Absence of major defects in non-homologous DNA end joining in human breast cancer cell lines Philippe Me´rel 1 , Alexandre Prieur 1 , Petra Pfeiffer 2 and Olivier Delattre* ,1 1 Laboratoire de Pathologie Mole ´culaire des Cancers, INSERM U509, Institut Curie, 26 rue d’Ulm, 75248 Paris Cedex 05, France; 2 FB9, GenetikUniversitaet, GH Essen Universitaetsstr, 5D-45117, Essen, Germany Structural abnormalities of chromosomes, including translocations and deletions, are extremely frequent in human cancer cells and particularly in breast cancer cells. One hypothesis to account for these alterations is a deficiency in the repair of DNA double-strand breaks (DSB). This repair process relies on two distinct pathways, homologous recombination (HR) and non- homologous DNA end joining (NHEJ). To investigate this latter pathway, we have studied the ability of cell- free extracts from a variety of human cells to rejoin different types of DSBs. The end joining activity of eleven sporadic breast cancer cell lines (BCCLs) was compared with that of control cells including primary human fibroblasts and cells harbouring a limited number of chromosome abnormalities. In vitro rejoining activity was not detected in extracts from MO59J DNA-PKcs- deficient cells and was strongly inhibited by wortmannin in control extracts. In contrast, most sporadic BCCLs and BRCA1 or BRCA2 deficient cells demonstrated similar efficiencies and accuracies of in vitro NHEJ than control cells. Only two BCCLs, SKBR3 and MDA-MB- 453 exhibited decreased in vitro NHEJ. This study therefore indicates that a major defect in the NHEJ pathway is unlikely to account for the high number of chromosomes abnormalities observed in sporadic and hereditary BCCLs. Oncogene (2002) 21, 5654 – 5659. doi:10.1038/sj.onc. 1205742 Keywords: breast cancer; non-homologous end joining; DNA repair; chromosome instability Introduction Breast cancer is the major type of malignancy affecting women from western industrialized countries. Five – ten per cent of the breast cancers occur in genetically predisposed families, and germline mutations in two tumour suppressor genes (BRCA1 and BRCA2) account for more than 65% of these familial forms (Ford et al., 1998). Two major arguments suggest that breast cancer cells might be deficient in DNA double-strand break (DSB) repair. First, FISH, standard karyotype and more recently multicolour analysis of breast cancer cells have revealed a very high frequency of chromosome rearrangements (Davidson et al., 2000; Forozan et al., 2000; Kytola et al., 2000; Loveday et al., 2000). Second, recent advances in the study of the functions of BRCA1 and BRCA2 tumour suppressor genes indicate that their products play important roles in DSB repair and chromosome stability (Khanna and Jackson, 2001; Patel et al., 1998; Shen et al., 1998; Moynahan et al., 1999, 2001; Snouwaert et al., 1999; Yu et al., 2000). Two main pathways exist in eukaryotic cells to repair DNA DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). While HR requires extensive homology, and is conducted by proteins from the Rad52 epistasis group (reviewed in Khanna and Jackson, 2001). NHEJ does not require homology and can rejoin broken DNA ends directly end-to-end. DNA-PKcs, Ku70, Ku80, XRCC4 and DNA ligase IV have been shown to be major components of this pathway (Khanna and Jackson, 2001). BRCA1 or BRCA2 loss-of-function mutations have been reported only in familial breast cancers but not in sporadic cases. This observation suggests that other genes, distinct from BRCA1 or BRCA2 but also involved in DSB repair pathways might be targets of inactivating mutations in non-hereditary cases of breast cancer. The screening for mutations of candidate genes, involved in HR, have presently not identified recurrent alterations (Bell et al., 1999; Hiramoto et al., 1999; Matsuda et al., 1999). Recent reports also suggest that the NHEJ pathway plays a major role in the maintenance of genome integrity. Indeed, mouse cells deficient in DNA – PKcs, XRCC4, DNA ligase IV, Ku70 or Ku80 exhibit spontaneously high degrees of chromosome instability suggesting that these genes also function as caretakers that maintain genome integrity (Karanjawala et al., 1999; Difilippantonio et al., 2000; Ferguson et al., 2000; Gao et al., 2000; d’Adda di Fagagna et al., 2001). To further evaluate a possible involvement of a deficient NHEJ in breast oncogenesis, we have used an in vitro assay to test this pathway in cell-free extracts. Indeed, extracts from various species are able to convert in vitro linearized plasmid substrates into various repaired molecules (RM) including monomeric Received 15 March 2002; revised 22 May 2002; accepted 7 June 2002 *Correspondence: O Delattre; E-mail: Olivier.Delattre@curie.fr Oncogene (2002) 21, 5654 – 5659 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc