TIMP-2、MT1-MMP、MMP-2的表达对人类单核细胞白血病细胞系SHI-1细胞侵袭力影响的研究
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摘要
目的和意义
急性白血病常伴有髓外浸润(EMI)的发生,急性髓细胞白血病(AML)中EMI的发生率约为20%–40%,并且多见于FAB分型中的M4、M5亚型。伴有EMI的患者大多数诱导化疗后的缓解率低,总体生存时间短,预后差。阐明白血病髓外浸润的病理发生过程及机制,有助于预防白血病的复发,改善白血病的预后。研究表明AML的EMI如同肿瘤的侵袭和转移一样,需要白血病细胞克服ECM屏障。已有研究报道急性白血病患者高表达基质金属蛋白酶(MMPs),并且提示这些高表达可能与EMI的特性有关,但是MMPs及其抑制剂在AML的EMI中确切作用和机制仍不清楚。SHI-1细胞是本研究所建立的一株具有高浸润特性的人类急性单核细胞白血病细胞系。本实验室前期研究中已用SHI-1细胞在裸鼠体内建立了白血病多脏器浸润的动物模型,并报道上调组织型基质金属蛋白酶抑制剂2 (TIMP-2)促进了SHI-1细胞在裸鼠体内的浸润。在此基础上,本课题将研究TIMP-2、Ⅰ型膜型基质金属蛋白酶(MT1-MMP)、基质金属蛋白酶2(MMP-2)的表达对SHI-1细胞体内外侵袭力的影响,探索白血病髓外浸润的机制。
方法
第一部分体外培养SHI-1、NB4、K562、U937、THP-1细胞和骨髓基质细胞(BMSCs)。定量PCR检测五株细胞的TIMP-2、MT1-MMP、MMP-2的mRNA表达水平,Western blot检测蛋白表达水平。RNA干扰分别沉默SHI-1细胞的TIMP-2、MT1-MMP、MMP-2。构建TIMP-2逆转录病毒载体,转染SHI-1细胞,G418筛选出多克隆细胞,RT-PCR鉴定有外源性TIMP-2基因整合后,经有限稀释法挑选出单克隆细胞S1、S2、S3。PCR鉴定单克隆细胞的外源性TIMP-2基因整合。定量RT-PCR检测单克隆细胞TIMP-2、MT1-MMP、MMP-2的mRNA表达水平,Western blot检测蛋白表达水平。明胶酶谱法测定五种细胞株、单克隆细胞、干扰后的细胞单独培养或和BMSCs共培养24小时后上清中MMP-2酶原(proMMP-2)分泌和活化的MMP-2的表达。收集BMSCs单独无血清培养24h、48h、72h后的上清检测proMMP-2的分泌和活化的MMP-2。跨膜侵袭实验观察各种细胞单独培养或和BMSC共培养24小时后侵袭人工基质膜的能力。
第二部分对4周龄的BALB/c裸鼠进行脾脏切除(splenectomy)、环磷酰胺腹腔注射(cytoxan intraperitoneal injection)及全身亚致死剂量辐照(sublethal irradiation)等预处理(SCI预处理)后随机分为:空白对照组(n=5)、实验组(SHI-1组、MOCK组、S1组、S2组、S3组,每组n=15)。经尾静脉接种1×107含不同细胞的IMDM液0.2ml/只后,在层流柜中无菌饲养观察至注射后60天。于裸鼠濒死前或死亡后即行解剖,留取骨髓涂片,瑞氏染色观察有无异常的白血病细胞;各脏器常规病理切片,观察异常白血病细胞在各脏器中的浸润情况;RT-PCR检测裸鼠各脏器中人白血病细胞MLL/AF6融合基因的表达。流式细胞仪(FCM)检测裸鼠股骨骨髓中人CD45阳性细胞比例。
结果
