Role of Survivin, Whose Gene Is Mapped to 17q25, in Human Neuroblastomaand Identification of a Novel Dominant-Negative Isoform, Survivin-b/2B

Ashraful Islam, MD,1,2 Hajime Kageyama,1 Kohei Hashizume, MD, PhD,2

Yasuhiko Kaneko, MD, PhD,3 and Akira Nakagawara, MD, PhD1*

Procedure. We investigated the expressionof survivin (SVV) and its isoform (SVV-b/2B)during different biological properties in neuro-blastoma (NBL). Results. High levels of SVVmRNA expression were significantly associatedwith advanced stages of NBL, diagnosis at over1 year of age, low levels of TrkA expression,and sporadic tumors. Expression of a novel iso-form, SVV-b/2B, which had an insertion of 23amino acids within the unique BIR domain waspredominant in some favorable NBLs, while itwas low and ubiquitous in most normal andmalignant tissues. The SVV expression was-down-regulated during apoptosis induced by

retinoic acid (RA) in CHP134 NBL cells, whichwas inhibited by forced expression of SVV. Incontrast, SVV-b was constantly expressed dur-ing apoptosis. Like SVV ,SVV-b was also highlyexpressed during G2/M in a cell cycle-dependent manner, and was associated withbut competed against SVV for binding with po-lymerized tubulin. Conclusion. These data sug-gest that expression of SVV is a poor prognosticindicator in human NBL, and it promotesgrowth and survival by regulating the levels ofboth isoforms. Med. Pediatr. Oncol. 35:550–553, 2000. © 2000 Wiley-Liss, Inc.

Key words: survivin; neuroblastoma; survivin-b/2B; 17q gain

INTRODUCTION

Neuroblastoma (NBL), one of the most common em-bryonal solid tumors of childhood, originates from post-ganglionic sympathetic neurons [1]. The disease has vari-able clinical outcomes, ranging from spontaneousregression to malignant progression with acquirement ofresistance to therapy. The distal deletion of the short armof chromosome 1p andMYCNamplification were shownto be associated with aggressive disease [2]. Recently,gain of the distal region of 17q was noted to have closecorrelation with the aggressiveness of NBL [3–4]. Theregion has been narrowed down to 17q21-qter, whichincludes thenm23andSurvivin(SVV) genes (5).SVV,anovel antiapoptosis gene, was mapped to 17q25, and itsexpression was restricted to neural crest-derived cellsincluding dorsal root ganglion neurons in mouse embryoat embryonic day 15 to postnatal day 21 [6]. It has re-cently been investigated for its potential contribution tothe cell cycle checkpoint through its interaction with po-lymerized tubulin. A potential opposing role for alterna-tive splicing in apoptosis was implicated by the discov-ery of functionally active, alternatively spliced variantsof major apoptosis regulators, such as Bcl-x, Ced-4, Ich-1, and caspase-9 [7]. In this study, we found that thelevels of SVVmRNA were high in advanced stages ofNBL. The low levels ofSVV-b/2B expression in normaltissues appeared to be switched to high levels ofSVVexpression along with carcinogenesis.SVV-b/2B boundwith polymerized tubulin and competed against the func-

tion of SVV,suggesting that twoSVVisoforms functionby regulating each other.

MATERIALS AND METHODSTumor Tissues, Cell Lines and Cell Culture

Fresh NBL tissues were obtained at surgery and storedat −80°C. Human cell lines we used were NBL cell lines(CHP134, IMR32, SH-SY5Y, CHP901, and SK-N-AS),the colon cancer cell line (Caco-2), the breast cancer cellline (MOA-MB-453), and the lung cancer cell line(A549). Cells were cultured at 37°C in 5% CO2-atmosphere as described previously [8]. Full-lengthcomplementary DNA (cDNA) forSVV and SVV-b/2Bwere obtained by reverse transcriptase polymerase chainreaction (RT-PCR). The PCR products of the correct sizewere digested with BamH1 and EcoR1, cloned into

1Division of Biochemistry, Chiba Cancer Center Research Institute,Chiba, Japan2Department of Pediatric Surgery, The University of Tokyo, Tokyo,Japan3Department of Cancer Chemotherapy, Saitama Cancer Center Hos-pital, Saitama, Japan

Grant sponsor: Ministry of Health and Welfare for a New Compre-hensive 10-year Strategy for Cancer Control, Japan; Grant sponsor:Ministry of Education, Science, and Culture of Japan.

*Correspondence to: A. Nakagawara, Division of Biochemistry, ChibaCancer Center Research Institute, 666-2 Nitona, Chiba 260-8717, Ja-pan. E-mail: akiranak@chiba-cc.pref.chiba.jp

Medical and Pediatric Oncology 35:550–553 (2000)

© 2000 Wiley-Liss, Inc.

pcDNA3 (Invitrogen), and were then confirmed by re-striction enzyme mapping and DNA sequencing.

Northern Analysis

Total RNA from the primary tumors or cultured cellswas prepared according to the method of Chomczynskiand Sacchi [9]. Twentymg of total RNA were subjectedto Northern analysis. TheSVV mRNA expression wasnormalized by that ofb-actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as reported previ-ously [10]. The values of expression levels ofSVVwerecalculated as arbitrary density units.

Transfection of SVV into CHP134 Cells

The SVVconstruct for transfection was generated bysubcloning the 936 ntSVVcDNA at the EcoRI site intothe pcDNA3 mammalian expression vector (Invitrogen,San Diego, CA). CHP134 cells cultured in 6-cm diametertissue culture plates were transfected with 10mg of theSVVcDNA construct or the vector alone using Lipofect-AMINE (Life Technologies, Inc.). Cells were selected inthe medium containing 400mg/ml geneticin (Sigma).The clones stably expressingSVV were confirmed byNorthern analysis.

PAC Screening and FISH Analysis

PAC clones containing theSVVgene were isolated byPCR-based three-dimensional screening of the RPCI hu-man PAC library [11] and used as FISH probes. Thes e q u e n c e o f t h e f o r w a r d p r i m e r w a s 58 -GTCTTTCTCAGCCATTCC-38, and that of the reverseprimer 58-AAAACCGAGTTCCAGGAG-38. PAC DNAwas prepared manually by the alkaline-SDS method [12]from 1 l of E. coli culture. To detect the copy numbers ofchromosome 1 or 17, the presence or absence of 1p de-letion, and the gains of chromosome arm 17q, the two-color FISH experiments were carried out by nick trans-lation as previously described [13].

RT-PCR

Two mg of total RNA was used to synthesize cDNAusing humanSVV- and GAPDH-specific primers withsuperscript II reverse transcriptase (GIBCO-BRL, Gai-thersburg, MD). For the analysis of the expression ofhumanSVV and SVV-b/2B, we used a primer pair of58-GACCACCGCATCTCTACATTC-38 and 58-GCACTTTCTTCGCAGTTTCC-38. The values werenormalized by the expression levels of GAPDH.

Cell-Cycle Synchronization

HeLa cells were grown to 50–70% confluence andarrested in G1, S, and G2/M phases of the cell cycle bymimosine, thymidine, and nocodazole, as reported pre-viously [14]. Phase fractionation of DNA content was

analyzed on the Becton-Dickinson FACScan flow cy-tometer by DNA Cell-Cycle Analysis Software-Ver C.

In Vitro Microtubule Binding and Competition Assay

Two hundred ninety-three cell (human embryonic re-nal cell carcinoma) extracts (200ml, 400 mg/ml) or brainpurified tubulin (5mg/ml, cytoskeleton, Denver) in G-PEM buffer (80 mM PIPES; 0.5 mM MgCl2; 1 mMEGTA; 1 mM GTP; pH 6.9) were treated with 20mM or10 mM Taxol (Sigma) for 20 min at 32°C. Polymeriza-tion of tubulin was confirmed by measuring the absor-bance of the solution (340 nm). [35S] Methionine-labeledSVVandSVV-b/2B were synthesized in a volume of 50ml by TNT Quick In Vitro Translation Kit (Promega,Madison, WI) according to manufacturer’s protocol. Af-ter addition ofSVV, SVV-b/2B with polymerized micro-tubule for 30 min at 22°C samples were centrifuged [15]for 5 min at 75,000 rpm and 37°C in a TLA100.2 rotor(Beckman Instruments) through 30ml glycerol cushion(50% glycerol in G-PEM buffer). For competition ex-periments, 10ml radio-labeledSVV/SVV-µ/2Bwas incu-bated with Taxol-stabilized polymerized tubulin in thepresence of 0–20ml radiolabeledSVV-µ/2Bor SVV be-fore cosedimentation. Proteins from pellets and superna-tant were separated by SDS-PAGE and were analyzed bycoomassie blue staining or autoradiography.

RESULTSIdentification and Expression Pattern of SVV-b

We performed RT-PCR on HeLa cell mRNA with twoSVV-specific primers amplifying the BIR domain. In ad-dition to the size corresponding toSVV, we identified along amplification product (418 kb). Sequence analysisreveled that the long PCR product encoded a 23 aa in-sertion into the BIR domain (Fig. 1). Following theSVVnomenclature, this isoform was designatedSVV-b/2B,which had five exons and four introns, and the size waspredicted to be about 18.0 kDa. RT-PCR analysis re-vealed that a 418 kb amplification product correspondingto the expression ofSVV-b was positive in fetal brain,fetal liver, thymus, brain, spinal cord, thyroid, heart,liver, colon, and prostate at low levels.

Expression of SVV and SVV-b in Human NBL

A human NBL cell line (NB1) seemed to grow slowlyin complete culture medium and appeared to have almostidentical expression of bothSVVandSVV-b. In 34 pri-mary NBLs at different stages, high levels ofSVVmRNAexpression (Northern blot) were significantly associatedwith advanced stages of NBL (P 4 0.0136), diagnosis atover 1 year of age (P 4 0.0003), low levels ofTrkAexpression (P < 0.0030), and sporadic tumors (P <0.0027). There was a tendency to be associated withDNA diploidy and MYCN amplification. Though pre-

Role of Survivin in Neuroblastoma 551

dominant expression ofSVV-b did not always signifi-cantly coincide with favorable NBLs, several tumors offavorable groups showed significant levels ofSVV-b ex-pression.

Biological Role of SVV and SVV-b in Human NBL

In a FISH experiment, theSVVPAC clone was probedfor the metaphase and interphase nuclei obtained fromthe fresh tumor specimen of a patient (case No. 342) withstage 3 NBL, whoseMYCN gene was amplified (>100copies). The experiment showed that the distal region of17q containing theSVVgene was gained and translocatedto the chromosome 1p, which had a deleted distal region.In an apoptosis model system using NBL cell lines, theexpression ofSVV-b remained steady state or ratherslightly up-regulated on day 7.SVV was dramaticallydown-regulated at that time. In a differentiation modelsystem, gradual down-regulation ofSVV-b expressionwas associated with moderate down-regulation ofSVVexpression (Fig. 2).

Interaction of SVV and SVV-b with Microtubule

The up-regulation ofSVV mRNA was observed inG2/M synchronized cells [14]. Our RT-PCR analysis re-vealed thatSVV-b was also abundantly expressed inG2/M cells. In an in vitro translation and cosedimenta-tion experiment, autoradiographic bands correspondingto bothSVVandSVV-b were detected in pellets of Taxol-stabilized microtubules from HeLa cell extracts or intubulin purified from the brain. Both were also detected

by coomassie-blue stain. We then performed in vitrotranslation and a competition experiment betweenSVVandSVV-b. Preincubation ofSVV, SVV-b, and polymer-ized tubulin or HeLa cell extracts caused a gradual de-crease in the amount ofSVV-b or SVVin a concentration-dependent manner in the pellets when the concentrationof SVVor SVV-b was gradually increased.

