Of note, analysis of Keap1 expression in these datasets showed no significant

However our data reveal a tumor sup pressor role for Nrf2 since its down regulation contributes to cellular transformation and in vivo tumor growth. Microarray comparison studies support our experimental data, indicating that expression ABT-888 Veliparib of Nrf2 is down regulated in many tumors. Moreover, analysis of available survival datasets obtained from GEO and TCGA databases shows that increased Nrf2 expression correlates with better survival in patients with melanoma, kidney and prostate cancers, further supporting our in vivo findings where restoration of Nrf2 expression in transformed MSC improved survival. Conclusions Overall our results indicate that defects in the cellular antioxidant capacity contribute to ROS accumulation during transformation, and that oncogene induced Nrf2 repression is an adaptive response for certain cancer cells that favors in vivo tumor expansion and poorer sur vival.<br><br> We also show that rescue of Nrf2 function in fully transformed cells is an effective strategy to tackle in vivo tumor growth, as Nrf2 expression sensitizes transformed cells to apoptosis and impairs the angiogenic response through destabilization of HIF 1. Methods Cell culture and generation of stable cell lines Culture conditions, retrovirus production and gener ation of cell AEB071 ic50 lines were previously described. Briefly, primary human MSC previously isolated from the bone marrow of a healthy donor according to institu tional guidelines were serially transduced with retrovi ruses encoding hTERT, E6 and E7 from HPV 16, ST antigen from SV40, and H RasV12.<br><br> For the generation of tMSC over expressing Nrf2, we amplified the Nrf2 gene from human cDNA using the following primers. Nrf2 was later cloned into pWZL hygro and used to infect tMSC AG-1478 Tyrphostin AG-1478 where H RasV12 had been previously introduced with pWZL blast. Detection of intracellular ROS ROS levels were quantified by staining the cells with MitoSOX Red and CM H2DCFDA dyes. After 30 minutes incubation with the dyes at 5 uM final concentration, cells were collected and analyzed by flow cytometry using either a FACSCali bur instrument or a CyAN flow cytometer. Data were analyzed using either CellQuest V or Summit software. Cell incubation with MitoSOX was performed in serum depleted media. Transformation assays Soft agarose colony formation by anchorage independent growth and tumor xenografts were previously described.<br><br> The animal experiments were conducted in accord ance with institutional guidelines under the approved pro tocols. For the in vivo tumor growth experiments, Kaplan Meier survival plots were generated, and from the survival data a log rank test was used to demonstrate significant differences between groups. Antibodies and reagents The following antibodies were used for immunoblot tingrabbit polyclonal C 20, mouse monoclonal clone 1 F3, and rabbit monoclonal EP1808Y for Nrf2. Actin was from CalbiochemMerckMillipore. NQO1 was from Novus Biologicals. G6PD was from Bethyl. HIF 1 was from BD Biosciences. Cleaved PARP, total AKT, phosphorylated AKT. total ERK12, phosphorylated ERK12. Cul3, Keap1, HSP90 and Lamin AC antibodies were all from Cell Signaling Technology.