Abrupt telomere shortening in normal human fibroblasts Nikolina Škrobot Vidac ˇek a , Andrea C ´ ukušic ´ a , Milena Ivankovic ´ a , Hrvoje Fulgosi b , Miljenko Huzak c , James R. Smith d , Ivica Rubelj a, * a Department of Molecular Biology, Laboratory of Molecular and Cellular Biology, Ru - der Boškovic ´ Institute, Bijenic ˇka cesta 54, 10000 Zagreb, Croatia b Department of Molecular Biology, Laboratory for Electron Microscopy, Ru - der Boškovic ´ Institute, Bijenic ˇka cesta 54, 10000 Zagreb, Croatia c Division for Probability Theory and Mathematical Statistics, Department of Mathematics, University of Zagreb, 10000 Zagreb, Croatia d Department of Cellular and Structural Biology, Sam and Ann Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center San Antonio, 15355 Lambda Drive, San Antonio, TX 78245-3207, USA article info Article history: Received 28 August 2009 Received in revised form 28 December 2009 Accepted 8 January 2010 Available online 18 January 2010 Keywords: Telomeres Cell senescence Aging Telomere shortening Human fibroblasts abstract Aging is one of the most basic properties of living organisms. Abundant evidence supports the idea that cell senescence underlies organismal aging in higher mammals. Therefore, examining the molecular mechanisms that control cell and replicative senescence is of great interest for biology and medicine. Sev- eral discoveries strongly support telomere shortening as the main molecular mechanism that limits the growth of normal cells. Although cultures gradually approach their growth limit, appearance of individ- ual senescent cells is sudden and stochastic. A theoretical model of abrupt telomere shortening has been proposed in order to explain this phenomenon, but until now there was no reliable experimental evi- dence supporting this idea. Here, we have employed novel methodology to provide evidence for the gen- eration of extrachromosomal circular telomeric DNA as a result of abrupt telomere shortening in normal human fibroblasts. This mechanism ensures heterogeneity in growth potential among individual cells, which is crucial for gradual progression of the aging process. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Telomeres are specialized structures found at the ends of eukaryotic chromosomes consisting of evolutionary conserved hexanucleotide repeats such as TTAGGG in all vertebrates. Their primary role is to maintain chromosomes and genome stability. They achieve this goal through formation of specialized t-loop structure in which G-rich single strand-3 0 protruding end is hidden from nucleases and recombinases within D-loop structure at the telomere border region (Griffith et al., 1999). Telomeres are dy- namic structures, constantly changing their length as cell division progresses. In normal cells, telomeres progressively shorten with each cell division (Harley et al., 1990). It is believed, when telo- meres shorten beyond a critical threshold, cell senescence is in- duced. This process is perceived as mitotic clock (Shay and Wright, 2007). It should be emphasized that senescent cells appear in the culture in a sudden and stochastic fashion so that the num- ber of population doublings (PD) reached by two daughter cells of a single mitosis may differ anywhere between 0 and >8 PD (Smith and Whitney, 1980). As cell culture divisions advance, this process is accelerated and the fraction of nondividing cells gradually accu- mulates until the entire culture ceases further expansion (Smith and Whitney, 1980). Since simple gradual telomere shortening (Olovnikov, 1973) cannot explain the observed unpredictable and sudden onset of cell senescence, a theoretical model has been pro- posed introducing abrupt telomere shortening (ATS) as a stochastic trigger for these phenomena (Rubelj and Vondracek, 1999). The model predicts t-loop formation and abrupt loss of all or most telo- mere repeats through recombination events, generating an extra short telomere end as well as extrachromosomal circular telomere DNA fragments (Rubelj and Vondracek, 1999). Such catastrophic telomere deletion can cause rapid onset of senescence even in cells with long telomeres. Although subsequent studies confirmed important structural or functional features of the model (Griffith et al., 1999; Wang et al., 2004) convincing evidence for its presence in normal cycling cells has been missing. In this work we detected, isolated and analyzed extrachromosomal circular telomere DNAs from normal human fibroblasts at different PDs and used quantita- tive PNA-FISH (Q-FISH) to analyze differences in PNA-FISH signals among their sister telomeres. Furthermore, our results suggest revision of the theoretical ATS mechanism in senescing cultures, demonstrating that this is not predominantly a single-step process, although single loss of (nearly) all telomere repeats cannot be ex- cluded. Most frequently telomeres loose 2 kb of repeats allowing possibility that more than one abrupt shortening can occur at sin- gle telomere in multi-step fashion during consecutive cell divisions (Multi-step Abrupt Telomere Shortening or MATS). Since MATS 0531-5565/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2010.01.009 * Corresponding author. Tel.: +385 1 456 1093; fax: +385 1 456 1177. E-mail address: rubelj@irb.hr (I. Rubelj). Experimental Gerontology 45 (2010) 235–242 Contents lists available at ScienceDirect Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero