Carcinogenesis Advance Access originally published online on June 12, 2007
Carcinogenesis 2007 28(11):2382-2390; doi:10.1093/carcin/bgm128
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Redox effector factor-1, combined with reactive oxygen species, plays an important role in the transformation of JB6 cells
1 Chao Family Comprehensive Cancer Center
2 Departments of Biological Chemistry and Medicine, University of California Irvine, Orange, California 92868
* To whom correspondence should be addressed. Tel: +714 456 6310; Fax: +714 456 2240; Email: flmeyske{at}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 transformants [by ultraviolet light B (UVB), H2O2 or Cd] to study this phenomenon.
Significant increases of reactive oxygen species (ROS) were observed in JB6P+ 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 JB6P+ cells. Ref-1 depletion also inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced anchorage-independent growth of JB6P+ by 40% and reduced the colony numbers of JB6P+/H2O2 and JB6P+/Cd cells. Mechanistic 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 JB6P+/H2O2 cells.
This is the first report of the novel role of Ref-1 in cellular transformation. 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.
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-
B, nuclear factor kappa B; Ref-1, redox effector factor-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
Received February 6, 2007; revised April 13, 2007; accepted May 17, 2007.
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