Skip to main content

Advertisement

SpringerLink
Log in

Isoform-specific silencing of the Livin gene by RNA interference defines Livin β as key mediator of apoptosis inhibition in HeLa cells

  • Original Article
  • Published:
Journal of Molecular Medicine Aims and scope Submit manuscript

Abstract

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 α and Livin β, 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 β, but not of Livin α, blocked the growth of HeLa cells in clonogenic survival assays. In addition, silencing of Livin β, but not of Livin α, sensitized HeLa cells to different proapoptotic stimuli such as UV irradiation, tumor necrosis factor α, or etoposide. These events were linked to activation of caspase-3 and increased poly(ADP-ribose) polymerase cleavage, specifically upon silencing of Livin β. The proapoptotic sensitization of HeLa cells upon RNAi-mediated silencing of the endogenous livin gene was specifically reverted by ectopic expression of Livin β but not of Livin α. We conclude that the Livin β 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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70

    Article  PubMed  CAS  Google Scholar 

  2. Evan GI, Voudsen KH (2001) Proliferation, cell cycle and apoptosis in cancer. Nature 411:342–348

    Article  PubMed  CAS  Google Scholar 

  3. Igney FH, Krammer PH (2002) Death and anti-death: tumour resistance to apoptosis. Nat Rev Cancer 2:277–288

    Article  PubMed  CAS  Google Scholar 

  4. Schmitt CA (2003) Senescence, apoptosis and therapy—cutting the life-lines of cancer. Nat Rev Cancer 3:286–295

    Article  PubMed  CAS  Google Scholar 

  5. Salvesen GS, Duckett CS (2002) IAP proteins: blocking the road to death’s door. Nat Rev Mol Cell Biol 3:401–410

    Article  PubMed  CAS  Google Scholar 

  6. Liston P, Fong WG, Korneluk RG (2003) The inhibition of apoptosis: there is more to life than Bcl2. Oncogene 22:8568–8580

    Article  PubMed  CAS  Google Scholar 

  7. Vucic D, Stennicke HR, Pisabarro MT, Salvesen GS, Dixit VM (2000) ML-IAP, a novel inhibitor of apoptosis that is preferentially expressed in human melanomas. Curr Biol 10:1359–1366

    Article  PubMed  CAS  Google Scholar 

  8. Lin J-H, Deng G, Huang Q, Morser J (2000) KIAP, a novel member of the inhibitor of apoptosis protein family. Biochem Biophys Res Commun 279:820–831

    Article  PubMed  CAS  Google Scholar 

  9. Kasof GM, Gomes B (2001) Livin, a novel inhibitor of apoptosis protein family member. J Biol Chem 276:3238–3246

    Article  PubMed  CAS  Google Scholar 

  10. Ashhab Y, Alian A, Polliack A, Panet A, Ben Yehuda D (2001) Two splicing variants of a new inhibitor of apoptosis gene with different biological properties and tissue distribution pattern. FEBS Lett 495:56–60

    Article  PubMed  CAS  Google Scholar 

  11. Gazzaniga P, Gradilone A, Giuliani L, Gandini O, Silvestri I, Nofroni I, Saccani G, Frati L, Agliano AM (2003) Expression and prognostic significance of LIVIN, SURVIVIN and other apoptosis-related genes in the progression of superficial bladder cancer. Ann Oncol 14:85–90

    Article  PubMed  CAS  Google Scholar 

  12. Tanabe H, Yagihashi A, Tsuji N, Shijubo Y, Abe S, Watanabe N (2004) Expression of survivin mRNA and livin mRNA in non-small-cell lung cancer. Lung Cancer 46:299–304

    Article  PubMed  Google Scholar 

  13. Quiping Z, Jei X, Youxin J, Wei J, Chun L, Jin W, Qun W, Yan L, Chunsong H, Mingzhen Y, Quingping G, Kejian Z, Zhimin S, Qun L, Junyan L, Jinquan T (2004) CC chemokine ligand 25 enhances resistance to apoptosis in CD4+ T cells from patients with T-cell lineage acute and chronic lymphocytic leukemia by means of livin activation. Cancer Res 64:7579–7587