Fig. 1. Structure ofsurvivin andsurvivinb/2B. a: The schemeshowing alignment ofSVV andSVV-b/2B. A part of intron 3 ofSVVwas transcribed as exon 3 ofSVV-b/2B. The BIR domain wasunderlined.b: Amino acid align-ment ofSVV, SVV-b/2B, and otherIAPs. There was an insertion of 23aa into the BIR domain. Consensusaa sequences are shown by aster-isks.

Fig. 2. Schematic representation of changes in expression ofSVVorSVV-b/2B mRNA during apoptosis and differentiation. Two NBL celllines (CHP134 and SH-SY5Y) were treated with 5mM RA for 5–7days and mRNA from cells was subjected to Northern and RT-PCRanalysis. CHP134 cells underwent apoptosis on day 5–7. Induction ofapoptosis was not accompanied by changes inSVV-b/2B expression,but there was complete down-regulation ofSVVexpression. SH-SY5Ycells were induced to extend neurites and survived for at least 7 daysafter treatment with RA. In this condition, the expression ofSVV-b/2Bdecreased markedly whereasSVVdecreased moderately but still main-tained a reasonable level for at least 5 days.

552 Islam et al.

DISCUSSION

In this study, high expression ofSVVwas significantlyassociated with the older age of the patients, advancedstages of NBL, sporadic tumors, and low levels ofTrkAexpression, suggesting that increased expression ofSVVwas a poor prognostic indicator in NBLs. TheSVVgenewas mapped to 17q25 [16], which was shown to be theearly cytogenetic abnormality in NBL [17], associatingwith other poor prognostic factors such as 1p deletionand MYCN amplification.SVVwas highly expressed inG2/M phase of the cell cycle. In addition, its gain on thechromosome in NBL seemed to be often associated withits translocation to the distal 1p with the terminal dele-tion. These results suggested that high expression ofSVVin aggressive NBL was due to the gene dosage effect.However, the expression levels were often extremelyhigh, suggesting that theSVVexpression was transcrip-tionally activated. This should further be pursued.

The molecular mechanism of 17q gain and its contri-bution to spontaneous regression and differentiation isstill unclear. However, we have shown in NBL cells thatthe expression ofSVVis completely down-regulated dur-ing apoptosis and slightly decreases as the cells differ-entiate. On the other hand, expression ofSVV-b tends tobe steady and slightly down-regulated during apoptosisand differentiation, respectively.SVV-b expression didnot coincide with differentiation, which could be one ofthe reasons why its predominant expression was not al-ways associated with favorable NBL.

SVV-b had a 22 aa insertion within the BIR domain,which contained a possible serine/threonine phosphory-lation site (RX2S/TF), following 17 residues downstreamby an invariant glycine and then DX3CX2C and HX6Cconsensus sequences [18]. This led us to hypothesize thatSVV-b might impair or oppose the function ofSVVin adominant-negative form. Molecules of structural ana-logue were reported as competitive antagonists [19]. Wedemonstrated thatSVV-b was highly expressed duringthe G2/M phase of the cell cycle, and colocalized andassociated with Taxol-induced polymerized tubulin. Inaddition, our competition assay demonstrated the func-tional interference ofSVV by SVV-b. Thus, SVV andSVV-b might act as competitive antagonists for bindingto the polymerized tubulin.

ACKNOWLEDGMENTS

The authors thank Keizo Takenaga, Toshinori Ozaki,and Shigeru Sakiyama for their helpful discussion.

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