Mammalian cells have mechanisms to counteract the effects of metabolic and exogenous stresses, many of that would be mutagenic if ignored. Damage arising during DNA replication is a major source of mutagenesis. The extent of damage dictates whether cells undergo transient cell cycle arrest and damage repair, senescence or apoptosis. Existing dogma defines these alternative fates as distinct choices. Here we show that immortalised breast epithelial cells are able to survive prolonged S phase arrest and subsequently re-enter cycle after many days of being in an arrested, senescence-like state. Prolonged cell cycle inhibition in fibroblasts induced DNA damage response and cell death. However, in immortalised breast epithelia, efficient S phase arrest minimised chromosome damage and protected sufficient chromatin-bound replication licensing complexes to allow cell cycle re-entry. We propose that our observation could have implications for the design of drug therapies for breast cancer.

] Faculty of Life Sciences and Wellcome Trust Centre for Cell Matrix Research University of Manchester Oxford Road Manchester M13 9PT UK [2] Genome Integrity Unit Danish Cancer Society Research Centre Copenhagen Denmark

] Genome Integrity Unit Danish Cancer Society Research Centre Copenhagen Denmark [2] Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University CZ 779 00 Olomouc Czech Republic

Faculty of Life Sciences and Wellcome Trust Centre for Cell Matrix Research University of Manchester Oxford Road Manchester M13 9PT UK

Genome Integrity Unit Danish Cancer Society Research Centre Copenhagen Denmark

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Kuilman T, Michaloglou C, Mooi WJ, Peeper DS. The essence of senescence. Genes Dev. 2010;24:2463–2479. PubMed PMC

Hayflick L. Limited in vitro lifetime of human diploid cell strains. Exp Cell Res. 1965;37:614. PubMed

Matsumura T, Zerrudo Z, Hayflick L. Senescent human diploid cells in culture—survival, DNA synthesis and morphology. J Gerontol. 1979;34:328–334. PubMed

Kong Y, Cui H, Ramkumar C, Zhang H. Regulation of senescence in cancer and aging. J Aging Res. 2011;2011:963172. PubMed PMC

Campisi J. Aging, cellular senescence, and cancer. Ann Rev Physiol. 2013;75:685–705. PubMed PMC

Campisi J, di Fagagna FD. Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cel Biol. 2007;8:729–740. PubMed

Harley CB. Telomere loss—mitotic clock or genetic time bomb. Mut Res. 1991;256:271–282. PubMed

Aubert G, Lansdorp PM. Telomeres and aging. Physiol Rev. 2008;88:557–579. PubMed

di Fagagna FD, Reaper PM, Clay-Farrace L, Fiegler H, Carr P, Von Zglinicki T, et al. A DNA damage checkpoint response in telomere-initiated senescence. Nature. 2003;426:194–198. PubMed

di Fagagna FD. Living on a break: cellular senescence as a DNA-damage response. Nat Rev Cancer. 2008;8:512–522. PubMed

Roos WP, Kaina B. DNA damage-induced cell death: from specific DNA lesions to the DNA damage response and apoptosis. Cancer Lett. 2013;332:237–248. PubMed

Chandler H, Peters G. Stressing the cell cycle in senescence and aging. Curr Opin Cell Biol. 2013;25:765–771. PubMed

Brown JP, Wei WY, Sedivy JM. Bypass of senescence after disruption of p21(CIP1/WAF1) gene in normal diploid human fibroblasts. Science. 1997;277:831–834. PubMed

Stein GH, Drullinger LF, Soulard A, Dulic V. Differential roles for cyclin-dependent kinase inhibitors p21 and p16 in the mechanisms of senescence and differentiation in human fibroblasts. Mol Cell Biol. 1999;19:2109–2117. PubMed PMC

Hara E, Smith R, Parry D, Tahara H, Stone S, Peters G. Regulation of p16(CDKN2) expression and its implications for cell immortalization and senescence. Mol Cell Biol. 1996;16:859–867. PubMed PMC

Kiyono T, Foster SA, Koop JI, McDougall JK, Galloway DA, Klingelhutz AJ. Both Rb/p16(INK4a) inactivation and telomerase activity are required to immortalize human epithelial cells. Nature. 1998;396:84–88. PubMed

Beausejour CM, Krtolica A, Galimi F, Narita M, Lowe SW, Yaswen P, et al. Reversal of human cellular senescence: roles of the p53 and p16 pathways. Embo J. 2003;22:4212–4222. PubMed PMC

Stingl J, Caldas C. Opinion—Molecular heterogeneity of breast carcinomas and the cancer stem cell hypothesis. Nat Rev Cancer. 2007;7:791–799. PubMed

Mumcuoglu M, Bagislar S, Yuzugullu H, Alotaibi H, Senturk S, Telkoparan P, et al. The Ability to Generate Senescent Progeny as a Mechanism Underlying Breast Cancer Cell Heterogeneity. PLoS One. 2010;5:e11288. PubMed PMC

