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The intrabody targeting of hTERT attenuates the immortality of cancer cells

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Abstract

hTERT (human telomerase reverse transcriptase) plays a key role in the process of cell immortalization. Overexpression of hTERT has been implicated in 85% of malignant tumors and offers a specific target for cancer therapy. In this paper, we describe an effective approach using a single-chain variable fragment (scFv) intrabody derived from monoclonal hybridoma directed against hTERT to attenuate the immortalization of human uterine cervix and hepatoma cells. The scFv we constructed had a high affinity to hTERT, and specifically neutralized over 70% of telomere synthesis activity, thereby inhibiting the viability and proliferation of the cancer cells. Our results indicate that this anti-hTERT intrabody is a promising tool to target hTERT and intervene in the immortalization process of cancer cells.

Abbreviations

DAPI:

diamidino-phenyl-indole

ECL:

enhanced chemiluminescence

ELISA:

enzyme linked immunosorbent assay

FITC:

fluorescein isothiocyanate

HRP:

horseradish peroxidase

mAb:

monoclonal antibody

MIFs:

mouse intraperitoneal fluid

MTT:

monotetrazolium

PI:

propidium iodide

References

  1. 1.

    Morin, G.B. The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell 3 (1989) 521–529.

  2. 2.

    Blackburn, E.H. Structure and function of telomeres. Nature 6319 (1991) 569–573.

  3. 3.

    Kim, N.W., Piatyszek, M.A., Prowse, K.R., Harley, C.B., West, M.D., Ho, P.L., Coviello, G.M., Wright, W.E., Weinrich, S.L. and Shay, J.W. Specific association of human telomerase activity with immortal cells and cancer. Science 5193 (1994) 2011–2015.

  4. 4.

    Meeker, A.K., Hicks, J.L., Platz, E.A., March, G.E., Bennett, C.J., Delannoy, M.J. and De Marzo, A.M. Telomere shortening is an early somatic DNA alteration in human prostate tumorigenesis. Cancer Res. 22 (2002) 6405–6409.

  5. 5.

    van Heek, N.T., Meeker, A.K., Kern, S.E., Yeo, C.J., Lillemoe, K.D., Cameron, J L., Offerhaus, G.J., Hicks, J.L., Wilentz, R.E., Goggins, M.G., De Marzo, A.M., Hruban, R.H. and Maitra, A. Telomere shortening is nearly universal in pancreatic intraepithelial neoplasia. Am. J. Pathol. 5 (2002) 1541–1547.

  6. 6.

    Meeker, A.K., Hicks, J.L., Gabrielson, E., Strauss, W.M., De Marzo, A.M. and Argani, P. Telomere shortening occurs in subsets of normal breast epithelium as well as in situ and invasive carcinoma. Am. J. Pathol. 3 (2004) 925–935.

  7. 7.

    Minev, B., Hipp, J., Firat, H., Schmidt, J.D., Langlade-Demoyen, P. and Zanetti, M. Cytotoxic T cell immunity against telomerase reverse transcriptase in humans. Proc. Natl. Acad. Sci. U.S.A. 9 (2000) 4796–4801.

  8. 8.

    Hytiroglou, P. and Theise, N.D. Telomerase activation in human hepatocarcinogenesis. Am. J. Gastroenterol. 4 (2006) 839–841.

  9. 9.

    Harrington, L., Zhou, W., McPhail, T., Oulton, R., Yeung, D.S., Mar, V., Bass, M.B. and Robinson, M.O. Human telomerase contains evolutionarily conserved catalytic and structural subunits. Genes Dev. 23 (1997) 3109–3115.

  10. 10.

    Weinrich, S.L., Pruzan, R., Ma, L., Ouellette, M., Tesmer, V.M., Holt, S.E., Bodnar, A.G., Lichtsteiner, S., Kim, N.W., Trager, J.B., Taylor, R.D., Carlos, R., Andrews, W.H., Wright, W.E., Shay, J.W., Harley, C.B. and Morin, G.B. Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nat. Genet. 4 (1997) 498–502.

  11. 11.

