Thus, we have investigated the mechanism behind HMOX1 induction in the adaphostin sensitive lung tumor cell line NCI H522, and demon strated an enhancement of adaphostin toxicity following inhibition of Nrf2 nuclear translocation with the PI3K inhibitor wortmannin. Methods Drugs and Cell Culture Adaphostin and wortmannin were obtained from the repository of the National Cancer Institutes Developmental Therapeutics Program. Desferrioxamine and N acetyl cysteine were purchased from Sigma. NCI H522, and the leukemia cell lines, were obtained from the NCI 60 Human Tumor Cell Line Screen. Transcriptional Profiling Microarray Technology Human OperonV2, 20K arrays, were utilized according to pub lished protocols.

Using compet itive hybridization of treated versus untreated samples chemically coupled to a Cy 3 or Cy 5 fluorescently labeled dye and fluorescence was read on a GenePix 4100A microarray scanner purchased from Axon Instruments. Data was ana lyzed using the Axon GenePix Pro 4. 1 software and data and image files were then uploaded to the National Can cer Institute/Cancer Center for Research Microarray Center mAdB Gateway for analysis and comparison of multiple arrays. Real Time RT PCR Five hundred nanograms of total RNA for each sample was reverse transcribed using the GeneAmp PCR System 9700 and TaqMan Reverse Transcription Reagents kit. Quantitative real time PCR reactions were conducted and measured using the ABI Prism 7700 Sequence Detection System and TaqMan chemistries using published prim ers. Samples were tested in triplicate wells for the genes of interest and for the endogenous control, 18 S.

Data was analyzed using the comparative Ct method as described in the Perkin Elmer User Bulletin 2 and expressed as a fold induction of the gene in the adaphostin treated sam ples compared to the untreated control samples, and sig nificant differences were calculated using a paired two sample t test. Western Blot Whole cell and nuclear Entinostat extracts were made for protein analysis by western blot. Nuclear extracts were prepared from cells in 100 mm dishes that were lysed using a hypo tonic buffer. The nuclei were pelleted at 13,000 g for 15 minutes, and then after the supernatant was aspirated, the nuclei were lysed using 1x RIPA lysis buffer containing protease inhibitors. Protein was quantitated using Bradford Protein Assay, and approximately 50 ug of each sample was resolved by SDS PAGE on 10% Tris glycine gels and probed with anti Nrf2 and anti HMOX1 antibodies. Proteins were visualized using chemiluminescence and imaged using a Kodak X OMAT 2000A Processor. Measurement of adaphostin induced ROS Intracellular ROS were measured after 2 and 4 hours exposure to 1 uM adaphostin using 2,7 dichlorofluores cein diacetate.