Characterization of rainbow trout CHK2 and its potential as a genotoxicity biomarker Jessica D. Steinmoeller, Kazuhiro Fujiki, Aman Arya, Kirsten M. Müller, Niels C. Bols, Brian Dixon ⁎, Bernard P. Duncker ⁎ Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1 abstract article info Article history: Received 24 June 2008 Received in revised form 7 November 2008 Accepted 9 November 2008 Available online 24 November 2008 Keywords: Biomarker Checkpoint CHK2 DNA damage Rainbow trout Checkpoint kinase 2 (CHK2) plays a central and conserved role in the eukaryotic DNA damage response. Few cell cycle checkpoint proteins have been examined in aquatic organisms, and this study is the first to characterize CHK2 expression in a fish species. CHK2 was cloned from Oncorhynchus mykiss, the rainbow trout. The coding region extends over 5741 nucleotides in the genome, including 13 introns, and specifies a predicted 533 amino acid protein. Southern blot analysis revealed that CHK2 exists as a single copy in the rainbow trout genome. Recombinant protein representing the FHA domain was used to generate polyclonal anti-CHK2 antibodies. While CHK2 transcript levels were relatively low in gill and high in brain, the opposite was true for protein levels. Both gill and brain cell cultures were treated with bleomycin, which induces double-strand DNA breaks. There was no effect on levels of CHK2 in gill cells, suggesting that the protein is constitutively active in this tissue. In contrast, brain cells upregulated CHK2 in a dose-dependent manner. The tissue specific expression of CHK2 and its ability to respond to bleomycin treatment suggests that some checkpoint proteins may serve as suitable biomarkers for DNA damage in rainbow trout and other fish species. © 2008 Elsevier Inc. All rights reserved. 1. Introduction Cell cycle checkpoints are critical for maintaining genomic stability, and function by monitoring DNA integrity and the successful completion of cell cycle events (Whitfield et al., 2006). Numerous checkpoint proteins work together to sense and respond to DNA damage, resulting in either DNA repair or programmed cell death depending on the severity and duration of the genetic injury. The expression level and/or phosphorylation state of these proteins changes in response to DNA damage, which provides a means of identifying cells that have activated a checkpoint response (Kwak et al., 2006). A key component of the checkpoint machinery is checkpoint kinase 2 (CHK2). CHK2 is the mammalian ortholog of Rad53 and Cds1 in budding and fission yeast, respectively. The formation of DNA double strand breaks (DSBs) leads to the activation of CHK2 (Ahn et al., 2004). CHK2 then phosphorylates a variety of downstream targets including CDC25C. Phosphorylated CDC25C is sequestered in the cytoplasm, preventing the activation of CDC2 and halting the G2 to M phase transition. Additional downstream targets of CHK2 include CDC25A, BRCA1 and p53. ATM (ataxia telangiectasia-mutated) regulates CHK2 activity and, in some circumstances, p53 has a role in regulating CHK2 expression (Chin and Li, 2003). Overall, CHK2 plays an important role in all the major checkpoints (G1/S, intra-S, G2/M) of the cell cycle causing cell cycle arrest, DNA repair or apoptosis (Hirao et al., 2000). Mutations in CHK2 are associated with malignancies in a variety of tissues, including brain tumors (Sallinen et al., 2005). Owing to its important role in cell cycle control and tumorigenesis, CHK2 has been studied in a wide assortment of eukaryotic species ranging from yeast to humans (Melo and Toczyski, 2002). However, few checkpoint proteins have been studied in fish, and no characterization of CHK2 has been reported. Therefore the conservation of the DNA damage response across teleost species remains largely unknown. Of the checkpoint genes that have been cloned and characterized, the majority have been from Danio rerio (zebrafish), including TP53 (Cheng et al., 1997; Berghmans et al., 2005), MPS1 (Poss et al., 2004), DTL (Sansam et al., 2006), separase (Shepard et al., 2007) and ATM (Garg et al., 2004; Imamura and Kishi, 2005). TP53 has also been cloned in Oryzias latipes (Japanese medaka; Krause et al., 1997), Platichthys flesus (European flounder; Cachot et al., 1998) and Oncorhynchus mykiss (rainbow trout; de Fromentel et al., 1992), and the RB gene has recently been isolated in Xiphophorus maculatus (southern platyfish; Butler et al., 2007). Sequence analysis by Krause et al. (1997). demonstrated a high degree of similarity between TP53 regions encoding functional p53 domains from Japanese medaka and other vertebrate species including rainbow trout, frog, chicken, rat, mouse, hamster, green monkey and human, suggesting that checkpoint genes are well conserved. Functional conservation of p53 was also shown among teleosts, in a study by Berghmans et al. (2005), as Comparative Biochemistry and Physiology, Part C 149 (2009) 491–499 ⁎ Corresponding authors. Duncker is to be contacted at Tel.: +1519 888 4567x33957; fax: +1519 746 0614. Dixon, Tel.: +1519 888 4567x32665; fax: +1519 746 614. E-mail addresses: bdixon@sciborg.uwaterloo.ca (B. Dixon), bduncker@sciborg.uwaterloo.ca (B.P. Duncker). 1532-0456/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2008.11.004 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc