Mechanism of Central Nervous System Leukemia Figure 4. Effects of MMP-2/MMP-9 knock-down on activity of gelatinase and rates of cell invasion by SHI-1 in the in vitro BBB model. (A) Transcription of specific mRNAs quantified by qRT-PCR 48 h after siRNA transfection. SHI-1 cells transfected with siRNAs targeting the expression of MMP-2 (KD-MMP-2) or (B) MMP-9 (KD- MMP-9). Control SHI-1 cells transfected with non-target-directed siRNA (set as 100%). (C) Gelatinase secretion from SHI-1 cells transfected with siRNAs. (D) Rates of cell invasion of SHI-1 cells 48 h after transfection with siRNA in in vitro model of BBB. Data represent the mean 6 SD of triplicate measurement representative for three independent experiments relative to SHI-1 cells (set as 100%). *P,.001. doi:10.1371/journal.pone.0020599.g004 claudin-5 and occludin showed the knock-down of MMP-2 or -9 gene partially relieved the degradation of TJ proteins (Fig. 3E). The morphological changes of BMEVCs also demonstrated this phenomenon (data not shown) Thus, these findings demonstrate that down-regulation of gene expression of MMP-2 or -9 in SHI-1 cells could partially reverse the disruption of ZO-1, claudin-5 and occludin and so decrease the permeability of the BBB. These results further confirm the roles of MMP-2 and -9 in disrupting the BBB by degrading TJ proteins. Fatal CNS leukemia in SCI-nu/nu mice caused by the human acute monocytic leukemia cell line SHI-1 In order to investigate whether the BBB breakdown in CNS leukemia was associated with changes in endothelial TJ proteins in vivo, we next established a mouse model of CNS leukemia with the highly invasive human acute monocytic leukemia cell line SHI-1. Of the 20 surviving mice (other 10 mice were sacrificed weekly) challenged with SHI-1 cells, 11 mice developed symptomatic CNS leukemia. The prominent clinical sign of CNS leukemia was muscle weakness in the legs with a slight lowering of the lower back. Subsequently, mice developed a rapidly progressive paralysis of the cranial nerve with or without sight loss and they were no longer able to obtain food or water at (32.5560.65) d (mean 6SD) after being challenged by SHI-1 (Fig. 5A). They were killed when mice were moribund. Intracerebral mass or chloroma with or without intracranial hemorrhage was found in the removed brains (Fig. 5B). Histopathology examination showed that the CNS was extensively involved in these mice, with leukemic cell infiltrating the brain parenchyma (Fig. 5C1–C3) and subarachnoid spaces or accumulated on the surface of the cerebrum (data not shown). Bone marrow smear demonstrated a lot of leukemia blasts, infiltrating the bone marrow (Fig. 5C4). Histopathology also demonstrated the infiltration by SHI-1 cells in livers, lungs, kidneys, testes, hearts and other organs (data not shown). SHI-1 cells have t (6; 11) (q27; q23) and the rearrangement of the MLL gene, expressing the MLL/AF6 fused gene product [22]. MLL/AF6 was initially amplified in the brain at day 21 after challenged SHI-1 in some mice. The transcription of MLL/AF6 fusion gene also could be detected in the brains and bone marrows of killed mice with paralysis (Fig. 5D1). In control mice and mice without CNS leukemia, we did not examine for MLL/AF6 fusion gene in brains. All of our data demonstrate that the animal model of PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e20599