DNA Repair 12 (2013) 38–45 Contents lists available at SciVerse ScienceDirect DNA Repair j ourna l ho me pag e: www.elsevier.com/locate/dnarepair Inhibition of homologous recombination by hyperthermia shunts early double strand break repair to non-homologous end-joining Judith W.J. Bergs a,1 , Przemek M. Krawczyk b,1 , Tijana Borovski a , Rosemarie ten Cate a , Hans M. Rodermond a , Jan Stap b , Jan Paul Medema a , Jaap Haveman a , Jeroen Essers c , Chris van Bree a , Lukas J.A. Stalpers a , Roland Kanaar c , Jacob A. Aten b , Nicolaas A.P. Franken a,∗ a Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine, Department of Radiation Oncology, Academic Medical Center, University of Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands b van Leeuwenhoek Centre for Advanced Microscopy – AMC, Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, The Netherlands c Department of Cell Biology & Genetics, Cancer Genomics Center, Department of Radiation Oncology, Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands a r t i c l e i n f o Article history: Received 10 November 2011 Received in revised form 11 October 2012 Accepted 12 October 2012 Available online 11 December 2012 Keywords: Hyperthermia Inhibition of homologous recombination NU7441 Chromosomal translocations RAD51 foci GammaH2AX foci a b s t r a c t In S and G2 phase mammalian cells DNA double strand breaks (DSBs) can potentially be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). Results of several studies sug- gest that these two mechanistically distinct repair pathways can compete for DNA ends. Because HR and NHEJ differ with respect to error susceptibility, generation of chromosome rearrangements, which are potentially carcinogenic products of DSB repair, may depend on the pathway choice. To investigate this hypothesis, the influence of HR and NHEJ inhibition on the frequencies of chromosome aberrations in G2 phase cells was investigated. SW-1573 and RKO cells were treated with mild (41 ◦ C) hyperthermia in order to disable HR and/or NU7441/cisplatin to inactivate NHEJ and frequencies of chromosomal fragments (resulting from unrepaired DSBs) and translocations (products of erroneous DSB rejoining) were stud- ied using premature chromosome condensation (PCC) combined with fluorescence in situ hybridization (FISH). It is shown here that temporary inhibition of HR by hyperthermia results in increased frequency of ionizing-radiation (IR)-induced chromosomal translocations and that this effect is abrogated by NU7441- or cisplatin-mediated inhibition of NHEJ. The results suggest that in the absence of HR, DSB repair is shifted to the error-prone NHEJ pathway resulting in increased frequencies of chromosomal rearrangements. These results might be of consequence for clinical cancer treatment approaches that aim at inhibition of one or more DSB repair pathways. © 2012 Elsevier B.V. All rights reserved. 1. Introduction DNA double strand breaks (DSBs) are potentially lethal lesions that can be induced by endogenous DNA-related processes as well as by exogenous agents, including ionizing radiation (IR) and chem- icals. In mammalian cells, DSBs trigger a complicated cascade of reactions initiated by recognition of the breaks by the ATM kinase and the Mre11/Rad50/NBS1 (MRN) complex and subsequent phos- phorylation of the histone protein H2AX at chromatin domains surrounding DSB sites [1]. Phosphorylated H2AX (-H2AX) is con- sidered to be one of the earliest markers of DSBs [1–4]. Damaged chromatin attracts other repair- and checkpoint activation-related proteins, including MDC1 and 53BP1 [5,6] which form so-called ∗ Corresponding author. Tel.: +31 20 5663641. E-mail address: n.a.franken@amc.uva.nl (N.A.P. Franken). 1 These authors have contributed equally. ionizing radiation induced foci (IRIF). IRIF are considered to be markers of ongoing repair activities and can be visualized by (immuno)fluorescence microscopy [7]. Failure of repair proteins to form IRIF has been linked to damage response deficiencies [8,9]. After initial recognition, repair of the DSBs is performed by homol- ogous recombination (HR) or non-homologous end-joining (NHEJ) [10]. HR requires a DNA sequence homologous to the broken strand and thus acts predominantly during the S and G2 phases of the cell cycle when a DNA template is available in the form of a sister chromatid [11]. Major HR factors include Rad51, Rad51 paralogs, Rad52, Rad54, BRCA2 and RPA [12,13]. NHEJ mediates rejoining of DSBs without the requirement of a homologous sequence. After induction of a DSB, the KU het- erodimer, consisting of the KU70 and KU80 proteins, binds DNA ends and recruits the DNA-dependent protein kinase catalytic sub- unit (DNA-PKcs) leading to formation of the DNA-PK holo-enzyme [11]. DNA-PK then forms a functional complex with Artemis, which 1568-7864/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.dnarep.2012.10.008