the oncogenic function of LMO3, which has been discovered based on sequence homology with LMO1 (11).

The nuclear LMO proteins, which lack intrinsic DNA-binding activity, have been considered to be involved in transcriptional regulation (2), raising a possibility that they alter the transcription of target genes by forming a complex with other transcription factors with DNA-binding activity. Indeed, in T-cell acute lymphoblastic leukemia in children, a basic helix-loop-helix transcription factor, TAL1, is physically associated with LMO1 or LMO2 and enhances their oncogenic activities (12, 13). Interestingly, the neuronal-specific basic helix-loop-helix transcription factors, HEN1 and HEN2, were identified based on cross-hybridization with TAL1 (14, 15). Their expression was restricted to the developing nervous system and a human neuroblastoma cell line. However, the role of HEN1 and HEN2 in tumorigenesis has long been elusive.

Neuroblastoma is one of the most common childhood cancers and is originated from sympathoadrenal lineage of the neural crest (16). It is clinically and cytogenetically divided into two major subgroups with favorable and unfavorable prognosis (17). The recent molecular and cellular analyses have revealed that amplification of MYCN and DDX1 as well as loss of heterozygosity at the region of chromosome 1p36 are strongly associated with a poor outcome, whereas high levels of expression of the neurotrophin receptors TrkA, CD44, and Fyn, are well correlated with favorable prognosis (16–23). However, we still do not know many other genes that play important roles in the genesis and progression of neuroblastoma. To identify the other genes closely involved in neuroblastoma, we have constructed several cDNA libraries from different subsets of neuroblastoma and randomly cloned 4,200 genes (24). Screening of the genes differentially expressed between favorable and unfavorable subsets of the tumor has identified Nbla3267 as one of the genes expressed at higher levels in unfavorable than favorable neuroblastomas (25).

In the present study, we found that Nbla3267 encoded the human LMO, LMO3, and that high expression of LMO3 as well as HEN2 was strongly associated with a poor prognosis of neuroblastoma. Furthermore, LMO3 interacted with HEN2 in mammalian cell nucleus, and enforced expression of LMO3 in human neuroblastoma-derived cell line SH-SY5Y markedly enhanced tumor growth in nude mice, supporting the oncogenic role of LMO3 in neuroblastoma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patient population. The RNA samples obtained from 87 patients with neuroblastoma were subjected to semiquantitative and quantitative real-time reverse transcription-PCR (RT-PCR) analyses. All patients were diagnosed clinically as well as pathologically and tested for DNA ploidy, MYCN amplification, and TrkA expression. Tumors were staged according to the International Neuroblastoma Staging System criteria (26). Thirty-four patients were stage I, 14 were stage II, 8 were stage III, 26 were stage IV, and 5 were stage IVS. Stages I, II, and IVS were considered as favorable and stages III and IV as unfavorable. The patients were treated following the protocols proposed by the Japanese Infantile Neuroblastoma Cooperative Study (27) and the Study Group of Japan for Treatment of Advanced Neuroblastoma (28). The clinical follow-up ranged from 4 to 58 months, with a median of 36 months. We have a precise list of patient characteristics, including age, stage, and clinical follow-up time, and this list will be provided upon request.

Cloning of human LMO3, HEN1, and HEN2. To obtain a complete human LMO3 cDNA, a cDNA library derived from human fetal brain (Stratagene, La Jolla, CA) was screened with a 32P-labeled Nbla3267 cDNA. Plaques showing positive signals were picked up and rescreened twice. To construct the expression plasmid for hemagglutinin (HA)–tagged LMO3-A, the cDNA fragment encoding the entire LMO3-A protein was amplified by PCR from the phage clone as a template using the primers designed to add a synthetic linker encoding the HA epitope on the NH2-terminal side of LMO3-A (forward 5'-GGTACCATGGCTTACCCATACGATGTTCCAGATTACGCTAGCCTCTCAGTCCAGCCAGACAC-3' and reverse 5'-TCAGATATCATTAGATCAGCGAACCTGGG-3'). The PCR product was digested with KpnI and EcoRV and subcloned into the identical restriction sites of pcDNA3 expression plasmid to give pcDNA3-HA-LMO3-A. cDNA encoding human HEN1 (amino acid residues 1-133) or HEN2 (amino acid residues 1-135) was generated by reverse transcribing total RNA isolated from neuroblastoma cell line, IMR32, using a forward primer (5'-AAGGAATTCATGCTCAACTCAGACACCATG-3') and a reverse primer (5'-ATAAGAATGCGGCCGCTCAGACGT-3') for HEN1 and a forward primer (5'-AAGGAATTCATGCTGAGTCCGGACCAAGCA-3') and a reverse primer (5'-ATAAGAATGCGGCCGCCTACACGTCCAGGACGTGGTT-3') for HEN2. The amplified PCR products were digested with EcoRI and NotI and subcloned into the identical restriction sites of pcDNA3-FLAG expression plasmid to give pcDNA-FLAG-HEN1 and pcDNA3-FLAG-HEN2.

Generation of a polyclonal anti-LMO3 antibody. The polyclonal anti-LMO3 and anti-HEN2 antibodies were raised against a peptide "Cys" plus containing the amino acid sequence between positions 127 and 145 of LMO3 and the amino acid sequence between positions 1 and 19 of HEN2, respectively. The peptides and the polyclonal antibodies were produced by Biologica Co. (Nagoya, Japan).

Cell culture and transfection. Human neuroblastoma (SK-N-AS, SH-SY5Y, NB69, OAN, SK-N-BE, NGP, NLF, IMR32, NB1, and KP-N-NS), ALL (RPMI, KOPT, HSB, and MOLT), osteosarcoma (OST, SAOS-2, and U2OS), rhabdomyosarcoma (RMS-MK), colon cancer (COLO-320), breast cancer (MCF-7 and MDA-MB-453), melanoma (G361, G32TG, and A875), thyroid cancer (TTC11), small cell lung carcinoma (H1299), and cervical cancer (HeLa) cell lines and COS7 cells were maintained in RPMI 1640 or DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 µg/mL streptomycin at 37°C in an atmosphere of 5% CO2 in the air. For transient transfection, COS7 cells were transfected with the indicated expression plasmids using FuGene 6 transfection reagent as recommended by the manufacturer (Roche Molecular Biochemicals, Mannheim, Germany). Stable transfections of SH-SY5Y cells were done with the empty plasmid (pcDNA3, Invitrogen, Carlsbad, CA) or with the expression plasmid for FLAG-tagged LMO3-A using LipofectAMINE Plus transfection reagent according to the manufacturer's instructions (Invitrogen). The transfected cells were cultured in the presence of G418 at a final concentration of 400 µg/mL (Sigma Chemical Co., St. Louis, MO). Thereafter, the selection medium was replaced every 3 days. Three weeks after the selection in G418, drug-resistant clones were isolated and allowed to proliferate in medium containing G418.

Reverse transcription-PCR analysis. Total RNA was prepared from cultured cells and human tissues by using Trizol reagent (Life Technologies, Grand Island, NY) or the RNeasy Mini kit (Qiagen, Valencia, CA). Reverse transcription was carried out using random primers and SuperScript II (Invitrogen). Following the reverse transcription, the resultant cDNA was subjected to PCR-based amplification. Oligonucleotides used to amplify LMO3-A, LMO3-B, LMO1, LMO2, LMO4, Ldb1, Ldb2, TAL1, HEN1, HEN2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs were as follows: LMO3-A: forward 5'-ACTGTGCTTACTGAACGGCCTC-3' and reverse 5'-CCGGTCCTTGATCTTTCGGTTG-3'; LMO3-B: forward 5'-TGCAACTCAGACACGCCTAAG-3' and reverse 5'-CCGGTCCTTGATCTTTCGGTTG-3'; LMO1: forward 5'-GCTCCACCCTCTACACCAAG-3' and reverse 5'-CTGCCCTTCCTCATAGTCCA-3'; LMO2: forward 5'-AATGCGGGTGAAAGACAAAG-3' and reverse 5'-CCCCAAAGTGCCTAAGAGTG-3'; LMO4: forward 5'-GCAAGGCAATGTGTATCATCT-3' and reverse 5'-GCATTCTGCATTACTCTGACC-3'; Ldb1: forward 5'-CCAGGGAGCAGAAGACAGAA-3' and reverse 5'-AGAGGCCCAGGTTCCAAG-3'; Ldb2: forward 5'-TAGCCCAAGTGCTGAAACAA-3' and reverse 5'-TAAACTGCCCACAAAACCAA-3'; TAL1: forward 5'-GTTCTTAGGCTGCTGGGATG-3' and reverse 5'-GATTTGGGACTGAGGGAAGA-3'; HEN1: forward 5'-AGAGACTGAGTCGGGCTTCA-3' and reverse 5'-CAGGCGCAGAATCTCAATCT-3'; HEN2: forward 5'-CCCCAAGGGTTGTGGTTTTA-3' and reverse 5'-TCTGAACTTCTGCCCTCATTCTTT-3'; and GAPDH: forward 5'-ACCTGACCTGCCGTCTAGAA-3' and reverse 5'-TCCACCACCCTGTTGCTGTA-3'. Amplified products were electrophoretically separated on agarose gels and visualized by ethidium bromide staining. The gels were photographed under UV illumination.

Northern analysis. A human MTN blot (Clontech, Palo Alto, CA), a nylon membrane on which poly(A)+ RNAs extracted from various human normal tissues were blotted, was used for analysis of the distribution of LMO3 expression in human normal tissues. 32P-labeled probe was prepared by random priming of the 2.5-kb restriction fragment of LMO3 cDNA. The membrane was hybridized overnight at 65°C in a solution containing 7.5% dextran sulfate, 1 mol/L NaCl, 1% N-lauroyl sarcosine, 100 µg/mL heat-denatured salmon sperm DNA, and the radiolabeled probe. The membrane was washed twice in 0.5 x SSC/0.1% N-lauroyl sarcosine at 50°C. Specific signals were obtained by autoradiography.

Section in situ hybridization. Section in situ hybridization was done as described previously (29). A riboprobe was synthesized with digoxygenin-UTP and T3 or T7 polymerase (Roche Molecular Biochemicals). The alkaline phosphatase reaction was done with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Roche Molecular Biochemicals). The riboprobe used for the section in situ hybridization were transcripts of the human cDNA fragments of the LMO3 gene.

Immunohistochemistry. Neuroblastoma tissues were stained with immunoperoxidase method using anti-HEN2 antibody. They included unfavorable neuroblastomas with MYCN gene amplification and favorable neuroblastomas with a single copy of MYCN gene. Neuroblastoma specimens were fixed in 10% buffered formalin and embedded in paraffin, and 3 µm sections were applied to the immunostaining. Before incubation with anti-HEN2 antibody, the sections were treated with 0.05% Pronase in 0.05 mol/L Tris-HCl (pH 7.6) for 5 minutes. The sections were incubated with anti-HEN2 antibody, which was diluted to 1:200 at 4°C overnight. The biotin-streptavidin method (Nichirei, Tokyo, Japan) was done, and the sections were visualized with diaminobenzidine solution. The nuclei were counterstained with hematoxylin.

Immunofluorescent staining. COS7 cells were doubly transfected with the expression plasmids for HA-LMO3-A and FLAG-HEN2. Forty-eight hours after transfection, cells were fixed for 30 minutes with 3.7% formaldehyde in PBS and permeabilized with 0.2% Triton X-100 for 5 minutes, and nonspecific epitopes were blocked for 1 hour in PBS containing 3% bovine serum albumin. The cells were then incubated with a polyclonal anti-HA antibody (1:200 dilution, Medical and Biological Laboratories, Nagoya, Japan) and a monoclonal anti-FLAG antibody (1:50, M2, Sigma Chemical). After three washes with PBS, cells were stained with a FITC- or a rhodamine-conjugated secondary antibody (1:200, Invitrogen). The coverslips were mounted onto glass slides, and the stained cells were viewed using a confocal laser scanning microscope (Olympus, Tokyo, Japan).

Western blot analysis and immunoprecipitation. After transfection, cells were rinsed twice with ice-cold PBS and then lysed immediately with SDS sample buffer. Equal amounts of proteins were separated under denaturing conditions by electrophoresis in 15% polyacrylamide gel containing SDS-PAGE and electrotransferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore, Bedford, MA). After blocking in a solution containing 5% skim milk, the membrane was incubated with a monoclonal anti-FLAG, a polyclonal anti-HA, a polyclonal anti-LMO3, or a polyclonal anti-actin antibody (20-33, Sigma Chemical) and then incubated with a horseradish peroxidase–conjugated goat anti-mouse or anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Protein bands were visualized with an enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). For immunoprecipitation, transfected cells were lysed in EBC buffer [50 mmol/L Tris-HCl (pH 7.5), 120 mmol/L NaCl, 0.5% NP40, 1 mmol/L phenylmethylsulfonyl fluoride] containing protease inhibitor mixture (Sigma Chemical). The precleared soluble supernatants were mixed with a polyclonal anti-HA or a monoclonal anti-FLAG antibody and incubated for 2 hours at 4°C. Protein A-Sepharose beads were then added to the reaction mixtures and incubated for 1 hour at 4°C. The immune complexes were washed with the lysis buffer thrice at 4°C. The bound proteins were resuspended in SDS sample buffer, resolved by SDS-PAGE, and analyzed by Western blotting.

Cell proliferation and soft agar assay. Cells were seeded in triplicate in 24-well plates (5 x 103 per well) in culture medium containing 10% or 1% FBS. Cells were allowed to adhere to the bottom of the cell culture dish for 24 hours. At the indicated times, cells were trypsinized and cell counting was carried out using a Coulter Counter (Coulter Electronics Ltd., Hialeah, Finland). For soft agar assay, 2.5 x 103 cells of the stable transfectants or the parental SH-SY5Y cells were seeded in triplicate in 35-mm cell culture plates containing 0.2% agar and RPMI 1640 supplemented with 10% FBS. After 21 days, colonies with diameters >300 µm were scored as positive.

Tumor formation in nude mice. For tumor formation, 6-week-old female athymic nu/nu mice (Charles River Laboratory, Sulzfeld, Germany) were injected into the femur with 5 x 106 parental SH-SY5Y cells or SH-SY5Y cells transfected with the empty plasmid or with the expression plasmid encoding LMO3-A suspended in 100 µL PBS. Tumor size and body weight were measured once weekly and mice were sacrificed 7 weeks after injection. For histologic examinations, tumor tissues were fixed in fresh 10% buffered formalin and embedded in paraffin. The handling of animals was in accordance with the guidelines of the Chiba Cancer Center Research Institute (Chiba, Japan).

Quantitative real-time PCR. Total RNA prepared from primary neuroblastomas was reverse transcribed into cDNA (SuperScript II kit) and subjected to the real-time PCR. The expression level of GAPDH was measured in all samples to normalize LMO3 and HEN2 expression according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). Oligonucleotide primers and TaqMan probes, which were labeled at the 5' end with the reporter dye 6-carboxyfluorescein (FAM) and at the 3' end with the quencher dye 6-carboxytetramethylrhodamine (TAMRA), were as follows: LMO3: forward 5'-TCTGAGGCTCTTTGGTGTAACG-3', reverse 5'-CCAGGTGGTAAACATTGTCCTTG-3', and probe 5'-FAM-AAACTGCGCTGCCTGTAGTAAGCTCATCC-TAMRA-3' and HEN2: forward 5'-CCCCAAGGGTTGTGGTTTTA-3', reverse 5'-TCTGAACTTCTGCCCTCATTCTTT-3', and probe 5'-FAM-TTGAGTTCTCCTACATTCATCCGCCACAA-TAMRA-3'. Amplification and detection were done using the ABI Prism 7700 Sequence Detection System (Applied Biosystems).

Statistical analysis. Student's t tests were used to explore possible associations between LMO3 expression and other factors. Because the values of the LMO3 expression were skewed, a log transformation was used to achieve the normality in the analyses using t test and Cox regression. The distinction between high and low levels of LMO3 expression was based on the median value (low, LMO3 < 0.2493 e.u.; high, LMO3 > 0.2493 e.u.) regardless of tumor stage, MYCN copy number, or survival. The distinction between high and low levels of HEN2 expression was based on the distribution of the values (low, undetectable; high, detectable). {chi}2 tests were used to examine possible associations between HEN2 expression and other factors, such as tumor stage. Kaplan-Meier survival curves were calculated, and survival distributions were compared using the log-rank test. Cox regression models were used to explore associations among LMO3 expression, HEN2 expression, age, MYCN amplification, mass screening, origin, and survival. Statistical significance was declared if P < 0.05. The statistical analysis was done using Stata Statistical Software Release 7.0 (Stata Corp., College Station, TX, 2001).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of the human LMO3 gene. To identify the genes specifically involved in the genesis and progression of neuroblastoma, we have previously constructed cDNA libraries from the primary neuroblastomas and screened for the differentially expressed genes between the tumors with good and poor clinical outcome (25). One of the cDNA clones, Nbla3267, significantly overexpressed in the poor prognostic neuroblastomas contained a partial nucleotide sequence encoding a LMO family protein, LMO3. To obtain the missing 5' part of the LMO3 cDNA, we screened a cDNA library derived from human fetal brain. From ~6 x 105 recombinant phage clones, 10 independent phage clones were isolated. Sequence analysis revealed that they were divided into two types, designated LMO3-A (145 amino acids) and LMO3-B (156 amino acids), with the different translation initiation sites. The NH2-terminal region of LMO3-A was identical to that of the previously reported LMO3 protein (11). As shown in Fig. 1A, the putative translation initiation sites of LMO3-A and LMO3-B were located within exons 4 and 3, respectively. Because LMO3 is a single gene, it is likely that LMO3-A and LMO3-B arise from differential splicing or alternative promoter usage. Amino acid sequence alignment of LMO3 with the other LMO family proteins (LMO1, LMO2, and LMO4) showed a significant homology among them (Fig. 1B). LIM domains of LMO3 presented 98%, 60%, and 55% amino acid homology with those of LMO1, LMO2, and LMO4, respectively.



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  		Figure 1. Identification of human LMO3-A and LMO3-B and their relation to the other LMO family members. A, schematic representation of the exons of human LMO3 gene. Solid and open boxes, coding and untranslated regions, respectively. B, deduced amino acid sequences of human LMO3-A and LMO3-B and their alignments with those of human LMO1, LMO2, and LMO4. Asterisks, identical amino acid residues. Two LIM domains are boxed. C, tissue-specific expression of LMO3. Human multiple tissue Northern blots containing poly(A)+ RNA were hybridized with a radiolabeled human LMO3 cDNA (top) or with a radioactive probe derived from human ß-actin cDNA (bottom). ß-actin was used as a control for equal loading. The 2-kb band was hybridized ubiquitously, and an additional 1.8-kb band was hybridized in heart and skeletal muscle with the ß-actin probe. D, coordinated expression of LMO3-A and LMO3-B in various human tissues. Total RNA isolated from the indicated human tissues was subjected to RT-PCR analysis to examine the expression levels of LMO3-A, LMO3-B, LMO1, LMO2, and LMO4. GAPDH expression is shown as an internal control.

 
To determine the expression pattern of human LMO3 mRNA, we did Northern blot analysis on a human multiple tissues blot using ß-actin as a control. As shown in Fig. 1C, LMO3 mRNA (~4 kb) was abundantly expressed in brain and at relatively low levels in the heart and lung but not in the other tissues examined. Similar to the adult tissues, LMO3 mRNA was expressed predominantly in fetal brain, with a lower level in fetal lung. We then compared the tissue distribution of LMO3-A expression with those of LMO3-B and the other LMO family gene expression in various human adult and fetal tissues by RT-PCR (Fig. 1D). The expression pattern of LMO3-A was similar to that of LMO3-B, with relatively higher levels in brain, cerebellum, and fetal brain. In contrast, LMO2 and LMO4 were expressed ubiquitously in human tissues, and LMO1 was expressed at higher levels in spleen and fetal brain.

Expression of LMO3 and HEN2 in aggressive neuroblastomas. As described previously, LMO family protein interacts with the nuclear LIM domain–binding protein 1 and 2 (Ldb1 and Ldb2), which act as adaptors for several LIM domain–containing proteins (30–32), and also binds to the basic helix-loop-helix transcription factor, TAL1, to regulate its transcriptional activity (12, 33, 34). Of interest, HEN1 and HEN2 were previously identified based on their homology with TAL1, and it was shown that LMO3 was associated with HEN1 (35). Furthermore, TAL1 was coexpressed with LMO1 or LMO2 in T-cell ALL (36), and double transgenic mice overexpressing TAL1 and LMO1 or LMO2 developed leukemia (37). As shown in Fig. 2A, LMO3 (A and B) and HEN2 were expressed at higher levels in unfavorable neuroblastomas compared with favorable tumors, whereas the levels of LMO1 expression were predominantly high in the favorable tumors. No significant changes in the expression levels of LMO2, Ldb1, and Ldb2 were detected between unfavorable and favorable neuroblastomas. LMO4, TAL1, and HEN1 showed extremely low levels of expression in both types of neuroblastoma. We then studied the expression of these genes in 10 neuroblastoma and 4 T-cell ALL cell lines to examine the presence or absence of the lineage specificity, neuronal or hematopoietic. Consistent with the previous reports (36), LMO2 and TAL1 were coexpressed in T-cell ALL-derived cell lines (RPMI, KOPT, HSB, and MOLT; Fig. 2B). However, of interest, LMO3 and Ldb2 were expressed predominantly in neuroblastoma cell lines compared with the leukemia-derived lines. In addition, HEN2 tended to be less highly expressed in leukemia cells compared with neuroblastoma cells. HEN1 expression was also restricted to neuroblastoma but limited to only a few cell lines. On the other hand, there was no difference in the expression of LMO4 and Ldb1 between neuroblastoma-derived and T-cell ALL-derived cell lines. Interestingly, coexpression of LMO3 and HEN2 was observed in the majority of neuroblastoma cell lines but not in the other tumor-derived cell lines with different origin (Fig. 2C). These results revealed that only LMO3 and HEN2 were expressed at high levels in aggressive neuroblastomas in a neuronal-specific pattern.



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  		Figure 2. Increased expression of LMO3 and HEN2 in unfavorable neuroblastomas and neuroblastoma-derived cell lines. A, expression of LMO3 and LMO-related genes in primary neuroblastomas with favorable (stage I, a single copy of MYCN and high expression of TrkA) and unfavorable (stages III and IV, MYCN amplification and decreased expression of TrkA) characteristics. Total RNA was isolated from the indicated neuroblastoma tissues, reverse transcribed, and amplified by PCR to examine the expression levels of LMO3, LMO3-A, LMO3-B, LMO1, LMO2, LMO4, Ldb1, Ldb2, TAL1, HEN1, and HEN2. Expression of GAPDH serves as an internal control. PCR products were visualized by ethidium bromide staining. B, expression of LMO3 and LMO-related genes in neuroblastoma cell lines without MYCN amplification (SK-N-AS, SH-SY5Y, NB69, and OAN), neuroblastoma cell lines with MYCN amplification (SK-N-BE, NGP, NLF, IMR32, NB1, and KP-N-NS), and ALL cell lines (RPMI, KOPT, HSB, and MOLT). Total RNA prepared from the indicated cultured cells was subjected to RT-PCR analysis. Expression of GAPDH serves as an internal control. C, expression of LMO3 and HEN2 in various tumor-derived cell lines. Total RNA prepared from the indicated culture cells was subjected to RT-PCR analysis as described above. D, section in situ hybridization of neuroblastoma with the LMO3 probe. Serial sections of the favorable neuroblastoma tissue (top left and inset) or the unfavorable one with MYCN amplification (top right and inset) were prepared, and expression of the LMO3 gene was examined by section in situ hybridization. The LMO3 transcripts are positive in unfavorable neuroblastoma. Immunohistochemical staining of HEN2 in primary neuroblastoma tissues. HEN2 is strongly positive in the nucleus of most tumor cells with MYCN amplification (bottom right), whereas it is negative in the favorable neuroblastoma tissue (bottom left).

 
Figure 2D shows the results of in situ hybridization for LMO3 in primary neuroblastomas. LMO3 mRNA was expressed in a stage IV neuroblastoma with MYCN amplification, whereas it was negative in a stage I tumor with a single copy of MYCN and high expression of TrkA. Unfortunately, our antibody raised against human LMO3 protein did not work for the immunohistochemical analysis. The immunostaining of HEN2 was also strongly positive in the nuclei of most tumor cells in MYCN-amplified neuroblastoma, albeit it was negative in favorable subset of the tumor (Fig. 2D).

LMO3 physically interacts with HEN2. Because LMO3 and HEN2 were coexpressed in the majority of unfavorable neuroblastomas as well as neuroblastoma cell lines, we examined whether LMO3 could interact with HEN2 in mammalian cells. Whole cell lysates prepared from COS7 cells transfected with the expression plasmids for HA-tagged LMO3 and FLAG-tagged HEN2 were immunoprecipitated with the anti-HA or with the anti-FLAG antibody followed by immunoblotting with the anti-FLAG or with the anti-HA antibody, respectively. As shown in Fig. 3A, FLAG-HEN2 was coimmunoprecipitated with HA-LMO3. We then examined the subcellular distribution of LMO3 and HEN2. COS7 cells were cotransfected with the expression plasmids for HA-LMO3 and FLAG-HEN2 and double stained with anti-HA and anti-FLAG antibodies. As shown in Fig. 3B, LMO3 as well as HEN2 appear exclusively nuclear. On closer inspection by merging two images, these two proteins colocalized in the nucleus. Consistent with the previous reports (35), HA-LMO3 was coimmunoprecipitated with FLAG-HEN1 under our experimental conditions (Fig. 3C).



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  		Figure 3. LMO3 interacts with HEN2 in mammalian cells. A, coimmunoprecipitation analysis. COS7 cells were transfected with the indicated expression plasmids. Forty-eight hours after transfection, whole cell lysates were prepared and subjected to the immunoprecipitation/Western analysis (top and top middle). Whole cell lysates were monitored on immunoblot for the expression of FLAG-HEN2 (bottom middle) and HA-LMO3-A (bottom). B, nuclear colocalization of LMO3 and HEN2 in cultured cells. COS7 cells were cotransfected with the expression plasmids for HA-LMO3-A and FLAG-HEN2. Forty-eight hours after transfection, cells were fixed and incubated with the polyclonal anti-HA and monoclonal anti-FLAG antibodies. Cells were then processed for double immunofluorescence using the FITC-conjugated anti-rabbit IgG (green) and with the rhodamine-conjugated anti-mouse IgG (red). The merged images (yellow) suggest the nuclear colocalization of LMO3 and HEN2. The phase-contrast images are also shown. C, coimmunoprecipitation of FLAG-HEN1 and HA-LMO3. Whole cell lysates prepared from COS7 cells transfected with the indicated combinations of the expression plasmids were immunoprecipitated with the anti-FLAG antibody followed by immunoblotting with the anti-HA antibody (top). Levels of FLAG-HEN1 and HA-LMO3 were also examined by immunoblotting with the anti-FLAG antibody (middle) and with the anti-HA antibody (bottom), respectively.

 
Overexpression of LMO3 accelerates growth of SH-SY5Y neuroblastoma cells. We addressed the question whether LMO3 could induce cell growth of neuroblastoma. To this end, we transfected the expression plasmid for FLAG-LMO3-A or the empty plasmid into SH-SY5Y neuroblastoma cells and established two stable transfectants overexpressing FLAG-LMO3-A (named as LMO3-15 and LMO3-20). As shown in Fig. 4A, the expression levels of FLAG-LMO3-A were higher in LMO3-15 and LMO3-20 cells than in the parental SH-SY5Y and the control transfectants (V-2 and V-12). LMO3-15 expressed FLAG-LMO3-A at the level comparable with that in LMO3-20. Similar results were also obtained by RT-PCR analysis (Fig. 4B). No obvious morphologic changes could be observed in LMO3-15 and LMO3-20 cells (data not shown). As shown in Fig. 4C, LMO3-15 and LMO3-20 cells proliferated at a much faster rate than the control transfectants and SH-SY5Y cells in culture medium containing 10% serum. More importantly, LMO3-15 and LMO3-20 cells continued to grow exponentially even in the low serum culture medium, whereas the growth of the vector-transfected cells as well as SH-SY5Y cells was significantly suppressed under this condition.



