Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Oncogenes Fusion Genes and Tumor Suppressor Genes

Effective treatment of leukemic cell lines with wt1 siRNA

Abstract

The expression of wt1 and bcl-2 is considered to have a proliferating and survival supporting effect in leukemia blast cells. Here we describe the use of siRNA against wt1 and bcl-2 in leukemic cell lines for successful growth inhibition. We have used two different sequences designated as siRNA-A and siRNA-B corresponding to positions within the wt1 coding sequence to downregulate wt1 and a commercially available siRNA kit to downregulate bcl-2. WT1 and bcl-2 gene expression in transfected leukemic cell lines were evaluated with RT-PCR and western blot analyses. MTT assay was used to measure the cell viability and flow cytometry using annexin V/PI-staining for apoptosis. K562 and HL-60 cell lines transfected with siRNA-A targeted to wt1 had greatly decreased levels of both wt1 mRNA and protein expression. In contrast, siRNA-B and control siRNA led almost to no effect on wt1 mRNA and protein expression. siRNA-A-reduced wt1 mRNA expression was associated with a decreased cell proliferation and increased number of apoptotic cells in K562 and HL-60 cells by 24 and 48 h after transfection. Combined treatment with wt1 siRNA and bcl-2 siRNA simultaneously was not able to override the cell growth and apoptosis effects compared to single treatment with wt1 siRNA. siRNAs targeted against human wt1 might be a valuable tool as antiproliferative agent against wt1 expressing leukemic cells.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Dey BR, Sukhatme VP, Roberts AB, Sporn MB, Rauscher F, Kim SJ . Repression of the transforming growth factor-beta 1 gene by the Wilms’ tumor suppressor WT1 gene product. Mol Endocrinol 1994; 8: 595–602.

    CAS  Google Scholar 

  2. Oh S, Song Y, Yim J, Kim TK . The Wilms’ tumor 1 tumor suppressor gene represses transcription of the human telomerase reverse transcriptase gene. J Biol Chem 1999; 274: 37473–37478.

    Article  CAS  Google Scholar 

  3. Hewitt SH, Hamada S, McDonell TJ, Rauscher FJ, Saunders GF . Regulation of the Proto-oncogenes bcl-2 and c-myc by the Wilms’ Tumor Suppressor Gene WT1. Cancer Res 1995; 55: 5386–5389.

    CAS  Google Scholar 

  4. Maurer U, Jehan F, Englert C, Hübinger G, Karakas T, Weidmann E et al. The Wilms’ tumor gene strongly enhances suppression of cell growth mediated by 1,25-dihydroxy-vitamin D3 by induction of the vitamin D receptor (VDR). J Biol Chem 2001; 276: 3727–3732.

    Article  CAS  Google Scholar 

  5. Malik K, Poirier V, Ivins S, Brown K . Autoregulation of the human WT1 gene promotor. FEBS Lett 1994; 349: 75–78.

    Article  CAS  Google Scholar 

  6. Rauscher I, Morris JF, Tournay JF, Cook DM, Curran T . Binding of the Wilms’ tumor locus zinc finger protein to the EGR-1 consensus sequence. Science 1990; 250: 1259–1262.

    Article  CAS  Google Scholar 

  7. Miwa H, Beran M, Saunders GF . Expression of the Wilms’ tumor gene (WT1) in human leukemias. Leukemia 1992; 6: 405–409.

    CAS  Google Scholar 

  8. Loeb DM, Evron E, Patel CB, Sharma PM, Niranjan B, Buluwela L et al. Wilms’ tumor suppressor gene (WT1) is expressed in primary breast tumors despite tumor–specific promotor methylation. Cancer Res 2001; 61: 921–925.

    CAS  Google Scholar 

  9. Oji Y, Miyoshi S, Maeda H, Hayashi S, Sugijama H . Overexpression of the Wilms’ tumor gene WT1 in de novo lung cancers. Int J Cancer 2002; 100: 297–303.

    Article  CAS  Google Scholar 

  10. Oji Y, Yamamoto H, Nomura M, Nakano Y, Sugijama H . Overexpression of the Wilms’ tumor gene WT1 in colorectal adenocarcioma. Cancer Sci 2003; 94: 712–717.

    Article  CAS  Google Scholar 

  11. Oji Y, Miyoshi Y, Koga S, Nakano Y, Sugijama H . Overexpression of the Wilms’ tumor gene WT1 in primary thyroid cancer. Cancer Sci 2003; 94: 606–611.

