(CANCER RESEARCH 55. 5445-5450, November 15, 19951

vesicles (1 1, 12) and is implicated in neurotransmitter release by

increasing the efficiency of translocation and docking of the vesiclesto the presynaptic plasma membrane (13). A putative target protein ofRab3A, Rabphilin-3A, has an ability to inhibit the Rab3A GTPaseactivating protein-stimulated GTPase activity of Rab3A (14—16).

GDI was first isolated from bovine brain as a factor that preventsthe dissociation of GDP from Rab3A (17, 18). Rab3A GDI also hasthe interesting property of being able to extract the GDP-bound formof Rab proteins from intracellular membranes (19). Rab3A GD! wassubsequently found to be active on a wide range of Rab proteins andrenamed Rab GDI (20). On the basis of these results, Rab GD! seemsto act as a chaperone to Rab proteins during their cycle betweencytosol and membranes.

Our previous study revealed the existence of two Rab GDI isoforms, a and (3, in rat (21). Another group also isolated two Rab GDIisoforms, mGDI-1 and -2, from mouse (22). GDI a gene is abundantlyexpressed in brain, whereas GD! f3 gene is ubiquitously expressed(21). In insulin-sensitive 3T3-L1 adipocytes, a strikingly highermGDI-2 content is associated with intracellular membrane compartments compared to that of mGDI-1 (23). Although the majority of Rabproteins are complexed with GD! a in rat brain and insulin-secretingRINm5F cells, they are complexed with GD! f3in CHO cells (24). Theproportion of Rab proteins complexed with either isoform mightdepend on the relative abundance and/or cellular localization of GDIa and GDI f3 in a particular cell type (24).

Neuroblastoma is a tumor in childhood that arises from neuralcrest-derived tissues. Neuroblastoma cells have an immature phenotype of peripheral neurons, and some neuroblastoma cell lines undergo neuronal differentiation in vitro. We have reported previouslythat the level of Rab3A mRNA was markedly increased duringneuronal differentiation of NB1 cells and might be related to thedifferentiation state of neuroblastoma (25, 26). In the present study,we have first isolated a brain-type isoform of human Rab GD! cDNAand have examined its expression in various human tissues andneuroblastoma.

MATERIALS AND METHODS

Materials. [a-32P]dCTP and Rediprime DNA labeling system were purchased from Amersham International (Buckinghamshire, United Kingdom).Human retina AgtlO cDNA library, human brain poly(A)@ RNA, humanmultiple tissue Northern blot, human brain multiple tissue Northern blot,human f3-actincDNA probe, and AgilOprimers were purchased from ClontechLaboratories (Palo Alto, CA). pT7Blue plasmid was purchased from Novagen(Madison, WI). AutoRead Sequencing kit was purchased from PharmaciaBiotech (Uppsala, Sweden). 5'RACE and 3'RACE system kits were purchasedfrom GIBCO-BRL Life Technologies (Gaithersburg, MD). Bt2 cAMP waspurchased from Sigma Chemical Co. (St. Louis, MO). Neurofilament-L cDNAwas obtained from American Type Culture Collection (Rockville, MD). Othermaterials and chemicals were obtained from commercial sources.

RT-PCR Amplification of cDNA. The nucleotidesequencesof RabGDIswere highly homologous in their protein-coding region, especially betweenbovine Rab GDI and rat Rab GDI a or between hu ODI (3and rat Rab GDI (3(21). We supposed that the nucleotide sequence of a brain-type isoform of hu

GDI a is very similar to rat Rab GDI a. Therefore, we designed PCR primersbased on the nucleotide sequence of rat Rab GDI a (21) to amplify a part of

5445

Cloning of a Brain-type Isoform of Human Rab GD! and Its Expression in HumanNeuroblastoma Cell Lines and Tumor Specimens'

Noriyuki Nishimura, Junko Goji, Hajime Nakamura, Satoshi Orita, Yoshiini Takal, and Kimihiko Sano2

Department of Pediatrics. Kobe University School of Medicine, 7.5-1 Kusunoki-cho. Chuo-ku, Kobe 650 (N. N., J. G., H. N., K. S.]; Shionogi Institute for Medical Science,2.5.1 Mishima. Seusu 566 (S. 0.1: and Department ofMolecular Biology and Biochemistry. Osaka University Medical School. 2-2 Yamadaoka, Suita 565 (Y. TI. Japan

ABSTRACT

Rab proteins, a family of Ras-related small GTP-blndlng proteins, playa key role in regulating Intracellular vesicle trafficking. Rab GDP dissociation inhibitor (GDI3) forms a soluble complex with Rab proteins andthereby prevents the exchange of GDP for GTP. Recently, two isoforms ofRob GDI eDNA were isolated from rats and mice. In this study, we have

isolated a brain-type isoform of human Rab GDI cDNA and examined itsexpression in neuroblastoma. We tentatively designate it as human Rob

GDI a (hu GDI a) and another human Rob GDI, as human Rob GD! fi(hu GD! fi). Hu GD! a cDNA encodes a protein of 447 amino acids witha deduced molecular weight of 50,200. Northern blot analysis revealedthat hu GDI a gene is expressed abundantly in the brain but much less inother tissues, while hu GD! fl gene Is ubiquitously expressed. All human

neuroblastoma cell lines and tumor specimens examined express hu GD!a gene to various extents, while a human T cell leukemia cell line,MOLT3, does not. The levels of both hu GD! a and @3mRNA wereconstant in a human neuroblastoma cell line, NB!, during its neuronaldifferentiation, while Rab3A and neurofllament-L gene expression and thenumber of neurosecretory granules were elevated at this condition. These

results suggest that hu GD! a gene expression is not related to thedifferentiation state of neuronal cells.

INTRODUCTION

Rab proteins appear to be essential for intracellular vesicle trafficking and are likely to play a key role in regulating vesicle targeting

and/or fusion at distinct stages in endocytic and exocytic pathways(1—3).Rab proteins are highly conserved from yeast to human andcomprise a Ras-related small GTP-binding protein family that consistsof more than 30 members. Although the specific function of individual Rab protein in the trafficking events is not completely understood,recent evidence has suggested that Rab proteins are required to

assemble the general docking/fusion machinery of the N-ethylmaleimide-sensitive fusion protein/soluble N-ethylmaleimide-sensitive fu

sion protein attachment protein/soluble N-ethylmaleimide-sensitive

fusion protein attachment protein receptor system using a yeast secretion mutant system as a model (4—9).

It is proposed that Rab proteins cycle between a cytosolic GDPbound form and a membrane-associated, GTP-bound form. This cyclecould specify accurate docking and/or fusion of transport vesicleswith their acceptor membranes. Several accessory proteins that regulate the nucleotide state of Rab proteins have been identified. Theyinclude GTPase-activating proteins that accelerate GTP hydrolysis,guanine nucleotide releasing factors that stimulate the exchange ofGDP for GTP, and GDI3 that inhibit the exchange of GDP for GTP(10). Among Rabproteins,Rab3Ais highly concentratedon synaptic

Received 6/1 2/95; accepted 9/18/95.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 with18 U.S.C. Section 1734 solely to indicate this fact.

4 This work was supported in part by a grant for Cancer Research from the Hyogo

Total Health Association (1994).2 To whom requests for reprints should be addressed, at Department of Pediatrics,

Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan.Phone: 81-78-341-7451 (cxl. 5722); Fax: 81-78-371-6239.

3 The abbreviations used arc: GDI, GDP dissociation inhibitor; Bt2 cAMP, dibutyrylcAMP; hu GDI, human Rab GDI; RT-PCR, reverse transcription-PCR; 3'RACE or5'RACE, rapid amplification of 3' or 5' cDNA ends, respectively.

