containing 100 ng/ml SDF-1a as a source of chemoattractants. The coated filter inserts with or without seeded BMVECs (2.06104 cells) were placed into the wells forming the upper compartment. The chambers were incubated for 24 h at 37uC in a 5% CO2 atmosphere. Cells that had migrated into the lower compartment were counted and the invasion rate was calculated by the ratio of the number of cells in the medium of lower compartment to the total number of leukemic cells loaded in the upper compartment. Morphologic changes of BMVECs on matrigel were observed under inverted phase contrast microscopy after washed with PBS. Photographs were captured using a camera (Canon A1000, Malaysia). Each invasion experiment was performed in triplicate. Murine CNS leukemia model and drug administration For CNS leukemia animal model, a total of 1.26107 SHI-1 cells in 0.01 M PBS for a total volume of 200 ml were inoculated into 46 SCI-nu/nu mice via the dorsal tail vein after receiving sublethal irradiation. 46 SCI-nu/nu mice were randomized into 4 groups: 30 SCI-nu/nu mice were only inoculated with SHI-1 cells, 8 SCI-nu/ nu mice were injected intraperitoneally with GM6001 (1.5 mg per mouse) daily until day 21, another 6 SCI-nu/nu mice were injected with PBS without SHI-1 cells and 2 mice were injected with the vehicle DMSO as control. Mice were monitored daily for survival and clinical appearance of nerve palsy and killed when becoming moribund. In order to identify the initial time of leukemic cells entering the CNS, 2 mice inoculated with SHI-1 cells and one control mouse were sacrificed weekly after inoculation. Reverse transcription-polymerase chain reaction (RT-PCR) analysis and quantitative real-time polymerase chain reaction (qRT-PCR) Animals were anaesthetized with chloral hydrate and perfused with PBS prior to sacrifice in order to avoid RNA contamination from blood cells. Brains were immediately removed after perfusion and total RNA was extracted using Trizol reagent according to the manufacturer’s instruction, and cDNA were synthesized by using MMLV reverse transcriptase (Gibco, USA). The oligonucleotide primer sequences were designed as the follows: human MMP-2, forward 59-GCT ATG GAC CTT GGG AGA A-39, reverse 59- TGG AAG CGG AAT GGA AAC-39, product 260 bp; human MMP-9, forward 59-TCC CTG GAG ACC TGA GAA CC-39, reverse 59-CGG CAA GTC TTC CGA GTA GTT T-39, product 287 bp; human GAPDH, forward 59- AGA AGG CTG GGG CTC ATT TG -39, reverse 59- AGG GGC CAT CCA CAG TCT TC -39,product 258 bp. MLL/AF6 fusion genes were detected by nested PCR performed as previously described [26]. The amplified products were analyzed by 2% agarose gel electropho- resis and photographed under UV light. Levels of MMP-2 and MMP-9 mRNA were analyzed with the TaqMan Gene Expression Assays (Applied Biosystems). Glyceral- dehyde-3-phosphatedehydrogenase (GAPDH) housekeeping gene was used as an endogenous control to correct potential variation in RNA loading or efficiency of the amplification procedure. Primers and probe specific for human MMP-2, MMP-9 and GAPDH used as reported previously [24,27]. Real-time PCR was performed on a MJ Research OpticonTM2. The PCR thermal cycle conditions and calculation of the relative expression of mRNA species were performed as described previously [24]. All samples were measured in triplicate. Histology and immunofluorescence Bone marrow smears of mouse femurs were stained with Wright’s-Giemsa for observing leukemic blast cells. Brains were Mechanism of Central Nervous System Leukemia immediately removed after perfusion with 4% paraformaldehyde (PFA) in PBS, pH 7.4, fixed for 3 h in 4%PFA in PBS at 4uC, and incubated overnight in PBS with 20% sucrose at 4uC. Brains were cut into 6 or 20 mm thick sections with a cryostat. Sections of 6 mm thick were stained with hematoxylin and eosin (H&E). The remaining slides were used for immunofluorescence. Immunofluorescence analysis determined the changes of TJ proteins in brain sections and BMVECs on Matrigel. Cell monolayers cultured on inserts were washed in PBS and fixed with 4% PFA. After blocking with 1% or 5% BSA, cells or brain sections were stained with primary monoclonal antibodies against ZO-1, occludin and claudin-5, conjugated to FITC or Alexa 488 (Invitrogen, USA) overnight at 4uC. The primary antibodies were used in a 1:50 dilution. For double labels, anti-fibrinogen (1:200, Abcam, USA) goat monoclonal antibody or anti-CD31 (1:50, Abcam, USA) rat monoclonal antibody were incubated with these primary antibodies respectively. Incubation with the secondary antibody Cy3-labelled anti-goat or anti-rat IgG (1:750) lasted for 1 h at 37uC. Nuclei were stained with DAPI (Molecular Probes, Invitrogen) for 3 min. Staining was analyzed with a laser-scanning confocal microscope (Nikon A1, Japan). In situ gelatin zymography The frozen, nonfixed sections from brain tissues were incubated in a humidity chamber for 3 h at 37uC in a reaction buffer (0.05 M Tris–HCl, 0.15 M NaCl, 5 mM CaCl, 0.2 mM NaN3) containing 20 mg/ml of fluorescein isothiocyanate (FITC)-labeled DQ-gelatin (EnzCheck Collagenase Kit, Invitrogen, USA). As a negative control, 50 mM of 1, 10-phenanthroline, a metalloproteinase inhibitor, was added to the reaction buffer before applying the FITC-labeled DQ gelatin to frozen sections. After incubation, fluorescence was detected by laser scanning fluorescence confocal microscopy. Quenched FITC molecules contained within the gelatin emit fluorescence when the gelatin is enzymatically cleaved by MMP-2/-9. Immunoblotting Equal amounts of protein (80 mg) were loaded on 6 or 10% polyacrylamide gels and SDS-PAGE was performed. After separation by electrophorese, proteins were electroblotted on polyvinyl difluoride membranes. The membranes were incubated with primary monoclonal mouse antibodies against against ZO-1 (2 mg/ml; Inritrogen), Claudin-5 (3 mg/ml; Inritrogen), occludin (2.5 mg/ml; Inritrogen) at 4uC overnight. After the blots were incubated with secondary antibodies, bands were visualized by using the ECL chemiluminescence detection system. The experi- ment was repeated at least twice with independent protein samples. Data analysis Statistical analysis was carried out using ANOVA followed by Tukey’s post hoc tests (GraphPad 4.0 Software, San Diego, CA). Significance was assumed for p value less than 0.05. Results MMP-2 and -9 are expressed by different leukemic cell lines and correlates with their invasive capacity The qRT-PCR determination of mRNA expressions in SHI-1, HL-60 and U937 cells revealed that the transcriptional level of MMP-2 and -9 in SHI-1 cells were all markedly higher than that in other leukemic cell lines (P,.001) (Fig. 1A). Furthermore, the zymographic analysis of MMP-2 and -9 activities of the cell culture supernatants were consistent with their mRNA levels. Moreover, SHI-1 cells released a small portion of activated MMP-9 into culture supernatants (Figure 1B). Cell migration assay was used to PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e20599