Telomere Dynamics in a Human Cancer Cell Line Carl N. Sprung,* Laure Sabatier,² and John P. Murnane* ,1 *Radiation Oncology Research Laboratory, University of California at San Francisco, 1855 Folsom Street, MCB 200, San Francisco, California 94103; and ²Laboratoire de Radiobiologie et Oncologie, Commissariat a` l’Energie Atomique, Fontenay-aux Roses, France Telomere maintenance is thought to be essential for immortalization of human cancer cells to compensate for the loss of DNA from the ends of chromosomes and to prevent chromosome fusion. We have investigated telomere dynamics in the telomerase-positive squa- mous cell carcinoma cell line SCC-61 by marking the ends of chromosomes with integrated plasmid se- quences so that changes in the length of individual telomeres could be monitored. Despite having very short telomeres, SCC-61 has a relatively stable genome and few telomere associations. The marked telomeres in different SCC-61 clones have similar mean lengths which show little change with increasing time in cul- ture. Thus, each marked telomere is maintained at a specific length, which we term the equilibrium mean length (EML). The Gaussian distribution in the length of the marked telomeres demonstrates that telomeres continuously fluctuate in length. Consistent with this observation, the mean lengths of the marked telomere in subclones of these cell lines initially differ, but then gradually return to the EML of the original clone with increasing time in culture. The analysis of a clone with two marked telomeres demonstrated that changes in telomere length can occur on each marked telomere independently or coordinately on both telomeres. These results suggest that the short telomeres in many tumor cell lines do not result from an inability to properly maintain telomeres at a specific length. © 1999 Academic Press Key Words: telomerase; telomere dynamics; telomere homeostasis. INTRODUCTION Telomeres are DNA–protein complexes containing short repeat sequences added on to the ends of chro- mosomes by the enzyme telomerase [1]. Telomeres serve multiple functions, including protecting the ends of chromosomes [1], preventing chromosome fusion [2– 4], and facilitating chromosome segregation [5, 6]. Telomeres are maintained in the germ line but shorten with age in most somatic cells at a rate of 30 to 120 bp per cell division [3]. Telomere shortening has been proposed to be the signal for senescence in mammalian cells [7], which involves a permanent block in the G1 phase of the cell cycle [8, 9]. Consistent with this hy- pothesis, the introduction of telomerase into normal human cells results in an increase in their capacity to continue to divide in culture [10]. Primary human cells that have lost the ability to senesce continue to show telomere shortening and eventually display increased telomere associations, an- euploidy, cell crisis, and death [2]. Thus, it has been proposed that for cells to become immortal they must not only avoid senescence but also regain the ability to maintain telomeres to avoid crisis [2, 7]. Evidence for a requirement for continuous telomere maintenance in cell immortalization is provided from experiments demonstrating that the telomeres of immortal cell lines do not continue to shorten [2, 11], and cancer cells and immortal cell lines commonly have telomerase activity [2, 12, 13]. However, addition of telomeric repeat se- quences by telomerase is not the only mechanism by which immortal human cells can maintain telomeres, because some tumor-derived cell lines [14] and many immortal human cell lines established in culture [13, 15, 16] show no detectable telomerase activity. Telomere homeostasis involves continuous adjust- ments in telomere length achieved through the nega- tive regulation of telomerase activity by proteins asso- ciated with the telomere. In yeast, telomere length is determined by monitoring the number of Rap1p pro- teins bound to the telomere [17]. Alterations in the telomeric repeat sequence itself [18], or mutations or altered expression of the Rap1p protein [19 –22], can result in either increases or decreases in the length of the telomere. This regulation of telomere length is mediated through other proteins that interact with Rap1p, including Rif1p and Rif2p [23, 24]. Similar pro- teins are involved in regulation of telomere length in other organisms [25, 26]. In humans, the product of the TERF1 gene (formerly TRF1), which binds specifically to telomeric repeat sequences [26], has been demon- strated to have a role in regulation of telomere length similar to that of Rap1p [27]. The regulation of telo- mere length is therefore a highly complex process 1 To whom reprint requests should be addressed. 0014-4827/99 $30.00 29 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. Experimental Cell Research 247, 29 –37 (1999) Article ID excr.1998.4293, available online at http://www.idealibrary.com on