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A BRAIN.TYPE ISOFORM OF HUMAN RAB GDI

-54 gd 9C9919aag gag gag gag gag ccg age gggcgc tgg cac cga ggc ctg sec ATGGf@C@GGtGTACc@TGiGATCGTGCTGGGGft@C061 @C@1 Met Asp Glu Glu Tyr Asp Val lieVal Lou Gly Thr Gly Leu Thr

46 Gb-ATGCATCCrG1-cctxx:ATCATGTCAGTGMC066MGMGGiGCTGCAC*16OX @GGMAOX TACTAC @iG013CGAGMICicc @ccATCfr@A0@16 Glu Cys lie Leu Ser Gly lie Met Ser Vai Mn Guy Lys Lys Val Leu His Met Gly Arg Lys Pro Tyr Tyr Gly Gly Glu Ser Ser Ser lie Thr Pro

145 CTGt@G&‘@GCTGTATMG EXiTm CAGfl'G CTG @GflOGaX tXT GAGTcGATGGGC @AGGCOGAt@CTGGUT 6ff GACCTGAn tXC MA ftC @C49 Leo Giu Giu Leu Tyr Lys Arg Phe Gin Leu Leu Giu Gly Pro Pro flu Ser @t Gly Arg Giy Arg Asp Trp Asn Val Asp Leu lie Pro Lys Pbs Leu

244@82 Met Ala Mn Giy Gin Leu Val Lys @tLeu Leu Tyr Thr Glu Val Thr Arg Tyr Leo Asp Phe Lys Val Val Glu Giy Set Pbs Val Tyr Lys Gly Gly

343@115 Lys lieTyr Lys Val Pro Ser Thr Giu Thr Giu Ala Leu Ala Ser Asn Leu @tGiy Met Pbs Glu Lys Arg Arg Phe Arg Lys ‘heLeu Vai Pta Vai

442 GCAGGA @TTATGAGUT C@CtIC MG MC m 6413GGCGil @CtXC CAGICT AIXAGCATGIGT GACGTCTAC@XlGMG m @TCTGOGCCAG @T148 Ala Gly Thr Tyr Glu Am Asp Pro Lys Thr Phe Glu Gly Val Asp Pro Gin Thr Thr Ser Met Arg Asp Val Tyr Arg Lys Pbs Asp Leu Gly GIn Asp

541@181 VaI Ile Asp Phe Thr Giy Hia Ala Leu Ala Leu Tyr Arg Thr Asp Asp Tyr Leu AspGin Pro Cys Leu GIu Thr VaI Mn Arg IIe Lys Leu Tyr Ser

640 GAGTt@CCTGc@c@ TATG@X@MGAGC @ATAThA TAC @GCTCTACGOCflG GGCGAGCTG @CCAGGGTIn GCAAGAUG frET0cCATCTATGGG214 Glu Ser Leo Ala Arg Tyr Gly Lys Ser Pro Tyr Leo Tyr Pro Leu Tyr Gly Leu Gly Gb Leu Pro Gin Gly Phe Ala Arg Leo Ser Ala lieTyr Gly

139 GGGM@ATATATGCTGMCMAtXTGIGG'@TGACATCATCATGGtiGMCmc MGGTGGTGGOCGiGMGTCTtW3GGA6@GGTGGIX @CTGCMGCAG241GiyThrTyrMetLeuAsnLysProValAspAsplielieMetGluAsnGlyLysVaiVaiGlyValLysSerGluGlyGiuValAlaArgCysLysGin

838 CTGATCTGTG@Cax;@ TACATCa@Gt@CWt GTGQ@iMGOCTG@CAGGil ATCIXICATCATCTGTATCCiTAGCCAC @CATCMGMC@ MC280 Leu lie Cys Asp Pro Ser Tyr lie Pro Asp Arg Val Arg Lys Ala Gly Gin Val lie Arg lie lie Cys lie Leu Ser His Pro lie Lys Asn Thr Asn

937 GAC0CCMCTcCTGCCMATAATCATCW CAGMCCAGGICMCAGGMGTCAG@CATCTACGIGTGCATGATCTcCTATGCACACMCGIGGIG0CC313 Asp Ala Asn Ser Cys Gin lie lie lie Pro Gin Asn Gin Val Asn Arg Lys Ser Asp lie Tyr Val Cys Met lie Ser Tyr Ala His Asn Vat Ala Ala

1036@346 Gin Gly Lys Tyr lieAla lieAla Ser Thr Thr Vat Glu Thr Thr Asp Pro Glu Lys Glu Vat Giu Pro Ala Leu Giu Leu Leu Glu Pro lieAsp Gin

