NPM1突变基因调控K562白血病细胞侵袭表型的相关机制

目的探讨CXCR4、Angs在NPM1突变参与调控的白血病细胞浸润转移中的作用,以期进一步明确NPM1突变在白血病浸润转移中的调控机制。方法通过基因转染构建稳定表达NPM1突变蛋白的K562白血病细胞株（K562-mA）。qRT—PCR检测各组细胞CXCR4、Ang-1／2的mRNA表达水平;Western免疫印迹和流式细胞仪分别检测细胞CXCR4总蛋白和膜蛋白的表达。结果建立了稳定表达NPM突变基因的K562-mA细胞株。与未处理组和空载体转染组相比,K562-mA细胞CXCR4的mRNA和蛋白表达水平显著增高;Ang-1mRNA表达水平明显降低、Ang-2mRNA表达水平明显增高。结论CXCR4、Ang-1／2可能在MPM1突变调控白血病细胞的浸润转移中发挥重要作用。     

NPM1基因突变对急性白血病细胞系体外侵袭能力的影响

核仁磷酸蛋白基因(nucleophosmin,NPM1)突变是目前急性髓系白血病发生突变率最高的基因改变,与白血病的发生发展密切相关。为探讨NPM1突变对白血病细胞体外侵袭能力的影响,将载体pEGFPC1-NPM1-mA转染THP-1白血病细胞系,筛选稳定表达NPM A型突变蛋白(NPM1-mA)的白血病细胞株(THP-1-mA)。通过transwell迁移实验、Matrigel侵袭实验以及细胞粘附实验来观察THP-1-mA细胞体外浸润转移能力的改变。结果发现,THP-1-mA细胞的体外迁移能力和侵袭能力明显高于亲代THP-1细胞;此外,THP-1-mA细胞对纤维连接蛋白的粘附能力也显著高于THP-1细胞。因此,我们的研究结果提示,NPMI突变可增强白血病细胞的体外侵袭能力,这有利于进一步明确NPM1突变基因在白血病细胞恶性转化中的调控作用。     

NPM1突变基因表达抑制K562白血病细胞体外增殖和侵袭

核仁磷酸蛋白(nucleophosmin,NPM1)突变是近年发现的在急性髓系白血病中发挥重要作用的基因改变,为探讨NPM1突变对K562白血病细胞体外增殖和侵袭能力的影响,将载体pEGFPC1-NPM1-mA转染K562细胞系,构建稳定表达NPM1突变蛋白的白血病细胞株(K562-mA)。利用细胞生长曲线观察细胞体外增殖能力;流式细胞仪检测细胞周期进程改变;细胞粘附、Transwell实验分别用以观察细胞体外粘附、迁移及侵袭能力。结果发现,NPM1突变转染后K562细胞体外增殖能力明显减弱;同时G1期细胞比例明显增高,S期细胞比例显著减低。与未处理组和空载体转染组细胞相比,K562-mA细胞体外迁移能力有所增加,但细胞粘附及侵袭能力却明显减弱。提示NPM1突变基因的表达能够抑制白血病细胞体外增殖和侵袭能力,为进一步深入探讨NPM1突变在白血病发生发展中的调控机制奠定了良好的基础。     

核仁磷酸蛋白基因突变对K562白血病细胞分化的影响

核仁磷酸蛋白基因（nucleophosmin,NPM1）突变在急性髓系白血病的发生发展中发挥着重要作用,而与白血病分化阻滞的关系尚未完全阐明。为探讨NPM1基因突变对白血病细胞体外分化的影响,将携带NPM1 A型突变（NPM1-mA）的表达质粒载体pEGFPC1-NPM1-mA转染白血病K562细胞系,构建稳定表达NPM1-mA蛋白的细胞株（K562 mA）,同时设立野生型NPM1转染组（K562 wt）、空载体转染组（K562 C1）和未处理组（K562）为对照。利用豆蔻酰佛波醇乙酯（PMA）诱导各组细胞分化,瑞氏–吉姆萨染色观察细胞分化的形态改变,计算诱导分化率;相差显微镜计数贴壁细胞数量;流式细胞术分析细胞表面分化抗原CD41的表达。结果显示,PMA作用72 h后,与对照组相比,K562 mA组细胞的诱导分化率及贴壁细胞数明显降低（P〈0.05）;同时,CD41的表达受到显著抑制（P〈0.01）。提示NPM1基因突变能够阻滞白血病细胞系K562的体外分化。     

干扰PML对人髓系白血病THP-1细胞增殖和凋亡的影响及机制探讨

早幼粒白血病(promyelocytic leukemia,PML)基因与维甲酸受体α(retinoic acid receptorα,RARα)基因形成PML-RARα融合基因是急性早幼粒细胞白血病(acute promyelocytic leukemia,APL)发生的分子基础,而PML在除APL外的其他髓系白血病亚型中的作用研究少见报道。为探讨PML在非APL的髓系白血病恶性表型中的调控作用及潜在机制,该文首先通过q PCR和Western blot检测了5株非APL来源的髓系白血病细胞中PML的表达水平。接着将靶向PML基因的sh RNA慢病毒(sh PML group)感染高表达PML的THP-1细胞;同时,设立未处理对照组(Mock group)和阴性对照组(Scramble group),嘌呤霉素筛选稳定表达sh PML的细胞株。q PCR和Western blot检测sh RNA干扰效率;CCK-8检测细胞体外增殖活性,克隆形成实验检测细胞体外克隆形成能力;流式细胞术检测细胞凋亡率;Western blot检测凋亡相关蛋白质Bcl-2、Bax水平和AKT及下游靶分子Foxo3a磷酸化蛋白质水平的改变。结果显示,5株髓系白血病细胞中PML m RNA和蛋白质水平不同,其中以THP-1细胞中的PML表达水平较高。靶向PML基因的sh RNA慢病毒感染THP-1细胞后,PML m RNA和蛋白质水平均明显下降,提示成功构建稳定表达sh PML的THP-1细胞株。与Mock组和Scramble组相比,sh PML组的细胞增殖能力显著增强(P0.05),细胞凋亡率显著降低(P0.05);同时,抗凋亡蛋白Bcl-2水平升高、促凋亡蛋白Bax水平降低;此外,干扰PML表达可增加p AKT(S473)及下游靶分子p Foxo3a(S253)的蛋白质水平,而总AKT和Foxo3a的蛋白质水平未见明显变化。该研究的结果提示,PML基因可能通过调控AKT/Foxo3a信号通路活性抑制白血病的恶性表型,表明PML在不同白血病型别中发挥着不同的作用。     

丹参单体IH764-3通过下调H2O2刺激的肝星状细胞FAK水平影响MMP-13和TIMP-1的表达

目的：研究丹参单体IH764—3对H2O2刺激的肝星状细胞（HSC）基质金属蛋白酶-13（MMP-13）、基质金属蛋白酶组织抑制因子-1（TIMP-1）表达的影响以及此过程中粘着斑激酶（FAK）的变化。方法：应用RT-PCR方法检测MMP-13及FAKmRNA表达,原位杂交方法检测TIMP-1mRNA水平,Western blotting技术检测FAK及TIMP-1蛋白表达。结果：IH764—3干预组的MMP-13mRNA在2h的表达强度明显上调,而TIMP-1mRNA表达明显受抑,FAKmRNA表达强度明显下调;IH764—3干预24h组FAK及TIMP-1蛋白表达受抑制。结论：丹参单体IH764—3可以诱导MMP-13表达,抑制TIMP-1表达,下调FAK表达是其中的机制之一。     

葛根素对糖尿病大鼠肾组织MMP-2的表达及活性的影响

为探讨葛根素对糖尿病大鼠肾组织基质金属蛋白酶2(MMP-2)及活性表达的影响,采用单侧肾切除大鼠ip链脲佐菌素诱发糖尿病模型的方法,每日ip葛根素注射液,共16周。采用原位杂交法检测肾小球MMP-2、TIMP-2mRNA表达,流式细胞术和免疫组织化学检测肾皮质MMP-2、TIMP-2及Ⅳ型胶原表达;酶谱分析检测肾皮质MMP-2活性变化。结果发现糖尿病组较对照组肾小球MMP-2mRNA及蛋白表达降低而TIMP-2mRNA及蛋白表达升高,Ⅳ型胶原表达亦增加,MMP-2活性降低,肾功能恶化;葛根素用药组较糖尿病组MMP-2mRNA及蛋白表达升高而TIMP-1、Ⅳ型胶原表达减少,MMP-2活性部分恢复,肾功能改善。表明葛根素可能部分是通过调节肾小球MMP-2蛋白表达及活性的改变从而减轻肾小球细胞外基质沉积,保护糖尿病大鼠的肾功能和形态。     

MMP-2, -9, -14及其抑制剂TIMP-1, -2, -3在恒河猴周期黄体中的时空表达

用原位杂交和免疫组织化学方法研究了基质金属蛋白酶MMP-2, -9, -14及其组织抑制因子TIMP-1, -2, -3在恒河猴周期黄体发育不同阶段的协同表达. 结果显示: MMP-2 mRNA及其蛋白主要表达在早中期发育黄体的内皮细胞上, 在晚期黄体发生萎缩时则大量表达于黄体细胞; MMP-9, -14及其TIMP-1, -2, -3主要表达于黄体细胞; MMP-14 mRNA在早期和晚期黄体中高表达, MMP-9蛋白只在晚期黄体中高表达; TIMP-3蛋白在早、中、晚三期黄体中表达均较高, 但很明显晚期表达降低. 结果提示: MMP/TIMP系统参与灵长类黄体发育的调控, MMP-2, -14及其TIMP-1, -3可能参与黄体的形成和功能维持, 同时MMP-2, -9, -14及其TIMP-1, -2, -3在黄体萎缩期的协同表达, 提示它们可能在黄体发生萎缩时发挥作用.     

PTEN蛋白表达和明胶酶及其抑制因子在乳腺癌侵袭转移中的关系

目的研究乳腺癌中明胶酶(MMP-2、MMP-9)及其抑制物(TIMP-1)与抑癌基因PTEN产物PTEN蛋白的表达在肿瘤侵袭转移中的关系.方法应用免疫组织化学S-P法检测98例浸润性乳腺癌组织MMP-2、MMP-9及TIMP-1和PTEN蛋白的表达.结果乳腺癌MMP-2、MMP-9与PTEN表达呈显著负相关,而TIMP-1与PTEN蛋白表达呈显著正相关(P<0.05).在PTEN低表达组中,MMP-2、MMP-9的表达与腋窝淋巴结受累呈正相关;在PTEN高表达组中,MMP-2、MMP-9的表达与腋窝淋巴结受累呈正相关,TIMP-1与腋窝淋巴结受累呈负相关.结论在乳腺癌侵袭转移中MMP-2、MMP-9、TIMP-1与PTEN蛋白表达显著相关.MMP-2、MMP-9和TIMP-1对肿瘤细胞侵袭转移的作用可能在一定程度上受到PTEN的调控.     

缺血后处理对缺血／再灌注大鼠心肌间质保护效应机制的探讨

目的：观察缺血后处理（IPIC）对缺血／再灌注（I／R）大鼠心肌基质金属蛋白酶-2（MMP-2）和基质金属蛋白酶抑制剂-2（TIMP-2）变化的影响,探讨IPTC保护I／R心脏间质的机制。方法：24只健康雄性SD大鼠随机分为3组（n：8）：假手术组（SC组）、I／R组和IPTC组。记录各组左室血流动力学变化,观察心肌胶原含量,测定血浆中肌酸激酶（CK）和乳酸脱氢酶（LDH）浓度。以Westernblot法测定心肌组织中MMP-2和TIMP-2蛋白表达水平,以实时定量PCR（RT-PCR）法检测MMP-2和TIMP-2的表达水平。结果：与sC组相比,I／R组心肌胶原含量和左室舒缩功能明显降低,血浆cK、LDH活力和心肌MMP-2蛋白表达及mRNA水平明显升高,TIMP-2蛋白及mRNA水平明显降低;而IPTC组,大鼠心肌胶原含量和左室舒缩功能明显升高,血浆cK、LDH活力和心肌MMP-2蛋白表达及mRNA水平降低,TIMP-2蛋白及mRNA水平升高。结论：IPTC对再灌注损伤心肌间质有保护作用,其机制可能与抑制心肌中MMP-2表达,促进TIMP-2表达有关。     

Nucleophosmin Mutants Promote Adhesion,Migration and Invasion of Human Leukemia THP-1 Cells through MMPs Up-regulation via Ras/ERK MAPK Signaling

Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1) has been defined as a unique subgroup in the new classification of myeloid neoplasm, and the AML patients with mutated NPM1 frequently present extramedullary infiltration, but how NPM1 mutants regulate this process remains elusive. In this study, we found that overexpression of type A NPM1 gene mutation (NPM1-mA) enhanced the adhesive, migratory and invasive potential in THP-1 AML cells lacking mutated NPM1. NPM1-mA had up-regulated expression and gelatinolytic matrix metalloprotease-2 (MMP-2)/MMP-9 activity, as assessed by real-time PCR, western blotting and gelatin zymography. Following immunoprecipitation analysis to identify the interaction of NPM1-mA with K-Ras, we focused on the effect of NPM1-mA overexpression on the Ras/Mitogen-activated protein kinase (MAPK) signaling axis and showed that NPM1-mA increased the MEK and ERK phosphorylation levels, as evaluated by western blotting. Notably, a specific inhibitor of the ERK/MAPK pathway (PD98059), but not p38/MAPK, JNK/MAPK or PI3-K/AKT inhibitors, markedly decreased the cell invasion numbers in a transwell assay. Further experiments demonstrated that blocking the ERK/MAPK pathway by PD98059 resulted in reduced MMP-2/9 protein levels and MMP-9 activity. Additionally, NPM1-mA overexpression had down-regulated gene expression and protein production of tissue inhibitor of MMP-2 (TIMP-2) in THP-1 cells. Furthermore, evaluation of gene expression data from The Cancer Genome Atlas (TCGA) dataset revealed that MMP-2 was overexpressed in AML patient samples with NPM1 mutated and high MMP-2 expression associated with leukemic skin infiltration. Taken together, our results reveal that NPM1 mutations contribute to the invasive potential of AML cells through MMPs up-regulation via Ras/ERK MAPK signaling pathway activation and offer novel insights into the potential role of NPM1 mutations in leukemogenesis.     

Differential regulation of MMP-1/9 and TIMP-1 secretion in human monocytic cells in response to Mycobacterium tuberculosis.

In tuberculosis, matrix metalloproteinase (MMP) secretion is involved in leukocyte migration to sites of infection but in excess may contribute to tissue destruction. We demonstrate that human monocytic THP-1 cells and primary monocytes secrete MMP-1 (52 kD collagenase) when phagocytosing live, virulent M. tuberculosis but not inert latex. The magnitude of MMP-1 secretion was approximately 10-fold less when compared to MMP-9 (92 kD gelatinase) secretion. MMP-1 secretion was also relatively delayed (detected at 24 h vs. 4 h). M. tuberculosis, zymosan or latex stimulate similar TIMP-1 secretion within 8 h and increasing over 24 h. MMP-1/9 secretion was decreased by inhibitors of protein kinase (PK) C, PKA or tyrosine kinases (PTK) in a concentration-dependent manner. In contrast, TIMP-1 secretion was not affected by PKC or PTK blockade and only somewhat reduced by high level PKA inhibition. In summary, M. tuberculosis-infected monocytes secrete MMP-1 at lower concentrations than MMP-9 and such MMP secretion is regulated by multiple upstream signalling pathways which do not control TIMP-1 secretion. Divergent effects of i on MMP and TIMP secretion from monocytes may be important in influencing matrix degradation in vivo.     

Matrix metalloproteinases 2 and 9 as the factors of head and neck tumor metastases

The levels of metalloproteinases (MMP-2,-9), their tissue inhibitors (TIMP-1,-2) and extracellular matrix metalloproteinase inducer (EMMPRIN) were studied in tumor tissue and blood serum from patients with head and neck squamous cell carcinoma. Immunohistochemical investigation showed much higher expression of MMP-9 and TIMP-1 in tumor tissue compared with MMP-2 and TIMP-2. There was different distribution of the investigated parameters (except TIMP-1) in cancer cells and stroma. Accumulation of MMP-2, MMP-9, and TIMP-2 was found mainly in cell elements (fibrocytes, leukocytes, etc.) and in stromal extracellular space. Expression of EMMPRIN was significantly higher in tumor cells than in stromal cells. It is possible that carcinoma cells express EMMPRIN, which may increase MMP production by surrounding cells. There was significant decrease of TIMP-1 expression in carcinoma cells with N1 grade of metastasis than in tumors without metastasis. The level of TIMP-1 in blood serum from patients with tumor metastases to regional lymph nodes was lower than in serum from patients without metastases. Thus, MMP-9 and TIMP-1 play an important role in the development of head and neck squamous cell carcinoma and the TIMP-1 level in blood serum and cancer tissues is linked to the first grade of regional lymph node metastasis.     

The calpastatin-derived calpain inhibitor CP1B reduces mRNA expression of matrix metalloproteinase-2 and -9 and invasion by leukemic THP-1 cells

The ubiquitous proteases mu- and m-calpain are Ca(2+)-dependent cysteine endopeptidases. Besides involvement in a variety of physio(patho)logical processes, recent studies suggest a pivotal role of calpains in differentiation of hematopoietic cells and tumor cell invasion. However, the precise actions of calpains and their endogenous inhibitor, calpastatin, in these processes are only partially understood. Here we have studied the role of the calpain/calpastatin system in the invasion of leukemic cells under basal and differentiation-stimulating conditions. To further differentiate the human leukaemic cell line THP-1 (monocytic), the cells were treated for 24 hours with the differentiation-stimulating reagents phorbol 12-myristate 13-acetate (PMA) and dimethyl sulfoxide (DMSO). Macrophage- and granulocyte-like differentiation was confirmed by induction of vimentin expression as well as by microscopic and fluorescence-assisted cytometric analysis. Extracellular matrix (ECM) invasion of both the basal and differentiation-stimulated cells in a Matrigel assay was inhibited by pre-incubation of the cells with the specific calpain inhibitor CP1B for 24 hours. Inhibition of invasiveness correlated with decreased mRNA expression and secretion of the matrix metalloproteinases MMP-2 and MMP-9. In contrast, addition of CP1B only during the invasion process did neither influence transmigration nor MMP release. This is the first report showing that the calpain/calpastatin system mediates MMP-mRNA expression of the leukemic THP-1 cells and as a consequence their invasiveness.     

Cellular activation of MMP-2 (gelatinase A) by MT2-MMP occurs via a TIMP-2-independent pathway

The role of membrane-type (MT) 2-matrix metalloproteinase (MMP) in the cellular activation of MMP-2 and the tissue inhibitor of matrix metalloproteinase (TIMP) requirements for this process have not been clearly established. To address these issues a TIMP-2-free cell line derived from a Timp2-/- mouse was transfected for stable cell surface expression of hMT2-MMP. Untransfected cells did not activate endogenous or exogenous TIMP-2-free MMP-2 unless both TIMP-2 and concanavalin A (ConA) were added. Transfected cells expressing hMT2-MMP efficiently activated both endogenous and exogenous MMP-2 (within 4 h) via the 68-kDa intermediate in the absence of TIMP-2 and ConA. In contrast, activation of MMP-2 by Timp2-/- cells expressing recombinant hMT1-MMP occurred more slowly (12 h) and required the addition of 0.3-27 nm TIMP-2. Addition of TIMP-2 or TIMP-4 did not enhance MMP-2 activation by MT2-MMP at any concentration tested; furthermore, activation was inhibited by both TIMPs at concentrations >9 nm, consistent with the similar association rate constants (k(on)) calculated for the binding of TIMP-4 and TIMP-2 to MT2-MMP (3.56 x 10(5) m(-1) s(-1) and 6.52 x 10(5) m(-1) s(-1), respectively). MT2-MMP-mediated activation involved cell surface association of the MMP-2 in a hemopexin carboxyl-terminal domain (C domain)-dependent manner: Exogenous MMP-2 hemopexin C domain blocked activation, and cells expressing hMT2-MMP did not bind or activate a truncated form of MMP-2 lacking the hemopexin C domain. These studies demonstrate the existence of an alternative TIMP-2-independent pathway for MMP-2 activation involving MT2-MMP, which may be important in mediating MMP-2 activation in specific tissues or pathologies where MT2-MMP is expressed.     

Epithelial and bacterial metalloproteinases and their inhibitors in H. pylori infection of human gastric cells

To test the hypothesis that Helicobacter pylori regulates gastric cell secretion of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), culture media from infected and uninfected human gastric adenocarcinoma (AGS) cells were analyzed by zymography, MMP activity assays, and immunoblotting. AGS cells secreted gelatinolytic (prominently 90 kDa) and caseinolytic (110 kDa) activity together with MMP-1, MMP-3, and TIMP-1, TIMP-2, and TIMP-3 isoforms. H. pylori secreted caseinolytic activity (60 kDa), MMP-3-like enzyme activity, and TIMP-3 immunoreactivity. H. pylori infection increased the 110-kDa caseinolytic activity and induced new gelatinolytic (~35 kDa) and caseinolytic (22 kDa) activities. Infection also increased both basal secretion and activation of MMP-1 and MMP-3, enhanced TIMP-3 secretion, and increased the formation of MMP-3/TIMP-3 complexes. TIMP-1 and TIMP-2 secretion were unchanged. Normal AGS cells showed a pancellular distribution of TIMP-3, with redistribution of immunoreactivity toward sites of bacterial attachment after H. pylori infection. The data indicate that MMP and TIMP secretion by AGS cells is modulated by H. pylori infection and that host MMP-3 and a TIMP-3 homolog expressed by H. pylori mediate at least part of the host cell response to infection.