Romanov SR, Kozakiewicz BK, Holst CR, Stampfer MR, Haupt LM, Tlsty TD, et al. Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes. Nature. 2001;409:633–637. PubMed

Salama R, Sadaie M, Hoare M, Narita M. Cellular senescence and its effector programs. Genes Dev. 2014;28:99–114. PubMed PMC

Goligorsky MS, Chen J, Patschan S. Stress-induced premature senescence of endothelial cells: a perilous state between recovery and point of no return. Curr Opin Hematol. 2009;16:215–219. PubMed

Tyson JJ, Novak B. Temporal organization of the cell cycle. Curr Biol. 2008;18:R759–R768. PubMed PMC

Gerard C, Goldbeter A. Temporal self-organization of the cyclin/Cdk network driving the mammalian cell cycle. Proc Natl Acad Sci USA. 2009;106:21643–21648. PubMed PMC

Lukas J, Lukas C, Bartek J. Mammalian cell cycle checkpoints: signalling pathways and their organization in space and time. DNA Repair. 2004;3:997–1007. PubMed

Jackson SP, Bartek J. The DNA-damage response in human biology and disease. Nature. 2009;461:1071–1078. PubMed PMC

Masai H, You ZY, Arai K. Control of DNA replication: regulation and activation of eukaryotic replicative helicase, MCM. IUBMB Life. 2005;57:323–335. PubMed

Alfieri R, Barberis M, Chiaradonna F, Gaglio D, Milanesi L, Vanoni M, et al. Towards a systems biology approach to mammalian cell cycle: modeling the entrance into S phase of quiescent fibroblasts after serum stimulation. BMC Bioinformatics. 2009;10 (Suppl 12:S16. PubMed PMC

Jung YS, Qian YJ, Chen XB. Examination of the expanding pathways for the regulation of p21 expression and activity. Cell Signal. 2010;22:1003–1012. PubMed PMC

Rudolph J. Cdc25 phosphatases: structure, specificity, and mechanism. Biochemistry. 2007;46:3595–3604. PubMed

Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem. 2004;73:39–85. PubMed

Vousden KH, Lane DP. p53 in health and disease. Nat Rev Mol Cell Biol. 2007;8:275–283. PubMed

Hamada H, Tashima Y, Kisaka Y, Iwamoto K, Hanai T, Eguchi Y, et al. Sophisticated framework between cell cycle arrest and apoptosis induction based on p53 dynamics. PLoS One. 2009;4:e4795. PubMed PMC

Maya-Mendoza A, Petermann E, Gillespie DAF, Caldecott KW, Jackson DA. Chk1 regulates the density of active replication origins during the vertebrate S phase. Embo J. 2007;26:2719–2731. PubMed PMC

Ge XQ, Blow JJ. Chk1 inhibits replication factory activation but allows dormant origin firing in existing factories. J Cell Biol. 2010;191:1285–1297. PubMed PMC

Ge XQ, Jackson DA, Blow JJ. Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress. Genes Dev. 2007;21:3331–3341. PubMed PMC

Blow JJ, Ge XQ, Jackson DA. How dormant origins promote complete genome replication. Trends Biochem Sci. 2011;36:405–414. PubMed PMC

Ilves I, Petojevic T, Pesavento JJ, Botchan MR. Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins. Mol Cell. 2010;37:247–258. PubMed PMC

Blow JJ, Dutta A. Preventing re-replication of chromosomal DNA. Nat Rev Mol Cell Biol. 2005;6:476–486. PubMed PMC

Arias EE, Walter JC. Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells. Genes Dev. 2007;21:497–518. PubMed

Truong LN, Wu XH. Prevention of DNA re-replication in eukaryotic cells. J Mol Cell Biol. 2011;3:13–22. PubMed PMC

Chuang LC, Teixeira LK, Wohlschlegel JA, Henze M, Yates JR, Méndez J, et al. Phosphorylation of Mcm2 by Cdc7 promotes pre-replication complex assembly during cell-cycle re-entry. Mol Cell. 2009;35:206–216. PubMed PMC

Streuli CH. Integrins and cell-fate determination. J Cell Sci. 2009;122:171–177. PubMed PMC

Jeanes AI, Maya-Mendoza A, Streuli CH. Cellular microenvironment influences the ability of mammary epithelia to undergo cell cycle. PLoS One. 2011;6:e18144. PubMed PMC

Soule HD, Maloney TM, Wolman SR, Peterson Jr, WD, Brenz R, McGrath CM, et al. Isolation and characterization of a spontaneously immortalized human breast eoithelial cell line, MCF-10. Cancer Res. 1990;50:6075–6086. PubMed

Cowell JK, LaDuca J, Rossi MR, Burkhardt T, Nowak NJ, Matsui S. Molecular characterization of the t(3;9) associated with immortalization in the MCF10A cell line. Cancer Genet Cytogenet. 2005;163:23–29. PubMed

