The Role of Apoptotic Inducing Factor (AIF) in Acute Myeloid Leukemia (AML).

Abstract

Apoptosis is a “physiological cell suicide program” and plays an integral role in a variety of biological events. Numerous genes are involved: Apoptosis inducing factor (AIF) is a newly discovered apoptogenic flavoprotein with NADH oxidase activity that is located in the intermembrane space of mitochondria. The mechanism of induction of cell death by AIF remains unclear. Some data suggest that AIF may play a central role in the regulation of caspase-independent cell death and participate in multiple cell death paradigms. When cells are entering apoptosis, AIF will translocate from the mitochondria to the nuclei causing DNA fragmentation and chromatin condensation. AIF also induces cell death in normal lymphocytes and leukemia cells, but the data is very limited. In this study, we investigate 1) whether AIF inducing cell death is caspase-independent and related to MMP, 2) whether AIF expression levels can be prognostic in primary AML. To determine AIF nuclear translocation in cells undergoing apoptosis is caspase-dependent or caspase-independent, OCI/AML3 cells were treated with several chemotherapeutic agents (Paclitaxol, Vincristine, Ara-C, and Doxorubicin with/without pan-caspase inhibitor IDN-1529). Cells were then fixed, immuno-stained, and analyzed. Results demonstrate that AIF translocated from mitochondria to the nucleus when cell undergo apoptosis, and that it is largely caspase-independent than caspase-dependent. To examine how the mitochondrial membrane potential (MMP) is involved in AIF's nuclear translocation, OCI/AML3 cells was stained with CMXRos and MitoTrack-green after drug treatment with/without caspase inhibitors and then analyzed by flow cytometry. 90% of untreated cells retained normal MMP. Vincristine and paclitaxol treated cells had a more complete depolarization of mitochondria (75.6% ± 4.3% and 79.3% ± 5.4%, respectively), even when caspase inhibitor was added (51.4% ± 3%; p=0.025; and 52.7% ± 5.1%; p=0.006, respectively). These results suggest that the higher ratio of AIF nuclear-translocation observed in cells undergoing apoptosis in Vincristine and paclitaxel treated cells is due to changes in MMP. To further test the function of AIF in AML, small interference RNA (siRNA) was employed to knockdown AIF gene expression in U937 cells and treated with the triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO). AIF siRNA was delivered by electroporation. Results suggest that nuclear translocation of AIF is prevented by silencing AIF expression. Down-regulation of AIF prior to CDDO exposure significantly reduced CDDO-induced growth inhibition (p<0.002). CDDO-induced apoptosis was decreased by approximately 50% in TUNEL assay. To test AIF expression in AML patients, we use immunobloting of bone marrow (BM) and peripheral blood (PB) cells. Twenty-five samples from AML patients were analyzed and the results demonstrated the correlation between blast count and AIF levels (p=0.04). Comparing PB from leukemia patients with normal PB, the p volume is 0.02. Taken together, these results suggest that 1) AIF is inducing cell death through a caspase-independent and caspase-dependent pathway related to MMP, 2) The difference of AIF expression levels in AML suggest a role in the regulation of apoptosis. It may be used as a prognosis marker.