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  		Figure 4. Growth-promoting activity of LMO3 in SH-SY5Y cells. A, stable SH-SY5Y transfectants expressing exogenous FLAG-LMO3-A. SH-SY5Y cells were stably transfected with the empty plasmid or with the expression plasmid for FLAG-LMO3-A and maintained in the presence of G418 (at a final concentration of 400 µg/mL) for 3 weeks. Whole cell lysates prepared from the indicated drug-resistant cell clones in addition to the parental SH-SY5Y cells were subjected to Western blot analysis using the anti-FLAG (top), anti-LMO3 (middle), or anti-actin (bottom) antibody. B, RT-PCR analysis of LMO3 in the indicated stable transfectants along with the parental SH-SY5Y cells. Expression of GAPDH serves as an internal control. C, effects of LMO3 overexpression on cell growth in SH-SY5Y cells. SH-SY5Y cells and the indicated transfectants were grown in the culture medium containing 10% (top) or 1% (bottom) FBS. Cells were harvested at 48-hour time intervals and number of cells was counted in triplicate. Points, means from three independent experiments; bars, SE. D, anchorage-independent growth of LMO3-overexpressing transfectants. The parental SH-SY5Y cells and the indicated transfectants (2.5 x 103 cells per dish) were grown in soft agar medium. After 3 weeks of culture, cells were examined by phase-contrast microscopy (top), and the numbers of colonies with a diameter of >300 µm were counted (bottom). Columns, means from three independent experiments; bars, SE.

 
To examine whether the LMO3-A-overexpressing cells have an ability to grow in soft agar medium, each transfectants were cultured in soft agar medium for 3 weeks. The numbers of colonies with diameters >300 µm formed by each transfectants in soft agar were scored. LMO3-15 and LMO3-20 cells formed large distinct colonies and showed a statistically significant increase in the number of colonies compared with the vector-transfected cells and SH-SY5Y cells (Fig. 4D). These results strongly suggest that overexpression of LMO3 is sufficient to induce malignant transformation in neuroblastoma cells. We also tried to obtain the cells stably transfected with HEN2 but never been successful with unknown reason.

LMO3 induces marked tumor growth in nude mice. SH-SY5Y cells with a single copy of MYCN form tumors in nude mice, although the growth rate is slow compared with that of the other neuroblastoma cell lines with MYCN amplification (38). To examine whether overexpression of LMO3 in SH-SY5Y cells could affect the tumor growth in vivo, we injected the each transfectants into the left flank of athymic nude mice, and the tumor volumes were measured weekly. V-2 and V-12 cells slowly formed tumors with similar kinetics and of similar sizes 35 to 42 days after injection (Fig. 5A). In contrast, the tumors grew rapidly in nude mice implanted with LMO3-15 or LMO3-20 cells. The sizes of the excised tumors from the LMO3-15-implanted mice on day 49 were >10-fold larger than those of control mice (Fig. 5B) and showed histologically undifferentiated neuroblastoma with small round cell shapes and small amounts of stromal components (Fig. 5C).



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  		Figure 5. Tumor growth in nude mice. A, nude mice were injected s.c. with 5 x 106 of SH-SY5Y cells or the indicated stable transfectants and tumor volumes were estimated weekly. Points, mean of 8 to 11 independent tumors. B, photographs of the tumors 49 days after s.c. injection of V-2 (left) and LMO3-15 cells (right) into nude mice. C, paraffin sections of the tumors arising from V-2 (left) and LMO3-15 cells (right) were stained with H&E.

 
Expression of LMO3 and HEN2 is associated with a poor outcome of neuroblastoma. To verify whether a significant relationship could be observed between the expression of LMO3 and/or HEN2 in primary neuroblastomas and the patients' survival, we quantitatively measured the expression levels of LMO3 and HEN2 mRNA in 87 primary tumors by using a quantitative real-time RT-PCR. The values of the levels of LMO3 and HEN2 expression were normalized to that of GAPDH expression [relative expression values (REV)]. The high level of LMO3 expression was significantly associated with high expression of HEN2 (Student's t tests, mean ± SE: 1.43 ± 0.27 REV, n = 48 versus 0.54 ± 0.17 REV, n = 39; P = 0.001), older age (≥1-year-old: 1.37 ± 0.29, n = 32 versus <1-year-old: 0.84 ± 0.21, n = 55; P = 0.008), advanced disease stages (stages III + IV: 1.83 ± 0.35, n = 34 versus stages I + II + IVS: 0.52 ± 0.14; P < 0.00005; Fig. 6A), low levels of TrkA expression (low TrkA: 1.63 ± 0.34, n = 37 versus high TrkA: 0.59 ± 0.15, n = 50; P = 0.0003), MYCN amplification (amplification: 1.91 ± 0.44, n = 27 versus single copy: 0.64 ± 0.13, n = 60; P = 0.0002), and sporadic cases of neuroblastoma (sporadic: 1.68 ± 0.32, n = 39 versus mass screening: 0.51 ± 0.14, n = 48; P < 0.00005). The high level of HEN2 expression was also significantly correlated with high expression of LMO3 ({chi}2 tests: P = 0.001), older age (P < 0.0005), advanced stages (P < 0.0005; Fig. 6B), low TrkA expression (P < 0.0005), MYCN amplification (P < 0.0005), and sporadic cases of neuroblastoma (P < 0.0005). Thus, high expression of LMO3 and HEN2 was well associated with conventional markers indicating the poor prognosis of neuroblastoma.



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  		Figure 6. Expression of LMO3 and HEN2 mRNA in 87 primary neuroblastomas. A, expression levels of LMO3 (left) and HEN2 (right) transcripts in 87 primary neuroblastoma samples categorized by the patient's clinical stage were examined by a quantitative real-time RT-PCR. Relative expression levels of LMO3 or HEN2 mRNA were determined by calculating the ratio between GAPDH and LMO3 or HEN2. Bars, median levels of LMO3 or HEN2 expression in each stage; open and closed circles, samples from patients who are alive and dead, respectively. B and C, Kaplan-Meier survival curves of patients with neuroblastomas based on high or low expression of LMO3, HEN2 (B), or LMO3 and HEN2 (C).

 
We next tested if expression levels of LMO3 and HEN2 could have prognostic significance in primary neuroblastomas. The results for log-rank tests showed that high expression of LMO3 or HEN2 was significantly associated with poor survival (P = 0.0002 and 0.0005, respectively; Fig. 6C and D). Remarkably, the combination of high expression of both LMO3 and HEN2 showed the significantly worse prognosis compared with the other combinations of LMO3 and HEN2 expression levels as shown in Fig. 6E. As expected, older patients and the patients with advanced tumors, low expression of TrkA, amplified MYCN, and the tumors found by mass screening were associated with short time to survival (P < 0.00005). However, the adrenal origin of the tumor was not associated with the outcome (P = 0.19; data not shown).

The univariate analysis suggested that LMO3 expression (P < 0.0005), HEN2 expression (P = 0.004), age (P < 0.0005), MYCN amplification (P < 0.0005), and mass screening (P = 0.001) were of prognostic importance, supporting the results of the log-rank test (Table 1). Furthermore, the multivariate analysis showed that LMO3 expression was significantly associated with survival after controlling HEN2 expression (P = 0.005), age (P = 0.005), mass screening (P = 0.044), and origin (P < 0.0005), suggesting that LMO3 expression was an independent prognostic factor from the other factors (Table 2). LMO3 expression was marginally associated with survival after controlling MYCN amplification (P = 0.066). On the other hand, because HEN2 expression was highly associated with age, MYCN amplification, and mass screening, it was not significantly associated with survival after controlling age, MYCN amplification, and mass screening in the corresponding multiple Cox regression models (data not shown).


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  		Table 1. Simple Cox regression models using LMO3 expression and dichotomous factors of HEN2 expression, age, MYCN amplification, mass screening, and origin (n = 87)

 

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  		Table 2. Multiple Cox regression models using LMO3 expression and dichotomous factors of HEN2 expression, age, MYCN amplification, mass screening, and origin (n = 87)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have identified that both LMO3 and HEN2 are expressed at higher levels in aggressive neuroblastomas especially with MYCN amplification than those with favorable prognosis. Coexpression of LMO3 and HEN2 has been observed almost exclusively in neuroblastoma cell lines, not the other lines, suggesting that their expression and function are neuronal specific. Furthermore, LMO3 physically interacted with HEN2 in mammalian cells. The functional significance of LMO3 expression was shown by a stable transfection into SH-SY5Y neuroblastoma cells, colony formation in soft agar, and tumor growth in nude mice, all of which have suggested that LMO3, probably by interacting with endogenous HEN2, markedly promotes the tumor growth. Indeed, the tumors with high expression of both LMO3 and HEN2 have shown the worst prognosis in the analysis of 87 primary neuroblastomas. Thus, our results suggested that, in concert with HEN2, the neuronal specifically expressed LMO3 plays an important role in the tumorigenesis of neuroblastoma. Our observation is strikingly intriguing because that LMO1 or LMO2 is already known to be the oncogene in T-cell acute lymphoblastic leukemia and that LMO4 has recently been implicated in the genesis of breast cancer (4, 9).

We have identified a Nbla3267/LMO3 clone from the screening of differentially expressed genes between favorable and unfavorable subsets of neuroblastoma. LMO3 was one of the genes expressed at higher levels in the latter than the former (24), like MYCN oncogene and DDX1, a DEAD box gene coamplified with MYCN in aggressive neuroblastomas. In the development of hematopoietic system, LMO1 and LMO2 form a transcriptional complex with Ldb1, a LIM domain–binding protein, and a basic helix-loop-helix protein TAL1, which was identified as an oncogene at the translocation breakpoint in T-cell ALL (4–7). From the analogy with the LMO1 or LMO2 transcriptional machinery in T-cell ALL, we searched for the similar complex in the neuronal system by using the different subsets of primary neuroblastoma and the cell lines in comparison with the T-cell ALL cell lines. As a result, the neuronal-specific pattern of expression was observed in LMO3, Ldb2, HEN1, and HEN2, among which LMO3 and HEN2 were significantly highly expressed in the unfavorable subset of neuroblastomas with MYCN amplification compared with the favorable subset. This result strongly suggested that LMO3 may function in collaboration with HEN2 in advanced stages of neuroblastoma. Indeed, both genes were coexpressed only in neuroblastoma derived-cell lines, not in other tumor-derived ones, suggesting that their expression is lineage specific. Furthermore, LMO3 and HEN2 physically interacted in mammalian cells, albeit with weak interaction between LMO3 and HEN1 (35). Thus, these results also suggest that LMO3 and HEN2 form a neuronal cassette mimicking the hematopoietic complex composed of LMO2 and TAL1 and regulate the growth of neuroblastoma.

The neuronal-specific basic helix-loop-helix transcription factors, HEN1 and HEN2, were originally identified from the cDNA library of a neuroblastoma cell line based on cross-hybridization with TAL1 (14, 15). Their expression was restricted to the developing nervous system and a neuroblastoma cell line. However, their function has long been unclear. Recently, Bao et al. have reported that HEN1 interacts with LMO proteins by yeast two-hybrid screen and that Xenopus HEN1, in concert with XLMO3, is a critical regulator of neurogenesis (35). This prompted us to test our hypothesis both in vitro and in vivo. As the results, we found that the SH-SY5Y neuroblastoma cells stably overexpressing LMO3, presumably by acting with endogenous HEN2, gained rapid cell growth in the culture medium with 10% or 1% serum, in the soft agar medium, and in nude mice. These suggested that LMO3 is a neuronal-specific oncogene in neuroblastoma, without any rearrangement of the LMO3 gene (data not shown). However, we failed to establish a stable SH-SY5Y cell line transfected with HEN2. It is presumed that overexpression of HEN2 might have caused cell death or growth arrest in the cells, albeit the reason is elusive.

The double transgenic mice overexpressing LMO2 and TAL1 displayed a more rapid development of leukemia compared with those overexpressing LMO2 alone, suggesting that LMO2 and TAL1 act synergistically through their complex formation in the development of leukemia (13). Of note, Ono et al. reported that LMO2 and TAL1 act as cofactors for GATA3 to induce the expression of the retinaldehyde dehydrogenase 2 gene in T-cell ALL (39). On the other hand, a stable complex comprising LMO2, TAL1, and GATA1 was required to promote erythroid differentiation (32). Therefore, LMO3 and HEN2 may also form a nuclear complex, including family members of GATA to regulate cell growth and differentiation in neuroblastoma. Our preliminary data have suggested that GATA2, GATA3, GATA4, and GATA6 are highly expressed in neuroblastoma cell lines, among which GATA4 and GATA6 are predominantly coexpressed in neuroblastoma cell lines compared with T-cell ALL lines. Thus, LMO3 and HEN2, in collaboration with GATA and Ldb families, may play a role in determining cell fate in both neural development and neuroblastoma genesis, although this hypothesis needs to be elucidated. Recently, it has been shown that LMO3 enhanced the ability of HEN1 through the physical interaction to transactivate the expression of Neurogenin-1 as well as NeuroD and thereby induced the neuronal differentiation in frog embryos (35). We tested if this is the case in the neuroblastoma cells. However, our preliminary results suggested that the LMO3/HEN2 complex does not transactivate the Neurogenin-1 as well as NeuroD promoter in neuroblastoma cell lines,5 although it is unclear if the complex could work in normal neuronal development. Thus, like LMO2, alterations in the LMO3-containing transcriptional complex might differentially regulate expression of the downstream target genes closely involved in neuronal differentiation or tumor formation.

It is striking that high levels of expression of both LMO3 and HEN2 are significantly associated with the poor prognosis in primary neuroblastomas. This clearly reflects how importantly both genes are functioning in the progression of neuroblastoma in vivo. Of interest, expression of either gene is well correlated with MYCN amplification, raising the possibility that they might be the downstream targets of MYCN. However, we could not confirm it in human neuroblastoma cell line SH-EP in which MYCN was regulated under the control of the rTet-inducible expression system (40). In agreement with this, cDNA microarray-based screening for the genes induced in the MYCN-amplified neuroblastoma cells thus far failed to detect either LMO3 or HEN2 (41, 42). The link between LMO family molecules and the other oncogenes or tumor suppressor genes is also important. Despite the lack of prognostic significance, LMO4 overexpressed in breast cancer seems to be indispensable in the mammary carcinogenesis because it interacts with both BRCA1 and CtIP to repress the BRCA1 function (10). This suggests that, similarly to LMO4, LMO3 may also have the interacting partners related to the tumorigenesis. Thus, LMO3 and HEN2 as well as their associated molecules might be good candidates for the future targets of the therapy against aggressive neuroblastomas.


   Acknowledgments
 
Grant support: Grant-in-Aid from the Ministry of Health, Labour and Welfare for Third Term Comprehensive Control Research for Cancer, Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science. M. Aoyama is an awardee of the Research Resident Fellowship from the Foundation for Promotion of Cancer Research in Japan.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank S. Sakiyama for critical reading of the article; A. Morohashi, N. Kitabayashi, H. Murakami, and N. Sugimitsu for excellent technical assistance; and the following institutions and hospitals for supplying the tumor samples: Department of Pediatric Surgery, Iwaki Kyoritsu Hospital; Departments of Pediatrics and Pediatric Surgery, Aichi Medical University; Department of Pediatrics, Nara Hospital; Department of Pediatrics, Kyoto Prefectural University of Medicine; Department of Pediatric Surgery, Kimitsu Central Hospital; Department of Surgery, Gunma Children's Medical Center; Department of Pediatrics, Sapporo National Hospital; Departments of Pediatrics, Pediatric Surgery, and General Surgery, Jichi Medical School; Departments of Pediatrics and Pediatric Surgery, Kagoshima University; Department of Pediatrics, Juntendo University; Department of Pediatric Surgery, Showa University; Department of Pediatric Surgery, Niigata University; Departments of Surgery and Pathology, Chiba Children's Hospital; Department of Pediatric Surgery, Chiba University; Department of Pediatric Surgery, Osaka City General Hospital; Department of Pediatric Surgery, Tsukuba University; Department of Pediatric Surgery, Tokai University; Department of Surgery, Tokyo Metropolitan Kiyose Children's Hospital; Department of Pediatric Surgery, Tohoku University; Tomor Board, Hyogo Prefectural Kobe Children's Hospital; and First Department of Surgery, Hokkaido University.


   Footnotes
 
5 Unpublished data. Back

Received 12/28/04. Revised 3/ 9/05. Accepted 3/23/05.


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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