The BRCA1 alternative splicing variant Δ14-15 with an in-frame deletion of part of the regulatory serine-containing domain (SCD) impairs the DNA repair capacity in MCF-7 cells Jan Sevcik a, b, ⁎, Martin Falk c , Petra Kleiblova a , Filip Lhota a , Lenka Stefancikova c , Marketa Janatova a , Lenka Weiterova c , Emilie Lukasova c , Stanislav Kozubek c , Petr Pohlreich a , Zdenek Kleibl a, ⁎ a Institute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University in Prague, U Nemocnice 5, 128 53 Prague 2, Czech Republic b Prague Burn Centre, Charles University, Third Faculty of Medicine and Teaching Hospital Kralovske Vinohrady, Srobarova 50, 10034 Prague 10, Czech Republic c Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 61265 Brno, Czech Republic abstract article info Article history: Received 12 December 2011 Accepted 28 December 2011 Available online 3 January 2012 Keywords: BRCA1 Alternative splicing Breast cancer DNA damage Homologous recombination Non-homologous end joining BRCA1-serine-containing domain (SCD) The BRCA1 gene codes for a protein involved in the DNA double strand break (DDSB) repair. Alongside the dominant full-length splicing form of BRCA1, numerous endogenously expressed alternative splicing variants of unknown significance have been described in various tissues. Some of them retain the original BRCA1 reading frame but lack several critical BRCA1 structural domains, suggesting an altered function of the result- ing protein in the BRCA1-regulated processes. To characterize the effect of the BRCA1Δ14-15 splicing variant (with an in-frame deletion affecting the reg- ulatory serine-containing domain) on the DDSB repair, we constructed the MCF-7 clones stably expressing the analyzed variant with/without a shRNA-mediated downregulation of the endogenous full-length wild- type BRCA1 expression. Our results show that the expression of the BRCA1Δ14-15 variant delays the γ-radiation-induced DDSB re- pair, alters the kinetics of irradiation-induced foci formation/decomposition and reduces the non- homologous end-joining capacity in MCF-7 cells. Therefore, the BRCA1Δ14-15 is not able to functionally replace the full-length wt BRCA1 in the DDSB repair. Our findings indicate that the endogenously expressed BRCA1 alternative splicing variants may negatively influence genome stability and support the growing evidence of the pathological potential of the sequence variants generated by an altered or misregulated alternative splicing in the process of mammary malignant transformation. © 2011 Elsevier Inc. All rights reserved. 1. Introduction Breast cancer (BC) is the most common malignancy among women worldwide [1]. Inactivating alterations in the tumor sup- pressor genes BRCA1 (MIM# 113705) and BRCA2 (MIM# 600185) represent major genetic BC-predisposition factors [2]. Mutations in these genes account for 3–5% of all BC cases. The proportion between mutations in BRCA1 and BRCA2 varies in different popu- lations. The Czech Republic, together with other Central European countries, belongs to regions with a prevailing incidence of muta- tions in BRCA1 [3]. Besides clearly pathogenic BRCA1 gene alter- ations, a large group of sequence variants of uncertain clinical significance (VUS) has been identified. At present, a growing in- terest is focused on VUS that generate unbalanced or aberrant BRCA1 pre-mRNA splicing, while the effects and generation of naturally-occurring alternative BRCA1 pre-mRNA splicing variants remain largely neglected. The most frequent BRCA1 transcription variant (NM_007294) contains 23 exons coding for a protein con- sisting of 1863 amino acids (220 kDa); however, many other al- ternative BRCA1 mRNA splicing variants have been identified in various healthy or pathological tissues and cell lines, but their significance, tissue specificity, and biological activities are almost unknown [4]. Cellular Signalling 24 (2012) 1023–1030 Abbreviations: ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3-related; AURKA, Aurora kinase A; BC, breast cancer; BRCA1/2, breast cancer- associated gene 1/2; BRCT, BRCA1 C-terminal domain; B2M, beta-2-microglobulin; Cdk2, cyclin-dependent kinase 2; Chk2, check-point kinase 2; CtIP, CTBP-interacting protein; DDSB, DNA double-strand break; GAPDH, glyceraldehyde-3-phosphate dehy- drogenase; HR, homologous recombination; IRIF, ionizing radiation-induced foci; NHEJ, non-homologous end joining; NLS, nuclear localization signal; PARP, poly(ADP- ribose)polymerase; PBMCs, peripheral blood mononuclear cells; PI, post-irradiation; RNAi, RNA interference; RPA, replication protein A; SCD, serine-containing domain; shRNA, short-hairpin RNA; VUS, variants of uncertain significance. ⁎ Corresponding authors at: Institute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University in Prague, U Nemocnice 5, 128 53, Prague 2, Czech Republic. Tel.: + 420 22496 5745; fax: + 420 22496 5732. E-mail addresses: jsevc@lf1.cuni.cz (J. Sevcik), zdekleje@lf1.cuni.cz (Z. Kleibl). 0898-6568/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2011.12.023 Contents lists available at SciVerse ScienceDirect Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig