Methylated arsenic metabolites bind to PML protein but do not induce cellular differentiation and PML-RARα protein degradation.

Arsenic trioxide (As2O3) is one of the most effective therapeutic agents used for patients with acute promyelocytic leukemia (APL). The probable explanation for As2O3-induced cell differentiation is the direct targeting of PML-RARα oncoprotein by As2O3, which results in initiation of PML-RARa degradation. However, after injection, As2O3 is rapidly methylated in body to different intermediate metabolites such as trivalent monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII), therefore, it remains unknown that which arsenic specie is actually responsible for the therapeutic effects against APL. Here we have shown the role of As2O3 (as iAsIII) and its intermediate metabolites (i.e., MMAIII/DMAIII) in NB4 cells. Inorganic iAsIII predominantly showed induction of cell differentiation, while MMAIII and DMAIII specifically showed to induce mitochondria and endoplasmic reticulum-mediated apoptosis, respectively. On the other hand, in contrast to iAsIII, MMAIII showed stronger binding affinity for ring domain of PML recombinant protein, however, could not induce PML protein SUMOylation and ubiquitin/proteasome degradation. In summary, our results suggest that the binding of arsenicals to the ring domain of PML proteins is not associated with the degradation of PML-RARa fusion protein. Moreover, methylated arsenicals can efficiently lead to cellular apoptosis, however, they are incapable of inducing NB4 cell differentiation.


Preparation of monomethylarsonous acid (MMA III ) and Dimethylarsinous Acid (DMA III )
MMA III and DMA III were prepared by reducing monomethylarsonic acid (MMA V ) and dimethylarsinic acid (DMA V ) respectively with 5 molar equivalents of L-cysteine in distilled water at 90°C for 1 h. The trivalent forms were confirmed by comparison to their iodide forms using gel filtration HPLC − ICP MS.

Isolation of pure mitochondria from rat liver
All experiments on animal were carried out according to the "Principles of Laboratory Animal Care" (NIH version, revised 1996) and the Guidelines of the Animal Investigation Committee, School of Medicine, Zhejiang University, China (Permit No: ZJU2010101033).
Six week old male SD rats were purchased from National Rodent Laboratory Animal, Resource, Shanghai, China. The rats were housed in a humidity-controlled room, maintained at 22~25°C with a 12 h light dark cycle.
The animals were fed commercial diet and tap water was provided. Following a one-week acclimatization period, rats at 7 weeks of age (body weight, 180-220 g) were used for experiments. Whole liver perfusion was performed following the method developed in our laboratory. Briefly, rats under sodium pentobarbital anesthesia were dissected to expose the heart, and then 0.2 mL of heparin was injected into the left ventricle. The remaining blood was perfused through the portal vein by a roller pump with phosphate buffer saline (PBS) at a flow rate of 6 mL/min. Blood-free liver was minced in ice-cold homogenization buffer A (230 mM Mannitol, 70 mM sucrose, 10 mM Tris-HCl, EDTA-2Na and 0. 5% bovine serum albumin (BSA), pH 7.4) by using a Dounce homogenizer to make 20% (w/v) liver homogenates. The homogenates were then kept on ice for 5 min to remove unbroken cells and connective tissues. After removing the unbroken cells, the supernatant was centrifuged twice at 700 g for 10 min at 4°C to obtain the cellular nuclear fraction and supernatant fraction. Mitochondria were isolated by subjecting the supernatant to centrifugation at 12, 000 g for 10 min at 4°C to obtain pellet. The pellet was washed twice and re-suspended in the isolation buffer B (230 mM Mannitol, 70 mM sucrose, 10 mM Tris-HCl, 1 mM EDTA• 2Na) with a protein concentration of 0.5 mg protein/mL. Isolated mitochondrial activity was determined by cytochrome c oxidase (CCO) assay kit.

Preparation of recombinant PML-R
The cDNAs encoding residue 49-104 of PML Zinc finger reign ring domain (R) was amplified according to the published paper, and inserted into pET32a using restriction sites Kpn I and Xho I to construct pET32a PML-R. Moreover, prokaryotic pET32M PML-R was transformed into E.coli BL21 (DE3) cells. Cells were grown in M9 minimal media and induced by 100 μM IPTG and 20 μM ZnCl 2 . The Trx tag of Trx-PML-R was removed by Enterokinase (EK) cleavage. PML-R was further purified by Superdex-75 column chromatography and confirmed by SDS-PAGE.

Removing unbound arsenic compounds in supernatants of cells
The supernatants (500 μL) were dialyzed two times for 6 h each in a Slide-A-Lyzer Dialysis Cassette against 500 mL of 50 mM ammonium acetate buffer (pH 7.4) at 4°C, and the arsenic concentration in the supernatants was determined by ICP MS after wet-digestion with a mixture of concentrated nitric acid and 30% H 2 O 2 (v/v = 1:1) at 150°C for 2 days.

Incubation of recombinant PML-R with the three arsenic compounds
Recombinant PML-R protein was diluted in 10mM Tris-HNO 3 buffer (pH7.4, dissolved oxygen was purged by bubbling with99.999% nitrogen gas), and incubated with 3 μM of iAs III , MMA III and DMA III for 30 min at 37°C. Later 20 μL of each sample was subjected to HPLC-ICP MS analysis on GS-220 gel filtration columns with 50 mM ammonium acetate (pH7.0) at the flow rate of 0.8 mL/min. Arsenic (As) was monitored at m/z75.