1135@379 Lys Pbs Val Ala lieSer Asp Leo Tyr Glu Pro lieAsp Asp Gly Cys Glu Ser Gin Vat Pta Cys Ser Cys Ser Tyr Asp Ala Thr Thr His Phe Glu

1234 ACAN@CTGCMC GACATCMA GACATCTACMA aic ATGOCT @iC@G0CCm 6@Cm G4@GMC ATGMG@ MA CAGMC GACCrC TCTGGA64@A412 Thr Thr Cys Asn Asp lie Lys Asp lie Tyr Lys Arg Met Ala Gly Thr Ala Pbs Asp Pbs Glu Asn Met Lys Arg Lys Gin Asn Asp Vat Ser Gly Glu

1333 OCTGIiGCAGTGAttg tgg ccg ccc cca gcc oct get gcc cca gcc tgt gtc tgt tct oct cga gggctc rag cat cct ctg ctt ccc cca cca cgt tcc445 Ala Glu Gin ***

Fig. 1. Nucleotide and deduced amino acid sequences of hu GDI a. Nucleotide residues are numbered on the lop; amino acid residues are numbered on the bottom. Residue 1 isthe putative initiator methionine.

bovine -1- @EEYDVIVLGTG1TECILSGIISVNGkKVLH@@PYYGGESSSITPLEELYKRF0LLEGPPE1MGRGFI)@P4VD1IPKFIJANG0LVKMLLYTEVTRYLhunan-a -1- G K Srat-a -1- Shunan-@ —1- D K@ Srat-Il -1- N 0 D KIP 0 AS F Mmouse-It -1-N U 0 AN D PGOASSS F Mmouse-2 -1- N Fl A D KIPGA AS F

bovine -101- DFKVVEGSFVYKGGKIYKVPSTETEALASNLMGMFEKRRFRKFLVFVANFDENDPKTFEGVDPONTSMRDVYRKFDI000VIDFTGHALALYRTDOVLDOhtsnan-a -101- OTY Irat-a -101- 1 Vhunan-@ -101- 1 A S L V K R I KItI Krat-n -101- 1 A S L V K R V KIt Kmouse-@-1O1- I A S L S Y KS Kl( S K M Smouse-2 -101- T A S L V R I KX E K

bovine -201- PCLETINRIKLYSESLARYGKSPVLYPLVGIGELPQGFARLSAIYGGTYMLNKPVOOIIMENGKVVGVKSEGEVARtKtLIcDPSVVPDRVRKAGOVIRIhuman-a -201- V Irat-a -201- Ihunan-@ -201- V lEE@ I Fl K E V Vrat-n -201- C@TELtF P, lEE ‘A@ I KV EV Vmouse-@-2O1- C S S S N IE@ I K EV SVmouse-2 -201 - C lEE 0 I I K E V V

bovine -301- ICILSHPIKNTNDANSCOII IPONQVNRKSDIYVCNISYAHNVAAOGKYIAIASTTVETTDPEKEVEPALELLEPIDOKFVAISDLVEPIDDGSESOVFChunan-a -301- N N Crat-a -301-hunan-@ -301- F V KE IR E E S LV K L I I Irat-ti -301- F V KE IR E S FV K L 10 I Imouse-n -301- F V KE ISP E S FV K L ID I Imouse-2 -301- S KE IR E S LVK L I I I

bovine -401 - SCSYDATTHFETTCNDIKDIYKRNAGSAF0FENNKI@(0NDVFGEAD0*-447-hunan-a -401- 1 5 E * -447-rat-a -401- $ -447-hunan-@ -401- RI D N I E E K IV Os -445-rat-n -401- RA D I E E K IV D* -445-mouse-p -401- RA 0 ST E E K IV D* -445-mouse-2 -401- RI D N E E K IV Es -445-

Fig. 2. Alignment of deduced amino acid sequences of bovine Rab GDI, hu GDI a, rat Rab ODI a, hu GDI @,rat Rab ODI @,mouse Rab GDI 2, and mouse Rab ODI (3. Aminoacid sequence of bovine Rab GD! is shown by single letter on the top lane. Only replacements of other Rab GDIs are indicated by the corresponding residue (one letter code).

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hu GDI a cDNA. Sequences of primers were 5'-CCCFAC1@ATGGGGGTGAGAGC-3' (110-130) and 5'-CTGmGCGCVFCATGUCFC-3' (1,314—1,294) (21). Human brain poly(A)@RNA was used as a template forRT-PCR by a standard method (27). This RT-PCR product was used to screena human retina cDNA library.

cDNA Cloning. A human retina cDNA library was screened with alabeled hu GDI a cDNA as described previously (21). After three rounds ofscreenings, the insert DNAs of positive clones were amplified by PCR usingAgtlOprimers and subcloned into pT7Blue vectors for DNA sequencing. DNAsequencing was performed using A.L.F. DNA sequencer (Pharmacia Biotech)with AutoRead Sequencing kit according to the manufacturer's instructions.

5'RACE and 3'RACE. We tentatively designated a brain-type isoform ofhuman Rab GDI as hu GDI a and renamed human Rab GDI3, isolated by M.Asada et a!.,4 as hu GDI (3.The 5' end of hu GDI a and the 3' end of hu GDI13cDNAwereamplifiedbyusingthe5'RACEor3'RACEkit,respectively.Two gene-specific antisense primers for 5'RACE [5'-'VFGGGGTCATFCTCATAAGTF-3' (467-446) and 5'-CG1TFCFCAAACATGCCCATC-3'(414—393)] were designed based on the nucleotide sequence of hu Rab GD! a(Fig. 1). The gene-specific sense primer for 3'RACE [5'-CACCACTCAlTFTGAGACAAC-3' (1238—1258)] was designed based on the nucleotide sequence of hu GDI (33•Human brain poly(A)@RNA was used as a template forboth 5'RACE and 3'RACE. The products were subcloned into pT7BIue vectorfor DNA sequencing.

Cell Lines and Tumor Specimens. A human neuroblastomacell line,NB1, was obtained from Japanese Cancer Research Resources Bank. SMSKAN was a kind gift from Dr. J. Biedler (Memorial Sloan Kettering Institute).KU-YS-C and KU-YS-N were established in our laboratory. A human T-celllymphoma cell line, MOLT3, was a kind gift from Dr. E. Tatsumi (Departmentof Laboratory Medicine, Kobe University School of Medicine). All cell lineswere grown in RPMI 1640 supplemented with 10% heat-inactivated fetalbovine serum, 100 units/mI penicillin G, and 100 p.g/ml streptomycin in ahumidified atmosphere of 95% air and 5% CO2. Tumor specimens wereobtained at primary surgery prior to chemotherapy and kept at —80°C.

RNA Preparation from Differentiated Neuroblastoma Cell Line (NB!).NB1 cells were small and dense cells with scant cytoplasm and a few shortprocesses, and treatment of NBI cells with 1 mMBt2 cAMP induced markedneurite extension and Rab3A gene expression (26). Total RNA was extractedfrom NB1 cells at 0, 1, 2, 4, and 7 days after treatment with 1 m@iBt2cAMPas described previously (28).

Northern Blot Analysis. Northern blotting was performed using 20 @goftotal RNA isolated from tumor specimens and various cell lines as described

previously (28). These blots, human multiple tissue Northern blot, and humanbrain multiple tissue Northern blot were hybridized with a 32P-labeledprobe asdescribed previously (28). The probe for Rab3A mRNA was a 0.6-kb EcoRIHindlll fragment of clone 9—14of the bovine Rab3A cDNA (18).

Quantitation of Neurosecretory Vesicles. The number of neurosecretoryvesicles in neurite-like processes of NB-i cells were counted on electronmicrographs and divided by area. Neurite-like processes were characterized bya longitudinal array of microtubles and intermediate filaments. Twenty-fivesections of the longitudinal and transverse sections of processes were exam

med in each determination. Statistical significance in the numbers of dense

core granules was assessed by paired t test.Other Procedures. The computerprograms,Mac VectorVersion4.1 and

Assembly LIGN Version 1.0 (International Biotechnologies, New Haven, CT),and Entrez data base Release 14.0 (National Center for Biotechnology Information, Bethesda, MD) were used for an analysis of the DNA sequencing data.

Nucleotide Sequence Accession Number. The nucleotidesequence dataof hu GDI a reported in this study will appear in the DDBJ, EMBL, andGenBank nucleotide sequence databases under accession number D45021.

RESULTS

Molecular Cloning and Determination of Nucleotide and Deduced Amino Acid Sequences of hu GD! a. To isolate a cDNAclone encoding human homologue of rat Rab GD! a, a part of hu GD!

4 M. Asada, K. Kaibuchi, and Y. Takai. GenBankIEMBIJDDBJ Nucleotide sequence

databases, accession no. D13988, 1993.

kb..ø@4•4

—2.4

kb

. —4.4

.. . .. .@••1Io•. —2.4

1@-kb

—4.4

..m@• *•p@@,@.l_ —2.45 —1.35

1 2 3 4 5 6 7 8 9 10111213141516

a cDNA was amplified by RT-PCR using human brain poly(A)@RNA as a template. Approximately 5 X 1O@recombinant phageplaques from a human retina cDNA library were screened using thisRT-PCR product as a probe. Seven positive clones were isolated fromthe first screening. Their nucleotide sequences overlapped each other

1 2 3 4 5 6 7 8 9 10111213141516

1 2 3 4 5 6 7 8 9 10111213141516

Fig. 3. Northern blot analysis of the hu GDI a and@ mRNA in various tissues. Humanmultiple tissue Northern blots and human brain multiple tissue Northern blots werehybridized with 32P-labeledhu GDI a and@ and @-actincDNA probes. The nucleotidesequences of hu GDI a and (3showed an extreme similarity in the protein coding regionbut not in the noncoding region. To avoid the cross-hybridization between hu GDI a and

@,we designed probes as follows. The probe for hu GDI a mRNA was designed to locate

at the 3' noncoding region of hu GDI a cDNA. The 476-bp fragment spanning from thenucleotide position of 1253 (COOH terminus of coding region) to 1675 (3' noncodingregion) of hu GDI a cDNA was amplified by PCR, and this fragment was used as a probefor hu GD! a mRNA. The probe for hu GDI 13mRNA was designed to locate at the 3'noncoding region of hu GDI@ cDNA. An approximately 600-bp fragment located at the3' noncoding region of hu GDI@ cDNA was amplified by 3'RACE, and this product wasused as a probe for hu GDI @3mRNA. Hu GDI a mRNA is shown on the top, and hu GDIf3mRNAisshowninthemiddle.Asacontrol,@-actinmRNAisshownonthebottom.Lane I, heart; Lane 2, brain; Lane 3, placenta; Lane 4. lung; Lane 5, liver; Lane 6, skeletalmuscle; Lane 7, kidney; Lane 8, pancreas; Lane 9. amygdala; Lane 10, caudate nucleus;Lane 11, corpus callosum; Lane 12, hippocampus; Lane 13, hypothalamus; Lane 14,substantia nigra; Lane 15, subthalamic nucleus; Lane 16, thalamus. The size of markersis shown at the right.

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human rab GD! a

human rab GD! f3

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Tissue Distribution of hu GD! a and @3mRNA Hu GD! a andf3mRNAlevelsin varioushumantissueswereexaminedby Northernblot analysis. Hu GD! a mRNA was detected as a single band ofapproximately 2.8 kb as shown in Fig. 3. Hu GD! a gene wasexpressed abundantly in cerebrum, but its mRNA level was very lowin other tissues including heart, placenta, lung, liver, skeletal muscle,kidney, and pancreas. In cerebrum, hu GD! a mRNA was detected inall regions examined including amygdala, caudate nucleus, corpuscallosum, hippocampus, substantia nigra, and thalamus. Hu GD! (3mRNA appeared as a single band of approximately 2.4 kb as shownin Fig. 3. In contrast to hu GD! a, hu GD! (3 mRNA was expressed in

A NF-L B Rab3A

C HumanRabGDIa

28S—@ •

18S-@

D HumanRabGDI@

28S —@

18S—@'

o'-I*F@

finFig. 5. Expression of NF-L (A), Rab3A (B), hu GD! a (C), and@ mRNA (D) in a

neuroblastoma cell line, NB1, during neuronal differentiation. NB! cells were grown asmonolayers in RPMI 1640 medium containing 1 msi Bt2 cAMP for the indicated period.Twenty g.@gof total RNA were extracted from NB1 cells at 0, 1, 2, 4, and 7 days aftertreatment with 1 mM Bt2 cAMP and were hybridized with 32P-labeled eDNA probespecific for neurofilament-L (NF-L), Rab3A, and hu GDI a and f3 as described in“Materialsand Methods.―Ethidium bromide staining of the gel is shown on the right toconfirm the equal loading of total RNA.

human rab GD! a

18S—@@

I >-)-@c#, I I

28S—.

lvii1-i-

28S—

18S—°@@

@ I >->-@soI I

Fig. 4. Expression of hu GDI a mRNA in human neuroblastoma specimens and celllines. Twenty jig of total RNA was extracted from various cell lines (Lanes 1-4) andtumor tissues (Lanes 5-15), electrophoresed, transferred to nylon membrane sheets, andhybridized with 32P-labeled hu GDI a and @-actincDNA probes. hu GD! a mRNA isshown on the top. As a control, @-actinmRNA is shown on the bottom. The size of thetranscript was estimated by using 18S and 28S rRNA as internal size markers.

and predicted one gene, but these clones lacked its 5 â€end. To obtainits 5' end, 5'RACE was performed. The resulting nucleotide sequencecontained a 1,341-bp open reading frame encoding a protein of 447amino acids with a calculated molecular weight of 50,200. Theneighboring sequence of the first ATG is consistent with the translation start site proposed by Kozak (29). This cDNA was tentativelynamed hu GD! a, and its nucleotide and deduced amino acid sequences are shown in Fig. 1. In analogy to rat Rab GD! a and (3, werename human Rab GD!3 as hu GD! (3.

Alignment of Mammalian Rab GDIS. The amino acid sequencesof hu GD! a and (3, bovine Rab GD! (18), rat Rab GD! a and (3 (21),and mouse Rab GD! 2 (22) and (3 (30) are compared in Fig. 2.Mammalian Rab GD!s were classified into two groups: the a-type,

which includes bovine, human-a, and rat-a, and has 447 amino acids;and the (3-type, which includes human-(3, rat-(3, mouse-2, andmouse-j3, and has 445 amino acids. The amino acid sequences of hu

GD! a had 97 and 97% identity with those of rat Rab GD! a andbovine Rab GD!, respectively. The amino acid identity between huGD! a and (3 was 86%.

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Fig. 6. Ultramicrostructure of neurite-like processes of NB1 cells with and without Bt2 cAMP treatment. NB1 cells were cultured for 7 days in the absence (A) or presence (B) ofI mM Bt2 cAMP. X20,000. Bar, 0.5 g.tm.

all tissues examined to various extents. Tissue distributions of hu GD!a and f3were consistent with our earlier observation with corresponding rat probes (21).

Expressionof hu GD! a in HumanNeurobla.stomaSpecimensandCellLines. Next,we examinedtheexpressionof huGDIa genein human neuroblastoma tumor specimens and cell lines includingSMS-KAN, KU-YS-C, and KU-YS-N as shown in Fig. 4. The KU-YScell line was established from metastatic bone marrow cells of aneuroblastoma patient. This cell line consisted of two types of cellshaving different phenotypes. We have established subclones fromKU-YS cells and designated them as KU-YS-N and -C cells. KUYS-N cells have a neuroblastic appearance, and their phenotype isneurofilament@/neuron-specific enolase @/cytokeratin 8 . KU-YS-Ccells show an epithelial morphology, and their phenotype is neurofilament/neuron-specific enolase@/cytokeratin 8@.The details of thecharacters of these cell lines will be described elsewhere. The 2.8-kbhu GD! a mRNA was detected in these neuroblastoma cell lines butnot in a T-cell lymphoma cell line, MOLT3. These results suggest thathu GD! a gene is expressed from an early stage of differentiation ofperipheral neuronal lineage. All of 11 human neuroblastoma speci

mens examined here expressed hu GD! a gene to a similar extent asshown in Fig. 4. There seemed to be no correlation between the huGD! a mRNA level and histological grade or clinical stage (data notshown).

Expression of hu GD! a and (3 mR.NA during Differentiation ofNB! Cells into Neuronal Cells. Bt2 cAMP induces neurite extensionin NB1 cells as reported before (26). The levels of neurofilament-L

and Rab 3A mRNA in NB1 cells were increased after the treatment

with Bt2 cAMP as shown in Figs. 5, A and B. The level of hu GD! a

mRNA in NB1 cells was not changed by Bt2 cAMP treatment (Fig.5C). Hu GD! (3 mRNA was barely detected in NB! cells and remained very low during Bt2 cAMP treatment (Fig. 5D). Neuroblastoma cells often contain dense core granules that are similar to thosefound in sympathetic neurons or chromaffin granules in adrenalmedulla. These granules contain catecholamines and proteins such asdopamine (3-hydroxylase, chromogranin A and B, and secretogranin.We have examined the numbers of dense core granules in NB1 cellsbefore and after treatment with Bt2 cAMP by morphometric analysis.Representative ultramicrographs are shown in Fig. 6. Bt2 cAMPtreated NB1 cells had a good number of dense core granules inneurite-like processes. The numbers of dense core granules in neuritelike processes of NB1 cells with and without Bt2 cAMP treatmentwere 54.0 ±17.5/100 gim2 and 5.2 ±2.0/100 p.m2 (mean ±SEM,n = 25), respectively (P < 0.05).

DISCUSSION

In this study, we have isolated a brain-type isoform of Rab GD!cDNA from a human retina cDNA library. We reported previouslythat two isoforms of Rab GD! exist in rat and that each cDNA encodesa protein of 447 or 445 amino acids (21). Adding to them, two miceGD! isoforms, mGD!-1 and -2 (22), and another mouse GD! isoform,mGD!-f3 (30), have been isolated. Although both mGD!-2 andmGD!-j3 encode 445 amino-acid proteins, the sequence of mGD!-(3 isdifferent from that of mGD!-2. Taken together, we propose here thatmammalian Rab GD!s can be classified into at least two types (Fig. 2).

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A BRAIN-TYPE ISOFORM OF HUMAN RAB GD!

One encodes an a-isoform of 447 amino acids, and the other encodesa (3-isoform of 445 amino acids.

As shown in Fig. 3, hu GD! a is predominantly expressed in brain.If hu GD! a is specifically involved in neuron-specific function, suchas sorting of neurosecretory vesicles, one might expect that hu GD! ais up-regulated during neuronal differentiation. Shisheva et a!. (22)reported that the levels of mouse Rab GDI-1 and -2 mRNA wereincrease during the differentiation of 3T3-L1 fibroblasts into highlyinsulin-responsive adipocytes. However, the level of the hu GD! a

mRNA in NB1 cells was not changed by Bt2 cAMP treatment, whilethe levels of Rab3A and neurofilament-L mRNA and the number ofneurosecretory granules increased at this condition (Figs. 5 and 6).

One possible explanation for our results is that hu GD! a plays ageneral role in intracellular trafficking events in neuronal cells, andthe basal level of hu GD! a is enough to deal with increased Rab3A

upon differentiation. Broad substrate specificity of bovine Rab GD!and rat Rab GD! a might support this idea (20, 21). There is anotherpossibility that hu GD! a is activated by unknown mechanisms suchas phosphorylation during neuronal differentiation (31, 32). Alternatively, there could exist another GD! specific for sorting of neurosecretory granules. Further investigation is required to clarify thesepossibilities.

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1995;55:5445-5450. Cancer Res   Noriyuki Nishimura, Junko Goji, Hajime Nakamura, et al.   SpecimensExpression in Human Neuroblastoma Cell Lines and Tumor Cloning of a Brain-type Isoform of Human Rab GDI and Its

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