Isoform-specific silencing of the Livin gene by RNA interference defines Livin beta as key mediator of apoptosis inhibition in HeLa cells.
Livin (alternatively called ML-IAP or KIAP) is a cancer-associated member of the antiapoptotic inhibitor of apoptosis protein family. Two splicing variants of Livin, designated Livin alpha and Livin beta, have been identified. The significance of these isoforms for Livin-mediated apoptosis inhibition is largely unclear. Using an isoform-specific RNA interference (RNAi) strategy, we silenced endogenous Livin expression in HeLa cells. We found that the targeted inhibition of Livin beta, but not of Livin alpha, blocked the growth of HeLa cells in clonogenic survival assays. In addition, silencing of Livin beta, but not of Livin alpha, sensitized HeLa cells to different proapoptotic stimuli such as UV irradiation, tumor necrosis factor alpha, or etoposide. These events were linked to activation of caspase-3 and increased poly(ADP-ribose) polymerase cleavage, specifically upon silencing of Livin beta. The proapoptotic sensitization of HeLa cells upon RNAi-mediated silencing of the endogenous livin gene was specifically reverted by ectopic expression of Livin beta but not of Livin alpha. We conclude that the Livin beta isoform plays the key role for the antiapoptotic protection of HeLa cells by the livin gene. Our results show that the Livin isoforms can strongly differ in their functional significance for the antiapoptotic resistance of tumor cells. Studies evaluating Livin as a novel diagnostic and prognostic tumor marker should benefit from isoform-specific expression analyses.
Crnković-Mertens
Irena
I
Deutsches Krebsforschungszentrum, Molekulare Therapie Virus-Assoziierter Tumore (F065), Im Neuenheimer Feld 242, 69120, Heidelberg, Germany. k.butz@dkfz.de
Semzow
Julia
J
Hoppe-Seyler
Felix
F
Butz
Karin
K
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D003114:Colony-Forming Units Assay; D005047:Etoposide / Q000494:pharmacology; D020868:Gene Silencing; D006367:HeLa Cells / Q000473:pathology; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics* / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism*; D020033:Protein Isoforms; D034622:RNA Interference; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014466:Ultraviolet Rays
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C419866:BIRC7 protein, human; C412403:DIABLO protein, human; D051034:Inhibitor of Apoptosis Proteins; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D009363:Neoplasm Proteins; D020033:Protein Isoforms; D014409:Tumor Necrosis Factor-alpha; D005047:Etoposide; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases