gms | German Medical Science

27. Deutscher Krebskongress

Deutsche Krebsgesellschaft e. V.

22. - 26.03.2006, Berlin

Inhibition of telomerase by mutant template telomerase RNA and anti-telomerase short interfering RNA induces ALT in immortalized human esophageal epithelial cells.

Meeting Abstract

  • corresponding author presenting/speaker Michaela Döbele - Department of Medicine and Institute for Molecular Medicine and Cell Research, University of Freiburg, Deutschland
  • Alexander von Werder - Department of Medicine and Institute for Molecular Medicine and Cell Research, University of Freiburg
  • Christine Fulda - Department of Medicine and Institute for Molecular Medicine and Cell Research, University of Freiburg
  • Steffen Heeg - Department of Medicine and Institute for Molecular Medicine and Cell Research, University of Freiburg
  • Gitta Gössel - Department of Medicine and Institute for Molecular Medicine and Cell Research, University of Freiburg
  • Michael Quante - Department of Medicine and Institute for Molecular Medicine and Cell Research, University of Freiburg
  • Hideki Harada - Gastroenterology Division, University of Pennsylvania
  • Yasir Suliman - Gastroenterology Division, University of Pennsylvania
  • Shang Li - Department of Biochemistry and Biophysics, University of California at San Francisco
  • Elizabeth H. Blackburn - Department of Biochemistry and Biophysics, University of California at San Francisco
  • Hubert E. Blum - Department of Medicine and Institute for Molecular Medicine and Cell Research, University of Freiburg
  • Oliver G. Opitz - Department of Medicine and Institute for Molecular Medicine and Cell Research, University of Freiburg

27. Deutscher Krebskongress. Berlin, 22.-26.03.2006. Düsseldorf, Köln: German Medical Science; 2006. DocPO212

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Veröffentlicht: 20. März 2006

© 2006 Döbele et al.
Dieser Artikel ist ein Open Access-Artikel und steht unter den Creative Commons Lizenzbedingungen (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.de). Er darf vervielf&aauml;ltigt, verbreitet und &oauml;ffentlich zug&aauml;nglich gemacht werden, vorausgesetzt dass Autor und Quelle genannt werden.


Gliederung

Text

Introduction: Immortalization is an important step in the malignant transformation of human cells and essentially depends on telomere maintenance. This is either mediated through activation of the ribonucleoprotein enzyme telomerase, which contains an intrinsic template RNA moiety (human telomerase RNA;hTER) and the core protein (hTERT) or through a recombination based alternative mechanism (ALT). Little is known about the regulation and coexistence of these two mechanisms in a single cell. Previously, we could demonstrate that the induction of the respective telomere maintenance mechanism is dependent on cell-cycle kinetics. Now we investigated, whether immortalized cells can switch from one mechanism to the other applying specific genetic telomerase inhibitors.

Methods: Cyclin-D1 (EPC-D1) or hTERT (EPC-hTERT) overexpressing cells were generated by retroviral transduction of normal human esophageal epithelial cells (EPC). Furthermore, these cells were transduced with mutated versions of hTER, anti-hTER siRNA or a combination of both using lentiviral gene transfer. Transduced cells were sorted by GFP-coexpression and their growth characteristics, telomerase activity (TRAP-assay), telomere length (PFGE-TRF) and indirect immunofluorescence (APBs) were assayed.

Results: Overexpression of MT-hTER, siRNA and the combination of both rapidly inhibits cell growth in immortalized human esophageal epithelial cells, in contrast to cells transduced with control vectors. A reduction in telomerase activity could be observed whereas the control cells showed a robust activity. TRF analysis revealed heterogeneous telomeres in telomerase(hTER)-inhibited cells, which is characteristic for ALT. In contrast, the control cells displayed stable and homogeneous telomeres characteristic for telomerase activity. Additionally, in indirect immunofluorescence ALT-associated PML bodies (APBs) were observed in a higher frequency in hTER-inhibited cells.

Summary: Genetically defined immortalized human esophageal epithelial cells are capable to elongate their telomeres using both telomere maintenance mechanisms. In our telomerase-positive cell populations ALT could be induced by the inhibition of telomerase using a lentiviral delivery system of mutant template telomerase RNA and anti-telomerase siRNA. These findings suggest that immortalized cells like cancer cells treated with telomerase inhibitors as potential anti-cancer strategy might find alternative ways to maintain their telomeres.