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Post-transcriptional silencing of Notch2 mRNA in chronic lymhocytic leukemic cells of B-CLL patients

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An Erratum to this article was published on 31 March 2012

Abstract

Environmental and genomic stresses induce different pathological conditions and one of them is blood cancer. This escalating load of disease with a constant threat to life requires an intensive comprehensive response. For our understanding about the cancer treatment capabilities, novel medicinal platforms should be strived to explore among the existing conventional and molecular approaches that have already been proven to be successful in fighting against genetic diseases. Several DNA therapeutics previously studied are currently in clinical settings. RNA interfering antisense oligonucleotide (AS-ODN) is the most experimentally advanced molecular therapeutic which has the potential to modify the gene activity resulting in the down regulation of particular protein. In this study, we focused on the inhibition of Notch2 function in B-cell chronic lymphocytic leukemia (B-CLL) by AS-ODN (phosphorothioate oligomers) targeted to the initiation codon region of the Notch2 mRNA. We investigated the in vitro ability of four such oligomers to reduce the expression of Notch2 gene in peripheral blood mononuclear cells from B-CLL patients. Our findings implicate that AS-ODNs specifically designed for the region of 314–333 neucleotides (AS1) of Notch2 inhibits its gene expression better than other AS-ODNs designed for other regions and respond in a dose dependent manner. The results of cell proliferation assay for the evaluation of AS1 in gene silencing, infer that the number of cells were reduced to 80% (P < 0.001). Our results implicate that using the AS-ODNs against specific Notch2 nucleotide sequence can be used as future therapeutic agent with the ability of Notch2 down regulation, which is the root problem in the pathogenicity of B-CLL.

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References

  1. Kay NE, Hamblin TJ, Jelinek DF, Dewald GW, Byrd JC, Farag S, Lucas M, Lin T (2002) Chronic lymphocytic leukemia. Hematology 2002(1):193–213

    Article  Google Scholar 

  2. Guipaud O, Deriano L, Salin H, Vallat L, Sabatier L, Merle-Béral H, Delic J (2003) B-cell chronic lymphocytic leukaemia: a polymorphic family unified by genomic features. Lancet Oncol 4(8):505–514

    Article  PubMed  Google Scholar 

  3. Chiorazzi N, Rai KR, Ferrarini M (2005) Chronic lymphocytic leukemia. N Engl J Med 352(8):804–815

    Article  PubMed  CAS  Google Scholar 

  4. Kipps TJ (2000) Chronic lymphocytic leukemia. Curr Opin Hematol 7(4):223–234

    Article  PubMed  CAS  Google Scholar 

  5. Caligaris-Cappio F, Hamblin TJ (1999) B-cell chronic lymphocytic leukemia: a bird of a different feather. J Clin Oncol 17(1):399–408

    PubMed  CAS  Google Scholar 

  6. Cuni S, Perez-Aciego P, Perez-Chacon G, Vargas J, Sanchez A, Martin-Saavedra F, Ballester S, Garcia-Marco J, Jorda J, Durantez A (2004) A sustained activation of PI3 K/NF-B pathway is critical for the survival of chronic lymphocytic leukemia B cells. Leukemia 18(8):1391–1400

    Article  PubMed  CAS  Google Scholar 

  7. Zaninoni A, Imperiali FG, Pasquini C, Zanella A, Barcellini W (2003) Cytokine modulation of nuclear factor-[kappa] B activity in B-chronic lymphocytic leukemia. Exp Hematol 31(3):185–190

    Article  PubMed  CAS  Google Scholar 

  8. Lagneaux L, Delforge A, Bron D, De Bruyn C, Stryckmans P (1998) Chronic lymphocytic leukemic B cells but not normal B cells are rescued from apoptosis by contact with normal bone marrow stromal cells. Blood 91(7):2387–2396

    PubMed  CAS  Google Scholar 

  9. Endo T, Nishio M, Enzler T, Cottam HB, Fukuda T, James DF, Karin M, Kipps TJ (2007) BAFF and APRIL support chronic lymphocytic leukemia B-cell survival through activation of the canonical NF-{kappa} B pathway. Blood 109(2):703–710

    Article  PubMed  CAS  Google Scholar 

  10. Ghia P, Caligaris-Cappio F (2000) The indispensable role of microenvironment in the natural history of low-grade B-cell neoplasms. Adv Cancer Res 79:157–173

    Article  PubMed  CAS  Google Scholar 

  11. Hubmann R, Schwarzmeier JD, Shehata M, Hilgarth M, Duechler M, Dettke M, Berger R (2002) Notch2 is involved in the overexpression of CD23 in B-cell chronic lymphocytic leukemia. Blood 99(10):3742–3747

    Article  PubMed  CAS  Google Scholar 

  12. Fournier S, Yang L, Delespesse G, Rubio M, Biron G, Sarfati M (1995) The two CD23 isoforms display differential regulation in chronic lymphocytic leukaemia. Br J Haematol 89(2):373–379

    Article  PubMed  CAS  Google Scholar 

  13. Ohishi K, Katayama N, Shiku H, Varnum-Finney B, Bernstein I (2003) Notch signalling in hematopoiesis. Semin Cell Develop Biol 14:143–150

    Article  CAS  Google Scholar 

  14. Artavanis-Tsakonas S, Rand MD, Lake RJ (1999) Notch signaling: cell fate control and signal integration in development. Science 284(5415):770–776

    Article  PubMed  CAS  Google Scholar 

  15. Milner LA, Bigas A (1999) Notch as a mediator of cell fate determination in hematopoiesis: evidence and speculation. Blood 93(8):2431–2448

    PubMed  CAS  Google Scholar 

  16. Leong KG, Karsan A (2006) Recent insights into the role of Notch signaling in tumorigenesis. Blood 107(6):2223–2233

    Article  PubMed  CAS  Google Scholar 

  17. Miele L, Osborne B (1999) Arbiter of differentiation and death: notch signaling meets apoptosis. J Cell Physiol 181(3):393–409

    Article  PubMed  CAS  Google Scholar 

  18. Allman D, Punt JA, Izon DJ, Aster JC, Pear WS (2002) An invitation to T and more: notch signaling in lymphopoiesis. Cell 109(2):S1–S11

    Article  PubMed  CAS  Google Scholar 

  19. Kopan R (2002) Notch: a membrane-bound transcription factor. J Cell Sci 115(Pt 6):1095–1097

    PubMed  CAS  Google Scholar 

  20. Kato H, Taniguchi Y, Kurooka H, Minoguchi S, Sakai T, Nomura-Okazaki S, Tamura K, Honjo T (1997) Involvement of RBP-J in biological functions of mouse Notch1 and its derivatives. Development 124(20):4133–4141

    PubMed  CAS  Google Scholar 

  21. Jarriault S, Brou C, Logeat F, Schroeter EH, Kopan R, Israel A (1995) Signalling downstream of activated mammalian Notch. Nature 377:355–358

    Article  PubMed  CAS  Google Scholar 

  22. Schroeter EH, Kisslinger JA, Kopan R (1998) Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain. Nature 393(6683):382–386

    Article  PubMed  CAS  Google Scholar 

  23. Berezovska O, Jack C, McLEAN P, Aster JC, Hicks C, Xia W, Wolfe MS, Weinmaster G, Selkoe DJ, Hyman BT (2000) Rapid Notch1 nuclear translocation after ligand binding depends on presenilin associated secretase activity. Ann N Y Acad Sci 920(1):223–226

    Article  PubMed  CAS  Google Scholar 

  24. Mumm JS, Kopan R (2000) Notch signaling: from the outside in. Dev Biol 228(2):151–165

    Article  PubMed  CAS  Google Scholar 

  25. Byrd JC, Stilgenbauer S, Flinn IW (2004) Chronic lymphocytic leukemia. Hematology 2004(1):163–183

    Article  Google Scholar 

  26. Institute NC (2011) Chronic lymphocytic leukemia (PDQ) treatment: stage information. National Institutes of Health. http://www.cancer.gov/cancertopics/pdq/treatment/CLL/healthprofessional/page2. Accessed 22 Feb 2011

  27. Lamanna N, Jurcic JG, Noy A, Maslak P, Gencarelli AN, Panageas KS, Heaney ML, Brentjens RJ, Golde DW, Scheinberg DA (2009) Sequential therapy with fludarabine, high-dose cyclophosphamide, and rituximab in previously untreated patients with chronic lymphocytic leukemia produces high-quality responses: molecular remissions predict for durable complete responses. J Clin Oncol 27(4):491–497

    Article  PubMed  CAS  Google Scholar 

  28. Tam CS, O’Brien S, Wierda W, Kantarjian H, Wen S, Do KA, Thomas DA, Cortes J, Lerner S, Keating MJ (2008) Long-term results of the fludarabine, cyclophosphamide, and rituximab regimen as initial therapy of chronic lymphocytic leukemia. Blood 112(4):975–980

    Article  PubMed  CAS  Google Scholar 

  29. Rai K, Peterson B, Appelbaum F, Tallman M, Belch A, Morrison V (2009) Long-term survival analysis of the North American Intergroup Study C9011 comparing fludarabine (F) and chlorambucil (C) in previously untreated patients with chronic lymphocytic leukemia (CLL). Blood 114(22):A-536

    Google Scholar 

  30. Hallek M, Fingerle-Rowson G, Fink AM, Busch R, Mayer J, Hensel M, Hopfinger G, Hess G, von Gruenhagen U, Bergmann MA (2009) First-line treatment with fludarabine (F), cyclophosphamide (C), and rituximab (R)(FCR) improves overall survival (OS) in previously untreated patients (pts) with advanced chronic lymphocytic leukemia (CLL): results of a randomized phase III trial on behalf of an international group of investigators and the German CLL study group clinically relevant abstract. Blood 144: 223–224

    Google Scholar 

  31. Developments in the treatment of lymphoproliferative disorders: rising to the new challenges of CLL therapy. A report of a symposium presented during the 48th American Society of Hematology Annual Meeting and Exposition, December 8, 2006, Orlando, Florida (2007). Clin Adv Hematol Oncol. 5(3):1–14; quiz 15–16

  32. Montserrat E, Moreno C, Esteve J, Urbano-Ispizua A, Gine E, Bosch F (2006) How I treat refractory CLL. Blood 107(4):1276–1283

    Article  PubMed  CAS  Google Scholar 

  33. Byrd JC, Lin TS, Dalton JT, Wu D, Phelps MA, Fischer B, Moran M, Blum KA, Rovin B, Brooker-McEldowney M (2007) Flavopiridol administered using a pharmacologically derived schedule is associated with marked clinical efficacy in refractory, genetically high-risk chronic lymphocytic leukemia. Blood 109(2):399–404

    Article  PubMed  CAS  Google Scholar 

  34. Lin TS, Ruppert AS, Johnson AJ, Fischer B, Heerema NA, Andritsos LA, Blum KA, Flynn JM, Jones JA, Hu W (2009) Phase II study of flavopiridol in relapsed chronic lymphocytic leukemia demonstrating high response rates in genetically high-risk disease. J Clin Oncol 27(35):6012–6018

    Article  PubMed  CAS  Google Scholar 

  35. Chanan-Khan A, Miller KC, Musial L, Lawrence D, Padmanabhan S, Takeshita K, Porter CW, Goodrich DW, Bernstein ZP, Wallace P (2006) Clinical efficacy of lenalidomide in patients with relapsed or refractory chronic lymphocytic leukemia: results of a phase II study. J Clin Oncol 24(34):5343–5349

    Article  PubMed  CAS  Google Scholar 

  36. Andritsos LA, Johnson AJ, Lozanski G, Blum W, Kefauver C, Awan F, Smith LL, Lapalombella R, May SE, Raymond CA (2008) Higher doses of lenalidomide are associated with unacceptable toxicity including life-threatening tumor flare in patients with chronic lymphocytic leukemia. J Clin Oncol 26(15):2519–2525

    Article  PubMed  CAS  Google Scholar 

  37. Pavletic SZ, Khouri IF, Haagenson M, King RJ, Bierman PJ, Bishop MR, Carston M, Giralt S, Molina A, Copelan EA (2005) Unrelated donor marrow transplantation for B-cell chronic lymphocytic leukemia after using myeloablative conditioning: results from the Center for International Blood and Marrow Transplant research. J Clin Oncol 23(24):5788–5794

    Article  PubMed  Google Scholar 

  38. Dreger P, Brand R, Milligan D, Corradini P, Finke J, Deliliers GL, Martino R, Russell N, Van Biezen A, Michallet M (2005) Reduced-intensity conditioning lowers treatment-related mortality of allogeneic stem cell transplantation for chronic lymphocytic leukemia: a population-matched analysis. Leukemia 19(6):1029–1033

    Article  PubMed  CAS  Google Scholar 

  39. Moreno C, Villamor N, Colomer D, Esteve J, Martino R, Nomdedéu J, Bosch F, López-Guillermo A, Campo E, Sierra J (2005) Allogeneic stem-cell transplantation may overcome the adverse prognosis of unmutated VH gene in patients with chronic lymphocytic leukemia. J Clin Oncol 23(15):3433–3438

    Article  PubMed  Google Scholar 

  40. Keating MJ, Flinn I, Jain V, Binet JL, Hillmen P, Byrd J, Albitar M, Brettman L, Santabarbara P, Wacker B (2002) Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study. Blood 99(10):3554–3561

    Article  PubMed  CAS  Google Scholar 

  41. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162(1):156–159

    Article  PubMed  CAS  Google Scholar 

  42. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82(13):1107–1112

    Article  PubMed  CAS  Google Scholar 

  43. Xie Z, Yuan H, Yin Y, Zeng X, Bai R, Glazer RI (2006) 3-Phosphoinositide-dependent protein kinase-1(PDK 1) promotes invasion and activation of matrix metalloproteinases. BMC Cancer 6(1):77

    Article  PubMed  Google Scholar 

  44. Agrawal S, Ikeuchi T, Sun D, Sarin PS, Konopka A, Maizel J, Zamecnik PC (1989) Inhibition of human immunodeficiency virus in early infected and chronically infected cells by antisense oligodeoxynucleotides and their phosphorothioate analogues. Proc Natl Acad Sci USA 86(20):7790–7794

    Article  PubMed  CAS  Google Scholar 

  45. Maillard I, Adler S, Pear W (2003) Notch and the immune system. Immunity 19(6):781–791

    Article  PubMed  CAS  Google Scholar 

  46. Vichai V, Kirtikara K (2006) Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc 1(3):1112–1116

    Article  PubMed  CAS  Google Scholar 

  47. Pannuti A, Foreman K, Rizzo P, Osipo C, Golde T, Osborne B, Miele L (2010) Targeting notch to target cancer stem cells. Clin Cancer Res 16(12):3141–3152. doi:10.1158/1078-0432.ccr-09-2823

    Article  PubMed  CAS  Google Scholar 

  48. Yuen AR, Halsey J, Fisher GA, Holmlund JT, Geary RS, Kwoh TJ, Dorr A, Sikic BI (1999) Phase I study of an antisense oligonucleotide to protein kinase C-(ISIS 3521/CGP 64128A) in patients with cancer. Clin Cancer Res 5(11):3357–3363

    PubMed  CAS  Google Scholar 

  49. Orr R (2001) Technology evaluation: fomivirsen, isis pharmaceuticals Inc/CIBA vision. Curr Opin Mol Ther 3(3):288–294

    PubMed  CAS  Google Scholar 

  50. Crooke ST (1999) Molecular mechanisms of action of antisense drugs. Biochim Biophys Acta 1489(1):31–44

    PubMed  CAS  Google Scholar 

  51. Vickers TA, Koo S, Bennett CF, Crooke ST, Dean NM, Baker BF (2003) Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. J Biol Chem 278(9):7108–7118

