Abstract
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 approximately 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.
Highlights
Epidemiological observations and animal experiments have shown that the process of carcinogenesis involves a complex series of multi-sequence events that result in a cell evolving from a healthy state to a precancerous condition and to an early stage of cancer
Both activator protein-1 (AP-1) and nuclear factor kappa B (NFjB) activation are required for transformation whether induced by TPA or by tumor necrosis factor and blocking reactive oxygen species (ROS)-mediated AP-1mitogen-activated protein kinase (MAPK) and NF-jB activation contributes to the anti-promotion effects of ROS scavengers [7,8]
5000-fold increase compared with JB6PÀ cells, which was significantly reduced by Redox effector factor-1 (Ref-1) depletion to a very low level (Figure 4E). It has been well documented in many studies of human cancer that alterations of Ref-1 expression levels and subcellular localization have contributed to progression of tumor cells and development of treatment resistance [27,28,29,30]
Summary
Epidemiological observations and animal experiments have shown that the process of carcinogenesis involves a complex series of multi-sequence events that result in a cell evolving from a healthy state to a precancerous condition and to an early stage of cancer. 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 reactions [2,3]; radical scavengers exhibited inhibitory effects on tumor promotion [4,5,6]. Both activator protein-1 (AP-1) and nuclear factor kappa B (NFjB) activation are required for transformation whether induced by TPA or by tumor necrosis factor and blocking ROS-mediated AP-1mitogen-activated protein kinase (MAPK) and NF-jB activation contributes to the anti-promotion effects of ROS scavengers [7,8]. Agents that interfere with the promotion stage have exhibited chemopreventive potential [10]
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