Abstract
Elevated reactive oxidative species (ROS) are cytotoxic, and chronic elevated levels of ROS have been implicated in multiple diseases as well as cellular transformation and tumor progression. However, the potential for a transient and minimally toxic episode of ROS exposure, or a minimal threshold dose of ROS, to initiate disease or cellular transformation is unclear. We examined both transcriptional and phospho-proteomic responses of murine embryonic stem (ES) cells to a single brief exposure of minimally toxic hydrogen peroxide (H(2)O(2)). The cellular response was distinct from those induced by either an acute exposure to H(2)O(2) or the topoisomerase II poison etoposide. Analysis of tumorigenesis-related transcripts revealed a significant up-regulation of oncogenes and down-regulation of tumor suppressors. Analysis of the phospho-proteomic response demonstrated insulin-signaling induction, including insulin receptor Y972 hypophosphorylation, similar to insulin-resistance mouse models and observed in diabetic patients. In addition, ES cells were more resistant to ROS than differentiated cells, and retained their transcriptional self-renewal signature, suggesting stem cells have a higher potential for ROS-mediated mutagenesis and proliferation in vivo. These results are a direct demonstration that even brief and non-toxic exposures to ROS may induce transduction of insulin resistance and transformation signaling in stem cells leading to diabetes and cancer.
Highlights
Reactive oxygen species (ROS) are produced by metabolizing molecular oxygen to produce hydroxyl free radicals (⋅OH), superoxide anions (O2-⋅), singlet oxygens (1O2), and hydrogen peroxide (H2O2)
Cells were exposed to 5mM hydrogen peroxide (H2O2) or 20 M etoposide for 30 min. 5mM H2O2 induces reactive oxidative species (ROS) levels similar to those observed in vivo during acute inflammatory reactions
Etoposide is an inhibitor of the topoisomerase II religation reaction and a known inducer of DNA double-strand breaks (DSBs). 20 M etoposide is in close agreement with pharmacokinetic studies demonstrating peak patient plasma levels [25]
Summary
Reactive oxygen species (ROS) are produced by metabolizing molecular oxygen to produce hydroxyl free radicals (⋅OH), superoxide anions (O2-⋅), singlet oxygens (1O2), and hydrogen peroxide (H2O2). Elevated ROS may promote tumorigenesis in more differentiated somatic cells indirectly through increased illegitimate repair of the ensuing DNA damage. It is not clear how susceptible stem cells are to a single brief exposure of ROS, at minimally toxic doses not expected to induce apoptosis. Parallel examination of transcriptional profiles with the posttranslational modifications of a significant though limited number of signaling molecules demonstrated that a single minimally toxic exposure to ROS is sufficient to induce significant increases in oncogenic and metastatic pathways and induce insulin signaling, similar to insulin-resistance mouse models and observed in diabetic patients. Despite the significant signaling changes induced by ROS, cells maintained their stem cell signatures suggesting a mechanism for maintenance, survival, and transformation in early stem cell pools
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