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PI3K信号通路通过Skp2、p27调节肝癌细胞的增殖   总被引:2,自引:0,他引:2       下载免费PDF全文
探讨磷脂酰肌醇3-激酶(PI3K)信号通路调节肝癌细胞增殖的机制.用LY294002特异性阻断PI3K信号通路后,人肝癌细胞(SMMC-7721)的增殖明显被抑制.RT-PCR及蛋白质印迹结果显示,LY294002增加了p27蛋白的表达,但不影响p27的mRNA表达.在LY294002处理的细胞中转入p27的RNAi质粒以干扰p27蛋白的表达后,肝癌细胞的增殖能力可部分恢复.放线菌酮(Chx)处理实验表明,阻断PI3K信号通路使p27蛋白的半衰期延长,稳定性增加.进一步研究发现,LY294002可抑制介导p27蛋白降解的关键分子Skp2的mRNA表达,还可缩短Skp2蛋白的半衰期,降低Skp2蛋白的稳定性.但在SMMC-7721中分别转染PI3K下游重要靶分子Akt的持续激活和失活突变体,却并不影响p27蛋白的表达.这些结果表明,PI3K信号通路在转录及翻译后水平调节Skp2的表达而影响p27蛋白的降解,从而调节肝癌细胞的增殖,但Akt并没有参与这种调节.  相似文献   

3.
Prostate cancer is the most frequently diagnosed tumor in men and the second most common cause of cancer-related death for males in the United States. It has been shown that multiple signaling pathways are involved in the pathogenesis of prostate cancer, such as androgen receptor (AR), Akt, Wnt, Hedgehog (Hh) and Notch. Recently, burgeoning amounts of evidence have implicated that the F-box protein Skp2 (S-phase kinase associated protein 2), a well-characterized oncoprotein, also plays a critical role in the development and progression of prostate cancer. Therefore, this review discusses the recent literature regarding the function and regulation of Skp2 in the pathogenesis of prostate cancer. Furthermore, we highlight that Skp2 may represent an attractive therapeutic target, thus warrants further development of agents to target Skp2, which could have significant therapeutic impact on prostate cancer.  相似文献   

4.
Cyclin-dependent kinase 2 (Cdk2) is dispensable for mitotic cell cycle progression and Cdk2 knockout mice are viable due to the compensatory functions of other Cdks. In order to assess the role of Cdk2 under limiting conditions, we used Skp2 knockout mice that exhibit increased levels of Cdk inhibitor, p27Kip1, which is able to inhibit Cdk2 and Cdk1. Knockdown of Cdk2 abrogated proliferation of Skp2−/− mouse embryonic fibroblasts, encouraging us to generate Cdk2−/−Skp2−/− double knockout mice. Cdk2−/−Skp2−/− double knockout mice are viable and display similar phenotypes as Cdk2−/− and Skp2−/− mice. Unexpectedly, fibroblasts generated from Cdk2−/−Skp2−/− double knockout mice proliferated at normal rates. The increased stability of p27 observed in Skp2−/− MEFs was not observed in Cdk2−/−Skp2−/− double knockout fibroblasts indicating that in the absence of Cdk2, p27 is regulated by Skp2-independent mechanisms. Ablation of other ubiquitin ligases for p27 such as KPC1, DDB1, and Pirh2 did not restore stability of p27 in Cdk2−/−Skp2−/− MEFs. Our findings point towards novel and alternate pathways for p27 regulation.  相似文献   

