Redox effector factor-1, combined with reactive oxygen species, plays an important role in the transformation of JB6 cells

Carcinogenesis vol.28 no.11 pp.2382–2390, 2007 doi:10.1093/carcin/bgm128 Advance Access publication June 12, 2007 Redox effector factor-1, combined with reactive oxygen species, plays an important role in the transformation of JB6 cells Sun Yang 1 , Bobbye J.Misner 1 , Rita J.Chiu 1 and Frank L.Meyskens Jr ,1,2 Chao Family Comprehensive Cancer Center and 2 Departments of Biological Chemistry and Medicine, University of California Irvine, Orange, California To whom correspondence should be addressed. Tel: þ714 456 6310; Fax: þ714 456 2240; Email: flmeyske@uci.edu Apurinic/apyrimidinic endonuclease/redox effector factor-1 (APE/ Ref-1) is a multifunctional protein involved both in DNA base excision repair and redox regulation. Studies have suggested that abnormal Ref-1 levels and/or activities are associated with tumor progression and sensitivities to treatment, but no direct evidence has yet been published regarding the role of Ref-1 in malignant transformation. We utilized the well-documented tumor promotor- sensitive JB6 mouse epithelial cell model as well as new trans- formants [by ultraviolet light B (UVB), H 2 O 2 or Cd] to study this phenomenon. Significant increases of reactive oxygen species (ROS) were observed in JB6Pþ and all the transformants compared with promotor-resistant JB6P cells. These increases were paralleled by a sustained elevation of Ref-1 expression. Further analysis exhibited a strong inverse correlation between oxidative DNA lesions [8-oxodeoxyguanosine (8-oxo-dG)] and Ref-1 levels in all JB6 cells. Notably, apoptosis occurred after knock-down of Ref-1 by small interfering RNA (siRNA)] demonstrated by a 2-fold increase of Annexin V-positive JB6Pþ cells. Ref-1 depletion also inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced anchorage-independent growth of JB6Pþ by 40% and reduced the colony numbers of JB6Pþ/H 2 O 2 and JB6Pþ/Cd cells. Mech- anistic studies revealed that Ref-1 reduction was associated with an increase of intracellular ROS levels and a marked decrease of activator protein-1 (AP-1) transcription activities in JB6Pþ/H 2 O 2 cells. This is the first report of the novel role of Ref-1 in cellular trans- formation. Based on the data presented here, we propose that induction of Ref-1, serving as an adaptive response to elevated ROS, plays a critical role in transformation and protects cells from excess ROS stresses through both DNA repair and activation of transcription factors such as activator protein-1. Introduction Epidemiological observations and animal experiments have shown that the process of carcinogenesis involves a complex series of mul- ti-sequence events that result in a cell evolving from a healthy state to a precancerous condition and finally to an early stage of cancer. The ‘initiation-promotion-progression’ model is a prototype for these stages. Many studies have shown that reactive oxygen species (ROS) are involved in all stages of carcinogenesis. The promotion stage is char- acterized by the clonal expansion of initiated cells and results in the formation of an identifiable focal lesion and involves reversible pro- Abbreviations: AP-1, activator protein-1; DCF, 2,#7#-dichlorofluorescein; FITC, fluorescein isothiocyanate; HE, dihydroethidium; MAPK, mitogen- activated protein kinase; Myb, Cellular homologue of avian myeloblastosis virus oncogene; NF-jB, nuclear factor kappa B; Ref-1, redox effector fac- tor-1; ROS, reactive oxygen species; SEAP, secreted alkaline phosphatase; si-NS, si-nonsense; si-Ref-1, small interfering RNA against Ref-1; TPA, 12- O-tetradecanoylphorbol-13-acetate; UVB, Ultraviolet light (290–320 nm); 8- oxo-dG, 8-oxodeoxyguanosine. cesses which require the continuous presence of the tumor promotion stimulus to be maintained. The promotion activity of TPA has been well documented (1) and extensive evidence has shown that tumor promotion by phorbol esters was, to a large degree, due to stimulation of oxygen radical generation and ROS-mediated inflammatory reac- tions (2,3); radical scavengers exhibited inhibitory effects on tumor promotion (4–6). Both activator protein-1 (AP-1) and nuclear factor kappa B (NF- jB) activation are required for transformation whether induced by TPA or by tumor necrosis factor and blocking ROS-mediated AP-1- mitogen-activated protein kinase (MAPK) and NF-jB activation con- tributes to the anti-promotion effects of ROS scavengers (7,8). It is also well documented that low levels of ROS can stimulate cell di- vision in the promotion stage and thus enhances tumor growth (9). Agents that interfere with the promotion stage have exhibited chemo- preventive potential (10). The JB6 mouse epidermal cell lines were established in the early 1980s by Nancy Colburn’s group and JB6Pþ cells (promotion sensitive) represent a well-developed cell culture system which have been extensively utilized as a model to study the molecular events that are associated with tumor promotion (11,12). In the presence of the tumor promoter TPA or epidermal growth factor, JB6Pþ cells undergo a re- sponse similar to second stage tumor promotion and are associated with anchorage-independent growth and tumourigenic transformation; this does not occur with JB6P cells. The basis for the transformation resistance has been associated with a deficiency of extracellular sig- nal-regulated kinase signaling and lack of AP-1 activation (12,13). Redox effector factor-1 (Ref-1) was first recognized by its DNA base excision repair activity and for this reason, it was initially named apurinic/apyrymidinic endonuclease. In addition to its DNA repair potential, Ref-1 also exhibits distinct redox regulation functions that facilitate the DNA-binding activities of many transcription factors (such as AP-1 dimers, NF-jB, p53 and Cellular homologue of avian myeloblastosis virus oncogene (Myb)) both in a redox-dependent and redox-independent manner. Because of its unique multiple functions, Ref-1 has attracted more attention recently and alterations of Ref-1 have been reported in many human tumors (14,15). Ref-1 expression has been shown to be well regulated both at the transcription and post- transcription levels. Apurinic/apyrimidinic endonuclease/Ref-1 is sensitive to alterations in redox status, and serves as a rapid adaptive response to subtoxic levels of a variety of ROS stresses and ROS generators such as H 2 O 2 , superoxide anion and c-rays (16–18). Stuart (2004) reported that the reduction of APE1 levels after lowering ROS production by mitochondrial DNA depletion was reversed by H 2 O 2 (19,20). Our previous study using human melanoma cells has also demonstrated remarkably elevated nuclear Ref-1 levels that are asso- ciated with abnormally increased intracellular ROS levels (15). The regulation of Ref-1 by ROS might occur via transcription activation, as many transcription factors such as AP-1, SP1, CREP and ATF bind to its promoter (14), which are also well known to be sensitive to oxida- tive stresses. In addition, ROS also affects the functions of Ref-1 by altering the redox status of this protein. Both DNA repair and redox regulation activities of Ref-1 were remarkably reduced either by oxi- dation of H 2 O 2 or cystein replacement by site-directed mutagenesis Many studies have also shown that Ref-1 deficiency rendered a wide range of cells more sensitive to apoptosis induced by alkylating and oxidative agents as well as ionizing radiation (14,23,24). However, the direct involvement of Ref-1 in tumourigenesis or cell transformation remains unclear. In the current paper, we characterized the role of Ref-1 in tumor transformation with the JB6Pþ/tumor promoter model, and have provided important insights into the mechanisms involved. O The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org