Overexpression of Apoptosis-Inducing Factor (AIF) and Pro-Survival Function in AML.

Apoptosis inducing factor (AIF) is a bifunctional flavoprotein with NADH oxidase activity with a vital function in bioenergetics and redox metabolism while also inducing apoptosis in cancer cells. The gene is localized on chromosome human X (q25–26). AIF is synthesized in the cytosol as a 67KDa precursor molecule and then imported into the intermembrane space of mitochondria. In response to pro-apoptotic stimuli, the change of mitochondrial membrane potential (MMP) results in release of mature AIF (57KDa) to the cytoplasm and translocation into the nuclei where AIF can induce caspase-independent large scale DNA fragmentation (~ 50 Kbp) and chromatin condensation. The mechanism of induction of cell death by AIF remains poorly understood. We have previously reported that AIF induced cell death is both caspase-dependent and independent and related to mitochondrial membrane depolarization in leukemic cells treated with chemotherapeutic agents. In this study, we investigate

1. the expression level and function of AIF and its relationship to Bcl-2 and Bcl-2 family members in leukemia cells; and

2. the expression levels of AIF in AML patient’s samples.

To study the function of AIF, we introduced AIF si-RNA into K562 CML cell line using lentiviral vectors and established permanent AIF knockdown cell lines. Western blot analysis showed that the expression levels of XIAP, MCL-1, Bcl-xl and suvivin were increased while the AIF expression level was reduced. Cell growth was diminished two-fold (siAIF-clone 2, cell doubling time ~52-hr) to four-fold (siAIF-clone 1, cell doubling time ~99-hr) in AIF knockdown cells compared to the parental cells (24-hr). This suggests that AIF is supporting cell growth.

Next, AIF knockdown and scrambled siRNA control cells were treated with Ara-C (10μM) or STI571 (Gleeevec) (2 μM). The frequency of apoptotic cells was determined by flow cytometry (table).

AIF-siRNA in K562 cells showed increased sensitivity to Ara-C and STI-571 (Gleevec). The results support again a pro-survival function of AIF in K562 cells.

To examine the expression levels of AIF in primary AML patients, 148 samples (newly diagnosed n=32, relapsed n=27, refractory n=18, CR n=5, and normal-donors n=31) were analyzed by western blot and 40 samples of newly diagnosed AML were analyzed by RT-PCR. Results show that in comparison with normal CD34+ cells, AIF mRNA levels in newly diagnosed AML patients were significantly higher (95.0 copies AIF per hundred copies ABL1 in AML, 57.4 copies AIF per hundred copies in NBM CD34+, p = 0.02). The AIF/actin protein ratios in AML (0.43±0.05) was increased compared with normal BM-CD34⁺ (0.12±0.02) (p=0.01). Comparison of AIF expression levels in normal CD34+ cells vs. AML with good (0.5±0.05), bad (0.34±0.05), and intermediate (0.39±0.06) cytogenetics respectively, were significantly different (p<0.002). The transcription levels of AIF also showed significant differences (p<0.04) in patients with different cytogenetics abnormalities.

Taken together, we conclude that

1. AIF transcript and protein levels are significantly higher in AML compared to normal CD34 cells.

2. AIF in K562 cells functions as pro-survival factor for drug sensitivity.

These results question the previously assumed predominant pro-apoptotic function of AIF.