Arsenic trioxide (As 2 O 3 ) is highly efficacious in acute promyelocytic leukemia (APL). Aquaglyceroporin 9 (AQP9) is a transmembrane protein that may be involved in arsenic uptake. In 10 of 11 myeloid and lymphoid leukemia lines, quantitative polymerase chain reaction (Q-PCR) and Western blotting showed that AQP9 expression correlated positively with As 2 O 3 -induced cytotoxicity. As a proof-of-principle, transfection of EGFPtagged AQP9 to the hepatoma line Hep3B, not expressing AQP9 and As 2 O 3 insensitive, led to membrane AQP9 expression and increased As 2 O 3 -induced cytotoxicity. Similarly, the chronic myeloid leukemia line K562 expressed low levels of AQP9 and was As 2 O 3 insensitive. The K562 EGFP-AQP9 transfectant accumulated significantly higher levels of intracellular arsenic than control K562 EGFP when incubated with As 2 O 3 , resulting in significantly increased As 2 O 3 -induced cytotoxicity. Pretreatment of the myeloid leukemia line HL-60 with all-trans retinoic acid (ATRA) up-regulated AQP9, leading to a significantly increased arsenic uptake and As 2 O 3 -induced cytotoxicity on incubation with As 2 O 3 , which might explain the synergism between ATRA and As 2 O 3 . Therefore, AQP9 controlled arsenic transport and might determine As 2 O 3 sensitivity. Q-PCR showed that primary APL cells expressed AQP9 significantly (2-3 logs) higher than other acute myeloid leukemias (AMLs), which might explain their exquisite As 2 O 3 sensitivity. However, APL and AML with maturation expressed comparable AQP9 levels, suggesting that AQP9 expression was related to granulocytic maturation. IntroductionArsenic trioxide (As 2 O 3 ) is a standard treatment for acute promyelocytic leukemia (APL) [1][2][3] and is showing promise in multiple myeloma and other hematologic malignancies. 4,5 The intracellular actions of As 2 O 3 include interaction with proteins possessing high cysteine and histidine contents, 6 generation of superoxides and reactive oxygen species, 7,8 disruption of the mitochondrial transmembrane potential with release of cytochrome c and caspases activation, and blockade of cell cycle at the G 1 /S and G 2 /M phases. 9 As 2 O 3 is detoxified by the glutathione redox system, comprising thiol-rich glutathione (GSH) carrier proteins, glutathione peroxidase, and glutathione S-transferase (GST-). 9-12 GSH forms a transient As(GSH) 3 complex with As 3ϩ , a process catalyzed by As(GSH) 3 binds the multi-drug-resistant protein 1 (MRP1), inducing a conformational change. 13,14 This in turn leads to an ATP-driven transmembrane transport of As(GSH) 3 to the extracellular space. 13,14 Free reduced GSH carrier protein is then replenished by glutathione peroxidase.The arsenic GSH redox system might be one of the mechanisms that account for the varying sensitivities of different cell types to As 2 O 3 . The sensitivities to As 2 O 3 had been shown to be inversely proportional to the cellular GSH content. 15 Furthermore, cell lines treated with ascorbic acid and buthionine sulfoxide (BSO), which decreased intra...