Z Gastroenterol 2008; 46 - P138
DOI: 10.1055/s-0028-1089514

Genetic inhibition of telomerase induces alternative lengthening of telomeres (ALT) in genetically defined human esophageal epithelial cells

A Queisser 1, M Thaler 1, A von Werder 1, EM Egenter 1, S Heeg 1, N Hirt 1, H Kunert 1, S Hauss 1, H Harada 2, H Nakagawa 2, S Li 3, EH Blackburn 3, HE Blum 1, OG Opitz 1
  • 1Universitätsklinik Freiburg, Institut für Molekulare Medizin und Zellforschung, Tumorzentrum Ludwig Heilmeyer-Comprehensive Cancer Center, Freiburg, Germany
  • 2Gastroenterology Division, University of Pennsylvania, Philadelphia, United States of America
  • 3Department of Biochemistry and Biophysics, University of California, San Francisco, United States of America

Introduction: Immortalization is an important step in malignant transformation of human cells depending on telomere maintenance. This is either mediated through activation of the enzyme telomerase containing a template RNA (hTER) and the core protein (hTERT) or through a recombination based alternative mechanism (ALT). Little is known about the regulation of these two mechanisms in a single cell. We investigated, whether genetically defined immortalized and malignant transformed cells can switch from one mechanism to the other using specific genetic telomerase inhibitors.

Methods: Immortalized Cyclin-D1 (EPC-D1) or hTERT (EPC-hTERT) overexpressing cells as well as malignant transformed cells overexpressing Cyclin-D1, dnp53, EGFR, c-myc (OKF6 D1/dnp53/EGFR/c-myc) were generated in normal human keratinocytes (EPC/OKF6). Both cell types were transduced with mutated versions of hTER, anti-hTER, siRNA or a combination of both. Transduced cells were sorted by GFP-coexpression. Telomerase activity (TRAP-assay), telomere length (PFGE-TRF and Q-FISH) and indirect immunofluorescence (APBs) were assayed.

Results: Overexpression of MT-hTER, siRNA and the combination of both showed a reduction of telomerase activity in all cell types, whereas TRF and Q-FISH analysis revealed in a subset of cells telomere elongation, characteristic for ALT. In contrast, control cells displayed a robust telomerase activity and a homogeneous telomere length. Additionally, in indirect immunofluorescence ALT-associated PML bodies (APBs) were observed in a higher frequency in immortalized hTER-inhibited cells.

Summary: Genetically defined immortalized and malignant transformed human oral-esophageal epithelial cells are able to elongate their telomeres using both telomere maintenance mechanisms. ALT can even be induced by the inhibition of telomerase with 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.