Intracellular thiol oxidation is linked with loss of Δψm and disease progression in acute promyelocytic leukaemia

Acute promyelocytic leukaemia (APL)is a type of myeloid malignancy defined by the chromosomal translocationt(15;17) and subsequent expression of the PML-RARα fusion protein. Long termremission in APL is achieved through a combination of high dose all-trans retinoic acid (ATRA) and arsenic trioxide(ATO), the latter of which has potential to induce mitochondrial derivedoxidative stress and initiate the intrinsic apoptosis pathway. ATO isparticularly effective in the treatment of APL when compared with acute myeloidleukaemia (AML), suggesting that the mitochondrial membrane potential (Δψm) ofAPL cells is compromised. Here we show that loss of Δψm is associated withintracellular thiol oxidation and disease progression in APL.

The mitochondrialprobe 3,3'-diethyloxacrbocyanine iodide (DiOC₂(3)) was used toevaluate MRP8-PML/RARA transgenic mice Δψm in bone marrow mononuclear cells(BMMC) at three disease stages: 1) pre-APL, 2) APL and 3) ATRA treated APL. Ourdata show that MRP8-PML/RARA BMMC cells during the APL stage accumulatesignificantly less DiOC₂(3) compared withpre-APL cells (0.05±0.02 vs 0.24±0.05,p<0.05; data are normalised FL1/FL3 MFI ratio). The DiOC₂(3) accumulation in BMMC from ATRA treated APL micewas equivalent to that of pre-APL cells (p>0.05). This finding indicates aloss of Δψm in APL. This may result in free radical leakage from themitochondria, which in turn could oxidise intracellular thiols.

To evaluateintracellular thiol oxidation, a previously optimised flow cytometric assaythat incorporates the thiol reactive probe fluorescein-5 maleimide (F5M) wasused. Our data show a significant decrease in F5M MFI for MRP8-PML/RARA BMMCcells during the APL stage, compared with pre-APL cells (219.01±47.67 vs 672.66±131.04, p<0.05; data are F5M MFIrelative to unstained controls). This finding signifies that there is morethiol oxidation at the APL stage. ATRA treated APL mice showed an equivalentF5M MFI to that of pre-APL cells. Confocal microscopy confirmed that the F5Msignal was intracellular. This observation shows that there is an increase inintracellular thiol oxidation in APL, which may be tracked during disease progressionand treatment course. This finding may explain why APL cells are more sensitiveto pro-oxidant treatments i.e. ATO.

To understand theseobservations further, the APL cell line (NB4) was subjected to glucose oxidase(GOX) mediated oxidative stress in cell culture over a 4 hour time-course. F5MMFI signal was compared with the THP1 and Kasumi-1 AML cell lines subjected tothe same treatment. All cell lines showed similar significant increases in F5MMFI after 1 hour GOX treatment relative to control (p<0.05), indicating anincrease in 'reduced' cellular thiol i.e. reductive stress. However, after 4hours NB4 cells showed a significant decrease in F5M MFI relative to control(p<0.05), indicating an increase in 'oxidised' cellular thiol i.e. oxidativestress. In contrast, THP1 cells showed no difference in MFI after 4 hours(p>0.05), whereas F5M MFI Kasumi-1 remained significantly increased after 4hours (p<0.05) relative to control. This finding indicates differences inthe sensitivity to oxidative stress between APL and AML cells.

Taken together,this study shows that APL cells are more sensitive to oxidative stress comparedwith AML cells. Our F5M flow cytometric assay illustrates that an increase incellular thiol oxidation is linked to disease progression in APL, with loss ofΔψm associated with this process. Our F5M flow cytometric assay may thereforehave clinical utility in monitoring treatment efficacy in APL.