    Vonderheide, R.H., Hahn, W.C., Schultze, J.L. and Nadler, L.M. The telomerase catalytic subunit is a widely expressed tumor-associated antigen recognized by cytotoxic T lymphocytes. Immunity 6 (1999) 673–679.

  12. 12.

    Meyerson, M., Counter, C.M., Eaton, E.N., Ellisen, L.W., Steiner, P., Caddle, S.D., Ziaugra, L., Beijersbergen, R.L., Davidoff, M.J., Liu, Q., Bacchetti, S., Haber, D.A. and Weinberg, R.A. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell 4 (1997) 785–795.

  13. 13.

    Graessmann, A., Graessmann, M. and Mueller, C. Microinjection of early SV40 DNA fragments and T antigen. Methods Enzymol. 1 (1980) 816–825.

  14. 14.

    Morgan, D.O. and Roth, R.A. Analysis of intracellular protein function by antibody injection. Immunol. Today 3 (1988) 84–88.

  15. 15.

    Valle, G., Jones, E.A. and Colman, A. Anti-ovalbumin monoclonal antibodies interact with their antigen in internal membranes of Xenopus oocytes. Nature 5887 (1982) 71–74.

  16. 16.

    Burke, B. and Warren, G. Microinjection of mRNA coding for an anti-Golgi antibody inhibits intracellular transport of a viral membrane protein. Cell 4 (1984) 847–856.

  17. 17.

    Marasco, W.A. Intrabodies as antiviral agents. Curr. Top. Microbiol. Immunol. (2001) 247–270.

  18. 18.

    Marasco, W.A. Intrabodies: turning the humoral immune system outside in for intracellular immunization. Gene Ther. 1 (1997) 11–15.

  19. 19.

    Williams, B.R. and Zhu, Z. Intrabody-based approaches to cancer therapy: status and prospects. Curr. Med. Chem. 12 (2006) 1473–1480.

  20. 20.

    Biocca, S., Pierandrei-Amaldi, P. and Cattaneo, A. Intracellular expression of anti-p21ras single chain Fv fragments inhibits meiotic maturation of xenopus oocytes. Biochem. Biophys. Res. Commun. 2 (1993) 422–427.

  21. 21.

    Biocca, S., Pierandrei-Amaldi, P., Campioni, N. and Cattaneo, A. Intracellular immunization with cytosolic recombinant antibodies. Biotechnology (N. Y.) 4 (1994) 396–399.

  22. 22.

    Duan, L., Bagasra, O., Laughlin, M.A., Oakes, J.W. and Pomerantz, R.J. Potent inhibition of human immunodeficiency virus type 1 replication by an intracellular anti-Rev single-chain antibody. Proc. Natl. Acad. Sci. U.S.A. 11 (1994) 5075–5079.

  23. 23.

    Mhashilkar, A.M., Bagley, J., Chen, S.Y., Szilvay, A.M., Helland, D.G. and Marasco, W A. Inhibition of HIV-1 Tat-mediated LTR transactivation and HIV-1 infection by anti-Tat single chain intrabodies. EMBO J. 7 (1995) 1542–1551.

  24. 24.

    Marasco, W.A., Haseltine, W.A. and Chen, S.Y. Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody. Proc. Natl. Acad. Sci. U.S.A. 16 (1993) 7889–7893.

  25. 25.

    Plumb, J.A., Bilsland, A., Kakani, R., Zhao, J., Glasspool, R.M., Knox, R.J., Evans, T.R. and Keith, W.N. Telomerase-specific suicide gene therapy vectors expressing bacterial nitroreductase sensitize human cancer cells to the pro-drug CB1954. Oncogene 53 (2001) 7797–7803.

  26. 26.

    Xiong, Y. and Eickbush, T.H. Origin and evolution of retroelements based upon their reverse transcriptase sequences. EMBO J. 10 (1990) 3353–3362.

  27. 27.

    Kalderon, D., Roberts, B.L., Richardson, W.D. and Smith, A.E. A short amino acid sequence able to specify nuclear location. Cell 3 Pt 2 (1984) 499–509.

  28. 28.

