data from 240 diffuse large B cell lymphomas (DLBCL) to test the extent to which redox-related genes predict outcome following chemotherapy. the data came from microarray analyses of frozen specimens. Patients with the worst prognosis had decreased antioxidant defense enzyme expression and increased thioredoxin system function. Translating these findings to the clinic is hindered by the impracticality of using microarrays in a clinical lab and difficulty obtaining frozen specimens. Here, we measured gene expression levels using paraffin-embedded, formalin-fixed tissues from standard pathology practice. the study used a quantitative nuclease protection-based assay and a custom redox gene array to test for differences in gene expression between non-neoplastic lymph nodes, follicular lymphomas (FL) and DLBCL. the results showed significantly lower expression of catalase, glutathione peroxidase 1, glutathione peroxidase 4, microsomal glutathione S-transferase 1 and thioredoxin interacting protein in both types of lymphoma, as compared to non-neoplastic lymph tissue. as expected for the two lymphoma subtypes, B cell markers were expressed at a significantly higher level compared to the levels found in the non- neoplastic lymph nodes. Gene expression measurements for the redox-related genes were combined to generate a redox score. the mean value obtained for the non-neoplastic tissue was significantly higher than the mean value obtained from FL and DLBCL. the redox scores for these two lymphoma subtypes were not significantly different. Our results are consistent with the concept of a more oxidized redox environment in neoplastic cells versus their normal counterparts. Furthermore, the study demonstrates a practical approach to assessing the cellular/tissue redox environment using readily-available patient specimens. This approach could be applied to investigate the redox environment as a biomarker in different disease states.  Catalytically Inactive Extracellular Superoxide Dismutase Reduces Pancreatic Tumor Cell Invasion Adam J. Case 1 , Brianne R. O'Leary 1 , Christopher S. Stipp 1 , Frederick E. Domann 1 , and James J. Mezhir 1 1 University of Iowa, United States Introduction: Extracellular superoxide dismutase (EcSOD) is a superoxide-scavenging enzyme that has been shown to be down regulated in various cancer cell types, and overexpression of EcSOD reduces tumor cell invasion in animal models of metastasis. Due to (F62'ｶV XQLTXH SURSHUW\ of being located extracellularly as well as binding heparan sulfate, we hypothesized that EcSOD may possess anti-metastasis properties independent of its superoxide-dismuting catalytic activity. We tested this hypothesis in a model of pancreatic cancer metastasis, where we have previous shown EcSOD expression is reduced compared to normal pancreatic cells. Methods: Using site directed mutagenesis, we created an enzymatically inactive mutant of EcSOD that targeted the residues responsible for channeling superoxide to the active site of the protein (i.e. N180 and R186). Wild type EcSOD (WT), mutant EcSOD (MUT), and vector control (VC) were stably overexpressed in a pancreatic cancer cell line (BxPC3) and the cells were characterized for mRNA, protein, and activity (EPR) of EcSOD. to quantify tumor cell invasive capacity, Matrigel invasion assays and 3D collagen invasion assays were performed. Results: WT and MUT cell lines demonstrated robust expression of EcSOD mRNA and intracellular and extracellular EcSOD protein compared to VC. Media from WT cells showed robust EcSOD activity by EPR, where MUT and VC cells did not. WT and MUT cell lines demonstrated a greater than 40% decrease in invasive capacity on Matrigel compared to VC (p=0.008). Conditioned media from cell lines used in a Matrigel assay on parent BxPC3 cells produced similar results. in 3D collagen invasion assays, expression of either WT or MUT EcSOD suppressed invasion into the collagen matrix compared to VC (p<0.001). Conclusions: Overexpression of WT and a catalytically inactive EcSOD reduced pancreatic tumor cell invasion in two assays of invasive capacity compared to VC. These data suggest that EcSOD has anti-cancer or metastasis properties independent of its catalytic activity. Studies are underway investigating a possible mechanistic link between the binding of EcSOD to heparan sulfate of the extracellular matrix, and how this may contribute to the metastatic potential of pancreatic cancer cells.  Biphasic Effects of NOS2-Derived NO on Breast Cancer Cell Bioenergetics Ching-Fang Chang 1 , Anne Diers 1 , and Neil Hogg 1 1 Department of Biophysics, Redox Biology Program, Medical College of Wisconsin, United States Nitric oxide (NO), a diatomic free radical signaling molecule, plays important roles in physiological and pathological processes, including carcinogenesis. Various studies have shown that NO can both promote and inhibit cancer progression, and its effects appear to depend on both the concentration and duration of NO exposure. Many cancers have altered energy metabolism to meet the needs for aberrant cell proliferation. Accumulating evidence shows that NO has a great impact on energy metabolism; for example, direct binding of NO to mitochondrial complex IV leads to an inhibition of mitochondrial respiration, and NO-dependent mitochondria biogenesis has also been detected in primary leukemia cells. However, how endogenous NO levels affect cellular energy metabolism in cancer has not yet been well characterized. in this study, human breast adenocarcinoma MCF7 cells were transduced with increasing amounts of lentivirus carrying human inducible nitric oxide synthase (NOS2) gene, and both cellular NOS2 protein and medium nitrite were measured to validate the generation of cells with different levels of NOS2 expression. to evaluate the effects of NO on bioenergetics, we measured oxygen consumption rate (OCR) and extracellular acidification rate (ECAR, a surrogate marker of glycolysis) using the Seahorse extracellular flux analyzer. a biphasic effect of NO on cellular respiration was observed in the NOS2 transduced cells. Increases of basal OCR, ATP-linked OCR and reserve capacity were measured in the cells with increasing levels of NOS2 expression and nitrite production rates of 0.9±0.3 to 2.5±0.1 nmol/mg/hr. However, bioenergetic parameters were decreased in the cells with highest NOS2 expression (6.6±0.9 nmol/mg /h nitrite production). Furthermore, expression of mitochondrial proteins was elevated with increasing levels of NOS2 expression, but decreased at the highest level. the results suggest that the endogenous NO effect on bioenergetics is concentration-dependent, and low concentrations of NO may stimulate mitochondria biogenesis. the highest level of NOS2, which approaches rates of nitrite formation observed in activated inflammatory cells, suppresses mitochondrial function. Supression of mitochondrial function was also observed with the NO donor DetaNONOate at concentrations above 100 μM. 6 6)5%0  doi: 10.1016/j.freeradbiomed.2013.10.416 doi: 10.1016/j.freeradbiomed.2013.10.417