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:

RNA interference is a functional pathway with therapeutic potential in human myeloid leukemia cell lines

Cancer Gene Therapy volume 10, pages 125–133 (2003)Cite this article

Abstract

Background: RNA interference (RNAi) is a cellular pathway of gene silencing in a sequence-specific manner at the messenger RNA level. The basic mechanism behind RNAi is the breaking of a double-stranded RNA (dsRNA) matching a specific gene sequence into short pieces called short interfering RNA, which trigger the degradation of mRNA that matches its sequence. In this study, we explored the effects of RNAi in reducing the target gene expression in human myeloid leukemia cell lines. Methods: Four myeloid leukemia cell lines (HL-60, U937, THP-1, and K562) were transfected with dsRNA duplexes corresponding to the endogenous c-raf and bcl-2 genes and the gene expression inhibition was assessed. The effect of RNAi on cell differentiation was studied; the apoptosis induction and the sensitization of the leukemia cell lines to etoposide and daunorubicin were quantified by flowcytometric methods. Results: Transfection of the myeloid leukemia cell lines with dsRNA corresponding to c-raf and bcl-2 genes decreased the expression of Raf-1 and Bcl-2 proteins. RNAi for c-raf gene blocked the appearance of the monocytic differentiation induced by treatment with TPA. Combined RNAi for c-raf and bcl-2 induced apoptosis in HL-60, U937, and THP-1 cells and increased chemosensitivity to etoposide and daunorubicin. Conclusions: RNAi is a functional pathway in human myeloid leukemia cell lines and combined RNAi of c-raf and bcl-2 genes may represent a novel approach to leukemia, providing a means to overcome the resistance to chemotherapeutic agents and ultimately to augment the efficacy of chemotherapy in myeloid leukemia.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

References

  1. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE . Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391:806 –811.

    Article  CAS  Google Scholar 

  2. Clemens JC, Worby CA, Siminson-Leff N, et al. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc Natl Acad Sci USA. 2000;97:6499–6503.

    Article  CAS  Google Scholar 

  3. Misquitta L, Peterson BM . Targeted disruption of gene function in Drosophila by RNA interference: a role for nautilus in embryonic somatic muscle formation. Proc Natl Acad Sci USA. 1999;96:1451–1456.

    Article  CAS  Google Scholar 

  4. Hammond SM, Bernstein E, Beach D, Hannon GJ . An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature. 2000;404:293–296.

    Article  CAS  Google Scholar 

  5. Kennerdell JR, Carthew RW . Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled2 act in the winglass pathway. Cell. 1998;95:1017–1026.

    Article  CAS  Google Scholar 

  6. Ngo H, Tschudi C, Gull K, Ullu E . Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc Natl Acad Sci USA. 1998;95:14687–14692.

    Article  CAS  Google Scholar 

  7. Sanchez-Alvarado A, Newmark PA . Double-stranded RNA specifically disrupt gene expression during planarian regeneration. Proc Natl Acad Sci USA. 1999;96:5049–5054.

    Article  CAS  Google Scholar 

  8. Lohmann JU, Endl I, Bosch TC . Silencing of developmental genes in Hydra. Dev Biol. 1999;214:211–214.

    Article  CAS  Google Scholar 

  9. Sharp PA . RNA interference 2001. Genes Dev. 2001;15:485–490.

    Article  CAS  Google Scholar 

  10. Marx S . Interfering with gene expression. Science. 2000;288:1370–1372.

    Article  CAS  Google Scholar 

  11. Nykanen A, Haley B, Zamore PD . ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell. 2001;107:309–321.

    Article  CAS  Google Scholar 

  12. Elbashir SM, Lendeckel W, Tuschl T . RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 2001;15:188–200.

    Article  CAS  Google Scholar 

  13. Hunter T, Hunt T, Jackson RJ, Robertson HD . The characteristics of inhibition of protein synthesis by double-stranded ribonucleic acid in reticulocyte lysates. J Biol Chem. 1975;250:409–417.