    Savre-Train, I., Gollahon, L.S. and Holt, S.E. Clonal heterogeneity in telomerase activity and telomere length in tumor-derived cell lines. Proc. Soc. Exp. Biol. Med. 4 (2000) 379–388.

  29. 29.

    Yang, N., Zhu, X., Chen, L., Li, S. and Ren, D. Oral administration of attenuated S. typhimurium carrying shRNA-expressing vectors as a cancer therapeutic. Cancer Biol. Ther. 1 (2008) 145–151.

  30. 30.

    Fu, W., Chu, L., Han, X., Liu, X. and Ren, D. Synergistic antitumoral effects of human telomerase reverse transcriptase-mediated dual-apoptosis-related gene vector delivered by orally attenuated Salmonella enterica Serovar Typhimurium in murine tumor models. J. Gene Med. 6 (2008) 690–701.

  31. 31.

    Strahl, C. and Blackburn, E.H. Effects of reverse transcriptase inhibitors on telomere length and telomerase activity in two immortalized human cell lines. Mol. Cell. Biol. 1 (1996) 53–65.

  32. 32.

    Seay, T.M., Peretsman, S.J. and Dixon, P.S. Inhibition of human transitional cell carcinoma in vitro proliferation by fluoroquinolone antibiotics. J. Urol. 2 (1996) 757–762.

  33. 33.

    Zhang, P.H., Zou, L. and Tu, Z.G. RNAi-hTERT inhibition hepatocellular carcinoma cell proliferation via decreasing telomerase activity. J. Surg. Res. 1 (2006) 143–149.

  34. 34.

    Holt, S.E., Aisner, D.L., Shay, J.W. and Wright, W.E. Lack of cell cycle regulation of telomerase activity in human cells. Proc. Natl. Acad. Sci. U.S.A. 20 (1997) 10687–10692.

  35. 35.

    Bonnin, E., Gruel, N., Moutel, S., Mantegazza, A.R., Barrio, M.M., Mordoh, J. and Teillaud, J.L. Generation of functional scFv intrabodies for triggering anti-tumor immunity. Methods 2 (2004) 225–232.

  36. 36.

    Cardinale, A., Lener, M., Messina, S., Cattaneo, A. and Biocca, S. The mode of action of Y13-259 scFv fragment intracellularly expressed in mammalian cells. FEBS Lett. 3 (1998) 197–202.

  37. 37.

    Strazisar, M., Mlakar, V. and Glavac, D. The expression of COX-2, hTERT, MDM2, LATS2 and S100A2 in different types of non-small cell lung cancer (NSCLC). Cell. Mol. Biol. Lett. 3 (2009) 442–456.

  38. 38.

    Harley, C.B. Telomere loss: mitotic clock or genetic time bomb? Mutat. Res. 2–6 (1991) 271–282.

  39. 39.

    Blackburn, E.H. Telomere states and cell fates. Nature 6808 (2000) 53–56.

  40. 40.

    Kelland, L.R. Overcoming the immortality of tumour cells by telomere and telomerase based cancer therapeutics-current status and future prospects. Eur. J. Cancer 7 (2005) 971–979.

  41. 41.

    Chang, J.T., Lu, Y.C., Chen, Y.J., Tseng, C.P., Chen, Y.L., Fang, C.W. and Cheng, A.J. hTERT phosphorylation by PKC is essential for telomerase holoprotein integrity and enzyme activity in head neck cancer cells. Br. J. Cancer 6 (2006) 870–878.

  42. 42.

    Wu, P., Meng, L., Wang, H., Zhou, J., Xu, G., Wang, S., Xi, L., Chen, G., Wang, B., Zhu, T., Lu, Y. and Ma, D. Role of hTERT in apoptosis of cervical cancer induced by histone deacetylase inhibitor. Biochem. Biophys. Res. Commun. 1 (2005) 36–44.

  43. 43.

    Ahmed, A. and Tollefsbol, T. Telomeres, telomerase, and telomerase inhibition: clinical implications for cancer. J. Am. Geriatr. Soc. 1 (2003) 116–122.

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Correspondence to Daming Ren.

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Key words

  • Cancer
  • Intrabody
  • Anti-hTERT ScFv
  • Immortality