AML3 cells were treated with increasing levels of obatoclax for various times and phosphatydil serine externalization was monitored by flow cytometry by staining with Annexin V APC. Cell volume was determined from the common size measured by the ViCell XR Dabrafenib ic50 analyzer. D, cells were treated with obatoclax for 1 h and washed twice in serum containing media. Cells were then cultured under standard conditions for 48 h, and cell viability and apoptosis were quantitated as described in Materials and Techniques. Obatoclax Induces Apoptosis in AML for 15 minutes followed by a cold centrifugation step and assessed the degrees of cytochrome c inside the matching supernatant and pellet. As shown in Fig. 2A, obatoclax encourages the release of cytochrome c from isolated mitochondria, indicating that, like ABT 737, this agent induces apoptosis through activation of the intrinsic apoptotic pathway. Similar results were obtained with U937 cell mitochondria. We then investigated if obatoclax induced activation of the intrinsic pathway involved the release of haemopoiesis Bak from the strong anti-apoptotic protein Mcl 1, a protein that we have previously noted mediates resistance to ABT 737. Treatment of OCI AML3 cells with obatoclax led to a quick and complete release of Bak from Mcl 1, and this was accompanied by increased expression of a conformationally altered Bak in a complex with Bax. Also, it was observed that obatoclax induced apoptosis was decreased, however not entirely abolished, in Bak cells, suggesting that Bak contributes to some degree to cytotoxicity induced by this agent. No more protection from cell death was seen in Bax/Bak MEFs. Eventually, we sought to find out if, just like ABT 737 induced apoptosis, obatoclax induced apoptosis proceeded in a Bim independent manner in leukemia cells. We discovered that Bim was efficiently Cediranib ic50 released from Bcl 2 and Mcl 1 in OCI AML3 cells treated with obatoclax, and most interestingly, cells devoid of Bim appearance were less susceptible to apoptosis induction by this BH3 mimetic. These results suggest that cells treated with obatoclax free Bim might cooperate with Bak to advertise the service of the intrinsic apoptotic pathway. Certainly, partial knockdown of both Bak and Bim by siRNA in HL 60 cells partially protected cells from apoptosis, although cells electroporated with Bak or Bim siRNA alone were minimally protected. Even though we were unable to achieve complete knockdown in notoriously hard to transfect leukemic cells, these data suggest other targets contributing to proapoptotic ramifications of this agent. In comparison, cell cycle analysis of wild type, Bax deficient, Bakdeficient, Bax/Bak deficient, or Bim deficient MEFs showed that obatoclax triggered an S G2 cell cycle block irrespective of the status of these proteins.