Borel F, Lacroix FB, Margolis RL. Prolonged arrest of mammalian cells at the G1/S boundary results in permanent S phase stasis. J Cell Sci. 2002;115:2829–2838. PubMed

Bartek J, Lukas J. DNA damage checkpoints: from initiation to recovery or adaptation. Curr Opin Cell Biol. 2007;19:238–245. PubMed

Di Micco R, Sulli G, Dobreva M, Liontos M, Botrugno OA, Gargiulo G, et al. Interplay between oncogene-induced DNA damage response and heterochromatin in senescence and cancer. Nat Cell Biol. 2011;13:292–U244. PubMed PMC

Lukas C, Savic V, Bekker-Jensen S, Doil C, Neumann B, Pedersen RS, et al. 53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress. Nat Cell Biol. 2011;13:243–U380. PubMed

Coller HA. What's taking so long? S-phase entry from quiescence versus proliferation. Nat Rev Mol Cell Biol. 2007;8:667–670. PubMed

Chien WW, Domenech C, Catallo R, Salles G, Ffrench M. S-phase lengthening induced by p16(INK4a) overexpression in malignant cells with wild-type pRb and p53. Cell Cycle. 2010;9:3286–3296. PubMed

Mirzayans R, Andrais B, Scott A, Murray D. New insights into p53 signaling and cancer cell response to DNA damage: implications for cancer therapy. J Biomed Biotechnol. 2012;2012:170325. PubMed PMC

Elmore LW, Di X, Dumur C, Holt SE, Gewirtz DA. Evasion of a single-step, chemotherapy-induced senescence in breast cancer cells: Implications for treatment response. Clin Cancer Res. 2005;11:2637–2643. PubMed

Roberson RS, Kussick SJ, Vallieres E, Chen SYJ, Wu DY. Escape from therapy-induced accelerated cellular senescence in p53-null lung cancer cells and in human lung cancers. Cancer Res. 2005;65:2795–2803. PubMed

Michishita E, Nakabayashi K, Ogino H, Suzuki T, Fujii M, Ayusawa D. DNA topoisomerase inhibitors induce reversible senescence in normal human fibroblasts. Biochem Biophys Res Commun. 1998;253:667–671. PubMed

Syljuasen RG. Checkpoint adaptation in human cells. Oncogene. 2007;26:5833–5839. PubMed

Petermann E, Orta ML, Issaeva N, Schultz N, Helleday T. Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair. Mol Cell. 2010;37:492–502. PubMed PMC

Debatisse M, Le Tallec B, Letessier A, Dutrillaux B, Brison O. Common fragile sites: mechanisms of instability revisited. Trends Genet. 2012;28:22–32. PubMed

Halazonetis TD, Gorgoulis VG, Bartek J. An oncogene-induced DNA damage model for cancer development. Science. 2008;319:1352–1355. PubMed

Di Micco R, Fumagalli M, Cicalese A, Piccinin S, Gasparini P, Luise C, et al. Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication. Nature. 2006;444:638–642. PubMed

Bartkova J, Rezaei N, Liontos M, Karakaidos P, Kletsas D, Issaeva N, et al. Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature. 2006;444:633–637. PubMed

Bester AC, Roniger M, Oren YS, Im MM, Sarni D, Chaoat M, et al. Nucleotide deficiency promotes genomic instability in early stages of cancer development. Cell. 2011;145:435–446. PubMed PMC

Stoeber K, Tlsty TD, Happerfield L, Thomas GA, Romanov S, Bobrow L, et al. DNA replication licensing and human cell proliferation. J Cell Sci. 2001;114:2027–2041. PubMed

Shetty A, Loddo M, Fanshawe T, Prevost AT, Sainsbury R, Williams GH, et al. DNA replication licensing and cell cycle kinetics of normal and neoplastic breast. Br J Cancer. 2005;93:1295–1300. PubMed PMC

Harmes DC, DiRenzo J. Cellular quiescence in mammary stem cells and breast tumor stem cells: got testable hypotheses. J Mammary Gland Biol Neoplasia. 2009;14:19–27. PubMed PMC

Lee MJ, Ye AS, Gardino AK, Heijink AM, Sorger PK, MacBeath G, et al. Sequential application of anticancer drugs enhances cell death by rewiring apoptotic signaling networks. Cell. 2012;149:780–794. PubMed PMC

Taylorpapadimitriou J, Stampfer M, Bartek J, Lewis A, Boshell M, Lane EB, et al. Keratin expression in human mammary epithelial cells cultured from normal and malignant tissue—relation to in vivo phenotypes and influence of medium. J Cell Sci. 1989;94:403–413. PubMed

Jackson DA, Pombo A. Replicon clusters are stable units of chromosome structure: Evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells. J Cell Biol. 1998;140:1285–1295. PubMed PMC

Chromatin architecture changes and DNA replication fork collapse are critical features in cryopreserved cells that are differentially controlled by cryoprotectants