NKX3.1 contributes to S phase entry and regulates DNA damage response (DDR) in prostate cancer cell lines Burcu Erbaykent-Tepedelen a , Besra Özmen a , Lokman Varisli a , Ceren Gonen-Korkmaz b , Bilge Debelec-Butuner a,c , Hamid Muhammed Syed a , Ozgur Yilmazer-Cakmak d , Kemal Sami Korkmaz a,⇑ a Ege University, Engineering Faculty, Department of Bioengineering, Cancer Biology Laboratory, Faculty of Pharmacy, Bornova, Izmir, Turkey b Ege University, Faculty of Pharmacy, Department of Pharmacology, Bornova, Izmir, Turkey c Ege University, Faculty of Pharmacy, Department of Biotechnology, Bornova, Izmir, Turkey d Izmir Institute of Technology, Biotechnology and Bioengineering Central Research Laboratories, Gulbahce, Izmir, Turkey article info Article history: Received 31 August 2011 Available online 14 September 2011 Keywords: DNA damage response Topoisomerase inhibitor CPT-11 Prostate cancer cH2AX (S139) foci pATM (S1981) NKX3.1 abstract NKX3.1 is an androgen-regulated homeobox gene that encodes a tissue-restricted transcription factor, which plays an important role in the differentiation of the prostate epithelium. Thus, the role of NKX3.1 as a functional topoisomerase I activity enhancer in cell cycle regulation and the DNA damage response (DDR) was explored in prostate cancer cell lines. As an early response to DNA damage fol- lowing CPT-11 treatment, we found that there was an increase in the cH2AX (S139) foci number and that total phosphorylation levels were reduced in PC-3 cells following ectopic NKX3.1 expression as well as in LNCaP cells following androgen administration. Furthermore, upon drug treatment, the increase in ATM (S1981) phosphorylation was reduced in the presence of NKX3.1 expression, whereas DNA-PKcs expression was increased. Additionally, phosphorylation of CHK2 (T68) and NBS1 (S343) was abrogated by ectopic NKX3.1 expression, compared with the increasing levels in control PC-3 cells in a time-course experiment. Finally, NKX3.1 expression maintained a high cyclin D1 expression level regardless of drug treatment, while total cH2AX (S139) phosphorylation remained depleted in PC-3, as well as in LNCaP, cells. Thus, we suggest that androgen regulated NKX3.1 maintains an active DDR at the intra S progression and contributes to the chemotherapeutic resistance of prostate cancer cells to DNA damaging compounds. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction Although androgen ablation therapy for prostate cancer leads to decreased tumor cell proliferation and regression of tumor size [1], the disease recurs within 2–3 years in most organ confined prostate cancer cases [2,3]. This recurrence may be due to developing tu- mors retaining functional androgen receptors (AR), which allow the tumors to respond to AR signaling events [4,5]. In these tumors, ARs can be induced via promiscuous growth factors, which results in transactivation of androgen-regulated genes that might signifi- cantly affect tumor progression. The NKX3.1 gene is upregulated by androgens via an AR dependent mechanism in normal prostate epithelium [6,7]. It encodes a prostate- and testis-specific nucleo- protein with two conserved domains in its wild type isoform (234 aa long). These domains are required for the interaction of NKX3.1 with specific nuclear proteins and DNA [8]. The binding of NKX3.1 negatively modulates AR transcription (putatively via tar- get promoter association) and subsequent signaling events in pros- tate cells. Its expression also contributes to both cell cycle and cell death machinery by increasing p53 acetylation and through subse- quent stabilization of the MDM2-dependent mechanism [9]. How- ever, as part of the Groucho complex, NKX3.1 is required to repress transcription with its loss resulting in prostatic epithelial dysplasia and benign hyperplasia of the rat prostate [10,11]. Ouyang et al., have shown that NKX3.1 suppresses tumor initiation by protecting the cell against oxidative damage via transcriptional regulation of the pro-oxidant enzyme levels [12,13]. TOPORS, a strong E3 ubiqui- tin ligase, promotes proteasomal degradation of NKX3.1 through di- rect interaction, predisposing cells to oncogenic transformation by providing an irreversible growth advantage as the first steps in prostate carcinogenesis [14]. Recently, it has been reported that NKX3.1 relocates to the nucleus and enhances the cleavage and re-ligation abilities of the topoisomerase I (topo I) enzyme through direct association [15,16]. ATM and ATR activities are influenced by NKX3.1 expression, which protects cells against IR- and mitomycin C-induced DNA damage [17]. Thus, the DNA damage response is more efficient in cells in the presence of NKX3.1 expression; this re- port provides useful insights into our understanding of the DDR as well as therapy resistance of LNCaP cells upon DNA damage. We 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.09.035 ⇑ Corresponding author. E-mail address: ks_korkmaz@yahoo.com (K.S. Korkmaz). Biochemical and Biophysical Research Communications 414 (2011) 123–128 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc