| 【Objects】The central nervous system (CNS) is one of the most frequent extramedullary locations of acute leukemia. The CNS leukemia represents a shorter overall survival and confers a poor prognosis to the patient. Despite its clinical importance, the mechanisms of CNS involvement are poorly understood. In acute leukemia, the leukemic cells dissociate from the bone marrow and enter the peripheral blood in the early stage of the disease. Like tumor invasion and metastasis, leukemia also needs leukemic cells to overcome all kinds of normal barriers. The blood-brain barrier (BBB) exists between the systemic circulatory system and the CNS to regulate the substances entering the brain tissue. Reports showed an association of matrix metalloproteinase (MMP)-2/MMP-9 expression with invasive behaviour of leukemia cells in acute leukemia. Moreover, considerable evidence has accumulated that in some CNS diseases MMP-2 and -9 are involved in the permeability of the BBB by disrupting the tight junction of brain microvascular endothelial cells (BMVECs). Little is known the effect of leukemic cell-derived MMP-2 and -9 to the BBB in the infiltration of leukemic cell to the CNS. In the present work, we examined the relationship of MMP-2 and -9 secreted by leukemic cells with the tight junction and the BBB dysfunction in a vitro BBB model and in an animal model of CNS leukemia.【Methods】[Section 1] The mRNA of MMP-2 and MMP-9 in SHI-1, HL-60 and U937 were detected by quantitative RT-PCR. Zymography was used to analyze the secretion of proMMP-2 and MMP-9 and expression of activated MMP-2 and MMP-9 in the supernatant of cultured different cells. Cell invasion through a barrier of reconstituted human basement membrane was assayed to measure the invasive capacity of different leukemic cell lines. All kinds of leukemic cell lines were treated with the broad-spectrum MMP inhibitor GM6001, then the levels of MMP-2 and MMP-9 secreted by different leukemic cell lines were detected by zymography and the cell invasion were also measured. RNA interference (RNAi) was used to knock down the expression of MMP-2 and MMP-9 in SHI-1 cell line and the invasive capacity of SHI-1 was measured. An in vitro BBB model composed of primary human BMVECs was developed in transwell culture chambers. GM6001 treated or without treated different leukemic cell lines or RNAi-treated SHI-1 cells were seeded with BMVECs into the upper compartment of the BBB models respectively. After cocultured with leukemic cells, morphologic changes of BMVECs were observed under inverted phase contrast microscopy and the changes of tight junction proteins ZO-1、claudin-5 and occludin in BMVECs were examined by laser scanning fluorescence confocal microscopy. Cell invasion through the barrier of the BBB reflected the invasion capacity of leukemic cells and the permeability of the BBB.[Section 2] All 44 BALB/c nu/nu mice pre-treated by splenectomy, cytoxan intraperitoneal injection, and sublethal irradiation (SCI nu/nu mice). For CNS leukemia animal model, a total of 1.2×107 SHI-1 cells in 0.01 M PBS for a total volume of 200μl were inoculated into SCI-nu/nu mice via the dorsal tail vein. All SCI-nu/nu mice were randomized into 3 groups: 28 SCI-nu/nu mice were only inoculated with SHI-1 cells, 8 SCI-nu/nu mice were injected intraperitoneally with GM6001 (5mg/kg/d) daily until day 21, another 8 SCI-nu/nu mice were injected with PBS without SHI-1 cells 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 to detect the human MLL/AF6 fuse gene. All nu/nu mice were bred in a laminar flow cabinet for 60 days. When the mice were moribund or killed immediately, the various organs were tracked to detect the expression of human MLL/AF6 fusion gene by RT-PCR. Bone marrow slides of nu/nu mice were stained by Wright’s to observe the infiltration of leukemic cells. The pathological section of different organ was done by routine method to survey the infiltration of leukemia. Human MMP-2 and MMP-9 mRNA were analyzed with the assay of RT-PCR. Gelatin in situ zymography was used to detected the activities of MMP-2 and -9 secreted by SHI-1 with or without treated with GM6001 in the brain of the mice. Tight junction proteins ZO-1, claudin-5 and occludin were observed by confocal imaging and western blot, respectively.【Results】[Section 1] The expression of MMP-2 and MMP-9 in SHI-1 cells were higher than in other leukemic cells at both mRNA and protein levels (P<0.01). The in vitro invasive capacity of SHI-1 cells were higher than other cells (P<0.01). The confluent BMVECs with neighbouring cells tightly packed against each other left no gaps. After incubation 24 h with leukemic cells the tight BMVECs disrupted to lose cell-cell contacts and the intrercellular gaps emerged. Confocal imaging demonstrated that in human BMVECs cultures, these proteins remained were localized on the plasma membrane in areas of cell-cell contact. 