Mechanism of Central Nervous System Leukemia Figure 6. Activities of MMP-2 and -9 secreted by SHI-1 with or without GM6001 treatment in CNS leukemia mice. (A) RT-PCR analysis of human MMP-2 and -9 gene transcription in normal brain tissues, brain tissues infiltrated with SHI-1 cells with or without GM6001 treatment. (B, C and D) In situ zymographic analysis of MMP-2 and -9 activities mouse tissues. (B) Normal brain tissues; (C) Brain tissues infiltrated with SHI-1 cells; (D) Brain tissues after treatment with GM6001. Results shown are from individual animals and are representative of findings from 3 experiments with 6 animals per condition per experiment. (Scale bars: 10 mm). doi:10.1371/journal.pone.0020599.g006 normal control mice, fewer fluorescent spots were seen in these brain sections (Fig. 6B, D). These data demonstrate that MMP-2 and -9 secreted by SHI-1 cells play important roles in leukemic cell infiltration to CNS and other extramedullary infiltration. BBB breakdown with the disruption of endothelial TJ proteins in CNS leukemia and GM6001 reversed these changes To investigate whether BBB breakdown in CNS leukemia is associated with changes in endothelial TJ proteins, brain tissue sections were immunostained for ZO-1, claudin-5 and occludin, plus fibrinogen as a marker of the BBB disruption. Fibrinogen is a serum protein which is absent in the CNS under normal physiological conditions. Confocal imaging of the brain cortex from normal control mice showed that ZO-1, claudin-5 and occludin localized to linear profiles, were confirmed as vessels using the CD31 antigen (Fig. 7A, B1). In brains of mice with leukemic cell infiltration, confocal imaging showed widespread BBB breakdown as assessed by parenchymal immunoreactivity for fibrinogen (Fig. 7B2). Importantly, ZO-1, claudin-5 and occludin immunoreactivity also appeared reduced in these areas (Fig. 7B2). Moreover, immunoblotting for ZO-1, claudin-5 and occludin showed that the protein levels were down-regulated in brains with CNS infiltration than in normal mice brains (Fig. 7C). However, these three TJ proteins did not change in the brain of mice with GM6001 treatment compared with normal control mice (data not shown). In accordance with the results of immunoreactivity, the protein levels did not change after MMP-2 and -9 were inhibited by GM6001 (Fig. 7C). Collectively, these results demonstrate that the loss of endothelial TJ proteins ZO-1, claudin-5 and occludin leaded by MMP-2 and -9 is a central event in the opening of the BBB in CNS leukemia. Discussion In this study, we have investigated the mechanism underlying the BBB breakdown which makes leukemic cell enter the CNS during the process of leukemic cell extramedullary infiltration. We conclude that MMP-2 and -9 secreted by leukemic cells have been implicated in the response. The targets of the MMP-2 and - 9 are the endothelial TJ proteins in addition to the well-known ECM. MMP-2 and -9 distinguishably disrupted the TJ proteins ZO-1, claudin-5 and occludin as the BBB breakdown. We also have shown that these alterations can be inhibited by the broadband MMP inhibitor GM6001 and the knock-down of MMP-2/MMP-9 genes. Furthermore, GM6001 could effectively protect the mice against CNS leukemia. These findings may PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e20599