N-乙酰氨基葡萄糖转移酶Ⅴ过表达对人肝癌细胞粘着斑复合物介导的信号转导作用

为了探讨过表达N 乙酰氨基葡萄糖转移酶Ⅴ (GnT Ⅴ )后 772 1细胞侵袭、迁移等行为改变的机制 ,检测了GnT Ⅴ 772 1及pcDNA3 772 1两组细胞中与恶性表型密切相关的粘着斑激酶 (focalad hesionkinase ,FAK)、PTEN蛋白、蛋白激酶B(PKB)等重要信号分子的表达水平 ,同时测定了 2组细胞非贴壁依赖生长的能力 .利用Western印迹方法检测FAK、PTEN、PKB的表达或磷酸化水平 .利用poly hema使细胞非贴壁生长 ,2组细胞悬浮无血清培养 2 0h ,采用流式细胞仪方法检测细胞的失巢凋亡 (anoikis) .研究发现 ,转染GnT Ⅴ后的肝癌细胞的FAK表达无明显变化 ,FAK的酪氨酸磷酸化水平增高 70 %;而PTEN的表达下降了 4 9%;PKB的磷酸化增加 2 0 0 %;pcDNA3 772 1细胞已有明显凋亡 ,而转染GnT Ⅴ的 772 1细胞未发生凋亡 .结果提示 ,转染GnT Ⅴ后的肝癌细胞迁移力增强 ,可能与其FAK的磷酸化程度升高 ,激酶活力增强有关 ;而能逃逸失巢凋亡是因为PTEN的表达下降 ,PTEN蛋白的磷酸酶活性降低 ,细胞Akt PKB磷酸化水平保持在较高水平 .     

整合蛋白α5β1过表达诱导人肝癌细胞SMMC-7721“失巢凋亡”机理

为了进一步阐明整合蛋白α5 β1过表达诱导非粘着状态的人肝癌细胞SMMC 772 1失巢凋亡的机理 ,利用poly HEME阻断细胞贴壁 ,诱导失巢凋亡 ,在poly HEME包被的 96孔板中检测细胞生存率 .利用Western印迹法检测了与细胞信号转导密切相关的粘着斑激酶 (FAK)、蛋白激酶B(PKB)及生存蛋白survivin的表达水平 .α5 β1过表达细胞株在去粘附状态下的生存率明显低于对照细胞 .在生长 8d时 ,以整合蛋白不同亚单位转染的α5 β1 772 1、β1 772 1和α5 772 1细胞 ,活细胞数分别降至对照组的 4 0 %、4 5 %和 83% .这说明整合蛋白α5 β1过表达细胞株 ,特别是α5 β1 772 1和 β1 772 1细胞的失巢凋亡明显加快 .细胞悬浮生长 2 4h后 ,α5 β1过表达细胞株都有不同程度的凋亡发生 ;同时粘着斑激酶 (FAK)和蛋白激酶B(PKB)表达量及其磷酸化水平均低于对照细胞 ,而survivin表达水平在α5 β1 772 1中最高 ,在 β1 772 1中最低 .在给予天然细胞外基质纤粘连蛋白的条件下 ,α5 β1 772 1和 β1 772 1细胞的FAK和PKB的磷酸化水平却呈升高趋势 .结果说明 ,FAK和PKB的变化与细胞的粘附状态密切相关 ,并且参与整合蛋白α5 β1过表达细胞在去粘附状态下失巢凋亡的发生 .     

PTEN对SMMC—7721人肝癌细胞迁移增殖及粘着斑激酶磷酸化水平的影响

抑癌基因PTEN编码产物具有双专一性磷酸酶活性 ,并与细胞骨架张力蛋白同源。PTEN可参与粘着斑的形成和解聚而影响细胞迁移。现用PTEN表达质粒转染SMMC 772 1肝癌细胞 ,研究SMMC 772 1细胞运动能力的变化及PTEN与粘着斑激酶 (FAK)酪氨酸磷酸化水平之间的关系。PTEN过表达能够显著抑制细胞在Fn基质上的活动 :细胞在Fn基质上的迁移下降了 35 % ;在 30min和 6 0min两个时间点 ,Fn基质上细胞铺展分别降低了 2 9%和 2 6 % ;而在多聚赖氨酸基质上细胞铺展并没有变化。运用免疫沉淀和Western印迹方法 ,分析FAK及其酪氨酸磷酸化水平 ,发现PTEN过表达不影响FAK表达 ,但显著降低Fn诱导的FAK酪氨酸磷酸化水平 ,两者水平呈负相关。流式细胞仪分析细胞周期结果表明 ,PTEN抑制细胞 ,S期细胞下降了 16 %。上述结果提示 ,PTEN抑制肝癌细胞迁移铺展和增殖 ;PTEN对细胞运动的影响可能通过调节FAK酪氨酸磷酸化水平而实现。     

人肺癌细胞抑癌基因PTEN的表达与失巢凋亡的关系

应用Northern印迹、Western印迹和DNA梯形片段方法 ,研究 8株不同细胞类型的人肺癌细胞中抑癌基因PTEN的表达与失巢凋亡 (anoikis)的关系 ,并分析在此过程中蛋白激酶B(proteinkinaseB ,PKB)和粘着斑激酶(focaladhesionkinase ,FAK)的作用。发现 8株人肺癌细胞PTEN均有mRNA表达 ,且mRNA水平比较接近。但PTEN的蛋白质水平不一致 ,其中 95C、95D和A1株的PTEN蛋白未检测到 ;A549、A4、A7和L1株的PTEN蛋白有表达 ,但较低 ;而H460 株的PTEN表达较强。PTEN缺失的 95D和高表达的H460 细胞株中PTENcDNA序列分析均未发生片断缺失或点突变。RNA稳定性分析表明 ,95DmRNA稳定性较H460 明显下降。在无血清且去粘附培养条件下 ,高表达PTEN的细胞株H460 可被诱导发生失巢凋亡现象 ,在 10 %血清培养条件下可保护其免于失巢凋亡 ,而PTEN缺失的 95D等和其他PTEN低表达的细胞株却没有发生诱导失巢凋亡现象。进一步研究发现PTEN表达可降低PKB的磷酸化 ,下调FAK蛋白质的表达。结果提示各种人肺癌细胞株中PTEN蛋白表达存在显著差异。PTEN参与了失巢凋亡的发生。     

粘着斑激酶在TNF-α作用下影响SMMC-7721细胞PKB的表达水平

 探讨在肿瘤坏死因子α(TNF-α)作用下 ,粘着斑激酶 (focal adhesion kinase,FAK)对蛋白激酶 B(PKB)蛋白水平的影响 .利用构建、转染 FAK反义质粒来特异性降低 SMMC- 772 1细胞的FAK含量 ,及用 Western杂交的方法来检测 PKB的蛋白含量 .文献报道 TNF- α能够激活磷脂酰肌醇 3-激酶 (PI3K)而使 PKB发生磷酸化 .但是至于 TNF-α对 PKB蛋白水平的影响目前并无报道 .研究发现 ,当用 wortmannin特异性抑制 PI3K活性后可以显著降低 PKB的蛋白含量 .提示PI3K对维持 PKB的基础蛋白水平是必需的 .但是 TNF- α本身对 PKB的蛋白水平无明显影响 .而当用不同浓度的 TNF- α和 wortmannin处理 SMMC- 772 1细胞时 ,发现 PKB的蛋白含量随着TNF-α浓度的增加而降低 .提示 TNF-α可能除了通过 PI3K外 ,还可能通过另一条途径来下调PKB的表达 .而当用 FAK反义质粒转染 SMMC- 772 1细胞后 (FAK下降了 60 % ) ,发现在当用不同浓度的 TNF- α处理的情况下 ,FAK反义质粒转染株 AS- 772 1细胞的 PKB含量降低为对照的70 % ;而在用 TNF-α和 wortmannin处理的情况下 ,下降为对照的 40 %～ 60 % .TNF-α能够通过PI3K及另一未知途径来影响 PKB的蛋白水平 .而 FAK在 TNF- α作用下能够不通过 PI3K来影响PKB的蛋白水平 .     

超表达蛋白激酶B对SMMC 7721肝癌细胞增殖和凋亡的影响

采用脂质体转染的方法 ,将含持续激活蛋白激酶B的真核表达质粒转染到SMMC 772 1肝癌细胞中 ,研究蛋白激酶B对人肝癌细胞增殖和凋亡的影响 .用RNA印迹及蛋白激酶B测活鉴定 ,并获得稳定表达持续激活蛋白激酶B的细胞株 ,用MTT法、软琼脂克隆形成率及细胞周期测定等方法检测超表达蛋白激酶B的 772 1细胞增殖情况 ,结果显示超表达蛋白激酶B的 772 1细胞生长能力增强 ,软琼脂克隆形成率增高 ,S期细胞增多 ,p2 7Kip1表达下降 .用流式细胞术检测悬浮培养诱导的细胞失巢凋亡 ,发现超表达蛋白激酶B能抑制细胞失巢凋亡 .上述结果提示蛋白激酶B能促进肝癌细胞增殖 ,抑制细胞凋亡 .     

PTEN基因诱导人胚肾293细胞凋亡和细胞周期停滞

为了研究抑癌基因PTEN过表达对HEK293细胞凋亡和细胞周期停滞的作用,以野生型PTEN和PTEN突变子(T910G)表达质粒分别转染无PTEN表达的人胚肾293细胞,采用细胞质梯度DNA方法检测细胞凋亡,以流式细胞仪分析细胞周期.发现PTEN过表达能够诱导人胚肾293细胞质中出现梯度DNA,293细胞发生凋亡,PTEN过表达改变细胞周期分布,G0/G1期细胞增加13%,S期细胞下降15%.PTEN突变子对细胞凋亡和G1细胞停滞的影响略弱于野生型PTEN.PTEN基因过表达明显下调血小板衍生生长因子(PDGF)诱导的蛋白激酶B(PKB)和p42,p44-促分裂原活化蛋白激酶(MAPK)磷酸化水平,PTEN突变子对p42,p44-MAPK磷酸化水平的调节作用略弱于野生型PTEN.PTEN通过抑制细胞增殖,诱导细胞凋亡而影响细胞生长.     

蛋白质酪氨酸磷酸化在抗失巢凋亡的癌细胞中的失调变化

失巢凋亡是细胞与细胞外基质脱离发生的一种特定的凋亡方式 . 癌细胞抗失巢凋亡或失巢生存能力可以使之在转移过程中生存 . 业已发现癌细胞失巢生存与 PI3K-PKB/Akt 、 MAPK 这两条重要信号途径有关,但是 PI3K-PKB/Akt 、 MAPK 通路的上游酪氨酸激酶途径还不甚清楚 . 为此设计了一种基于 SH2-pTyr 特异性结合特性的功能性筛选方法,以期发现癌细胞失巢生存相关的酪氨酸磷酸化蛋白质,为最终明确酪氨酸激酶途径提供有力的实验依据 . 实验发现, MDCK 细胞悬浮培养后失巢凋亡,但癌细胞可以失巢生存 . 与这一现象相一致的是,悬浮培养后, MDCK 细胞中一系列 SH2 结合的酪氨酸磷酸化蛋白质水平急剧下降,而癌细胞中蛋白质酪氨酸磷酸化水平并不呈锚着依赖性 . 细胞悬浮培养后,随着培养时间的延长, MDCK 细胞中 Abl S SH2 结合的靶蛋白酪氨酸磷酸化水平逐渐降低,在 H460 肺癌细胞中经过短暂下降后升高, H1792 肺癌细胞随着培养时间的延长, Abl SH2 结合的靶蛋白酪氨酸磷酸化水平逐渐增加 . Fyn SH2 和 Crk SH2 结合的蛋白质分别为 FAK 和 p130Cas ,后者是重要的失巢生存信号 . 这些结果提示,酪氨酸磷酸化蛋白质可能赋予肺癌细胞失巢生存能力 . 结果也表明,功能性 SH2 筛查方法可以有效地发现肿瘤细胞中失巢生存相关的酪氨酸磷酸化蛋白质 .     

粘着斑相关非激酶对骨桥蛋白促血管平滑肌细胞迁移的抑制作用及分子机制

为阐明整合素 β3 粘着斑激酶 (FAK)信号途径在骨桥蛋白 (OPN)诱导血管平滑肌细胞(VSMC)迁移中的作用 ,用FAK磷酸化特异性抑制剂粘着斑相关非激酶 (FRNK)选择性阻断FAK磷酸化 ,观察对OPN 整合素 β3 相互作用所激活的FAK信号通路的影响及其与OPN诱导VSMC迁移之间的关系 .外源性FRNK在VSMC中的过表达可显著抑制OPN诱导的VSMC迁移 ,使跨膜迁移细胞数下降 5 0 5 8% (P <0 0 5 ) .OPN刺激不但明显诱导FAK表达 ,而且还促进其磷酸化 .外源性FRNK对OPN诱导的FAK磷酸化具有显著抑制作用 ,使磷酸化型FAK水平比相应对照细胞下降5 9 1% ,但其对FAK表达不产生明显的影响 .FRNK还具有下调整合素 β3 表达的作用 ,免疫荧光细胞化学分析结果显示 ,在转染FRNK的VSMC中 ,粘着斑蛋白的磷酸化水平降低 ,粘着斑数量明显减少 .结果提示 ,整合素 β3 FAK是介导VSMC迁移的重要信号途径 ,外源性FRNK通过下调 β3 表达、抑制FAK磷酸化和减少粘着斑蛋白磷酸化及粘着斑形成等机制 ,减弱OPN刺激信号的跨膜转导及沿胞内途径传递 ,发挥抑制OPN促VSMC迁移的效应 .     

不同亚细胞定位的黏着斑激酶对肿瘤的调控机制

黏着斑激酶(focal adhesion kinase, FAK)是一种非受体蛋白酪氨酸激酶。在细胞质黏着斑中,FAK通过调控细胞黏着斑的解聚与组装及多个蛋白质的磷酸化从而调节细胞的增殖和迁移。FAK也定位于细胞核和内体中。在细胞核中,FAK可导致p53的多聚泛素化及降解,从而影响正常细胞的存活和肿瘤细胞的生长。此外,核FAK还能够通过调节细胞因子和趋化因子的表达促进肿瘤免疫逃逸的发生。在内体中,FAK通过FERM结构域定位到内体并被激活。内体中整合素的信号转导时间比质膜上持续的时间更长,并且能够阻止肿瘤细胞发生失巢凋亡,因此,内体FAK可通过抵抗失巢凋亡促进肿瘤细胞的转移。现就不同亚细胞定位的FAK对肿瘤发生和发展的调控作用及机制进行讨论。     

TGF-beta 1 modulated the expression of alpha 5 beta 1 integrin and integrin-mediated signaling in human hepatocarcinoma cells

Integrins are a family of cell surface adhesion molecules which mediate cell adhesion and initiate signaling pathways that regulate cell spreading, migration, differentiation, and proliferation. TGF-beta is a multifunctional factor that induces a wide variety of cellular processes. In this study, we show that, TGF-beta 1 treatment enhanced the amount of alpha 5 beta 1 integrin on cell surface, the mRNA level of alpha 5 subunit, and subsequently stimulated cell adhesion onto a fibronectin (Fn) and laminin (Ln) matrix in SMMC-7721 cells. TGF-beta 1 could also promote cell migration. Furthermore, our results showed that TGF-beta1 treatment stimulated the tyrosine phosphorylation level of FAK, which can be activated by the ligation and clustering of integrins. PTEN can directly dephosphorylate FAK, and the results that TGF-beta 1 could down-regulate PTEN at protein level suggested that TGF-beta 1 might stimulate FAK phosphorylation through increasing integrin signaling and reducing dephosphorylation of FAK. These studies indicated that TGF-beta 1 and integrin-mediated signaling act synergistically to enhance cell adhesion and migration and affect downstream signaling molecules of hepatocarcinoma cells.     

Focal adhesion kinase affects the sensitivity of human hepatocellular carcinoma cell line SMMC-7721 to tumor necrosis factor-alpha/cycloheximide-induced apoptosis by regulating protein kinase B levels.

Most cell lines are resistant to tumor necrosis factor-alpha (TNF-alpha) cytotoxicity and require cotreatment of TNF-alpha with cycloheximide (Chx) to undergo apoptosis. Recently, the serine/threonine protein kinase, protein kinase B has been demonstrated to protect cells from apoptosis induced by TNF-alpha. In this study, we have shown that the human hepatocellular carcinoma cell line, SMMC-7721, was insensitive to TNF-alpha cytotoxicity and underwent apoptosis quickly in the presence of TNF-alpha and Chx. PKB levels decreased during TNF-alpha/Chx-induced apoptosis. No significant change in PKB levels was found in the presence of TNF-alpha or Chx alone. It seemed that the level of PKB closely correlated with apoptosis. The protein level of focal adhesion kinase (FAK) was reduced by 66% by transfecting FAK antisense cDNA recombinant vector into SMMC-7721 cells. We determined the apoptosis-induced effect of TNF-alpha/Chx on the FAK antisense cDNA transfectant cells. The results indicated that the percentage of apoptotic cells was enhanced at lower doses of TNF-alpha (10, 20 or 50 U.mL(-1)) and decreased at a higher dose of TNF-alpha (1000 U.mL(-1)) in the transfected cells as compared to the control. Correspondingly, in the FAK antisense cDNA transfectant cells treated with lower doses of TNF-alpha in presence of 10 microg.mL(-1) Chx, the PKB level was lower, but in the FAK antisense cDNA transfectants treated with higher doses of TNF-alpha in presence of 10 microg.mL(-1) Chx, the PKB level was higher. In response to TNF-alpha alone, FAK antisense cDNA transfectants showed a decrease in the level of PKB. However, in the case of TNF-alpha cotreated with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PtdIns3K), the FAK antisense cDNA transfectants produced significantly less amounts of PKB than the control. It seemed that FAK could stimulate PKB levels through a pathway not involving PtdIns3K. These results suggest that FAK can affect the sensitivity of SMMC-7721 cells to TNF-alpha/Chx-induced apoptosis in a biphasic manner by regulating PKB levels.     

An altered fibronectin matrix induces anoikis of human squamous cell carcinoma cells by suppressing integrin alpha v levels and phosphorylation of FAK and ERK

Fibronectin regulates many cellular processes, including migration, proliferation, differentiation, and survival. Previously, we showed that squamous cell carcinoma (SCC) cell aggregates escape suspension-induced, p53-mediated anoikis by engaging in fibronectin-mediated survival signals through focal adhesion kinase (FAK). Here we report that an altered matrix, consisting of a mutated, nonfunctional high-affinity heparin-binding domain and the V region of fibronectin (V⁺H⁻), induced anoikis in human SCC cells; this response was blocked by inhibitors of caspase-8 and caspase-3. Anoikis was mediated by downregulation of integrin alpha v in a panel of SCC cells and was shown to be proteasome-dependent. Overexpression of integrin alpha v or FAK inhibited the increase in caspase-3 activation and apoptosis, whereas suppression of alpha v or FAK triggered a further significant increase in apoptosis, indicating that the apoptosis was mediated by suppression of integrin alpha v levels and dephosphorylation of FAK. Treatment with V⁺H⁻ decreased the phosphorylation of extracellular signal-regulated kinase (ERK) 1 and 2, and direct activation of ERK by constitutively active MEK1, an ERK kinase, increased ERK1 and ERK2 phosphorylation and inhibited the increase in apoptosis induced by V⁺H⁻. ERK acted downstream from alpha v and FAK signals, since alpha v and FAK overexpression inhibited both the decrease in ERK phosphorylation and the increase in anoikis triggered by V⁺H⁻. These findings provide evidence that mutations in the high-affinity heparin-binding domain in association with the V region of fibronectin, or altered fibronectin matrices, induce anoikis in human SCC cells by modulating integrin alpha v-mediated phosphorylation of FAK and ERK.     

PTEN interactions with focal adhesion kinase and suppression of the extracellular matrix-dependent phosphatidylinositol 3-kinase/Akt cell survival pathway.

