Chemico-Biological Interactions 182 (2009) 173–182 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint Oxaliplatin-induced gamma-H2AX activation via both p53-dependent and -independent pathways but is not associated with cell cycle arrest in human colorectal cancer cells Shu-Jun Chiu a,∗ , Yi-Jang Lee b , Tzu-Sheng Hsu c , Wen-Shu Chen a a Department of Life Science, Tzu Chi University, Hualien 970, Taiwan b Department of Biomedical Imaging and Radiation Sciences, National Yang-Ming University, Taipei 100, Taiwan c Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien 970, Taiwan article info Article history: Received 18 May 2009 Received in revised form 15 August 2009 Accepted 31 August 2009 Available online 6 September 2009 Keywords: Oxaliplatin -H2AX p53 Apoptosis Colorectal cancer cells abstract Oxaliplatin, a chemotherapeutic drug, induces DNA double-strand breaks (DSBs) and apoptosis in colorec- tal cancer cells. It has been shown that -H2AX acts as a marker of DSBs. However, the molecular events associated with oxaliplatin-mediated cell cycle arrest and cell death remain unclear. In this study, we investigated the roles of p53 and -H2AX following oxaliplatin treatment, as they are important effector proteins for apoptosis and DSB repair, respectively. Both phosphorylated-p53 (Ser-15) and -H2AX were up-regulated and accumulated in the nuclei of p53-wild type human colorectal cancer HCT116 cells after exposure to oxaliplatin. Concomitantly, oxaliplatin-induced G 2 /M arrest was associated with a reduction in both cyclin B1 expression and phosphorylated-CDC2 (Thr-161). Release of G 2 /M arrest by caffeine was accompanied by a decrease in the levels of p53/p21; however, -H2AX levels were unchanged. Further- more, inhibition of p53 phosphorylation by pifithrin- was sufficient to reduce the oxaliplatin-induced up-regulation of -H2AX and apoptosis. Oxaliplatin-induced -H2AX via a p53-independent pathway but did not cause caspase-3 activation in p53-null HCT116 cells. Interestingly, no changes were observed in the H2AX gene knockdown with regards to oxaliplatin-induced G 2 /M arrest in p53-wild type and S phase arrest in p53-null HCT116 cells. Taken together, these data indicate that a molecular pathway involving p53, -H2AX and cell cycle arrest plays a pivotal role in the cellular response to oxaliplatin. © 2009 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Oxaliplatin is a third-generation platinum analog of the 1,2- diaminocyclohexane family of chemotherapy drugs and was developed more recently than cisplatin and carboplatin [1,2]. Although oxaliplatin produces DNA cross-links similar to those cre- ated by cisplatin, it confers a more effective spectrum of anti-tumor effects than cisplatin and is not cross-resistant with cisplatin and carboplatin [3]. A combination of oxaliplatin and 5-fluorouracil (5- FU)/leucovorin is clinically active in the treatment of colon and rectum carcinomas [4]. Oxaliplatin-induced DNA cross-links lead to DNA damage [5] and provoke G 0 /G 1 and G 2 /M cell cycle arrest, as well as increased apoptosis in cancer cells [6,7]; however, the molecular mechanisms of oxaliplatin-induced cell cycle arrest in human colorectal cancer cells are still unclear. ∗ Corresponding author at: Department of Life Science, Tzu Chi University, 701, Section 3, Chung-Yang Road, Hualien 970, Taiwan. Fax: +886 3 8572526. E-mail address: chiusj@mail.tcu.edu.tw (S.-J. Chiu). H2AX is a variant of the histone H2A family, which com- prises H2A1, H2A2, H2AZ and H2AX, and differs from the other members in terms of its conserved serine residue (Ser-139) in the four amino acid residues (S-Q-E/D-L/Y) of the C-terminal [8]. When cells are exposed to -irradiation (IR), Ser-139 is rapidly - phosphorylated [9]. Phosphorylated H2AX (-H2AX) forms nuclear foci at the sites of IR-induced double-strand breaks (DSBs) and is essential in the recruitment of repair factors to the damaged DNA sites [9,10]. In addition to environmental stress, DSBs induced by replication fork collision and dysfunctional telomeres also result in phosphorylation of H2AX [11–13]. Moreover, -H2AX is formed during apoptosis initiated by DNA damage [14]. However, it is not yet known whether -H2AX is involved in oxaliplatin- induced G 2 /M arrest and apoptosis in human colorectal cancer cells. p53 is a tumor suppressor protein that controls cell cycle arrest and apoptosis in cancer cells [15], and activation of p53 is associ- ated with protein phosphorylation on serine-15 via the ATM (ataxia telangiectasia mutated) signaling pathway [16]. The role of p53 in cancer cells after drug treatment varies considerably: in colorectal cancer cells expressing wild-type p53, oxaliplatin induces cell cycle 0009-2797/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2009.08.019