Annals of Hematology

, Volume 88, Issue 12, pp 1169–1176 | Cite as

Downregulation of telomerase activity in human promyelocytic cell line using RNA interference

Original Article
First Online:
Received:
Accepted:

Abstract

Telomerase is a ribonucleoprotein complex. It consists of two main components, human telomerase reverse transcriptase (hTERT) and human telomerase RNA. High telomerase activity is present in most malignant cells, but it is barely detectable in majority of somatic cells. The direct correlation between telomerase reactivation and carcinogens has made hTERT a key target for anticancer therapeutic studies. In this study, for the first time, we evaluated the ability of the new generation of short interfering RNA (siRNA) to regulate telomerase activity in the human promyelocytic leukemia cell line (HL-60). Transient transfection cell line by hTERT siRNAs resulted in statistically significant suppression of hTERT messenger RNAs which were detected by quantitative real-time polymerase chain reaction, while the expressed hTERT protein levels were measured by flow cytometry. The results of telomeric repeat amplification protocol showed that telomerase activity was significantly reduced upon transfection of the HL-60 cell line with hTERT siRNAs. The results of this study showed that telomerase activity and cell proliferation were efficiently inhibited in the hTERT siRNA-treated leukemic cell line.

Keywords

hTERT Telomerase siRNA HL-60 Cell proliferation 
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Notes

Acknowledgements

We thank Dr. Samie SH, Vaseli N, Arefi S, and Hayat P for their technical assistance and useful comments. We also thank Dr. Aliyari R and Dr. Shariati P for critical reading of the manuscript.

References

  1. 1.
    Zakian VA (1995) Telomeres: beginning to understand the end. Science 270:1601–1607. doi: 10.1126/science.270.5242.1601 CrossRefPubMedGoogle Scholar
  2. 2.
    McEachern MJ, Krauskopf A, Blackburn EH (2000) Telomeres and their control. Annu Rev Genet 34:331–358. doi: 10.1146/annurev.genet.34.1.331 CrossRefPubMedGoogle Scholar
  3. 3.
    Oulton R, Harrington L (2000) Telomeres, telomerase, and cancer: life on the edge of genomic stability. Curr Opin Oncol 12:74–81. doi: 10.1097/00001622-200001000-00013 CrossRefPubMedGoogle Scholar
  4. 4.
    Hoeijmakers JHJ (2001) Genome maintenance mechanisms for preventing cancer. Nature 411:366–374. doi: 10.1038/35077232 CrossRefPubMedGoogle Scholar
  5. 5.
    Wright WE, Shay JW (1992) The two-stage mechanism controlling cellular senescence and immortalization. Exp Gerontol 27:383–389. doi: 10.1016/0531-5565(92)90069-C CrossRefPubMedGoogle Scholar
  6. 6.
    Blackburn EH (1992) Telomerases. Annu Rev Biochem 61:113–129. doi: 10.1146/annurev.bi.61.070192.000553 CrossRefPubMedGoogle Scholar
  7. 7.
    Greider CW (1996) Telomere length regulation. Annu Rev Biochem 65:337–365. doi: 10.1146/annurev.bi.65.070196.002005 CrossRefPubMedGoogle Scholar
  8. 8.
    Weinrich SL, Pruzan R, Ma L, Ouellette M, Tesmer VM, Holt SE et al (1997) Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nat Genet 17:498–502. doi: 10.1038/ng1297-498 CrossRefPubMedGoogle Scholar
  9. 9.
    Nakayama JI, Tahara H, Tahara E, Saito M, Ito K, Nakamura H et al (1998) Telomerase activation by HTRT in human normal fibroblasts and hepatocellular carcinomas. Nat Genet 18:65–68. doi: 10.1038/ng0198-65 CrossRefPubMedGoogle Scholar
  10. 10.
    Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB et al (1998) Extension of life-span by introduction of telomerase into normal human cells. Science 279:349–352. doi: 10.1126/science.279.5349.349 CrossRefPubMedGoogle Scholar
  11. 11.
    Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PLC et al (1994) Specific association of human telomerase activity with immortal cells and cancer. Science 266:2011–2015. doi: 10.1126/science.7605428 CrossRefPubMedGoogle Scholar
  12. 12.
    Jiang XR, Jimenez G, Chang E, Frolkis M, Kusler B, Sage M et al (1999) Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nat Genet 21:111–114. doi: 10.1038/5056 CrossRefPubMedGoogle Scholar
  13. 13.
    Zhang TC, Schmitt MT, Mumford JL (2003) Effects of arsenic on telomerase and telomeres in relation to cell proliferation and apoptosis in human keratinocytes and leukemia cells in vitro. Carcinogenesis 24:1811–1817. doi: 10.1093/carcin/bgg141 CrossRefPubMedGoogle Scholar
  14. 14.
    Liu L, Berletch JB, Green JG, Pate MS, Andrews LG, Tollefsbol TO (2004) Telomerase inhibition by retinoids precedes cytodifferentiation of leukemia cells and may contribute to terminal differentiation. Mol Cancer Ther 3:1003–1009PubMedGoogle Scholar
  15. 15.
    Liu J-J, Wu X-Y, Peng J, Pan X-L, Lu H-L (2004) Antiproliferation effects of oridonin on HL-60 cells. Ann Hematol 83:691–695. doi: 10.1007/s00277-004-0919-y CrossRefPubMedGoogle Scholar
  16. 16.
    Yamaguchi T, Takahashi H, Harada Y, Yoshikawa M, Iyono S, Inomata M et al (2004) Telomere shortening in human HL60 cells by treatment with deoxyguanosine analogs. Nucleic Acids Symp Ser 48:187–188. doi: 10.1093/nass/48.1.187 CrossRefGoogle Scholar
  17. 17.
    Pizzimenti S, Briatore F, Laurora S, Toaldo C, Maggio M, De Grandi M et al (2006) 4-Hydroxynonenal inhibits telomerase activity and hTERT expression in human leukemic cell lines. Free Radic Biol Med 40:1578–1591. doi: 10.1016/j.freeradbiomed.2005.12.024 CrossRefPubMedGoogle Scholar
  18. 18.
    Wang F, Sun L, Sun H, Fan Q, Le X, Zhang Q (2007) Inhibition hTERT antisense oligodeoxynucleotide changes proliferation and telomerase activity of HL-60 cell. Life Sci J 4:17–21Google Scholar
  19. 19.
    Hannon GJ (2002) RNA interference. Nature 418:244–251. doi: 10.1038/418244a CrossRefPubMedGoogle Scholar
  20. 20.
    Hammond SM, Caudy AA, Hannon GJ (2001) Post-transcriptional gene silencing by double-stranded RNA. Nat Rev Genet 2:110–119. doi: 10.1038/35052556 CrossRefPubMedGoogle Scholar
  21. 21.
    Zhang PH, Zou L, Tu ZG (2006) RNAi-hTERT inhibition hepatocellular carcinoma cell proliferation via decreasing telomerase activity. J Surg Res 131:143–149. doi: 10.1016/j.jss.2005.09.017 CrossRefPubMedGoogle Scholar
  22. 22.
    Gandellini P, Folini M, Bandiera R, De Cesare M, Binda M, Veronese S et al (2007) Down-regulation of human telomerase reverse transcriptase through specific activation of RNAi pathway quickly results in cancer cell growth impairment. Biochem Pharmacol 73:1703–1714. doi: 10.1016/j.bcp. 2007.01.035 CrossRefPubMedGoogle Scholar
  23. 23.
    De Souza Nascimento P, Alves G, Fiedler W (2006) Telomerase inhibition by an siRNA directed against hTERT leads to telomere attrition in HT29 cells. Oncol Rep 16:423–428PubMedGoogle Scholar
  24. 24.
    Zheng JN, Sun YF, Pei DS, Liu JJ, Chen JC, Li W, Sun XQ, Shi QD, Han RF, Ma TX (2006) Inhibition of proliferation and induction of apoptosis in human renal carcinoma cells by anti-telomerase small interfering RNAs. Acta Biochim Biophys Sin (Shanghai) 38:500–506. doi: 10.1111/j.1745-7270.2006.00182.x CrossRefGoogle Scholar
