Please cite this article in press as: B. Erbaykent-Tepedelen, et al., DNA damage response (DDR) via NKX3.1 expression in prostate cells, J. Steroid Biochem. Mol. Biol. (2014), http://dx.doi.org/10.1016/j.jsbmb.2014.01.001 ARTICLE IN PRESS G Model SBMB 4128 1–11 Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx Contents lists available at ScienceDirect Journal of Steroid Biochemistry and Molecular Biology jo ur nal home page: www.elsevier.com/locate/jsbmb DNA damage response (DDR) via NKX3.1 expression in prostate cells Burcu Erbaykent-Tepedelen a , Selda Karamil a , Ceren Gonen-Korkmaz b , Q1 Kemal S. Korkmaz a,∗ a Ege University, Faculty of Engineering, Department of Bioengineering, Cancer Biology Laboratory, Bornova, Izmir, Turkey b Ege University, Faculty of Pharmacy, Department of Pharmacology, Bornova, Izmir, Turkey a r t i c l e i n f o Article history: Received 23 August 2013 Received in revised form 3 January 2014 Accepted 6 January 2014 Keywords: H2AX ATM phosphorylation Irinotecan (CPT-11) Doxorubicin Etoposide a b s t r a c t It has been reported that NKX3.1 an androgen-regulated homeobox gene restricted to prostate and testi- cular tissues, encodes a homeobox protein, which transcriptionally regulates oxidative damage responses and enhances topoisomerase I re-ligation by a direct interaction with the ATM protein in prostate cells. In this study, we aimed to investigate the role of NKX3.1 in DNA double-strand break (DSB) repair. We demonstrate that the DNA damage induced by CPT-11 (irinotecan, a topo I inhibitor), doxorubi- cin (a topo II inhibitor), and H 2 O 2 (a mediator of oxidative damage), but not by etoposide (another topo II inhibitor), is negatively influenced by NKX3.1 expression. We also examined H2AX (S139) foci formation and observed that the overexpression of NKX3.1 resulted a remarkable decrease in the for- mation of H2AX (S139) foci. Intriguingly, we observed in NKX3.1 silencing studies that the depletion of NKX3.1 correlated with a significant decrease in the levels of p-ATM (S1981) and H2AX (S139) . The data imply that the DNA damage response (DDR) can be altered, perhaps via a decrease in the topoisomerase I re-ligation function; this is consistent with the physical association of NKX3.1 with DDR mediators upon treatment of both PC-3 and LNCaP cells with CPT-11. Furthermore, the depletion of NKX3.1 with siRNA resulted in a G1/S progression via the facilitation of an increase in E2F stabilization concurrent with the suppressed DDR. Thus, the topoisomerase I inhibitor-mediated DNA damage enhanced the physical association of NKX3.1 with H2AX (S139) on the chromatin in LNCaP cells, whereas NKX3.1 in the soluble fraction was associated with p-ATM (S1981) and RAD50 in these cells. Overall, the data sug- gest that androgens and NKX3.1 expression regulate the progression of the cell cycle and concurrently activate the DDR. Therefore, androgen withdrawal may facilitate the development of an error-prone phenotype and, subsequently, the loss of DNA damage control during prostate cancer development. © 2014 Published by Elsevier Ltd. 1. Introduction The NKX3.1 gene is transcriptionally upregulated by androgens in the prostate gland and in the androgen receptor (AR)-positive prostate tumor cell line, LNCaP [1,2]. It has been demonstrated that the loss of an Nkx3.1 allele results in epithelial dysplasia and benign hyperplasia in the rat prostate, and NKX3.1 expres- sion was found to be inversely correlated with high-grade prostate tumors in humans [3,4]. The NKX3.1 protein contains conserved domains in its wild-type isoform (234 amino acids long) called tin- man [5] and homeobox domains, which mediate interactions with ∗ Corresponding author. Tel.: +90 5383087613; fax: +90 2323884955. Q2 E-mail addresses: burcu erbaykent@yahoo.com (B. Erbaykent-Tepedelen), seldakaramil@yahoo.com (S. Karamil), korkmaz ceren@yahoo.com (C. Gonen-Korkmaz), ks korkmaz@yahoo.com (K.S. Korkmaz). several nuclear proteins and DNA, respectively. Consistent with the tumor suppressor function of NKX3.1, this factor supposedly represses transcription together with the Groucho complex sub- sequent to the recruitment of HDAC1 to target promoters, while NKX3.1 contributes to the cell cycle and cell death machinery via an MDM2-dependent mechanism [6]. Furthermore, the proteosomal degradation of NKX3.1 by TOPORS, which directly interacts with and acts as a robust E3 ubiquitin ligase, has also been shown in prostate cancer cells. However, the knockdown of TOPORS using an siRNA approach increases the steady-state level of NKX3.1 and the half-life in LNCaP [7]. These data suggest that the loss of NKX3.1 expression is an important molecular alteration in the development of dysplastic factors in prostate cells, which presumably occurs sub- sequent to the loss of NKX3.1 function: to protect the cells from oxidative DNA damage [6,7]. Hence, the protective role of NKX3.1 regarding the oxidative DNA damage in the cell cycle has not been fully investigated in prostate cancer development, and mechanistic studies are required. 0960-0760/$ – see front matter © 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.jsbmb.2014.01.001 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47