Mechanism of Central Nervous System Leukemia Figure 1. Expression of MMP-2 and -9, gelatinase secretion and invasion rate in different leukemic cells. (A) qRT-PCR analysis of MMP-2 (A1) and MMP-9 (A2) gene transcription in SHI-1, HL-60 and U937 leukemic cells. (B) Gelatinase secretion from culture supernatants of three leukemic cells. (C) Cell invasion rates of SHI-1, HL-60 and U937 leukemic cells. Cell invasion rates were determined by the ratios relative to SHI-1 cells. Data represent the mean 6 SD of triplicate measurement representative for three independent experiments relative to SHI-1 cells (set as 100%). Compare with SHI-1 cells *P,.001. doi:10.1371/journal.pone.0020599.g001 measure the invasive capacity of leukemic cells. The invasion rate of SHI-1 was significantly higher than that of HL-60 and U937 cells (P,.001) (Figure 1C). Increased permeability of the BBB by leukemic cells and down-regulation of endothelial TJ proteins via MMP-2 and -9 in vitro We hypothesized that MMP-2 and -9 secreted by leukemic cells induced the permeability of the BBB by disrupting the TJ proteins in CNS leukemia. To test this hypothesis, we first seeded SHI-1, HL-60 and U937 cells into the upper compartment of the in vitro BBB model, constituted by primary human BMVECs to form a co-culture system. The results showed that before co-culture with SHI-1, HL- 60 and U937 cells, BMVECs became confluent with neighbouring cells tightly packed against each other leaving no gaps. However, after incubation for 24 h with leukemic cells, the tightness of the BMVECs was disrupted, giving rise to a loss of cell-cell contact and the formation of intercellular gaps (Fig. 2A1). The number of leukemic cells across the barrier was calculated as the rate of invasive in order to reflect the degree of BBB breakdown. The rates of invasion of leukemic cells in this co-culture system were inconsistent with their secreted MMP-2 and -9 activities and their ability to invade. Where SHI-1 showed a higher invasion capacity than either HL-60 or U937 cells (Fig. 2B). Interestingly, the degree of morphological changes in BMVECs depicted in Figure 2A1 matched well with the rate of invasive of different leukemic cells. SHI-1 cells caused the strongest disruptive effects on BMVECs and the BBB. To further determine whether MMP-2 and MMP-9 secreted by leukemic cells played roles in increasing the permeability of BMVECs and the BBB, SHI-1, HL-60 and U937 cells were treated with the MMP inhibitor GM6001, which had been previously shown to inhibit MMP-2 and -9 [27], before co-culture with BMVECs in the in vitro BBB model. We found that the intercellular gap of BMVECs, led by leukemic cells, became tight (narrow) or even partially disappeared (Fig. 2A2). Consistently and significantly, the number of leukemic cells across the BBB decreased, reflecting a decided reversal in the impairment of the BBB. Gelatin zymography of supernatants from GM6001-treated leukemic cells demonstrated a distinct loss of MMP-2 and -9 enzyme activities, compared to the untreated supernatants, thereby confirming the ability of GM6001 to block MMP-2 and -9 activities (Fig. 2C). These results show that MMP-2 and -9 secreted by leukemic cells were closely related to the BBB breakdown by disrupting BMVECs. To define whether the breakdown of BBB induced by MMP-2 and -9 was led by changes in TJ in addition to the basement membrane, BMVECs were immunostained for the TJ proteins ZO-1, claudin-5 and occludin. Confocal imaging demonstrated that in human BMVECs cultures, these proteins remained were localized on the plasma membrane in areas of cell-cell contact (Fig. 3A). After incubation with leukemic cells, the fluorescent immunoreactivity for ZO-1, claudin-5 and occludin appeared patchy or even faded away in those areas accompanied by the same degree of BBB breakdown (Fig. 3B–D). On the contrary, the expression of three proteins, all up-regulated in company with the decreased BBB permeability when MMP-2 and -9 were secreted by leukemic cells was inhibited by GM6001 (Fig. 3B–D). Collectively, these results suggested that leukemic cells disrupted the BBB by down-regulating the TJ proteins ZO-1, claudin-5 and occludin in BMVECs. MMP-2 and -9 secreted by leukemic cells play important roles in this process. PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e20599