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 BBB breakdown. After 24 h coculture with BMVECs, the migration of SHI-1, HL-60 and U937 into the lower compartment was observed, indicating the occurrence of the BBB breakdown. SHI-1 cells displayed higher invasive rate than HL-60 and U937 cells across the barrier of the BBB in vitro BBB models and showed stronger disruptive ability to the ZO-1, claudin-5 and occludin and the BBB (P<0.01). GM6001-treated leukemic cells demonstrated a distinct loss of MMP-2 and -9 enzyme activities as compared to the untreated supernatants. when treated with GM6001, the injury of the BMVECs had an obviously improvement. In the same time the disruption of ZO-1, claudin-5 and occludin decreased, and the invasion rates of different leukemic cells which reflected the degree of BBB permeability were lower than those absence of GM6001 (P<0.01). Knock-down of MMP-2 or -9 gene impaired SHI-1 cells invasion and decrease the degradation of ZO-1, claudin-5 and occludin with the alleviative permeability of the BBB in vitro.[Section 2] The survival time of mice except the killed mice weekly in control group was more than 60 days. Of the 20 surviving mice (Other 10 mice were sacrificed weekly) challenged with SHI-1 cells in SHI-1 group, 11 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 after 32.55±0.65 d (mean±SD). 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. Some mice also had leukemic cell infiltration in many organs such as stomach, liver and urinary bladder. The transcription of human MLL/AF6 fusion gene also could be detected in the brains, bone marrows and other organs of killed mice with paralysis. In control mice and mice without CNS leukemia, we did not examine for MLL/AF6 fusion gene in brains. Bone marrow smear demonstrated leukemia blasts and histopathology examination showed that the CNS were extensively involved in these mice, with leukemic cell infiltrating the brain parenchymaand subarachnoid spaces or accumulated on the surface of the cerebrum. The mice in GM6001 group had no systems of CNS infiltration and no blasts were detected in bone marrow and brains. The number of other organs infitrated by SHI-1 cells and the degree of infiltration also decreased. The average survival time of mice in group GM6001 was (41.50±1.62) d. Consistent with MLL/AF6, the human MMP-2 and -9 transcriptions were initially detected at day 21 after being challenged by SHI-1 and amplified in 11 mice with CNS leukemia. But MMP-2 and -9 transcripts were not detected in the brains of mice treated with GM6001. Furthermore, we used in situ zymography to identify the regions with gelatinolytic activity. Bright ?uorescent spots were apparent in the areas of infiltration with leukemic cells and in the periphery of mouse brains with CNS leukemia. While after treatment with GM6001, gelatinolytic activities had not changed in normal control mice, fewer ?uorescent spots were seen in these brain sections. Confocal imaging of the brain cortex from normal control mice showed that ZO-1, claudin-5 and occludin localized to linear profiles. In brains of mice with leukemic cell infiltration, confocal imaging showed widespread BBB breakdown as assessed by parenchymal immunoreactivity for fibrinogen. Importantly, ZO-1, claudin-5 and occludin immunoreactivity also appeared reduced in these areas. 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. However, these three tight junction proteins did not change in the brain of mice with GM6001 treatment mice. In accordance with the results of immunoreactivity the protein levels did not change after MMP-2 and -9 were inhibited by GM6001.【Conclusion】(1) MMP-2 and MMP-9 were expressed in SHI-1、HL-60 and U937 leukemic cells, and the expression levels of MMP-2 and MMP-9 were consistent with their invasive capacity.(2) The tight junction proteins ZO-1、Claudin-5 and occludin in BMVECs decreased in brain tissues with leukemic cell infiltration.(3) The decrease of tight junction proteins ZO-1、Claudin-5 and occludin in BMVECs followed with the increase of the permeability of the BBB.(4) The disruptive degree of the BBB was consistent with the expression level of MMP-2 and MMP-9 in leukemic cells in central nervous system leukemia.(5) MMP-2 and -9-mediated down-regulation of tight junction proteins as a significant mechanism in the breakdown of the BBB in the leukemic cell infiltraion of central nervous system.In summary: MMP-2 and MMP-9 were expressed in leukemic cells, and the expression levels of MMP-2 and MMP-9 were consistent with their invasive capacity. We demonstrated that leukemic cell disrupted the BBB in the infiltration of the CNS and the MMP-2 and -9 secreted by leukemic cells were involved in. Leukemic cells impaired tight junction proteins ZO-1, claudin-5 and occludin in BMVECs resulting in increased permeability of the BBB and these alterations reduced when MMP-2 and -9 activities were inhibited by RNA interference strategy or by MMP inhibitor GM6001 in an in vitro BBB model. We also found that the disruption of the BBB in company with the down-regulation of ZO-1, claudin-5 and occludin and the up-regulation of MMP-2 and -9 in mouse brain tissues with leukemic cell infiltration. Besides, GM6001 protected all mice against CNS leukemia. Our findings suggest that the degradation of ZO-1, claudin-5 and occludin by MMP-2 and -9 secreted by leukemic cells constitutes an important mechanism in the BBB breakdown which contributes to the invasion of leukemic cells to the CNS in acute leukemia. |
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