Telomere Maintenance in Telomerase-Deficient Mouse Embryonic Stem Cells: Characterization of an Amplified Telomeric DNA

Abstract

Telomere dynamics, chromosomal instability, and cellular viability were studied in serial passages of mouse embryonic stem (ES) cells in which the telomerase RNA (mTER) gene was deleted. These cells lack detectable telomerase activity, and their growth rate was reduced after more than 300 divisions and almost zero after 450 cell divisions. After this growth crisis, survivor cells with a rapid growth rate did emerge. Such survivors were found to maintain functional telomeres in a telomerase-independent fashion. Although telomerase-independent telomere maintenance has been reported for some immortalized mammalian cells, its molecular mechanism has not been elucidated. Characterization of the telomeric structures in one of the survivor mTER^−/− cell lines showed amplification of the same tandem arrays of telomeric and nontelomeric sequences at most of the chromosome ends. This evidence implicatescis/trans amplification as one mechanism for the telomerase-independent maintenance of telomeres in mammalian cells.

ACKNOWLEDGMENTS

Y.S. is supported by the Japanese Foundation for Multidisciplinary Treatment of Cancer and the Cell Science Research Foundation. Research in the laboratory of P.M.L. is supported by NIH grants ROIAI29524 and GM56162 and by a grant from the National Cancer Institute of Canada with funds from the Terry Fox Run.

We thank Terumi Kohwi-Shigematsu and Dag H. Yasui for critical reading of the manuscript and Cheryl Helgason for experimental help.

H.N. and M.P.H. contributed equally to this work.