    CAS  PubMed  Google Scholar 

  14. Bass BL . The short answer. Nature. 2001;11:428–429.

    Article  Google Scholar 

  15. Minks MA, West DK, Benvin S, Baglioni C . Structural requirements of double-stranded RNA for the activation of 2′,5′-oligo (A) polymerase and protein kinase of interferon-treated HeLa cells. J Biol Chem. 1979;254:10180–10183.

    CAS  PubMed  Google Scholar 

  16. 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 

  17. Funato T, Kozawa K, Fujimaki S, Kaku M . Increased sensitivity to cytosine arabinoside in human leukemia by c-raf-1 antisense oligonucleotides. Anticancer Drugs. 2001;12:325–329.

    Article  CAS  Google Scholar 

  18. Miyashita T, Reed JC . BCL-2 gene transfer increases relative resistance of S49.1 and WEH17.2 lymphoid cells to cell death and DNA fragmentation induced by glucocorticoids and multiple chemotherapeutic drugs. Cancer Res. 1992;52:5407–5411.

    CAS  PubMed  Google Scholar 

  19. Miyashita T, Reed JC . Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a human leukemia cell line. Blood. 1993;81:151–157.

    CAS  PubMed  Google Scholar 

  20. Wang HG, Miyashita T, Takayama S, et al. Apoptosis regulation by interaction of Bcl-2 protein and Raf-1 kinase. Cancer Res. 1994;9:2751–2756.

    CAS  Google Scholar 

  21. Okuda K, Matulonis U, Salgia R, Kanakura Y, Druker B, Griffin JD . Factor independence of human myeloid leukemia cell lines is associated with increased phosphorylation of the proto-oncogene Raf-1. Exp Hematol. 1994;22:1111–1117.

    CAS  PubMed  Google Scholar 

  22. Collins SJ, Gallo RC, Gallagher RE . Continuous growth and differentiation of human myeloid leukemic cells in suspension culture. Nature. 1977;270:347–349.

    Article  CAS  Google Scholar 

  23. Sundstrom C, Nilsson K . Establishment and characterization of a human histocytic cell line (U-937). Int J Cancer. 1976;17:565–577.

    Article  CAS  Google Scholar 

  24. Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno T, Tada K . Establishment and characterization of a human acute monocytic leukemic cell line (THP-1). Int J Cancer. 1980;26:171–176.

    Article  CAS  Google Scholar 

  25. Lozzio CB, Lozzio BB . Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood. 1975;45:321–334.

    CAS  PubMed  Google Scholar 

  26. Monia BP, Johnston JF, Geiger T, Muller M, Fabbro D . Antitumor activity of a phosphorothioate antisense oligodeoxynucleotide targeted against C-raf kinase. Nat Med. 1996;2:668–675.

    Article  CAS  Google Scholar 

  27. Konopleva M, Tari AM, Estrov Z, et al. Liposomal Bcl-2 antisense oligonucleotides enhance proliferation, sensitive acute myeloid leukemia to cytosine-arabinoside, and induce apoptosis independent of other antiapoptotic proteins. Blood. 2000;95:3929–3938.

    CAS  PubMed  Google Scholar 

  28. Duriez PJ, Shah GM . Cleavage of poly (ADP-ribose) polymerase: a sensitive parameter to study cell death. Biochem Cell Biol. 1997;75:337–349.

    Article  CAS  Google Scholar 

  29. Zamzami N, Marchetti P, Castedo M, et al. Reduction in mitochondrial potential constitutes an early irreversible step of programmed cell death. J Exp Med. 1995;181:1661–1672.

    Article  CAS  Google Scholar 

  30. Kharbanda S, Saleem A, Emoto Y, Stone R, Rapp U, Kufe D . Activation of Raf-1 and mitogen-activated protein kinases during monocytic differentiation of human myeloid leukemia cells. J Biol Chem. 1994;269:872–878.

    CAS  PubMed  Google Scholar 

  31. Amarante-Mendes GP, Naekyung KC, Liu L, et al. Bcr-Abl exerts its antiapoptotic effect against diverse apoptotic stimuli through blockage of mitochondrial release of cytochrome c and activation of caspase-3. Blood. 1998;91:1700–1705.

    CAS  PubMed  Google Scholar 

  32. Benito A, Lerga A, Silva M, Leon J, Fernandez-Luna JL . Apoptosis of human myeloid leukemia cells induced by an inhibitor of protein phosphatases (okadaic acid) is prevented by Bcl-2 and Bcl-X(L). Leukemia. 1997;11:940–944.

    Article  CAS  Google Scholar 

  33. Stiewe T, Parssanedjad K, Esche H, Opalka B, Putzer BM . E1A overcomes the apoptosis block in BCR-ABL+ leukemia cells and renders cells susceptible to induction of apoptosis by chemotherapeutic agents. Cancer Res. 2000;60:3957–3964.

    CAS  PubMed  Google Scholar 

  34. Smith BD, Bambach BJ, Vala MS, et al. Inhibited apoptosis and drug resistance in acute myeloid leukemia. Br J Haematol. 1998;102:1042–1049.

    Article  CAS  Google Scholar 

  35. Wang HG, Rapp UR, Reed JC . Bcl-2 targets the protein kinase Raf-1 to mitochondria. Cell. 1996;87:629–638.

    Article  CAS  Google Scholar 

  36. Olivier R, Otter I, Monney L, Wartmann M, Borner C . Bcl-2 does not require Raf kinase activity for its death-protective function. Biochem J. 1997;324:75–83.

    Article  CAS  Google Scholar 

  37. Zhong J, Troppmair J, Rapp UR . Independent control of cell survival by Raf-1 and Bcl-2 at the mitochondria. Oncogene. 2001;20:4807–4816.

    Article  CAS  Google Scholar 

  38. Yang T, Kozopas KM, Craig RW . The intracellular distribution and pattern of expression of Mcl-1 overlap with, but are not identical, to those of Bcl-2. J Cell Biol. 1995;128:1173–1184.

    Article  CAS  Google Scholar 

  39. Kitada S, Andersen J, Akar S, et al. Expression of apoptosis-regulating proteins in chronic lymphocytic leukemia: correlations with in vitro and in vivo chemoresponses. Blood. 1998;91:3379–3389.

    CAS  PubMed  Google Scholar 

  40. Pepper C, Bentley P, Hoy T . Regulation of clinical chemoresistance by bcl-2 and bax oncoproteins in B-cell chronic lymphocytic leukemia. Br J Haematol. 1996;95:513–517.

    Article  CAS  Google Scholar 

  41. Krontiris TG . Oncogenes. N Engl J Med. 1995;333:303–306.

    Article  CAS  Google Scholar 

  42. Levitzki A, Gazit A . Tyrosine kinase inhibition: an approach to drug development. Science. 1995;267:1782–1788.

    Article  CAS  Google Scholar 

  43. Nakayama K, Negishi I, Kuika K, Sawa H, Loh D . Targeted disruption of Bcl-2αβ in mice: occurrence of gray hair, polycystic disease and lymphocytopenia. Proc Natl Acad Sci USA. 1994;91:3700–3704.

    Article  CAS  Google Scholar 

  44. Schimmer AD, Hedley DW, Penn LZ, Minden MD . Receptor- and mitochondrial apoptosis in acute leukemia: a translational view. Blood. 2001;98:3541–3553.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. The Second Department of Internal Medicine, Shinshu University School of Medicine, Matsumoto, Nagano, Japan

    Daniel P Cioca, Yuji Aoki & Kendo Kiyosawa

Authors
  1. Daniel P Cioca
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yuji Aoki
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kendo Kiyosawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Daniel P Cioca.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cioca, D., Aoki, Y. & Kiyosawa, K. RNA interference is a functional pathway with therapeutic potential in human myeloid leukemia cell lines. Cancer Gene Ther 10, 125–133 (2003). https://doi.org/10.1038/sj.cgt.7700544

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.cgt.7700544

Keywords

This article is cited by

Search

Advanced search

Quick links