Thus, BSO was suggested protein inhibitors to augment ATO induced cell death via mitochondrial injury charac terized by cytochrome c release, caspase 9 activation and MOMP reduction. BSO induces conformational change in BAX, but not in BAK Since the two proapoptotic BCL2 effector proteins, BAX and BAK, play central roles in oxidative stress mediated mitochondrial apoptosis, the effect of BSO addition on the conformational change of BAX and BAK in ATO Inhibitors,Modulators,Libraries treated cells was examined by immunoprecipita tion and immunoblotting. Immunoblotting analysis with anti whole BAX antibody demonstrated no significant difference in total BAX expression between ATO/BSO and ATO alone treatment. However, an antibody which defines conformationally changed BAX immunoprecipi tated much more BAX in ATO/BSO treated cells than ATO alone treated cells.

An anti whole BAX antibody immunoprecipitated a lower level of BAX from the supernatant fraction of ATO/ BSO treated cells. Therefore, BSO was suggested to trig ger conformational change of BAX in ATO/BSO treat ment. In addition, the conformational change of BAX in ATO/BSO treated cells was prevented by DTT as an antioxidant. Immunoprecipitation analysis using an antibody Inhibitors,Modulators,Libraries to whole BAK or conformationally changed BAK demon strated no presence of conformationally changed BAK in either Inhibitors,Modulators,Libraries ATO/BSO or ATO alone treatment. BSO induces phosphorylation of BIMEL and MCL1 in mitochondria A possibility was raised that the conformational change of BAX might be critical for BSO mediated mitochon drial injury. Therefore, we examined the expression and activation of a series of BCL2 family proteins, which affect the conformational change of BAX.

First, the expression of BIMEL, a proapoptotic protein in the BCL2 family, was analyzed by immunoblotting. Normal HL60 cells expressed a readily detectable level of Inhibitors,Modulators,Libraries the two major BIM isoforms, BIMEL and BIML whereas they expressed a low level of the smallest isoform, BIMS. BIMEL underwent an electrophoretic mobility shift following ATO/ BSO treatment Inhibitors,Modulators,Libraries whereas the smaller isoform, BIML did not. BSO addition caused a high level of S69 phosphorylated BIMEL. The enhanced BIMEL phosphorylation was abolished by NAC or DTT as antioxidants. In contrast, neither mobility shift nor phosphorylation of BIMEL was induced by ATO alone. There was no signifi cant difference in the expression of the other pro apoptotic proteins of the BCL2 family, BAD and BID between ATO/ BSO and ATO treatment.

Second, the effect of BSO addition on the expression and activation of MCL1, an anti apoptotic protein of the BCL2 family, was examined. BSO addition augmented the expression and phosphorylation of MCL1 at Ser159 and/or Thr163, whereas ATO alone did it only minimally. The BSO mediated augmentation of MCL1 expression especially and phosphorylation was abolished by antiox idants. Similar augmentation was seen in phosphoryl ation of BCLXL.