The tumor suppressor PTEN is a phosphatase with sequence homology to tensin. PTEN dephosphorylates phosphatidylinositol 3,4, 5-trisphosphate (PIP3) and focal adhesion kinase (FAK), and it can inhibit cell growth, invasion, migration, and focal adhesions. We investigated molecular interactions of PTEN and FAK in glioblastoma and breast cancer cells lacking PTEN. The PTEN trapping mutant D92A bound wild-type FAK, requiring FAK autophosphorylation site Tyr397. In PTEN-mutated cancer cells, FAK phosphorylation was retained even in suspension after detachment from extracellular matrix, accompanied by enhanced PI 3-K association with FAK and sustained PI 3-K activity, PIP3 levels, and Akt phosphorylation; expression of exogenous PTEN suppressed all five properties. PTEN-mutated cells were resistant to apoptosis in suspension, but most of the cells entered apoptosis after expression of exogenous PTEN or wortmannin treatment. Moreover, overexpression of FAK in PTEN-transfected cells reversed the decreased FAK phosphorylation and PI 3-K activity, and it partially rescued PIP3 levels, Akt phosphorylation, and PTEN-induced apoptosis. Our results show that FAK Tyr397 is important in PTEN interactions with FAK, that PTEN regulates FAK phosphorylation and molecular associations after detachment from matrix, and that PTEN negatively regulates the extracellular matrix-dependent PI 3-K/Akt cell survival pathway in a process that can include FAK.     

TGF—β1短时处理降低肝癌细胞与Fn的粘附及FAK的磷酸化

In order to investigate whether TGF-beta 1 could rapidly regulate integrin induced signaling, we treated SMMC-7721 human hepatocellular carcinoma cells with human recombinant TGF-beta 1 for 10 min, and examined cell adhesion, integrin amount and FAK tyrosine phosphorylation. We used cell adhesion assay to estimate the affinity of alpha 5 beta 1 integrin with fibronectin, and analyzed the amount of integrin alpha 5 and beta 1 subunits by performing FACS analysis. Then western blot analysis was carried out to examine tyrosine phosphorylation level of FAK. Our results showed that TGF-beta 1 could rapidly attenuated cell adhesion onto Fn without changing the expression of alpha 5 beta 1 integrin, and at the meantime dephosphorylated FAK. It suggested that TGF-beta 1 rapidly regulated the activation of integrin, and stimulated FAK dephosphorylation, which might induce depolarization in SMMC-7721 hepatocellular carcinoma cells, then facilitates the detachment of tumor cells at early stages of migration.     

Role of protein-tyrosine phosphatase SHP2 in focal adhesion kinase down-regulation during neutrophil cathepsin G-induced cardiomyocytes anoikis

Inflammatory cells and their proteases contribute to tissue reparation at site of inflammation. Although beneficial at early stages, excessive inflammatory reaction leads to cell death and tissue damage. Cathepsin G (Cat.G), a neutrophil-derived serine protease, has been shown to induce neonatal rat cardiomyocyte detachment and apoptosis by anoikis through caspase-3 dependent pathway. However the early mechanisms that trigger Cat.G-induced caspase-3 activation are not known. This study identifies focal adhesion kinase (FAK) tyrosine dephosphorylation as an early mechanism that regulates Cat.G-induced anoikis in cardiomyocytes. Both FAK tyrosine phosphorylation at Tyr-397 and kinase activity decrease rapidly upon Cat.G treatment and was associated with a decrease of FAK association with adapter and cytoskeletal proteins, p130(Cas) and paxillin, respectively. FAK-decreased tyrosine phosphorylation is required for Cat.G-induced myocyte anoikis as concurrent expression of phosphorylation-deficient FAK mutated at Tyr-397 or pretreatment with a protein-tyrosine phosphatase (PTP) inhibitor, pervanadate, blocks Cat.G-induced FAK tyrosine dephosphorylation, caspase-3 activation and DNA fragmentation. Analysis of PTPs activation shows that Cat.G treatment induces an increase of SHP2 and PTEN phosphorylation; however, only SHP2 forms a complex with FAK in response to Cat.G. Expression of dominant negative SHP2 mutant markedly attenuates FAK tyrosine dephosphorylation induced by Cat.G and protects myocytes to undergo apoptosis. In contrast, increased SHP2 expression exacerbates Cat.G-induced FAK tyrosine dephosphorylation and myocyte apoptosis. Taken together, these results show that Cat.G induces SHP2 activation that leads to FAK tyrosine dephosphorylation and promotes cardiomyocyte anoikis.