much difficult to penetrate, and infiltration through these barriers selects for leukemic cells that express the necessary extravasation functions. Although reports have shown that MMP-2 and -9 are closely associated with the BBB disruption in some CNS related diseases [20,31], little is known about the pathological role of MMP-2 and -9 secreted by leukemic cells in BBB breakdown in CNS leukemia. In order to investigate the relationship between leukemic cell- derived MMP-2 and -9 and the BBB in CNS leukemia, we first studied the effect of MMP-2 and -9 secreted by different leukemic cells on cultured BMVECs in the in vitro model of the BBB. Before transmembrane of leukemic cells, we seeded BMVECs onto the upper surface of matrigel-coated polycarbonate membranes in transwell culture chambers to simulate the barrier of the BBB. Cultured BMVECs retain many of the features of the BBB and have been used as in vitro models to study mechanisms that regulate the permeability of the BBB [32,33]. It is well- known that BBB is maintained primarily by TJs between adjacent microvascular endothelial cell images as a network of strands formed by intramembranous particles and occlude intercells more effectively [10,11,34]. The transmembrane proteins occludin and claudin-5 form the primary seal of the TJs. They bind to the ZO- 1 and other proteins that couple the TJs to the actin cytoskeleton of endothelial cells [10]. Studies have demonstrated a correlation between the reduction of TJ and tumor differentiation and placed TJ in the frontline as the structure that cancer cells must overcome in order to metastasize [28,35,36]. We also found that each of the different leukemic cell lines HL-60, SHI-1 and U937 all could disrupt the TJ proteins ZO-1, claudin-5 and occludin, accompanying the formation of intercellular gaps in co-cultured BMVECs and the breakdown of BBB, but the levels of disruption differed greatly. SHI-1 cells displayed a stronger capacity of disrupting TJ proteins than the other two leukemic cell lines, consistent with its higher rate of invasion which is closely related to the constitutive strong expression of MMP-2 and -9. GM6001 is a broad-spectrum MMP inhibitor to prevent the activities of MMP-2, -3, -8 and -9 and is routinely used to understand the pathogenic roles of MMP-2 and -9 [23,37]. We have shown that GM6001 blockaded the activities of MMP-2 and -9 and significantly decreased the disruption of the leukemic cells to the TJ proteins. Moreover, the knock-down of MMP-2 or MMP- 9 genes by iRNA in SHI-1 cells also reduce the disruption of those proteins and the permeability of BBB. These findings suggest that MMP-2 and -9 secreted by leukemic cells can degrade components of TJ in addition to basement membranes, which help the leukemic cells to infiltrate the brain. Our previous study has created an efficient and reproducible experimental model of AML CNS leukemia and multiorgan infiltration by using highly invasive SHI-1 cells in nu/nu mice [26]. However, a mouse model of CNS leukemia can not be successfully established with HL-60 and U937 cells. In this study we increased the number of inoculated SHI-1 cells. More than half of the mice developed CNS leukemia and 4 of them had SHI-1 cell infiltration in the brain parenchyma. In the section of infiltrarion with SHI-1 cells, we found a widespread BBB breakdown with a loss of the endothelial TJ proteins ZO-1, claudin-5 and occludin. The accompanying up-regulation of human MMP-2 and -9 showed that MMP-2 and -9 secreted by SHI-1 cells played an important role in this event. Surprisingly, GM6001 protected mice against CNS leukemia and as well, other organs were protected and no mice developed CNS leukemia. Mechanism of Central Nervous System Leukemia These results further defined that MMP-2 and -9 secreted by leukemic cells increased the permeability of the BBB by disrupting the TJ proteins ZO-1, claudin-5 and occludin in CNS leukemia. We also found that both MMP inhibitor GM6001 and the RNAi technology to inhibit the expression of MMP-2 and -9 in leukemic cells could not entirely reverse the disruption of the TJ proteins and the permeability of the BBB in vitro. In addition to MMP-2 and -9, other MMP family members such as MMP-1, MMP-3, MMP-11 and MMP-14 have all been shown to be secreted by tumor cells in tumor microenvironments, serving to mediate the breakthrough of tissue barriers comprising cell–cell adherent junctions, basement membranes, and interstitial tissue stroma [38–40]. Furthermore, the invasive behavior of tumor cells depends not only upon the secretion of a variety of degradative enzymes but also on the alterations in the expression of adhesion molecules (selectin–selectin ligand axis), the responses to cytokines and chemokines (CXCR4, CCR7, et al.) and the gene products regulating motility. Multiple mechanisms appear to contribute to the disease. The integral transmembrane proteins such as claudin-5 and occludin are the essential adhesion proteins responsible for correct assembly of the TJ structure of the BBB and controlling TJ functions via homotypic and heterotypic interactions [10,28]. There is an ongoing interest in the question of how the TJ proteins are modified by MMP-2 and -9 as it occurs in CNS leukemia. ZO-1, claudin-5 and occludin cleavage by MMPs is a possible mechanism underlying BBB impairment and has been detected in vivo models of focal cerebral ischemia [18,41,42]. Both occludin and claudin-5 consist of four transmembrane domains, intracellular N- and C-termini, and two extracellular domains that might interact with cell membranes of vicinal cells thus sealing the intercellular clefts [43,44]. The first extracellular loop displayed type IV collagenase sensitive motives, implying that claudin-5 and occludin cleavage might be caused by MMP-2 or - 9 [45,46]. In addition to the most important functions as a barrier to cell migration, TJs have been linked to various signaling mechanisms [44], hence possible mechanisms will be further investigated. In conclusion, our results established a correlation between MMP-2 and -9 secreted by leukemic cells and the disruption of the TJ proteins ZO-1, claudin-5 and occludin, which increases paracellular permeability of leukemic cell across the BBB in CNS leukemia. This information provides an important mecha- nism of leukemic cell infiltration of the CNS, which may provide additional insight into its potential target for therapeutic intervention. Future studies will have to clarify the precise mechanism or signal transduction pathway for the permeability- increasing effect of leukemic cell-derived MMP-2 and -9. Acknowledgments We would like to thank Dr. Tom Hazenberg (the professor emeritus Lakehead University, Canada) for polishing the language of this paper. Author Contributions Conceived and designed the experiments: SF YH ZC. Performed the experiments: SF JC HS LY YW. Analyzed the data: SF ZC. Contributed reagents/materials/analysis tools: SF JC HS. Wrote the paper: SF ZC. Provided the guides for immunofluorescence assay: YW. PLoS ONE | www.plosone.org 10 August 2011 | Volume 6 | Issue 8 | e20599