MnSOD knockdown cells, as determined by dihydroethidium levels. UV-induced EGFR activation was inhibited by knockdown of Nox4 expression by lentiviral transduction of shRNA targeting Nox4, indicating a role for Nox4 in regulation of inside-out signaling. UV-induced EGFR activation was also abrogated by treatment with the SOD mimetic MnTnBuOE-2-PyP 5+ , suggesting a potential for pharmacological intervention of inside-out signaling. Our results suggest a relationship between MnSOD, Src, and Nox4 in UV-induced EGFR activation, and may provide an important target pathway for cancer prevention.  Quercetin Induced Starvation-like Condition Is Followed by Autophagy in Rat Gastric Tumor Cells Hiroko P. Indo 1,2 , Hsiu-Chuan Yen 3 , Toshihiko Ozawa 4 , Aaron K. Holley 2 , Daret K. St. Clair 2 , and Hideyuki J. Majima 1 1 Kagoshima University, Japan, 2 University of Kentucky, United States, 3 Chang Gung University, Taiwan, 4 Yokohama College of Pharmacy, Japan The aim of this study is to achieve a mechanistic understanding of the influence of the quercetin in autophagy of the rat gastric mucosal tumor cells, RGK1. It has been reported that quercetin induces apoptosis, which is associated with caspase activation in tumor cells. However, our previous study showed that quercetin did not induce apoptosis but autophagy in RGK1. RGK1 were treated with 30, 100, 2 ȝ0 TXHUFHWLQ for 24 h or with Earle's balanced salt solution for 2 h to simulate starvation. Oxygen consumption rate and ATP production were dose dependently decreased by quercetin treatments. Tthe treatment significantly induced steady state mRNA levels of mTOR but suppressed BECN1 and bcl2 levels. The autophagy signaling pathway,PDK1 and AKT were similarly activated in both quercetin and EBSS treated cells. These results have led to a working model in which quercetin activates class I PI3 leading to the phosphorylation of PDK1 and activates PDK1 phosphorylate Akt leading to the suppression of mTOR, thus contributing to subsequent autophagy that is similar to starvation-induced autophagy.  Distinct ROS Control Mechanisms and their Mutagenic Consequences in Progenitor Subsets of Normal Human Mammary Gland Nagarajan Kannan 1 , Maisam Makarem 1 , Long Nguyen 1 , Jeff Dong 1 , Kingsley Shih 1 , Peter Eirew 1 , and Connie Eaves 1 1 Terry Fox Laboratory, BC Cancer Agency, Canada Reactive oxygen species (ROS) mediate DNA damage and are likely contributors to oncogenesis. However, little is known about mechanisms controlling ROS in normal human mammary cells, in spite of their being a major target of cancer development. to investigate this question, we isolated luminal progenitors (LPs) and basal cells (BCs) that are highly enriched in stem and bipotent progenitor cells at high purities by FACS from normal human mammary gland tissue and compared their levels of ROS and elements that positively and negatively regulate ROS, their responses to oxidative stressors and their content of ROS- associated DNA damaged nucleotides. The results show that LPs contain higher levels of superoxide (O2 x ) anions, and consume H2O2 and O2 at a higher rate than BCs. We also find that the elevated levels of these ROS elements in the LPs are associated with a higher content of mitochondria and higher levels of all 3 superoxide dismutases (SOD-1, 2 and 3). LPs are also highly resistant to glutathione depletion and express higher levels of both non-canonical non-glutathione hydroperoxidases and multiple enzymes that control ROS-induced nucleotide damage - thus providing a likely explanation for the differential ability of LPs to survive following GSH depletion. Interestingly, we found the mitochondrial glutathione peroxidase (GPX)-2 to be expressed almost exclusively and at high levels in the BCs. Moreover, depletion of this enzyme following transduction of BCs with a specific shRNA lentivirus, resulted in selective loss of basal progenitor activity. LPs also displayed greater resistance to acute oxidative insults (H2O2 and X-radiation) and an increased accrual of oxidative damage-induced (genomic 8-oxo-dGTP) mutations. Taken together, our findings reveal a major difference in the molecular machinery that controls ROS levels in normal human mammary bipotent and luminal progenitors. These correlate with an ability of the LPs to maintain and tolerate elevated levels of ROS accompanied by, and possibly a major cause of, a continuous acquisition of unrepaired ROS-induced DNA damage, resulting in a previously unanticipated mechanism of promoting their oncogenic transformation.  Endosomal Hydrogen Peroxide Production Leads to Localized Cysteine Sulfenic Acid Formation on Proteins during Lysophosphatidic Acid-Mediated Cell Signaling Chananat Klomsiri 1 , LeAnn C. Rogers 1 , Laura Soito 1 , Kimberly J. Nelson 1 , Anita K. McCauley 2 , S. Bruce King 3 , Leslie B. Poole 1 , and Larry W. Daniel 1 1 Department of Biochemistry, Wake Forest School of Medicine, United States, 2 Department of Biology, Wake Forest University, United States, 3 Department of Chemistry, Wake Forest University, United States Lysophosphatidic acid (LPA) is a growth factor that stimulates proliferation and survival in many cells including prostate and ovarian cancer-derived cell lines. We have previously shown that LPA stimulates ROS release in SKOV3 ovarian cancer cells and PC3 prostate cancer cells and that the addition of ROS modulators blocks LPA-dependent signaling. in an effort to clarify molecular changes in cells associated with localized production of signaling-relevant H2O2, we describe herein the use of a fluorescent oxidation-sensing probe, DCP-Rho1, to visualize sulfenic acids as they are formed in ovarian and prostate cancer- derived cells. We demonstrate that LPA signaling increases production of hydrogen peroxide, and leads to colocalization of internalized LPA receptors with an early endosome marker, consistent with the LPA-induced formation of receptor-bearing endosomes associated with activated NADPH oxidase (Nox) components (i.e., redoxosomes). for the first time we are able to show that protein cysteine oxidation is also increased around these LPAR-containing redoxosomes. by targeting the first intermediary product, sulfenic acid, for labeling by our fluorescent probe, our approach allows for a degree of temporal resolution as nascent sulfenic acids are detected whereas subsequently formed disulfide-bonded proteins, which will also have had time to diffuse away from the cellular site of oxidation, are not. Using a biotinylated version of our compounds we show that the protein 6 6)5%0  doi: 10.1016/j.freeradbiomed.2013.10.430 doi: 10.1016/j.freeradbiomed.2013.10.432 doi: 10.1016/j.freeradbiomed.2013.10.431