    Article  PubMed  CAS  Google Scholar 

  52. Rosati E, Sabatini R, Rampino G, Tabilio A, Di Ianni M, Fettucciari K, Bartoli A, Coaccioli S, Screpanti I, Marconi P (2009) Constitutively activated Notch signaling is involved in survival and apoptosis resistance of B-CLL cells. Blood 113(4):856–865

    Article  PubMed  CAS  Google Scholar 

  53. Gray GD, Basu S, Wickstrom E (1997) Transformed and immortalized cellular uptake of oligodeoxynucleoside phosphorothioates, 3′-alkylamino oligodeoxynucleotides, 2′-O-methyl oligoribonucleotides, oligodeoxynucleoside methylphosphonates, and peptide nucleic acids. Biochem Pharmacol 53(10):1465–1476

    Article  PubMed  CAS  Google Scholar 

  54. Bennett CF, Chiang MY, Chan H, Shoemaker J, Mirabelli CK (1992) Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides. Mol Pharmacol 41(6):1023–1033

    PubMed  CAS  Google Scholar 

  55. Stein C, Tonkinson JL, Zhang LM, Yakubov L, Gervasoni J, Taub R, Rotenberg SA (1993) Dynamics of the internalization of phosphodiester oligodeoxynucleotides in HL60 cells. Biochemistry 32(18):4855–4861

    Article  PubMed  CAS  Google Scholar 

  56. Dias N, Stein C (2002) Antisense oligonucleotides: basic concepts and mechanisms. Mol Cancer Ther 1(5):347–355

    PubMed  CAS  Google Scholar 

  57. Monia BP, Johnston JF, Geiger T, Muller M, Fabbro D (1996) Antitumor activity of a phosphorothioate antisense oligodeoxynucleotide targeted against C-raf kinase. Nat Med 2(6):668–675

    Article  PubMed  CAS  Google Scholar 

  58. Bacon TA, Wickstrom E (1991) Walking along human c-myc mRNA with antisense oligodeoxynucleotides: maximum efficacy at the 5′ cap region. Oncogene Res 6(1):13–19

    PubMed  CAS  Google Scholar 

  59. Dash P, Lotan I, Knapp M, Kandel ER, Goelet P (1987) Selective elimination of mRNAs in vivo: complementary oligodeoxynucleotides promote RNA degradation by an RNase H-like activity. Proc Natl Acad Sci USA 84(22):7896–7900

    Article  PubMed  CAS  Google Scholar 

  60. Walder RY, Walder JA (1988) Role of RNase H in hybrid-arrested translation by antisense oligonucleotides. Proc Natl Acad Sci USA 85(14):5011–5015

    Article  PubMed  CAS  Google Scholar 

  61. Sanz L, Garcia-Marco JA, Casanova B, de la Fuente MI (2004) Bcl-2 family gene modulation during spontaneous apoptosis of B-chronic lymphocytic leukemia cells. Biochem Biophys Res Commun 315(3):562–567

    Article  PubMed  CAS  Google Scholar 

  62. Patil SD, Rhodes DG, Burgess DJ (2005) DNA-based therapeutics and DNA delivery systems: a comprehensive review. AAPS J 7(1):E61–E77

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. NUST Centre of Virology and Immunology (NCVI), National University of Sciences and Technology (NUST), Sector H-12, Islamabad, Pakistan

    Kaneez Fatima & Rehan Zafar Paracha

  2. Biotech Park Faculty of Biological Sciences, Quaid-e-Azam University, 3rd Avenue, Islamabad, Pakistan

    Ishtiaq Qadri

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  1. Kaneez Fatima
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Correspondence to Ishtiaq Qadri.

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An erratum to this article can be found at http://dx.doi.org/10.1007/s11033-012-1613-0.

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Fatima, K., Paracha, R.Z. & Qadri, I. Post-transcriptional silencing of Notch2 mRNA in chronic lymhocytic leukemic cells of B-CLL patients. Mol Biol Rep 39, 5059–5067 (2012). https://doi.org/10.1007/s11033-011-1301-5

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