Restoration of senescent human diploid fibroblasts by modulation of the extracellular matrix Hae Ri Choi 1 *, Kyung A Cho 2 *, Hyun Tae Kang 1 , Jung Bin Lee 3 , Matt Kaeberlein 4 , Yousin Suh 5,6 , In Kwon Chung 7 and Sang Chul Park 1 1 Department of Biochemistry and Molecular Biology, Aging and Apoptosis Research Center, Institute on Aging, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea 2 Department of Biochemistry, Chonnam National University Medical School, Gwangju, South Korea 3 Department of Forensic Medicine, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea 4 Department of Pathology, University of Washington, Seattle, WA 98195, USA 5 Institute for Aging Research, Diabetes Research and Training Center and 6 Departments of Medicine and Genetics, Albert Einstein College of Medicine, Bronx, NY, USA 7 Departments of Biology and Biomedical Sciences, Yonsei University, Seoul, South Korea Summary Human diploid fibroblasts have the capacity to complete a finite number of cell divisions before entering a state of replicative senescence characterized by growth arrest, changes in morphology, and altered gene expression. Herein, we report that interaction with extracellular matrix (ECM) from young cells is sufficient to restore aged, senescent cells to an apparently youthful state. The identity of the restored cells as having been derived from senescent cells has been confirmed by a variety of meth- ods, including time lapse live cell imaging and DNA finger print analysis. In addition to cell morphology, phenotypic restoration was assessed by resumption of proliferative potential, growth factor responsiveness, reduction of intracellular reactive oxygen species levels, recovery of mitochondrial membrane potential, and increased telo- mere length. Mechanistically, we find that both Ku and SIRT1 are induced during restoration and are required for senescent cells to return to a youthful phenotype. These observations demonstrate that human cellular senes- cence is profoundly influenced by cues from the ECM, and that senescent cell plasticity is much greater than that was previously believed to be the case. Key words: Aging; cellular senescence; extra cellular matrix; Ku70; SIRT1. Introduction The microenvironment in which a cell resides has a profound effect on cellular function and physiology. The nature of this microenvironment is determined in large part by synthesis and secretion of a variety of factors, especially components of the extracellular matrix (ECM). The ECM is a complex, three-dimen- sional network of macromolecules that provides spatial informa- tion and an architectural scaffold for cells. Moreover, the ECM coordinates cell organization within tissues, and supports mani- fold activities of cells by modulating signal transduction (Hynes, 2009). Most human cells are capable of only a finite number of cell divisions before arresting growth and entering a nondividing state referred to as replicative senescence. Cell division potential is in general limited by telomere shortening, which occurs grad- ually with each mitotic division in cells not actively expressing telomerase (Harley et al., 1990). Telomeres provide essential protection for chromosome ends, and when telomere lengths are sufficiently reduced, a DNA damage response (DDR) is initi- ated that terminally arrests the cell cycle. In rare cases, telomer- ase-negative cells are able to escape replicative senescence by elongating their telomeres through a telomerase-independent alternative lengthening mechanism referred to as ALT (Henson et al., 2002). In mammals, several of the proteins involved in the nonhomologous end joining (NHEJ) DNA repair pathway are telomere bound and affect telomere length maintenance (Gas- ser, 2000). These include Ku70 and Ku80, which form a hetero- dimer (Ku) that can bind to free double-stranded DNA ends. Inactivation of Ku leads to various defects including telomere length deregulation and end-to-end chromosome fusions (Critchlow & Jackson, 1998; Indiviglio & Bertuch, 2009). Sirtuins are Nicotinami de adenine dinucleotide (NAD + )-dependent class III histone deacetylases that have been linked to the onset of senescence (Longo & Kennedy, 2006). Over expression of SIRT1 orthologs leads to lifespan extension in yeast, flies, and worms (Kaeberlein et al., 1999; Finkel et al., 2009). Yeast Sir proteins perform multiple biological functions, including localizing to sites of DNA double strand breaks (Kennedy et al. 1997) and modulating their repair by NHEJ (Tsukamoto et al., 1997; Haber, 1999), although there is some evidence that this finding is at least partially an indirect consequence of derepressing silent mating types loci (Astrom et al., 1999; Lee et al., 1999). The roles of SIRT1 in NHEJ are poorly understood in mammals; Correspondence Sang Chul Park, Department of Biochemistry and Molecular Biology, Aging and Apoptosis Research Center, Institute on Aging, Seoul National Univer- sity College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea. Tel.: 82 2 744 4534; fax: 82 2 744 4534; e-mail: scpark@ snu.ac.kr *These authors contributed equally to this work. Accepted for publication 1 November 2010 148 ª 2011 The Authors Aging Cell ª 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland Aging Cell (2011) 10, pp148–157 Doi: 10.1111/j.1474-9726.2010.00654.x Aging Cell