Advertisement

Journal of Neuro-Oncology

, Volume 72, Issue 1, pp 29–34 | Cite as

ΔNp73 antisense activates PUMA and induces apoptosis in neuroblastoma cells

  • A.P. Simões-Wüst
  • B. Sigrist
  • L. Belyanskaya
  • S. Hopkins Donaldson
  • R.A. Stahel
  • U. Zangemeister-Wittke
Laboratory Investigation

Abstract

The p73 gene codes for various different protein isoforms. They include proteins expressed under the control of the P1 promoter that contain a transactivation domain and are similar in function to p53 (TAp73 isoforms), as well as proteins regulated by the P2 promoter that lack this domain and function as dominant negative inhibitors of TAp73 and p53 (ΔNp73 isoforms). Whereas TAp73 functions as a tumor suppressor with pro-apoptotic function, ΔNp73 is likely to prevent the induction of apoptosis in tumor cells and to participate in oncogenesis. Here we used a loss-of-function strategy to assess the role of ΔNp73 in SH-SY5Y neuroblastoma cells. An antisense oligonucleotide designed to target ΔNp73 mRNA, but not TAp73, was used to effectively downregulate this transcript. ΔNp73 downregulation was accompanied by increased levels of the pro-apoptotic BH3 family member PUMA at the mRNA and protein level, and by conformational activation of BAX which translocated to mitochondria. These ΔNp73 antisense-mediated alterations led to the induction of apoptosis as detected by decreased cell viability, augmented DNA fragmentation and increased caspase-3 activity in cell lysates. Our results demonstrate the cytoprotective role of ΔNp73 in neuroblastoma and suggest its use as a target for molecular intervention therapy.

