The fluorescence was read on a Wallac Victor 2 I420 Multilabel Counter at excitation of 485 nm and emission of 535 nm and protein normalized using Brad ford Protein Assay. Results were expressed as percentage increase compared to control and significant differences calculated using a two sample t test assuming equal vari A ances. Modulation of growth inhibition Cells were inoculated onto 96 well plates and preincubated with DFX, NAC or wortmannin prior to addition of ada phostin for a further 96 h incubation. Growth inhibition was assessed by alamarBlue, fluorescence was read on a Tecan Ultra plate reader . and results ana lyzed using the average percent treated/control, with significant differences calculated using a paired two sample t test.

Immunofluorescence Cells were plated in Lab Tek chamber slides and treated 4 6 hours with 1 uM adaphostin, or pre treated 30 minutes with 500 nM wortmannin, followed by 4 hour incubation with 1 uM adaphostin where indicated. Cells were fixed using cold methanol. permeabilized with 0. 1% Triton X 100. blocked in 20% goat serum. incubated with Nrf2 antibody overnight. labeled using FITC conju gated secondary antibody. and nuclei were counter stained with DAPI. Prolong Anti Fade was used to mount coverslip overnight. Samples were visualized using a Leitz Laborlux D fluores cence microscope and images were captured by Leica DFC420 camera and analyzed in Adobe Photoshop Ele ments 2. 0. Results Although hematopoietic malignancies have been the major target of pre clinical studies with adaphostin, NCI H522, a solid tumor derived, non small lung cancer cell line, was also very sensitive to adaphostin in the NCI 60 human tumor cell line screen.

From four independent experiments in the NCI 60 screen, the 50% growth inhibitory concentration for the 6 leuke Brefeldin_A mia cell lines ranged from 40 nM 630 nM, and the GI50 for NCI H522 was 79 nM, which was 10 fold more sensi tive than the average response for the whole cell line panel. Transcriptional profiling of NCI H522 in response to 1 uM adaphostin showed one of the most highly upregulated genes to be HMOX1 fold increase after 24 h which encodes for an enzyme that protects against oxidative stress. This increase in HMOX1 expression was confirmed using Q RT/PCR which also corroborated the lack of significant change in expression of the NRF2 gene. Moreover, a small but significant increase in the Nrf2 transcriptional target gene, NAD H dehydrogenase, quinone 1 NQO1 was observed although there was no change in another Nrf2 target, the catalytic subunit of glutamate cysteine ligase GCLC. A significant increase in ROS production was observed as early as 2 h after adaphostin treatment which is confirmation of the presence of drug induced oxidative stress.