APAF-1-ALT,anovelalternativesplicingformofAPAF-1, potentiallycausesimpededabilityofundergoingDNA damage-inducedapoptosisintheLNCaP humanprostatecancercellline TakenoriOgawa, a,b KiyotoShiga, b ShoHashimoto, b ToshimitsuKobayashi, b AkiraHorii, a andToruFurukawa a, * a Department of Molecular Pathology, Tohoku University School of Medicine, Sendai, Miyagi 980-8575, Japan b Department of Otolaryngology, Tohoku University School of Medicine, Sendai, Miyagi 980-8575, Japan Received12May2003 Abstract Wehavefoundanovelalternativesplicingproductoftheapoptoticproteaseactivatingfactor1(APAF-1),termedAPAF-1-ALT, intheLNCaPhumanprostatecancercellline.APAF-1-ALTharborsthecaspaserecruitmentdomainandanincompleteCED-4like/ ATPasedomain,butlackstheWD-40repeatunits.TheLNCaPcellexpressedthefull-lengthAPAF-1weaklyandAPAF-1-ALT ratherabundantly,especiallyaftermycoplasmainfection.LNCaPunderwentaretardedDNAdamage-inducedapoptosis,whichwas independentofcaspase9activity.APAF-1-ALTfunctionedlesseffectivelyininducingapoptosisthandidAPAF-1-XL,thefull- length APAF-1, in transient transfection. Moreover, APAF-1-ALT interfered with APAF-1-XLÕs ability to induce apoptosis in transientdoubletransfectionexperiment.TheseresultsindicatethatAPAF-1-ALTisamoleculethatisdeficientandimpededfor mediatingapoptosisandthatitmaycontributetotheresistancetoDNAdamage-inducedtreatmentobservedinLNCaP. Ó 2003ElsevierScience(USA).Allrightsreserved. Keywords: APAF-1;APAF-1-XL;APAF-1-ALT;Alternativesplicing;Apoptosis;Prostatecancer;CARD;Caspase9 Apoptoticproteaseactivatingfactor1(APAF-1)was firstclonedasahumanhomologueofCED-4,aniniti- ator protein for apoptosis identified in Caenorhabditis elegans [1].APAF-1containsthreefunctionaldomains, a caspase recruitment domain (CARD), a CED-4 like/ ATPase domain, and WD-40 repeats. All of these do- mains are important in the ability of APAF-1 to con- structamultioligomerapoptosomebybindingthrough the WD-40 repeat with cytochrome c released from mitochondria.Thissequenceispoweredbyhydrolysisof ATPordATP,whichisinturnmediatedbytheCED-4/ ATPase domain in response to apoptosis-inducing stimulations [2]. The apoptosome then recruits procas- pase9throughCARDandcleavesitforactivation.The cleavedcaspase9activatesthesubsequentcaspasecas- cade, which results in commitment to apoptosis [2]. In APAF-1-knockoutmice,anomaliesdevelopinthebrain and craniofacial tissues mainly because of cellular overgrowthduetothefailureofappropriateapoptosis. This pattern indicates that APAF-1 is essential for normalorganogenesis[3].Ontheotherhand,APAF-1 deficient mouse embryonal fibroblasts can phenotypi- cally undergo a caspase-independent apoptosis by means of ‘‘mitochondrial dysfunction’’ induced by ar- tificially strong expressions of BH3-domain only mole- cules[4].APAF-1isadirecttranscriptionaltargetofthe tumor suppressor TP53 and has been nominated as a keyregulatorofTP53-dependentapoptosis[5].Lossof APAF-1 function promotes transformation and chemoresistanceinprimarymurinefibroblasts,whichis compatible with loss of TP53 [6]. Reduction or loss of APAF-1 expression by somatic hemizygous allelic de- letionormethylation,whichhasbeendetectedinsome BiochemicalandBiophysicalResearchCommunications306(2003)537–543 www.elsevier.com/locate/ybbrc BBRC * Correspondingauthor.Fax:+81-22-717-8047. E-mail address: furukawa@mail.cc.tohoku.ac.jp (T.Furukawa). 0006-291X/03/$-seefrontmatter Ó 2003ElsevierScience(USA).Allrightsreserved. doi:10.1016/S0006-291X(03)00995-1