第一部分SHI-1细胞株的TIMP-2、MT1-MMP、MMP-2的mRNA表达和蛋白表达均高于其它细胞株(P<.05)。SHI-1细胞无血清培养以及和BMSC共培养24h后上清中的proMMP-2(72KDa)和活化的MMP-2(62KDa)含量及体外侵袭率均高于其它细胞株(P<.05)。并且共培养24h后proMMP-2的分泌、MMP-2的活化和体外侵袭率均高于单独培养的(P<.05)。BMSC单独培养24h后的上清中检测不到proMMP-2的分泌和MMP-2的活化,直到48h才能检测到,72h后达高峰。RNA干扰沉默TIMP-2、MT1-MMP、MMP-2基因的效率为85%-98%。基因沉默SHI-1细胞TIMP-2、MMP-2、MT1-MMP后,细胞侵袭率均分别下降60%-70%、50%-60%、40%-50%(P<.05)。干扰后的条件培养基中检测不到活化的MMP-2片段。RT-PCR鉴定转染了TIMP-2 cDNA的多克隆细胞和单克隆细胞均有外源性TIMP-2基因的整合和表达。单克隆细胞S1、S2、S3 TIMP-2的mRNA表达水平分别是SHI-1细胞的3.2倍、2.2倍、1.7倍(P<.05),蛋白表达水平分别上调2.6倍、1.9倍、1.5倍(P<.01),体外侵袭率增加了1.5倍-2.5倍(P<.05),活化的MMP-2增加了1.5倍-3倍(P<.01)。单克隆细胞的MT1-MMP、MMP-2的表达和条件培养基中的proMMP-2表达和SHI-1细胞没有差异。
第二部分对照组裸鼠注射后1-2周进食减少、体重下降、消瘦,2周后逐渐恢复正常,生存期大于60天以上而无死亡。SHI-1组和MOCK组裸鼠尾静脉注射细胞后,同样1-2周进食减少、体重下降、消瘦,2周后渐渐恢复,但5-6周左右再次出现进食减少,体重下降,部分裸鼠出现双下肢的瘫痪,眼、头颅等部位肿块生长,并逐渐衰竭死亡。SHI-1组中位生存期45天(40~48天),MOCK组中位生存期44天(39~49天)。S1、S2和S3细胞注射组裸鼠发病时间较SHI-1组提前,注射细胞后4-5周即再次出现进食减少,体重下降,部分裸鼠双下肢瘫痪,眼、头颅等部位肿块生长,并逐渐衰竭死亡。中位生存期S1组35天(33~38天)、S2组38天(35~42天)、S3组39天(37~43天)。MOCK组裸鼠生存期与SHI-1组无差别,S1、S2和S3细胞注射组裸鼠较SHI-1组生存期缩短(P<.05)。对照组裸鼠的各脏器组织无异常病理改变,实验组各组胃、肝脏、肺、肾脏、脑等多脏器组织均出现明显的白血病细胞浸润。S1、S2和S3细胞注射组裸鼠骨髓涂片中人白血病细胞的比例大于SHI-1组(P<.05)。除空白对照组外,各组裸鼠体内形成的肿块、肝脏、肾脏、肺、睾丸、心脏、胃、脑、肠以及骨髓中均扩增出MLL/AF6融合基因,并且S1、S2和S3组裸鼠脏器累及多于SHI-1组和MOCK组(P<.05)。空白对照组裸鼠骨髓中未检测到人CD45阳性细胞,S1、S2和S3细胞注射组裸鼠中人CD45阳性细胞的表达高于SHI-1组和MOCK组(P<.05)。
结论
(1)本研究发现SHI-1细胞在mRNA水平和蛋白水平均高表达TIMP-2、MT1-MMP、MMP-2。
(2) TIMP-2、MT1-MMP、MMP-2这些分子的高表达通过促进MMP-2的活化,增强了SHI-1细胞的体外侵袭力。
(3)骨髓基质细胞和SHI-1细胞共培养增强了细胞的体外侵袭力。
(4)内源性上调1.5倍-2.5倍的TIMP-2不但增强了SHI-1细胞的体外侵袭力,而且使SHI-1细胞在裸鼠体内形成了更为广泛和严重的白血病浸润,从而导致裸鼠白血病的发病时间提前,生存期缩短。因此上调TIMP-2对SHI-1细胞的体内外侵袭力发挥的均是增强作用,而不是抑制作用。
综上所述:本研究表明SHI-1细胞在mRNA和蛋白水平结构性高表达MMP-2、MT1-MMP、TIMP-2。上调1.5倍-2.5倍TIMP-2不但和高表达TIMP-2、MMP-2、MT1-MMP一样在骨髓基质细胞存在的条件下通过促进MMP-2的活化增强了细胞的体外侵袭力,而且增强了SHI-1细胞的体内侵袭力,在裸鼠体内形成了更为广泛和严重的白血病浸润。本研究结果提示对伴有EMI的AML患者,尤其是高表达MMP-2和MT1-MMP的,增加TIMP-2反而导致负面的效果,需要仔细分析体内TIMP/MMP和其它细胞表面分子的恰当比例和平衡,才能更好地辨别和改进急性白血病髓外浸润的靶向治疗策略。
【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.
急性白血病常伴有髓外浸润(EMI)的发生,急性髓细胞白血病(AML)中EMI的发生率约为20%–40%,并且多见于FAB分型中的M4、M5亚型。伴有EMI的患者大多数诱导化疗后的缓解率低,总体生存时间短,预后差。阐明白血病髓外浸润的病理发生过程及机制,有助于预防白血病的复发,改善白血病的预后。研究表明AML的EMI如同肿瘤的侵袭和转移一样,需要白血病细胞克服ECM屏障。已有研究报道急性白血病患者高表达基质金属蛋白酶(MMPs),并且提示这些高表达可能与EMI的特性有关,但是MMPs及其抑制剂在AML的EMI中确切作用和机制仍不清楚。SHI-1细胞是本研究所建立的一株具有高浸润特性的人类急性单核细胞白血病细胞系。本实验室前期研究中已用SHI-1细胞在裸鼠体内建立了白血病多脏器浸润的动物模型,并报道上调组织型基质金属蛋白酶抑制剂2 (TIMP-2)促进了SHI-1细胞在裸鼠体内的浸润。在此基础上,本课题将研究TIMP-2、Ⅰ型膜型基质金属蛋白酶(MT1-MMP)、基质金属蛋白酶2(MMP-2)的表达对SHI-1细胞体内外侵袭力的影响,探索白血病髓外浸润的机制。
方法
第一部分体外培养SHI-1、NB4、K562、U937、THP-1细胞和骨髓基质细胞(BMSCs)。定量PCR检测五株细胞的TIMP-2、MT1-MMP、MMP-2的mRNA表达水平,Western blot检测蛋白表达水平。RNA干扰分别沉默SHI-1细胞的TIMP-2、MT1-MMP、MMP-2。构建TIMP-2逆转录病毒载体,转染SHI-1细胞,G418筛选出多克隆细胞,RT-PCR鉴定有外源性TIMP-2基因整合后,经有限稀释法挑选出单克隆细胞S1、S2、S3。PCR鉴定单克隆细胞的外源性TIMP-2基因整合。定量RT-PCR检测单克隆细胞TIMP-2、MT1-MMP、MMP-2的mRNA表达水平,Western blot检测蛋白表达水平。明胶酶谱法测定五种细胞株、单克隆细胞、干扰后的细胞单独培养或和BMSCs共培养24小时后上清中MMP-2酶原(proMMP-2)分泌和活化的MMP-2的表达。收集BMSCs单独无血清培养24h、48h、72h后的上清检测proMMP-2的分泌和活化的MMP-2。跨膜侵袭实验观察各种细胞单独培养或和BMSC共培养24小时后侵袭人工基质膜的能力。
第二部分对4周龄的BALB/c裸鼠进行脾脏切除(splenectomy)、环磷酰胺腹腔注射(cytoxan intraperitoneal injection)及全身亚致死剂量辐照(sublethal irradiation)等预处理(SCI预处理)后随机分为:空白对照组(n=5)、实验组(SHI-1组、MOCK组、S1组、S2组、S3组,每组n=15)。经尾静脉接种1×107含不同细胞的IMDM液0.2ml/只后,在层流柜中无菌饲养观察至注射后60天。于裸鼠濒死前或死亡后即行解剖,留取骨髓涂片,瑞氏染色观察有无异常的白血病细胞;各脏器常规病理切片,观察异常白血病细胞在各脏器中的浸润情况;RT-PCR检测裸鼠各脏器中人白血病细胞MLL/AF6融合基因的表达。流式细胞仪(FCM)检测裸鼠股骨骨髓中人CD45阳性细胞比例。
结果
第一部分SHI-1细胞株的TIMP-2、MT1-MMP、MMP-2的mRNA表达和蛋白表达均高于其它细胞株(P<.05)。SHI-1细胞无血清培养以及和BMSC共培养24h后上清中的proMMP-2(72KDa)和活化的MMP-2(62KDa)含量及体外侵袭率均高于其它细胞株(P<.05)。并且共培养24h后proMMP-2的分泌、MMP-2的活化和体外侵袭率均高于单独培养的(P<.05)。BMSC单独培养24h后的上清中检测不到proMMP-2的分泌和MMP-2的活化,直到48h才能检测到,72h后达高峰。RNA干扰沉默TIMP-2、MT1-MMP、MMP-2基因的效率为85%-98%。基因沉默SHI-1细胞TIMP-2、MMP-2、MT1-MMP后,细胞侵袭率均分别下降60%-70%、50%-60%、40%-50%(P<.05)。干扰后的条件培养基中检测不到活化的MMP-2片段。RT-PCR鉴定转染了TIMP-2 cDNA的多克隆细胞和单克隆细胞均有外源性TIMP-2基因的整合和表达。单克隆细胞S1、S2、S3 TIMP-2的mRNA表达水平分别是SHI-1细胞的3.2倍、2.2倍、1.7倍(P<.05),蛋白表达水平分别上调2.6倍、1.9倍、1.5倍(P<.01),体外侵袭率增加了1.5倍-2.5倍(P<.05),活化的MMP-2增加了1.5倍-3倍(P<.01)。单克隆细胞的MT1-MMP、MMP-2的表达和条件培养基中的proMMP-2表达和SHI-1细胞没有差异。
第二部分对照组裸鼠注射后1-2周进食减少、体重下降、消瘦,2周后逐渐恢复正常,生存期大于60天以上而无死亡。SHI-1组和MOCK组裸鼠尾静脉注射细胞后,同样1-2周进食减少、体重下降、消瘦,2周后渐渐恢复,但5-6周左右再次出现进食减少,体重下降,部分裸鼠出现双下肢的瘫痪,眼、头颅等部位肿块生长,并逐渐衰竭死亡。SHI-1组中位生存期45天(40~48天),MOCK组中位生存期44天(39~49天)。S1、S2和S3细胞注射组裸鼠发病时间较SHI-1组提前,注射细胞后4-5周即再次出现进食减少,体重下降,部分裸鼠双下肢瘫痪,眼、头颅等部位肿块生长,并逐渐衰竭死亡。中位生存期S1组35天(33~38天)、S2组38天(35~42天)、S3组39天(37~43天)。MOCK组裸鼠生存期与SHI-1组无差别,S1、S2和S3细胞注射组裸鼠较SHI-1组生存期缩短(P<.05)。对照组裸鼠的各脏器组织无异常病理改变,实验组各组胃、肝脏、肺、肾脏、脑等多脏器组织均出现明显的白血病细胞浸润。S1、S2和S3细胞注射组裸鼠骨髓涂片中人白血病细胞的比例大于SHI-1组(P<.05)。除空白对照组外,各组裸鼠体内形成的肿块、肝脏、肾脏、肺、睾丸、心脏、胃、脑、肠以及骨髓中均扩增出MLL/AF6融合基因,并且S1、S2和S3组裸鼠脏器累及多于SHI-1组和MOCK组(P<.05)。空白对照组裸鼠骨髓中未检测到人CD45阳性细胞,S1、S2和S3细胞注射组裸鼠中人CD45阳性细胞的表达高于SHI-1组和MOCK组(P<.05)。
结论
(1)本研究发现SHI-1细胞在mRNA水平和蛋白水平均高表达TIMP-2、MT1-MMP、MMP-2。
(2) TIMP-2、MT1-MMP、MMP-2这些分子的高表达通过促进MMP-2的活化,增强了SHI-1细胞的体外侵袭力。
(3)骨髓基质细胞和SHI-1细胞共培养增强了细胞的体外侵袭力。
(4)内源性上调1.5倍-2.5倍的TIMP-2不但增强了SHI-1细胞的体外侵袭力,而且使SHI-1细胞在裸鼠体内形成了更为广泛和严重的白血病浸润,从而导致裸鼠白血病的发病时间提前,生存期缩短。因此上调TIMP-2对SHI-1细胞的体内外侵袭力发挥的均是增强作用,而不是抑制作用。
综上所述:本研究表明SHI-1细胞在mRNA和蛋白水平结构性高表达MMP-2、MT1-MMP、TIMP-2。上调1.5倍-2.5倍TIMP-2不但和高表达TIMP-2、MMP-2、MT1-MMP一样在骨髓基质细胞存在的条件下通过促进MMP-2的活化增强了细胞的体外侵袭力,而且增强了SHI-1细胞的体内侵袭力,在裸鼠体内形成了更为广泛和严重的白血病浸润。本研究结果提示对伴有EMI的AML患者,尤其是高表达MMP-2和MT1-MMP的,增加TIMP-2反而导致负面的效果,需要仔细分析体内TIMP/MMP和其它细胞表面分子的恰当比例和平衡,才能更好地辨别和改进急性白血病髓外浸润的靶向治疗策略。
【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.
引文
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