Keywords

antisense apoptosis bax neuroblastoma ΔNp73 PUMA 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Vousden, KH, Lu, X 2002Live or let die: the cell’s response to p53Nat Rev Cancer2594604CrossRefPubMedGoogle Scholar
  2. 2.
    Yang, A, Walker, N, Bronson, R, Kaghad, M, Oosterwegel, M, Bonnin, J, Vagner, C, Bonnet, H, Dikkes, P, Sharpe, A, McKeon, F, Caput, D 2000p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumoursNature40499103Google Scholar
  3. 3.
    Melino, G, De, LV, Vousden, KH 2002p73: Friend or foe in tumorigenesisNat Rev Cancer2605615Google Scholar
  4. 4.
    Melino, G, Bernassola, F, Ranalli, M, Yee, K, Zong, WX, Corazzari, M, Knight, RA, Green, DR, Thompson, C, Vousden, KH 2003p73 induces apoptosis via PUMA transactivation and Bax mitochondrial translocationJ Biol Chem27980768083Google Scholar
  5. 5.
    Goldschneider, D, Blanc, E, Raguenez, G, Barrois, M, Legrand, A, Le Roux, G, Haddada, H, Benard, J, Douc-Rasy, S 2004Differential response of p53 target genes to p73 overexpression in SH-SY5Y neuroblastoma cell lineJ Cell Sci117293301Google Scholar
  6. 6.
    Nakagawa, T, Takahashi, M, Ozaki, T, Watanabe, K, Hayashi, S, Hosoda, M, Todo, S, Nakagawara, A 2003Negative autoregulation of p73 and p53 by ΔNp73 in regulating differentiation and survival of human neuroblastoma cellsCancer Lett197105109Google Scholar
  7. 7.
    Grob, TJ, Novak, U, Maisse, C, Barcaroli, D, Luthi, AU, Pirnia, F, Hugli, B, Graber, HU, De, LV, Fey, MF, Melino, G, Tobler, A 2001Human ΔNp73 regulates a dominant negative feedback loop for TAp73 and p53Cell Death Differ812131223Google Scholar
  8. 8.
    Ishimoto, O, Kawahara, C, Enjo, K, Obinata, M, Nukiwa, T, Ikawa, S 2002Possible oncogenic potential of ΔNp73: a newly identified isoform of human p73Cancer Res62636641Google Scholar
  9. 9.
    Stiewe, T, Zimmermann, S, Frilling, A, Esche, H, Putzer, BM 2002Transactivation-deficient ΔTA-p73 acts as an oncogeneCancer Res6235983602Google Scholar
  10. 10.
    Zaika, AI, Slade, N, Erster, SH, Sansome, C, Joseph, TW, Pearl, M, Chalas, E, Moll, UM 2002ΔNp73, a dominant-negative inhibitor of wild-type p53 and TAp73, is up-regulated in human tumorsJ Exp Med196765780Google Scholar
  11. 11.
    Vossio, S, Palescandolo, E, Pediconi, N, Moretti, F, Balsano, C, Levrero, M, Costanzo, A 2002DN-p73 is activated after DNA damage in a p53-dependent manner to regulate p53-induced cell cycle arrestOncogene2137963803Google Scholar
  12. 12.
    Allart, S, Martin, H, Detraves, C, Terrasson, J, Caput, D, Davrinche, C 2002Human cytomegalovirus induces drug resistance and alteration of programmed cell death by accumulation of ΔN-p73αJ Biol Chem2772906329068Google Scholar
  13. 13.
    Nakagawa, T, Takahashi, M, Ozaki, T, Watanabe Ki, K, Todo, S, Mizuguchi, H, Hayakawa, T, Nakagawara, A 2002Autoinhibitory regulation of p73 by ΔNp73 to modulate cell survival and death through a p73-specific target element within the ΔNp73 promoterMol Cell Biol2225752585Google Scholar
  14. 14.
    Noesel, MM, Versteeg, R 2004Pediatric neuroblastomas: genetic and epigenetic ‘Danse Macabre’Gene325115Google Scholar
  15. 15.
    Casciano, I, Mazzocco, K, Boni, L, Pagnan, G, Banelli, B, Allemanni, G, Ponzoni, M, Tonini, GP, Romani, M 2002Expression of ΔNp73 is a molecular marker for adverse outcome in neuroblastoma patientsCell Death Differ9246251Google Scholar
  16. 16.
    Ziegler, A, Simões-Wüst, AP, Zangemeister-Wittke, U 2000Optimizing efficacy of antisense oligodeoxynucleotides targeting inhibitors of apoptosisMeth Enzymol314477490Google Scholar
  17. 17.
    Simões-Wüst, AP, Olie, RA, Gautschi, O, Leech, SH, Haner, R, Hall, J, Fabbro, D, Stahel, RA, Zangemeister-Wittke, U 2000Bcl-xl antisense treatment induces apoptosis in breast carcinoma cellsInt J Cancer87582590Google Scholar
  18. 18.
    Gong, J, Traganos, F, Darzynkiewicz, Z 1994A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometryAnal Biochem218314319Google Scholar
  19. 19.
    Darzynkiewicz, Z, Juan, G, Li, X, Gorczyca, W, Murakami, T, Traganos, F 1997Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis)Cytometry27120Google Scholar
  20. 20.
    Matzura, O, Wennborg, A 1996RNAdraw: an integrated program for RNA secondary structure calculation and analysis under 32-bit Microsoft WindowsComput Appl Biosci12247249Google Scholar
  21. 21.
    McManus, MT, Sharp, PA 2002Gene silencing in mammals by small interfering RNAsNat Rev Genet3737747Google Scholar
  22. 22.
    Cory, S, Huang, DC, Adams, JM 2003The Bcl-2 family: roles in cell survival and oncogenesisOncogene2285908607CrossRefPubMedGoogle Scholar
  23. 23.
    Dias, N, Stein, CA 2002Antisense oligonucleotides: basic concepts and mechanismsMol Cancer Ther1347355Google Scholar
  24. 24.
    Braasch, DA, Corey, DR 2002Novel antisense and peptide nucleic acid strategies for controlling gene expressionBiochemistry4145034510Google Scholar
  25. 25.
    Levin, AA 1999A review of the issues in the pharmacokinetics and toxicology of phosphorothioate antisense oligonucleotidesBiochim Biophys Acta14896984Google Scholar
  26. 26.
    Mihara, M, Erster, S, Zaika, A, Petrenko, O, Chittenden, T, Pancoska, P, Moll, UM 2003p53 has a direct apoptogenic role at the mitochondriaMol Cell11577590CrossRefPubMedGoogle Scholar
  27. 27.
    Puthalakath, H, Strasser, A 2002Keeping killers on a tight leash: transcriptional and post-translational control of the pro-apoptotic activity of BH3-only proteinsCell Death Differ9505512Google Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • A.P. Simões-Wüst
    • 1
  • B. Sigrist
    • 1
  • L. Belyanskaya
    • 1
  • S. Hopkins Donaldson
    • 1
  • R.A. Stahel
    • 1
  • U. Zangemeister-Wittke
    • 1
  1. 1.Molecular Oncology LaboratoryUniversity Hospital ZurichZurichSwitzerland

Personalised recommendations