myelogenous leukemia (AML) [14–17]. Moreover, MMP-2/-9, appears to have prognostic impact in AML [16]. Considerable evidence has accumulated that in some CNS diseases MMP-2 and -9 are involved in the permeability of the BBB by disrupting the junction complexes such as occludin and claudin-5 [18–21]. We have thus hypothesized that MMP-2 and -9 secreted by leukemic cells may play critical roles in the BBB opening in CNS leukemia by disrupting TJ proteins. In order to investigate this hypothesis, we examined the relationship of MMP-2 and -9 secreted by leukemic cells with the TJ proteins and the BBB dysfunction in an in vitro model of BBB and in an animal model of CNS leukemia. We show that leukemic cell-derived MMP-2 and -9 disrupt the key TJ proteins ZO-1, claudin-5 and occludin and that this is correlated with the BBB opening. Down-regulation of MMP-2 and -9 secreted by leukemic cells reduces the disruption of these three proteins with decreased permeability of the BBB and also protect mice against CNS leukemia. These data implicate MMP-2 and -9-mediated down-regulation of TJ proteins as a significant mechanism in the breakdown of the BBB, which contributes to the metastatic dissemination of leukemic cells in CNS leukemia. Materials and Methods Ethics statement All animal work was done following an institutionally approved protocol in accordance with National Health Guides for the Care and Use of Laboratory Animals and the Law of Animal Experiments regulation of the University of Soochow (26/2008). All surgery was performed under chloral hydrate anesthesia, and all efforts were made to minimize suffering. Mice Four-week-old male BALB/c nu/nu mice were purchased from the Shanghai Laboratory Animals Center of the Chinese Academy of Science and maintained under specific pathogen-free condi- tions. All mice were pretreated as follows: splenectomized on day 0, 2.5 mg cytoxan intraperitoneal injection for three days, surgery after seven days and total body sublethal irradiation of 4.0 Gy at rate of 2 Gy/min per dose on day 10. Those mice that received splenectomy, cytoxan intraperitoneal injection, and were pretreat- ed with sublethal irradiation (SCI), were referred to as SCI-nu/nu mice. Cell culture, reagents and treatments Leukemic cell line SHI-1 was originally derived from the bone marrow mononuclear cells of a patient with acutemonocytic leukemia in relapse, and maintained as an in vitro cell line [22]. SHI-1, NB4 and U937 cells were available and carried in our laboratory. Cells were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM) (Gibco, USA) with a 10% fetal bovine serum (FBS) (Gibco, USA) at 37uC in a humidified atmosphere containing 5% CO2. Primary human BMVECs were purchased from the American Sciencell Research Laboratory and cultured in a complete serum of endothelial cell medium (Sciencell, USA) in tissue culture flasks pre-coated with human fibronectin (3 mg/cm, Gibco, USA). Endothelial cells achieved 70,80% confluence after 5 to 7 days. Following two passages digested with trypsin/EDTA solution, the cells were used to set experiments. The broad-spectrum MMP inhibitor GM6001 (Calbiochem, Germany) was dissolved in dimethyl sulfoxide (DMSO) as a stock solution and further supplemented in PBS immediately before administration or in IMDM before incubation with leukemic cells. Mechanism of Central Nervous System Leukemia For inhibition studies, cells were incubated with 20 mg/ml (the concentration that highly inhibited the activities of MMP-2 and -9 in leukemic cells but had no influence on cell viability) GM6001. Cell viability was analyzed by trypan staining, which indicated cell survival rates of .95%. Transfection of SHI-1 cells with siRNA MMP-2 and -9 genes of SHI-1 cells were knocked down by RNAi. A specific siRNA of MMP-2 was designed given the protocol suggested by Ries et al [23]. A specific siRNA of MMP-9 was designed by Genepharma (China). The sequences were as follows: sense, 59-GAC CUG AGA ACC AAU CUC ATT-39; antisense, 59-UGA GAU UGG UUC UCA GGU CTT-39. All oligonucleotides were synthesized by Genepharma. SiRNA transfection of SHI-1 was performed as previously described by us [24]. Zymography The activities of MMP-2 and -9 in the conditioned medium were determined by gelatin zymography as previously described [25]. Supernatants of different leukemia cell lines treated or untreated with GM6001, as well as the RNAi-treated cells for 24 h after transfection at the final density of 2.06105 cells/well suspended in serum-free medium (IMDM 100 ml) were seeded in 96-well plates. Supernatants were harvested after 24 h cultivation and 15 ml serum-free medium were collected and clarified by centrifugation to remove cells and debris. Samples were loaded under nonreducing conditions onto SDS-polyacrylamide gel polymerized with 1 mg/ml gelatin. Following electrophoresis, the gels were washed with 2.5% Triton X-100 to remove SDS and then incubated in a developing buffer [50 mM Tris–Cl pH 7.5, 10 mM CaCl2, 150 mM NaCl, 1 mM ZnCl2, 0.02%NaN3] overnight at 37uC. Gels were stained with 0.25% Coomassie Brilliant Blue R-250 and destained in the same solution without dye. Gelatinase activity was visualized as clear bands against the blue-stained gelatin background and analyzed in a computer system. Three individual experiments were conducted with independent protein samples. In vitro BBB model and evaluation of the barrier integrity An in vitro BBB model composed of BMVECs was developed on a matrigel-based insert with 8.0 mm pore size in a 24-well plate. Growth-arrested BMVECs of 26104 were seeded onto the upper surface of matrigel and fibronectin-coated polycarbonate mem- branes in transwell culture chambers. After the cells were incubated for 5–8 d to allow cell attachment, we carried out our experiments. The permeability of the barrier and the formation of the monolayer and its tightness were judged by measuring dextran-FITC (100 mg/ml, 40 Kd, Molecular Probes) flux across the barrier by flow cytometry analysis. The matrigel-based insert without BMVECs was used as control. When the dextran-FITC became absent in the lower compartment, the model was used to set experiments. Invasion and cell migration assay Twenty-four-well transwell inserts with 8 mm pores coated with matrigel (50 mL/well; BD Biosciences, Palo Alto, CA) were used to assess cell invasion. Different leukemia cell lines treated or untreated with GM6001, as well as the RNAi-treated cells for 24 h were suspended in 200 ml serum-free IMDM medium and seeded at a final density of 2.06105 cells/well in triplicate in the upper chamber. The lower compartment of the invasion chamber was filled with 800 ml IMDM (supplemented with 10% human serum) PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e20599