    Article  Google Scholar 

  14. Yagihashi A, Asanuma K, Tsuji N, Torigoe T, Sato N, Hirata K, Watanabe N (2003) Detection of anti-livin antibody in gastrointestinal cancer patients. Clin Chem 49:1206–1208

    Article  PubMed  CAS  Google Scholar 

  15. Yagihashi A, Asanuma K, Kobayashi D, Tsuji N, Shijubo Y, Abe S, Hirohashi Y, Torigoe T, Sato N, Watanabe N (2005) Detection of auto-antibodies to livin and survivin in sera from lung cancer patients. Lung Cancer 48:217–221

    Article  PubMed  Google Scholar 

  16. Yagihashi A, Ohmura T, Asanuma K, Kobayashi D, Tsuji N, Torigoe T, Sato N, Hirata K, Watanabe N (2005) Detection of auto-antibodies to survivin and livin in sera from patients with breast cancer. Clin Chim Acta 362(1–2):125–130

    Article  Google Scholar 

  17. Crnkovic-Mertens I, Hoppe-Seyler F, Butz K (2003) Induction of apoptosis in tumor cells by siRNA-mediated silencing of the livin/ML-IAP/KIAP gene. Oncogene 22:8330–8336

    Article  PubMed  CAS  Google Scholar 

  18. Brummelkamp TR, Bernards R, Agami R (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550–553

    Article  PubMed  CAS  Google Scholar 

  19. Chen C, Okayama H (1987) High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745–2752

    PubMed  CAS  Google Scholar 

  20. Southern PJ, Berg P (1982) Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet 1:327–341

    PubMed  CAS  Google Scholar 

  21. Butz K, Whitaker N, Denk C, Ullmann A, Geisen C, Hoppe-Seyler F (1999) Induction of the p53-target gene GADD45 in HPV-positive cancer cells. Oncogene 18:2381–23816

    Article  PubMed  CAS  Google Scholar 

  22. Butz K, Ristriani T, Hengstermann A, Denk C, Scheffner M, Hoppe-Seyler F (2003) siRNA targeting of the viral E6 oncogene efficiently kills human papillomavirus-positive cancer cells. Oncogene 22:5938–5945

    Article  PubMed  CAS  Google Scholar 

  23. Vucic D, Franklin MC, Wallweber HJA, Das K, Eckelman BP, Shin H, Elliott LO, Kadkhodayan S, Deshayes K, Salvesen GS, Fairbrother W (2005) Engineering ML-IAP to produce an extraordinarily potent caspase 9 inhibitor: implications for Smac-dependent anti-apoptotic activity of ML-IAP. Biochem J 385:11–20

    Article  PubMed  CAS  Google Scholar 

  24. Fulda S, Wick W, Weller M, Debatin K-M (2002) Smac agonists sensitize for Apo2L/TRAIL- or anticancer drug-induced apoptosis and induce regression of malignant glioma in vivo. Nat Med 8:808–815

    PubMed  CAS  Google Scholar 

  25. Altieri DC (2003) Validating survivin as a cancer therapeutic target. Nat Rev Cancer 3:46–54

    Article  PubMed  CAS  Google Scholar 

  26. Mahotka C, Wenzel M, Springer E, Gabbert HE, Gerharz CD (1999) Survivin-ΔEx3 and survivin-2B: two novel splice variants of the apoptosis inhibitor survivin with different antiapoptotic properties. Cancer Res 59:6097–6102

    PubMed  CAS  Google Scholar 

  27. Badran A, Yoshida A, Ishikawa K, Goi T, Yamaguchi A, Ueda T, Inuzuka M (2004) Identification of a novel splice variant of the human anti-apoptosis gene survivin. Biochem Biophys Res Commun 314:902–907

    Article  PubMed  CAS  Google Scholar 

  28. Caldas H, Honsey LE, Altura RA (2005) Survivin 2alpha: a novel Survivin splice variant expressed in human malignancies. Mol Cancer 4:11–20

    Article  PubMed  Google Scholar 

  29. Krieg A, Mahotka C, Krieg T, Grabsch H, Muller W, Takeno S, Suschek CV, Heydthausen M, Gabbet HE, Gerharz CD (2002) Expression of different survivin variants in gastric carcinomas: first clues to a role of survivin-2B in tumour progression. Br J Cancer 86:737–743

    Article  PubMed  CAS  Google Scholar 

  30. Meng H, Lu C, Mabuchi H, Tanigawy N (2004) Prognostic significance and different properties of survivin splicing variants in gastric cancer. Cancer Lett 216:147–155

    Article  PubMed  CAS  Google Scholar 

  31. Taubert H, Kappler M, Bache M, Bartel F, Köhler T, Lautenschläger C, Blümke K, Würl P, Schmidt H, Meye A, Hauptmann S (2005) Elevated expression of survivin-splice variants predicts a poor outcome for soft-tissue sarcomas patients. Oncogene 24:5258–5261

    Article  PubMed  CAS  Google Scholar 

  32. Fields S, Sternglanz R (1994) The two-hybrid system: an assay for protein–protein interactions. Trends Genet 10:286–292

    Article  PubMed  CAS  Google Scholar 

  33. Vucic D, Deshayes K, Ackerly H, Pisabarro MT, Kadkhodayan S, Fairbrother WJ, Dixit VM (2002) SMAC negatively regulates the anti-apoptotic activity of melanoma inhibitor of apoptosis (ML-IAP). J Biol Chem 277:12275–12279

    Article  PubMed  CAS  Google Scholar 

  34. Yang QH, Church-Hajduk R, Ren J, Newton ML, Du C (2003) Omi/HtrA2 catalytic cleavage of inhibitor of apoptosis (IAP) irreversibly inactivates IAPs and facilitates caspase activity in apoptosis. Genes Dev 17:1487–1496

    Article  PubMed  CAS  Google Scholar 

  35. Sanna MG, da Silva Correia J, Ducrey O, Lee J, Nomoto K, Schrantz N, Deveraux QL, Ulevitch RJ (2002) IAP suppression of apoptosis involves distinct mechanisms: the TAK1/JNK1 signalling cascade and caspases inhibition. Mol Cell Biol 22:1754–1766

    Article  PubMed  CAS  Google Scholar 

  36. Nachmias B, Ashhab Y, Bucholtz V, Drize O, Kadouri L, Lotem M, Peretz T, Mandelboim O, Ben Yehuda D (2003) Caspase-mediated cleavage converts livin from an antiapoptotic to a proapoptotic factor: implications for drug-resistant melanoma. Cancer Res 63:6340–6349

    PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr. X. Wang for the Smac expression vector. This work was supported by grants from the Wilhelm-Sander Stiftung (2001.052.2) and the Deutsche Krebshilfe (106213).

Author information

Authors and Affiliations

  1. Deutsches Krebsforschungszentrum, Molekulare Therapie Virus-assoziierter Tumore (F065), Im Neuenheimer Feld 242, 69120, Heidelberg, Germany

    Irena Crnković-Mertens, Julia Semzow, Felix Hoppe-Seyler & Karin Butz

Authors
  1. Irena Crnković-Mertens
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Julia Semzow
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Felix Hoppe-Seyler
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Karin Butz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Karin Butz.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Crnković-Mertens, I., Semzow, J., Hoppe-Seyler, F. et al. Isoform-specific silencing of the Livin gene by RNA interference defines Livin β as key mediator of apoptosis inhibition in HeLa cells. J Mol Med 84, 232–240 (2006). https://doi.org/10.1007/s00109-005-0021-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00109-005-0021-5

Keywords

Search

Navigation