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DNA AND CELL BIOLOGYVolume 24, Number 7, 2005© Mary Ann Liebert, Inc.Pp. 432–437

Differentially Expressed Genes in Endothelial Differentiation

HIDESHI ISHII,1 KOSHI MIMORI,2 MASAKI MORI,2 and ANDREA VECCHIONE3

ABSTRACT

By screening differentially expressed genes in mouse embryonic stem (ES) cells by subtractive hybridization,we identified three conserved but uncharacterized genes encoding bromodomain containing 3 (BRD3), pro-tein lysine methyltransferase (PLM), and kelch domain containing 2 (KLHDC2), which were downregulatedduring endothelial differentiation. An RNA blot study showed that these genes were markedly expressed inundifferentiated ES cells, whereas the expression was reduced upon endothelial differentiation; a study ofmouse endothelium showed a significant reduction in the expression of BRD3. A study of human BRD3, lo-cated on chromosome 9 at q34, a region susceptible to genomic rearrangement, showed an altered expressionin 4 of 12 patients with bladder cancer, compared with adjacent noncancerous tissues. Taken together withthe result of siRNA inhibition showing the positive regulation of cell proliferation by BRD3, it is suggestedthat this molecule plays a role in allowing cells to enter the proliferative phase of the angiogenic process.

INTRODUCTION

THE VESSELS in an embryo are assembled from endothelialprecursors in a process known as vasculogenesis (Coffin

and Poole, 1988; Risau et al.,1988). The most typical and ear-liest events of vasculogenesis occur in the yolk sac, where ex-tra-embryonic mesoderm develops blood islands composed of hemangioblasts, common precursors of endothelial andhematopoietic cells. Although vasculogenesis occurs typicallyat limited sites, the primitive network expands by sprouting, an-giogenesis, or intussusception at many stages of development(Folkman, 1995).

Pathological angiogenesis is a hallmark of cancer and vari-ous ischemic and inflammatory diseases (Carmeliet and Jain,2000). Different from the finely regulated physiological pro-cess, the growth of tumor vessels involves various mechanisms(Carmeliet and Jain, 2000): the host vascular network expandsthrough the budding of endothelial sprouts or formation ofbridges in tumor tissues, so-called angiogenesis; tumor vesselsremodel and expand by the insertion of interstitial tissuecolumns into the lumen of preexisting vessels, intussusception;and endothelial precursors are circulated from bone marrow intotumors, contributing to the endothelial lining of tumor vessels,vasculogenesis. Various molecules and genes are involved inthe different mechanisms promoting vascular growth, includ-

ing members of the vascular endothelial growth factor (VEGF)and angiopoietin (Ang) family, although exactly how is not en-tirely clear (Yancopoulos et al., 2000). In tumor angiogenesis,inhibitory molecules likely play an important role (Carmelietand Jain, 2000). The temporal and spatial expression of theseregulators is deregulated, and differs somewhat from the phys-iological function and structure; for example, tumor vesselsseem to lack functional perivascular cells, which protect ves-sels against alterations to the oxygen or hormonal balance (Ben-jamin et al., 1999); importantly, the vessel wall is not formedby a homogeneous layer of endothelial cells (Jain, 1988), and,instead, may be lined with cancerous cells or a mosaic of can-cer and endothelial cells. Indeed, it is reported that 15% of ves-sels in xenografted and spontaneous human colon carcinomasare mosaic in nature (Carmeliet and Jain, 2000), suggesting di-verse functions of angiogenic cells in physiological and can-cerous processes.

In this report, we performed subtractive hybridization tostudy preferentially expressed genes in mouse embryonic stem(ES) cells, compared with differentiated cells. Three uncharac-terized genes were identified including that encoding bromo-domain containing 3 (BRD3). A study of human BRD3, locatedat 9q34, a region susceptible to genomic rearrangement in tu-mors (Hopman et al., 2002; Williams et al., 2002), indicatedan altered expression in bladder cancer. This, together with the

1Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan.2Institute of Bioregulation, Kyushu University, Ohita, Japan.3Division of Pathology, University of Rome, La Sapienza, II Faculty of Medicine, Hospital “Santo-Andrea,” Italy.

finding that siRNA inhibition of BRD3 resulted in a reductionof cell proliferation, suggested that BRD3 is involved in al-lowing endothelial cells to enter the proliferative phase of theangiogenic process.

MATERIALS AND METHODS

Cell culture and tumor samples

Mouse vascular endothelium (SVEC) and mouse pancreaticendothelium (SVR) were purchased from the American TypeCulture Collection (ATCC, Manassas, VA). Human embryonickidney fibroblast 293 cells were purchased from Q-biogen(Irvine, CA). Mouse ES cells (Incyte Genomics, Palo Alto, CA)were cultured on a feeder cell layer of mouse embryonic ESQfibroblasts (Incyte Genomics), in DMEM supplemented with10% ES-certified serum (Invitrogen-Gibco, Carlsbad, CA) and1000 units/ml of leukemia inhibitory factor (LIF) (Chemicon,Temecula, CA). ES cells were trypsinized every second day forpassages on freshly prepared feeder cells; the feeders were pre-treated on 0.1% gelatin-coated dishes with 0.01 mg/ml of mito-mycin C (Sigma) for 3 h to induce growth arrest. For endothe-lial differentiation in vitro, 1 � 105 ES cells were cultured on theBalb/c 3T3 cells (ATCC) as alternative feeder cells with colla-gen type IV-coated dishes (BD Biosciences, Palo Alto, CA) inEGM-2 medium (Clonetics-Cambrex, Baltimore, MD) withoutLIF, but supplemented with Single Quots, a cocktail of growthfactors and cytokines (Clonetics-Cambrex) including vascular en-dothelial growth factor (VEGF), basic fibroblast growth factor(bFGF), epithelial growth factor (EGF), and insulin-like growthfactor-1 (IGF-1), as recommended by the manufacturer. After 4days, cells were harvested and then filtered through a nylon meshfilter (70 �m) to remove clumps prior to FACS analysis. Afterthe surface was stained with antimouse Flk-1 monoclonal anti-body (Chemicon), Flk-1–positive cells were sorted on a FACSFlow Cytometer (Becton Dickinson, Franklin Lakes, NJ). Cellswere further cultured in the same conditions for various periodsto extract RNA. The interference of gene expression by siRNAwas performed (Elbashir et al., 2002). Briefly, a set of compli-mentary RNA for the human Brd3/RINGL3/ORFX gene, 5�-GUAGCAGUGAGGAGAGCUCTT-3� and 5�-TTCAUCGU-CACUCCUCUCGAG-3�, was synthesized (Integrated DNATechnologies, Coralville, IA), annealed and transfected into cellsusing the TransIT-TKO transfection reagent (Mirus, Madison,WI). For transfection of D18 cDNA with a pcDNA expressionplasmid (Invitrogen-Gibco), the Fugene 6 reagent (Roche, Indi-anapolis, IN) was used, according to the manufacturer’s instruc-tions. Uncultured, paired primary tumors and adjacent nonneo-plastic tissues of the bladder (12 cases) and pancreas (10 cases)were obtained from the University of Rome, La Sapienza, II Fac-ulty of Medicine, Hospital “Santo-Andrea” and the Institute ofBioregulation, Kyushu University, after approval by the Institu-tional Review Boards.

Mitogenic assay

The mitogenic assay was performed by pulse labeling with[3H] thymidine. Briefly, 2 � 104 cells/well were seeded in a24-well plate in DMEM with 10% FCS. After incubation for24 h, cells were transfected with siRNA or plasmid. After ad-

ditional incubation for 24 h, the serum concentration of themedium was reduced to 0.1% for 24 h. Cells then were pulse-labeled with 0.5 �Ci/ml of [3H] thymidine (925 GBq/mmol;Amersham, Tokyo, Japan) in 10% FBS medium for 6 h. Thecells were subsequently washed with phosphate-buffered saline,and 10% trichloroacetic acid-insoluble radioactivity was deter-mined. Under the conditions used, the cells stayed alive duringthe [3H] thymidine incorporation assay as shown by trypan blueexclusion.

cDNA isolation

Differentially expressed genes were screened with a cDNAsubtraction system (BD Biosciences Clontech, Palo Alto, CA),according to the manufacturer’s instructions. Briefly, tester cDNAs from undifferentiated ES cells were subtracted by com-petitor cDNAs from endothelially differentiated ES cells. Af-ter two-step subtractive hybridizations, cDNAs were amplifiedby PCR and ligated to pST-Blue vector plasmid (EMD Bio-sciences Novagen, San Diego, CA). Plasmid clones were ran-domly removed to perform DNA sequencing with the RISA384 capillary DNA sequencing system (Shimadzu, Kyoto,Japan). Sequence data were analyzed with the NCBI nucleotidedatabases (http://www.ncbi.nlm.nih.gov/BLAST/ and http://www.ncbi.nlm.nih.gov/dbEST/) to classify redundant clones.Full-length cDNAs were cloned by screening a cDNA libraryand using a 5�-rapid-amplification cDNA-ends (RACE) system(BD Biosciences Clontech).

RNA expression

Total and polyA� RNAs were extracted with the Qiagen ex-traction system (Qiagen, Tokyo, Japan). The RNA blot studywas performed as described (Ausubel et al., 1989). Briefly, 3�g of polyA� RNA or 15 �g of total RNA was transferred toa nylon membrane (Amersham) and crosslinked under UV light.The DNA probes were D18, D10, and D20 cDNAs, which wereamplified by reverse transcription (RT)-PCR as described be-low, subcloned into the vector, and sequenced. The hybridiza-tion probes were radiolabeled by random primer extension. Un-incorporated nucleotides were removed by spin filtration in aG-50 column. Heat-denatured probes were added to filters inthe Perfect Hybridization Buffer (Sigma, St. Louis, MO). Af-ter incubation for 16 h at 65°C, filters were washed under highstringent conditions and exposed to X-ray film. For the RT-PCR, the following primers were used for PCR amplification:D10-RTPCR-1/F, 5�-ACA CAA GCT ATA CAT GAT GTCTTC AG-3�; D10-RTPCR-2/R, 5�-ACA CAT ATC CCA TAAAGG AAT CAG AG-3�; D18-RTPCR-1/F, 5�-TGC ACG ACTACC ACG ACA TCA TCA AG-3�; D18-RTPCR-2/R, 5�-TCCACG GGC TCA TCT GGC ATC TTG G-3�; D20-RTPCR-1/F,5�-GTG GAT GTG CCA ACA ACT TGC TTG TC-3�; D20-RTPCR-2/R, 5�-GTT TTG GAA GGC AGT TCC ATG AGTTGG-3�. The PCR profile was: a cycle of 94°C for 1 min, fivecycles of 94°C for 8 sec, 57°C for 10 sec, and 72°C for 1 min,and 30 cycles of 94°C for 8 sec, 59°C for 10 sec, and 72°C for1 min, followed by a cycle of 72°C for 7 min. The primers usedfor RT-PCR amplification encompass exon–intron junctions.As a negative control, no amplification occurred in the absenceof RTase and without cDNA templates. The accuracy of PCRamplification was confirmed by DNA sequencing.

DIFFERENTIALLY EXPRESSED GENES IN ENDOTHELIAL DIFFERENTIATION 433

Mutation search

Human genomic DNA was extracted from cancerous and ad-jacent noncancerous regions and subjected to PCR amplifica-tion with 11 sets of primers for D18 exons: the primer sequenceswill be provides upon request. PCR amplification was per-formed for 35 cycles, and the products were subjected to DNAsequencing.

Protein study

Immunoblot analysis was performed as described (Ausubel etal., 1989). Briefly, protein lysates were separated by SDS-PAGE,transferred to membranes, and probed with rabbit antihumanBrd3/RINGL3/ORFX serum, which was developed against a syn-thetic polypeptide, (KLH)-DPKQAKVVARRESGGR (Invitro-gen- Research Genetics, Carlsbad, CA). Antiactin monoclonalantibody (ICN, Irvine CA) was used as a control. Signal was de-tected with appropriate secondary antibodies in the ECL system(Amersham).

RESULTS

Isolation of differentially expressed genes

A central event in angiogenesis is the proliferation of bloodvessels, which also plays a major role in the progression of anumber of inflammatory and neoplastic diseases. The processis responsible for the switch of endothelial cells from an an-tiangiogenic to an angiogenic phenotype. To identify novel ac-tivated and proliferation-related molecules in endothelial cells,a subtractive hybridization was performed using RNA extractedfrom mouse ES cells, before and after culturing in the mediumfor endothelial differentiation, as described in the materials andmethods. After subtractive hybridization, we isolated and se-quenced 310 clones, which were classified by nucleotide se-quence analysis into six groups: (1) 141 clones (45%) identicalto mouse expressed sequence tags (ESTs) (�95% homologousover 200 bp); (2) 13 clones (4%) similar to mouse ESTs(40–95% homologous over 200 bp); (3) 0 clones (0%) identi-cal to ESTs in other species (�95% homologous over 200 bp);(4) 50 clones (16%) similar to ESTs in other species (40–95%homologous over 200 bp); (5) 70 unique clones (23%) (�40%homologous over 200 bp); (6) 36 short insert (12%) (�200 bp).In category 1, we identified three highly redundant clones, D18(48 clones), D10 (29 clones), and D20 (8 clones), and analyzedthem further.

Three isolated cDNAs

The RNA blot study with isolated cDNA fragments as probesshowed that the expression of D10, D20, and, more noticeably,D18, was downregulated during the endothelial differentiationof ES cells, while the control actin was constitutively expressed(Fig. 1). Extension of cDNA by the 5�-RACE method and thedatabase search indicated that the mouse D18 gene is locatedon chromosome 2 and encodes cDNA which is �98% homol-ogous to mouse bromodomain containing 3, Brd3 (accessionno. NM_023336, BC031536) and to mouse bromodomain-con-taining FSH-like protein, Fsrg2, which is a putative mouse ho-molog of the Drosophila gene female sterile homeotic (acces-

sion no. AF269193). The database search indicated that mouseD18/Brd3/Fsrg2 was �95% homologous in peptide sequenceto human bromodomain containing protein 3 (Brd3), to RING3-like gene (RINGL3), and to open reading frame X (ORFX) (ac-cession no. NP_031397, NM_007371, and BC032124).

Molecular cloning and a database search showed that mouseD10 cDNA was �98% homologous to the “D12Ertd771e” gene(accession no. NM_028262; AK011993), which is located onchromosome 12 at F1. The encoded protein is predicted to bea lysine methyltransferase enzyme with two SET domains,which appears to be involved in protein–protein interaction, likeproteins that display similarity with dual-specificity phos-phatases (dsPTPases). Mouse D10 /”D12Ertd771e” has 26%similarity to an Arabidopsis thaliana protein, putative ribulose-1,5 bisphosphate carboxylase/oxygenase large subunit N-methyltransferase.

Molecular cloning and a database search showed that mouseD20 cDNA was 99% homologous to mouse kelch domain con-taining 2, KLHDC2 (accession no. BC005581), which is lo-cated on chromosome 12 at C3. The kelch-repeat protein is arecently identified actin-binding protein. It is characterized bytandemly arranged motifs of about 50 amino acids. Previousstudy showed that most members of the kelch-repeat familywere cytoskeletal proteins implicated in various cellularprocesses, such as actin cytoskeleton interaction, cytoplasmicsequestration of transcription factors and cell morphology (Xuet al., 2003). Some of the family members play important rolesin tissue development (Xu et al., 2003). The study of pro-tein–protein interaction may be useful for elucidation of thefunction of those proteins.

Expression in various tissues and cell lines

To study the expression of the D10, D18 and D20 genes inmouse endothelial cell lines, an RNA blot analysis with en-dotheliums, SVEC and SVR, was performed. Results showedthat the expression of D18, and to a lesser extent D10, was re-duced in SVEC and SVR, compared to mouse ES cells; the

ISHII ET AL.434

FIG. 1. Differential expression of endothelially differentiatedES cells. The endothelial differentiation was induced as de-scribed in the text, and RNA was extracted to perform the RNAblot analysis. PolyA� RNA was separated by electrophoresis ina gel, transferred to a nylon membrane, and hybridized with ra-diolabeled probes. Lane 1, 0 days; lane 2, 2 days; lane 3, 4 days;and lane 4, 6 days after the induction of differentiation in vitro.Actin probe was used as a control.

D10, D18, and D20 genes were expressed substantially in un-differentiated ES cells, and the control actin was expressed con-stitutively (Fig. 2). No alteration of D20 expression in SVECand SVR was apparent. The RNA blot study with mouse em-bryonic and adult tissues indicated that the three genes were ex-pressed ubiquitously at various levels (Fig. 3), suggesting a spe-cific altered expression in endothelial cells and consistent withthe concept that the differentially expressed genes may be in-volved in the proliferation and differentiation of the endothe-lium.

Gene mutation study

The data base search showed that human Brd3/RINGL3/ORFX, a homolog of D18/Brd3/Fsrg2, was located at 9q34, aregion which is frequently rearranged in human tumors, in-cluding bladder cancer (Hopman et al., 2002; Williams et al.,2002), whereas the human homologs of mouse D10/“D12Ertd771e” and D20/KLHDC2 protein are located at 14q32and 14q21.3, respectively. To assess the contribution of D18gene to uncontrolled cell growth and tumor development, weperformed a mutation search and expression study. Although agenomic mutation search indicated no alterations in the DNA se-quence of the human Brd3/RINGL3/ORFX gene in primary tu-mors of bladder and pancreas, the RNA blot study showed thatthe expression of the gene was increased in 4 of 12 cases of blad-der tumor, compared with adjacent normal tissues (Fig. 4).

Effect of altered expression of Brd3

To examine whether the downregulation of Brd3 expressioncontributes to the regulation of cell growth, the expression ofhuman Brd3/RINGL3/ORFX was inhibited by siRNA interfer-ence. Immunoblot analysis with antihuman Brd3/RINGL3/ORFX serum indicated that the siRNA treatment resulted in aninhibition of the expression of Brd3/RINGL3/ORFX in 293cells, whereas the negative control siRNA did not affect the ex-pression, and actin was expressed constitutively, showing a spe-cific inhibition of Brd3/RINGL3/ORFX by the siRNA treat-

ment (Fig. 5A). Cell counts indicated that the treatment withBrd3/RINGL3/ORFX siRNA resulted in the inhibition ofgrowth, compared with that of control siRNA-treated cells; theeffect was apparent 4 or 5 days after transfection (Fig. 5B).Since the trypan blue exclusion indicated that the stained, deadpopulation in Brd3/RINGL3/ORFX siRNA-treated cells wassimilar to that of control siRNA-treated cells, it is suggestedthat the cell count reduction by Brd3/RINGL3/ORFX siRNAwas due to the inhibition of cell proliferation. To examinewhether the DNA synthesis was altered by inhibition of Brd3/RINGL3/ORFX protein, the uptake of [3H] thymidine was ex-amined. Results showed that the uptake was reduced after treat-ment with siRNA against Brd3/RINGL3/ORFX, but neitherwith control siRNA nor with the expression vector (Fig. 5C),suggesting that the Brd3 expression plays a role in the regula-tion of cell growth at least through involvement at a phase ofDNA synthesis.

DISCUSSION

In the present study, we isolated three uncharacterized geneswhose expression was altered during endothelial differentiation.Mouse D18/Brd3/Fsrg2 was expressed preferentially in undif-ferentiated ES cells, and the use of siRNA inhibition suggestedthat D18/Brd3/Fsrg2 regulates cell proliferation. As observedin D18/Brd3/Fsrg2 protein, bromodomains are �110 aminoacids long, and found in many chromatin-associated proteins.Bromodomains can interact specifically with acetylated lysineand, in many cases, regulate gene expression. Although thefunction of the encoded protein is not characterized, humanBrd3/RINGL3/ORFX, a homolog of mouse D18/Brd3/Fsrg2,lies at 9q34, a region which contains several major histocom-

DIFFERENTIALLY EXPRESSED GENES IN ENDOTHELIAL DIFFERENTIATION 435

FIG. 2. Expression analysis of identified cDNAs in endothe-lial cell lines. The RNA blot analysis was performed, as in Fig-ure 1. Lane 1, SVEC, vascular endothelium; lane 2, SVR, pan-creatic endothelium; lane 3, mouse ES cells. Actin probe wasused as a control.

FIG. 3. Expression analysis of identified cDNAs in mouseembryo and adult tissues. RNA blot membranes were hy-bridized with radiolabeled probes, as in Figure 1. Lanes 1–4:7; 11; 15; and 17-day-old embryos; lanes 5–12: heart, brain,spleen, lung, liver, skeletal muscle, kidney, and testis of adultmice.

patibility complex (MHC)-related genes (Thorpe et al., 1997);the region is also rearranged in human tumors, such as bladdercancer (Hopman et al., 2002; Williams et al. 2002). The pre-sent study indicated that the expression of human Brd3/RINGL3/ORFX was upregulated in bladder tumors (4 of 12cases), supporting the concept that the gene plays a role in cellproliferation. A bromodomain-containing protein was reportedto be expressed differentially in lymphocytes activated by mi-togens, which was detected with the RNA arbitrarily primed-PCR method (Kaneko et al., 2002), suggesting that bromod-omain-containing protein may be involved in the activation oflymphocytes induced by mitogens, through the involvement ofcytokines, surface molecules, and nuclear proteins (Kaneko etal., 2002). Human Brd3/RINGL3/ORFX has high homology tothe gene encoding RING3, a human MHC-linked homologous,serine/threonine kinase at 6p21.3. Structures of both Brd3/RINGL3/ORFX and RING3 are highly conserved and ubiqui-tously expressed in human adult and fetal tissues (Thorpe et al.,1997), which is consistent with the present observation. It issuggested that those genes may have arisen from an ancient du-plication in a common ancestral chromosome (Thorpe et al.,1997). Taken together with the present study, it is suggestedthat mouse and human homologous bromodomain-containingproteins exert multiple functions, which play a role in the acti-vation of lymphocytes and in the regulation of the proliferativephase of the angiogenic process through gene expression.

Interestingly, a recent subtractive immunization approach toidentifying novel proliferation-related proteins in human en-dothelial cells resulted in the identification of an 85-kDa pro-tein (p85); DNA sequencing of the clone corresponding to p85showed more than 93% homology to RING3 kinase (BelAibaet al., 2001), which is believed to be another member of thebromodomain-containing protein family that transactivates inthe nucleus the promoters of a number of the E2F family oftranscription factors (BelAiba et al., 2001). p85 may representa signaling target activated by a potent angiogenic cytokine,such as VPF/VEGF165 and bFGF, that allows endothelial cellsto enter the proliferative phase of the angiogenic process (Bel-Aiba et al., 2001). Thus, it is suggested that RING3 and its twohomologues, p85 and Brd3/RINGL3/ORFX, exert diverse andcomplimentary functions, which have a role in the growth anddifferentiation of endothelial cells, and are new signaling tar- gets of angiogenesis. More recently, Brd4 protein, which con-

tains a double bromodomain, was reported to bind to chromatinand to regulate cell cycle progression at multiple stages, throughinteraction with signal-induced proliferation-associated protein1 (SPA-1), a member of the Rap GTPase-activating protein(GAP) family, suggesting a novel link between Brd4 and a GT-Pase-dependent mitogenic signaling pathway (Farina et al.,2004). Our study did not detect any interaction between humanBrd3/RINGL3/ORFX and SPA-1 (data not shown), suggestingthat human Brd3 and Brd4 might be involved in distinct signaltransduction pathways.

In summary, our subtractive hybridization approach allowedthe identification of differentially expressed genes duringmouse endothelial differentiation. The differential expressionof Brd3 was more apparent than that of any of the other genes,and the siRNA experiment indicated that the gene may be in-volved in the growth of endothelial cells, suggesting that Brd3plays a role in switching on and off the growth and differenti-ation of endothelial cells.

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FIG. 4. Expression analysis of primary tumors and adjacentnoncancerous tissues of human bladder. Total RNA was ex-tracted, separated by electrophoresis and transferred to a mem-brane, which was hybridized with radiolabeled Brd3 (D18 infigure) cDNA as a probe. EtBr, stained with ethidium bromideto show ribosomal RNA. T, tumors; N, noncancerous tissues.

FIG. 5. Inhibition of Brd3 expression. The expression of theBrd3/RINGL3/ORFX gene was inhibited by specific siRNA in-terference in 293 cells. (A) Immunoblot analysis with rabbitanti-Brd3 antibody. Twenty-four hours after the transfection,cell lysates were subjected to immunoblotting with anti-Brd3(D18 in figure) serum or antiactin antibody. Lane 1, mock trans-fection; lane 2, treatment with luciferase siRNA; lanes 3 and 4,two independent experiments of Brd3/RINGL3/ORFX siRNA-treatment. (B) Cell growth curve. After treatment withBrd3/RINGL3/ORFX siRNAs (siRNA-D18 in figure) or withluciferase siRNAs (siRNA-luci.), the cell number was counted.(C) Uptake of [3H] thymidine. After transfection with siRNAs,with empty vector (vector in figure), or with the pcDNA ex-pression vector of Brd3/RINGL3/ORFX (D18 in figure), cellswere pulse-labeled with [3H] thymidine, and the uptake wasmeasured.

ACKNOWLEDGMENTS

This project was supported in part by Grants-in-Aid for Sci-entific Research from the Ministry of Education, Culture,Sports, Science and Technology of Japan, and by research fundsfrom the Takeda Science Foundation and the Mochida ResearchFoundation. The nucleotide sequences are shown as the Gen-Bank accession nos. AY513269, AY513270, AY513271, andAY513272.

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Address reprint requests to:Hideshi Ishii, M.D., Ph.D.

Center for Molecular MedicineJichi Medical School

Tochigi 329-0498, Japan

E-mail: hishii@ms.jichi.ac.jp

Received January 10, 2005; accepted March 14, 2005.

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