Studies On The Essential Roles Of Matrix Metalloproteinase-2, Membrane Type 1 Metalloproteinase And Tissue Inhibitor Of Metalloproteinase-2 In The Invasive Capacity Of Acute Monocytic Leukemia SHI-1 Cells

【Objects】Acute leukemia always accompany with extramedullary infiltration (EMI). The frequency of EMI in acute myeloblastic leukemia (AML) is reported to be between 20%–40% and EMI is found to highly associate with FAB classified M4 or M5 subgroups. In addition, patients with EMI have a lower complete remission rate following induction chemotherapy and a shorter overall survival. Exploring mechanism underlying EMI will help to prevent leukemia relapse and improve the prognosis of leukemia. Like tumor invasion and metastasis, EMI of AML also needs AML cells to overcome extracellular matrix (ECM) barriers. Some investigators have reported high expression of matrix metalloproteinases (MMPs) in acute leukemia and suggested that high expression correlated with the EMI property of AML. However, the underlying mechanism of MMPs and their inhibitors in EMI of AML is not clear. SHI-1 cells which demonstrate high invasive ability belong to human acute monocytic leukemic cell line. It was established and maintained in our institute. Our previous study has created an efficient and reproducible experimental model of central nervous system leukemia and multiorgan infiltration by using SHI-1 cell in nu/nu mice. Moreover, we have observed that up-regulation of TIMP-2 promoted SHI-1 cells infiltration in vivo. In the present work, we studied the essential roles of matrix metalloproteinase-2 (MMP-2), membrane type 1 metalloproteinase (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in the invasive capacity of acute SHI-1 cells both in vitro and in vivo to explore the mechanism underlying EMI. 【Methods】[Section 1] SHI-1, NB4, K562, U937 and THP-1 human leukemia cell lines and bone marrow stromal cells (BMSCs) were cultured in vitro. The mRNA and protein expressions of TIMP-2, MMP-2 and MT1-MMP in different cells were detected by quantitative RT-PCR and western blot, respectively. RNA interference (RNAi) was used to knock down the expression of MMP-2, MT1-MMP and TIMP-2. A retroviral vector carrying human TIMP-2 cDNA was constructed and transfected into the SHI-1 cells. After transfection, polyclonal cells were screened by G418. Following the integration of exogenous TIMP-2 cDNA was affirmed in polyclonal cells by RT-PCR, three subclone cells (S1, S2 and S3) cells were selected by limiting dilution. The integration of exogenous TIMP-2 in three subclone cells was also detected by RT-PCR. Zymography was used to analyze the secretion of proMMP-2 and expression of activated MMP-2 in the supernatant harvested 24 hours after different cells monoculture or co-culture with bone marrow stromal cells (BMSCs). The secretion of proMMP-2 and expression of activated MMP-2 in the supernatants harvested 24h, 48h and 72h after monoculture of BMSCs were detected by Zymography. Cell invasion through a barrier of reconstituted human basement membrane was assayed to measure the invasive capacity of different leukemic cell lines, three TIMP-2 transfectant subclone cells as well as RNAi-treated cells monocultured or co-cultured with BMSCs.[Section 2] 4-week-old Balb/C nu/nu mice pre-treated by splenectomy , cytoxan intraperitoneal injection, and sublethal irradiation (SCI nu/nu mice), were transplanted intravenously with 1×107 cells of different groups suspended in 0.2ml IMDM. These mice were randomizely divided into six groups including the control group (n=5), the SHI-1 transplanted group, the MOCK transplanted group, the TIMP-2 transfected S1, S2, S3 groups, (n=15 for each group). After cell infusion, the nu/nu mice were bred in a laminar flow cabinet for 60 days. Mice were dissected when they were moribund or dead immediately. If they could survive longer than 60 days, the dissection was performed in the 60d. Bone marrow slides of nu/nu mice were stained by Wright's to observe the infiltration of leukemia cells. The pathological section of different organ was done by routine method to survey the infiltration of leukemia. The leukemic cells engraftments in various organs were tracked by the expression of the MLL/AF6 fusion gene detected by RT-PCR. The ratio of leukemic cells in the bone marrow was determined by flow cytometer (FCM) using human CD45 antibody.【Results】[Section 1] The expression of MMP-2, MT1-MMP and TIMP-2 in SHI-1 cells were higher than in other leukemic cells at both mRNA and protein levels(P<.05). The in vitro invasive capacity of SHI-1 cells were higher than other cells both in presence and absence of BMSCs(P<.05).The level of proMMP-2 (72KDa) and activated MMP-2 (62KDa) released from SHI-1 cells were found higher than that in other leukemic cells both in presence and absence of BMSC(sP<.05). Furthermore, the amounts of proMMP-2 and activated MMP-2 in co-culture supernatants as well as the in vitro invasive capacity were significantly higher than that of in monoculture(P<.05). The culture supernatants taken at different time points during a 72h cultivation period of BMSCs in serum-free medium were analyzed. It showed that the gelatinase secretion was not detected in culture supernatants until BMSCs were cultured longer than 48 hours and the secretion was reached peak after 72 hours cultivation. The knock-down efficiency of siRNA was 85% to 98%. The down-regulation of TIMP-2, MMP-2 and MT1-MMP reduced the invasion rates of SHI-1 cells by 60%-70%, 50%-60% and 40%-50%, respectively(P<.05). No activated MMP-2 in the supernatants from any knock-down cells could be found. Both polyclonal cells and three monoclonal cells transfected with TIMP-2 were found the integration and expression of exogenous TIMP-2 by RT-PCR. The mRNA level of TIMP-2 increased about 3.2 fold, 2.2 fold and 1.7 fold in S1, S2 and S3 cells, respectively(P<.05), while the protein levels of TIMP-2 in S1, S2 and S3 cells were about 2.6 fold, 1.9 fold and 1.5 fold of SHI-1 cells, respectively (P<.01). The invasion rates of subclone cells demonstrated a 1.5 fold -2.5 folds'elevation (P<.05) and activated MMP-2 from their supernatants increased 1.5 fold -3.0 fold(P<.01). No difference in MMP-2 and MT1-MMP expression as well as in the secretion of proMMP-2 in the supernatants among three transfected subclone cells and SHI-1 cells could be found.[Section 2] The manifestation of foodintake reducing, body weight losing, emaciation have emerged in the mice of Control group one to two weeks after infusion, but disappeared two weeks later. The survival time of mice was more than 60 days. The same manifestation occurred in the mice of SHI-1 and MOCK groups during the first two weeks and they also recovered, but 5-6 weeks later the manifestation appeared again. Some mice were developed paralysis in both of the rear legs. Some mice have the neoplasm formed in the brain and eyes. All the mice become prostration gradually till dead. The median survival time of all mice in SHI-1 group was 45d (40-48d) while in MOCK was 44d (39-49d). The time of disease onset in mice of S1, S2 and S3 group were all earlier than that in SHI-1 group. The median survival time of all mice in S1, S2 and S3 group was 35d (33-38d), 38d (35-42d) and 39d (37-43d), respectively. The survival time of S1, S2 and S3 group were all shorter than SHI-1 group(P<.05), while that of mock group was similar to the SHI-1 group. Histopathological evidences demonstrated severally leukemic infiltration in many organs (such as liver, stomach intestine, lung, kidney, brain et al.) of the experimental group, while the Control group mice slices had no abnormal changes. Bone marrow slides of S1, S2 and S3 group showed more human leukemic cells than that of SHI-1 group(P<.05). RT-PCR detection showed that many organs in experimental groups expressed MLL/F6 fuse gene, moreover, the mount of organs which was infiltrated by leukemic cells in S1, S2 and S3 group was all more than that of SHI-1 group(P<.05). The ratio of human CD45 positive cells detected in the femur bone borrow of S1, S2 and S3 group was higher than that of SHI-1 group(P<.05), while it was not found in Control group.【Conclusion】(1) SHI-1 cells strongly expressed constitutive MMP-2, MT1-MMP and TIMP-2 at both mRNA and protein levels.(2) The high expression of MMP-2, MT1-MMP and TIMP-2 in the SHI-1 cells enhanced cell invasion by promoting the proMMP-2 activation.(3) The co-culture of BMSCs and SHI-1 cells increased the invasive capacity of SHI-1 cells.(4) A 1.5 to 2.5 folds up-regulation of TIMP-2 expression in SHI-1 cells not only increased the cell invasive capacity in vitro, but also made SHI-1 cells develop more severally leukemic infiltration in vivo in the nu/nu mice. Thus the onset time of mice which were infiltrated by leukemic cells was earlier with a shorter survival time. It indicated that up-regulation of TIMP-2 exhibited not a repressive but an activating effect on SHI-1 cells invasion.In summary: SHI-1 cells strongly expressed constitutive MMP-2, MT1-MMP and TIMP-2 at both mRNA and protein levels. The co-culture of BMSCs and SHI-1 cells increased the invasive capacity of SHI-1 cells. Up-regulation of TIMP-2 as well as constitutive strong expression of TIMP-2, MMP-2 and MT1-MMP in SHI-1 cells not only enhanced cell in vitro invasion by promoting proMMP-2 activation but also increased SHI-1 cell in vivo infiltration in the nu/nu mice. These results suggested that increasing the expression of TIMP-2 in AML patients with EMI may potentially cause adverse effects, particularly in those who have high levels of MMP-2 and MT1-MMP.