These data are consistent with the 10 uM adaphostin induced heme oxygenase 1 expression reported in glioblastoma cell lines, which did not appear until after 8 24 h. This adaphostin induced HMOX1 upregulation in NCI H522 cells and glioblastoma cell lines is in contrast to the response of hematologic cell lines where we have previ ously reported the major transcriptional response involved 10 fold induction of genes encoding for both heavy and light ferritin polypeptides. Moreover, even after treatment with 10 uM adaphostin, leukemia cell lines showed no increase in HMOX1 expression on the cDNA arrays after 6 h incubation 1. 24 0. 7, 1. 35 0. 39 and 1. 16 0. 28 respec tively compared to a 7. 4 and 30. 8 fold increase in HMOX1 expression in NCI H522 cells when measured on the same type of arrays following treatment with 1 and 4 uM adaphostin for 6 h.

Evidence that ROS are an important factor in determining sensitivity of NCI H522 to adaphostin was demonstrated by the ablation of ada phostin toxicity by the anti oxidant, N acetyl cysteine in a manner similar to that shown for the leukemia cell line Jurkat. However, in contrast to Jurkat, the iron chelating agent desferrioxamine did not attenu ate adaphostin toxicity in the NCI H522 cell line, and there was no measurable increase in either ferritin gene FTH 1. 09 0. 15 . FTL 1. 02 0. 24 indicating that release of excess free iron is not involved in the NCI H522 response to adaphostin. Thus, these data substantiate the difference between response of a solid tumor and that which we have shown in leukemia cell lines.

As the induction of HMOX1 appears to be unique to the response of solid tumors, we investigated the role of its putative regulatory transcription factor, Nrf2, in adaphostin treated Drug_discovery NCI H522 cells. Nrf2 protein, when activated is rapidly translocated into the nucleus, and in adaphostin treated NCI H522 cells, Nrf2 was rapidly induced in the nuclear fraction within 2 6 h, although there was no detectable Nrf2 expression in the cytosolic fraction over this time. Furthermore, translo cation of Nrf2 from the cytoplasm into the nucleus by adaphostin can be visualized using immunohistochemis try where nuclear localization of Nrf2 after 4 h and 6 h incubation of NCI H522 cells with 1 uM ada phostin was apparent compared to the more diffuse Nrf2 distribution in untreated cells. Wortmannin, a PI3 kinase inhibitor, has been shown to inhibit Nrf2 translocation into the nucleus and was successfully used as a tool to inhibit adaphostin , induced, nuclear translocation of Nrf2. Pre treatment of NCI H522 cells with 500 nM wortmannin was effective at inhibiting adaphostin induced nuclear localization of Nrf2, although wortman nin alone had no effect.