    Article  CAS  Google Scholar 

  12. Ueda T, Oji Y, Naka N, Nakano Y, Sugijama H . Overexpression of the Wilms’ tumor gene WT1 in human bone and soft-tissue sarcomas. Cancer Sci 2003; 94: 271–276.

    Article  CAS  Google Scholar 

  13. Oji Y, Nakamori S, Fujikawa M, Nakatsuka SI, Sugiyama H . Overexpression of the Wilms’ tumor gene WT1 in pancreatic ductal adenocarcinoma. Cancer Sci 2004; 95: 583–587.

    Article  CAS  Google Scholar 

  14. Miyoshi Y, Ando A, Egawa C, Taguchi T, Tamaki Y, Tamaki H et al. High expression of Wilms’ tumor suppressor gene predicts poor prognosis in breast cancer patients. Clin Cancer Res 2002; 8: 1167–1171.

    CAS  Google Scholar 

  15. Brieger J, Weidmann E, Fenchel K, Mitrou PS, Hoelzer D, Bergmann L . The expression of the Wilms’ tumor gene in acute myelocytic leukemias as a possible marker for leukemic blast cells. Leukemia 1994; 8: 2138–2143.

    CAS  Google Scholar 

  16. Brieger J, Weidmann E, Maurer U, Hoelzer D, Mitrou PS, Bergmann L . The Wilms’ tumor gene is frequently expressed in acute leukemias and may provide a marker for residual blast cells detectable by PCR. Ann Oncol 1995; 8: 811–816.

    Article  Google Scholar 

  17. Bergmann L, Miething CC, Maurer U, Brieger J, Karakas T, Weidmann E et al. High levels of Wilms’ tumor gene (wt1) mRNA in acute myeloid leukemias are associated with a worse long-term outcome. Blood 1997; 90: 1217–1225.

    CAS  Google Scholar 

  18. Inoue K, Sugiyama H, Ogawa H, Nakagawa M, Yamagami T, Miwa H . WT1 as a new prognostic factor and a new marker for the detection of minimal residual disease in acute leukaemia. Blood 1994; 84: 3071–3079.

    CAS  Google Scholar 

  19. Barragan E, Cervera J, Bolufer P, Ballester S, Martin G, Fernandez P et al. Prognostic implications of Wilms’ tumor gene (WT1) expression in patients with de novo acute myeloid leukemia. Haematologica 2004; 89: 26–33.

    Google Scholar 

  20. Chiusa L, Francia di Celle P, Campisi P, Ceretto C, Marmont F, Pich A . Prognostic value of quantitative analysis of WT1 gene transcripts in adult acute lymphoblastic leukemia. Haematologica 2006; 91: 270–271.

    CAS  Google Scholar 

  21. Yamagami T, Sugiyama H, Inoue K, Ogawa H, Tatekawa T, Hirata M et al. Growth inhibition of human leukemic cells by WT1 (Wilms’ tumor gene) antisense oligodeoxynucleotides: implications for the involvement of WT1 in leukemogenesis. Blood 1996; 87: 2878–2884.

    CAS  Google Scholar 

  22. Yamagami T, Ogawa H, Tamaki H, Oji Y, Soma T, Oka Y et al. Suppression of Wilms’ Tumor gene (WT1) expression induces G2/M arrest in leukemic cells. Leuk Res 1998; 22: 383–384.

    Article  CAS  Google Scholar 

  23. Huebinger G, Schmid M, Linortner S, Manegold A, Bergmann L, Maurer U . Ribozyme-mediated cleavage of wt1transcription suppresses growth of leukemia cells. Exp Hematol 2001; 29: 1226–1235.

    Article  Google Scholar 

  24. Hirose M, Kuroda Y . p53 may mediate the mdr-1 expression via the WT1 gene in human vincristine-resistant leukemia/lymphoma cell lines. Cancer lett 1998; 129: 165–171.

    Article  CAS  Google Scholar 

  25. Carrington D, Algar E . Overexpression of murine WT1 +/+ and −/− isoforms has no effect on chemoresistance but delays differentiation in K562 cell line. Leuk Res 2000; 24: 927–936.

    Article  CAS  Google Scholar 

  26. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T . Duplexes of 21 nucleotide RNAs mediate interference in cultured mammalian cells. Nature 2001; 411: 494–498.

    Article  CAS  Google Scholar 

  27. Kreuzer KA, Saborowski A, Lupberger J, Appelt C, Na I . Fluorescent 50-exonuclease assay for the absolute quantification of Wilms' tumour gene (WT1) mRNA: implications for monitoring human leukaemias. Br J Haematol 2001; 114: 313–318.

    Article  CAS  Google Scholar 

  28. Cilloni D, Gottardi E, Messa F, Fava M, Scaravaglio P, Bertini M et al. Significant correlation between the degree of WT1 expression and the international prognostic scoring system score in patients with myelodysplastic syndromes. J Clin Oncol 2003; 21: 1988–1995.

    Article  CAS  Google Scholar 

  29. Fisher G, Advani R, Wakelee H, Jacobs C, Gladysheva K, Fitzgerald AM . A phase I trial of oblimersen and gemcitabine in refractory and advanced malignancies. Proc Annu Meeting ASCO 2005; 23 (16S): 234s.

    Google Scholar 

  30. Marcucci G, Stock W, Dai G . Phase I study of oblimersen sodium, an antisense to Bcl-2, in untreated older patients with acute myeloid leukemia: pharmacokinetics, pharmacodynamics, and clinical activity. J Clin Oncol 2005; 23: 3404–3411.

    Article  CAS  Google Scholar 

  31. Campos L, Rouault JP, Sabido O, Oriol P, Roubi N, Vasselon C et al. High expression of bcl-2 protein in acute myeloid leukemia is associated with poor response to chemotherapy. Blood 1993; 81: 3091–3096.

    CAS  Google Scholar 

  32. Karakas T, Miething CC, Maurer U, Weidmann E, Ackermann H, Hoelzer D et al. The coexpression of the apoptosis-related genes bcl-2 and wt1in predicting survival in adult acute myeloid leukaemia. Leukemia 2002; 16: 846–854.

    Article  CAS  Google Scholar 

  33. Elmaagacli A, Koldehoff M, Peceny R, Klein-Hitpass L, Ottinger H, Beelen D et al. WT1 and BCR-ABL specific small interfering RNA have additive effects in the induction of apoptosis in leukemic cells. Haematologica 2005; 90: 326–334.

    CAS  Google Scholar 

  34. Del Bufalo D, Trisciuoglio D, Scarsella M, Zangemeister-Wittke U, Zupi G . Treatment of melanoma cells with a bcl-2/bcl-xL antisense oligonucleotide induces antiangiogenic activity. Oncogene 2003; 22: 8441–8447.

    Article  CAS  Google Scholar 

  35. Pandyra AA, Berg R, Vincent M, Koropatnik J . Combination siRNA targeting Bcl-2 antagonizes siRNA against thymidylate synthase in human tumor cell lines. J Pharmacol Exp Ther 2007; 322: 123–132.

    Article  CAS  Google Scholar 

  36. Klasa RJ, Gillum AM, Klem RE, Frankel SR . Oblimersen Bcl-2 antisense: facilitating apoptosis in anticancer treatment. Antisense Nucleic Acid Drug Dev 2002; 12: 193–213.

    Article  CAS  Google Scholar 

  37. Tolcher AW . Targeting Bcl-2 protein expression in solid tumors and hematologic malignancies with antisense oligonucleotides. Clin Adv Hematol Oncol 2005; 3: 635–642.

    Google Scholar 

  38. Kim R, Emi M, Matsuura K, Tanabe K . Antisense and nonantisense effects of antisense Bcl-2 on multiple roles of Bcl-2 as a chemosensitizer in cancer therapy. Cancer Gene Ther 2007; 14: 1–11.

    Article  CAS  Google Scholar 

  39. Daruka M, List A . Targeting the multidrug resistance-1 transporter in AML: molecular regulation and therapeutic strategies. Blood 2004; 104: 1940–1951.

    Article  Google Scholar 

  40. Cilloni D, Messa F, Gottardi E, Fava M, Arruga F, Defilippi I et al. Sensitivity to imatinib therapy may be predicted by testing Wilms’ tumor gene expression and colony growth after a short in vitro incubation. Cancer 2004; 101: 979–988.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Adolf-Messer-stiftung, Koenigstein, Germany.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to L Bergmann.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Glienke, W., Maute, L., Koehl, U. et al. Effective treatment of leukemic cell lines with wt1 siRNA. Leukemia 21, 2164–2170 (2007). https://doi.org/10.1038/sj.leu.2404878

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.leu.2404878

Keywords

Search

Quick links