  25. 25.
    Shammas MA, Koley H, Batchu RB, Bertheau RC, Protopopov A, Munshi NC, Goyal RK (2005) Telomerase inhibition by siRNA causes senescence and apoptosis in Barrett's adenocarcinoma cells: mechanism and therapeutic potential. Mol Cancer 4:24. doi: 10.1186/1476-4598-4-24 CrossRefPubMedGoogle Scholar
  26. 26.
    Ning S, Fuessel S, Kotzsch M, Kraemer K, Kappler M, Schmidt U et al (2004) siRNA-mediated down-regulation of survivin inhibits bladder cancer cell growth. Int J Oncol 25:1065–1071PubMedGoogle Scholar
  27. 27.
    Tebes SJ, Johnson NC, Fiorica JV, Kruk PA (2005) Inhibition of telomerase in ovarian cancer using siRNA technology. J Clin Oncol 23:3182 Meeting abstractsGoogle Scholar
  28. 28.
    Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A (2004) Rational siRNA design for RNA interference. Nat Biotechnol 22:326–330. doi: 10.1038/nbt936 CrossRefPubMedGoogle Scholar
  29. 29.
    Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159. doi: 10.1016/0003-2697(87)90021-2 CrossRefPubMedGoogle Scholar
  30. 30.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  31. 31.
    Lin Y, Miyamoto N, Fujinami K, Uemura H, Hosaka M, Iwasaki Y, Kubota Y (1996) Telomerase activity in human bladder cancer. Clin Cancer Res 2:929–932PubMedGoogle Scholar
  32. 32.
    Ohyashki J, Sashida G, Tauchi T, Ohyashiki K (2002) Telomeres and telomerase in hematologic neoplasia. Oncogene 21:680–687. doi: 10.1038/sj.onc.1205075 CrossRefGoogle Scholar
  33. 33.
    Kyo S, Takakura M, Taira T, Kanaya T, Itoh H, Yutsudo M, Ariga H, Inoue M (2000) Sp-1 cooperates with c-Myc to activate transcription of the human telomerase reverse transcriptase gene (hTERT). Nucleic Acids Res 28:669–677. doi: 10.1093/nar/28.3.669 CrossRefPubMedGoogle Scholar
  34. 34.
    Yamada O, Ozaki K, Nakatake M, Akiyama M, Kawauchi K, Matsuoka R (2008) Multistep regulation of telomerase during differentiation of HL-60 cells. J Leukoc Biol 83:1–9CrossRefGoogle Scholar
  35. 35.
    Guittat L, Alberti P, Gomez D, De Cian A, Pennarun G, Lemarteleur T et al (2004) Targeting human telomerase for cancer therapeutics. Cytotechnology 45:75–90. doi: 10.1007/s10616-004-5127-z CrossRefPubMedGoogle Scholar
  36. 36.
    Saretzki G (2003) Telomerase inhibition as cancer therapy. Cancer Lett 194:209. doi: 10.1016/S0304-3835(02)00708-5 CrossRefPubMedGoogle Scholar
  37. 37.
    Wang Q, Liu M, Kozasa T, Rothestein JD, Sternweis PC, Neubig RR (2004) Ribozyme and siRNA-mediated suppression of RGS-containing RhoGEF proteins. Methods Enzymol 389:244–265. doi: 10.1016/S0076-6879(04)89015-3 CrossRefPubMedGoogle Scholar
  38. 38.
    Lu PY, Xie FY, Woodle MC (2003) siRNA-mediated antitumorigenesis for drug target validation and therapeutics. Curr Opin Mol Ther 5:225–234PubMedGoogle Scholar
  39. 39.
    Kosciolek BA, Kalantidis K, Tabler M, Rowley PT (2003) Inhibition of telomerase activity in human cancer cells by RNA interference. Mol Cancer Ther 2:209–216PubMedGoogle Scholar
  40. 40.
    Hu B, Insel RA (1999) Up-regulation of telomerase in human B lymphocytes occurs independently of cellular proliferation and with expression of telomerase catalytic subunit. Eur J Immunol 29:3745–3753. doi: 10.1002/(SICI)1521-4141(199911)29:11<3745::AID-IMMU3745>3.0.CO;2-1 CrossRefPubMedGoogle Scholar
  41. 41.
    Nowak T, Januszkiewicz D, Zawada M, Pernak M, Lewandowski K, Rembowska J et al (2006) Amplification of hTERT and hTERC genes in leukemic cells with high expression and activity of telomerase. Oncol Rep 16:301–305PubMedGoogle Scholar
  42. 42.
    Casillas MA Jr, Brotherton SL, Andrews LG, Ruppert JM, Tollefsbol TO (2003) Induction of endogenous telomerase (hTERT) by c-Myc in WI-38 fibroblasts transformed with specific genetic elements. Gene 316:57–65. doi: 10.1016/S0378-1119(03)00739-X CrossRefPubMedGoogle Scholar
  43. 43.
    Zou L, Zhang P, Luo C, Tu Z (2006) shRNA-targeted hTERT suppress cell proliferation of bladder cancer by inhibiting telomerase activity. Cancer Chemother Pharmacol 57:328–334. doi: 10.1007/s00280-005-0056-x CrossRefPubMedGoogle Scholar
  44. 44.
    Zou L, Zhang PH, Luo CL, Tu ZG (2005) Transcript regulation of human telomerase reverse transcriptase by c-myc and mad1. Acta Biochim Biophys Sin (Shanghai) 37:32–38CrossRefGoogle Scholar
  45. 45.
    Lai SR, Cunningham AP, Huynh VQ, Andrews LG, Tollefsbol TO (2007) Evidence of extra-telomeric effects of hTERT and its regulation involving a feedback loop. Exp Cell Res 313:322–330. doi: 10.1016/j.yexcr.2006.10.014 CrossRefPubMedGoogle Scholar
  46. 46.
    Huh HJ, Huh JW, Yoo ES, Seong CM, Lee M, Hong KS, Chung WS (2005) hTERT mRNA levels by real-time RT-PCR in acute myelogenous leukemia. Am J Hematol 79:267–273. doi: 10.1002/ajh.20394 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • E. Miri-Moghaddam
    • 1
    • 2
  • A. Deezagi
    • 1
    • 3
  • Z. S. Soheili
    • 1
  1. 1.National Institute of Genetic Engineering and Biotechnology (NIGEB)TehranIran
  2. 2.Zahedan University of Medical SciencesZahedanIran
  3. 3.Department of BiochemistryNational Institute of Genetic Engineering and BiotechnologyTehranIran

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