5.
The cyclin inhibitory protein p27Kip1 (p27) plays a vital role in regulating cell proliferation in response to the extracellular growth environment. Active proliferation requires the suppression of p27 levels throughout the cell cycle. Late in the cell cycle, p27 degradation requires phosphorylation of Thr 187 by cyclin dependent kinase 2, leading to recognition by the SCF ubiquitin ligase containing the Skp2 F-box protein. Suppression of p27 is also essential for cell proliferation early in the cell cycle, but this occurs independently of Skp2, whose expression is suppressed during G1 phase. In this study, we use a time lapse and quantitative imaging approach to study the connection between proliferative signaling and the degradation of p27 during each cell cycle period in actively cycling cells. Ras activity was required for the suppression of p27 levels throughout the cell cycle, but separate pathways downstream of Ras signaling were required in different cell cycle periods. For example, inhibitors of MEK and phosphatidylinositol-3-kinase induced p27 expression primarily in G1 phase, while inhibitors of AKT activity stimulated these levels primarily in S phase. Skp2 was expressed in a Ras-dependent manner at higher levels late in the cell cycle. Its ablation resulted in higher p27 levels primarily in G2 phase as expected. The fact that separate signaling pathways downstream of Ras function in each cell cycle phase to suppress p27 levels helps explain the vital connection between proliferative signaling, cell cycle control, and p27 expression.  相似文献   

6.
Zheng WQ  Zheng JM  Ma R  Meng FF  Ni CR 《Steroids》2005,70(11):770-774
Estrogen receptor-negative breast carcinomas are more aggressive and are unresponsive to anti-estrogens. Thus, they clearly require new therapies targeted against specific genes and proteins actively engaged in the pathophysiology of cancer. The S-phase kinase-associated protein Skp2 is required for the ubiquitin-mediated degradation of the cdk-inhibitor p27 and is a bona fide proto-oncoprotein. We attempted to explore whether Skp2 may be a potential specific therapeutic target in the subset of aggressive breast carcinomas by investigating the possible relationship between expression of Skp2 and p27 proteins and estrogen receptor (ER). Immunohistochemical analysis of tumor tissues was employed to determine the expression of Skp2, p27, and ER proteins in 82 cases of primary breast carcinoma. Higher levels of Skp2 were detected more frequently in ER-negative tumors and tumors metastatic to the axillary lymph nodes. The expression of p27 was inverse with the histologic grade. Statistical analysis showed that the percentage of high Skp2 expressors was significantly greater in the group with low p27 expression than in the group with high p27 expression. The current study, together with the results from a previous study, demonstrated the existence of a subtype of high-grade, negative ER breast carcinomas with high Skp2 and low p27 levels. This implies that Skp2 may be a potential specific therapeutic target in a subset of aggressive breast carcinomas. Thus far, there is no specific therapy for the ER-negative and HER-2/neu resistant groups, which are among the subset of aggressive tumors.  相似文献   

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Integrins may play important roles in many cellular events, such as cell proliferation, differentiation, and apoptosis. We showed previously that overexpression of integrin beta1 inhibits cell proliferation in SMMC-7721 cells. Here we reported that one of the cyclin-dependent kinase (CDK) inhibitors, p27(Kip1) was involved in proliferation-inhibition induced by overexpression of integrin beta1. Overexpression of integrin beta1 upregulated p27(Kip1) at the protein level, but not mRNA level. The knock-down of p27(Kip1) expression restored cell growth in integrin beta1-overexpressing cells. Cycloheximide (Chx) treatment and pulse-chase experiments revealed that overexpression of integrin beta1 prolonged the half-life of p27(Kip1) by inhibiting its degradation. Proteasome inhibitor (MG132) treatment of the cells indicated that proteasome mediated degradation of p27, and Skp2-dependent degradation might be prevented. Overexpression of integrin beta1 decreased Skp2 at mRNA level, which was regulated by cell adhesion and the subsequent adhesion-dependent signaling. Overexpression of integrin beta1 reduced cell adhesion, accordingly, inactivated the phosphoinositide 3-kinase (PI3K) signaling. PI3K inhibitor LY294002 upregulated p27(Kip1) at post-translational level and downregulate Skp2 at mRNA level, which could mimic the effects of integrin beta1 overexpression on p27(Kip1) and Skp2. Together, these results suggested that overexpression of integrin beta1 inhibited cell proliferation by preventing the Skp2-dependent degradation of p27(Kip1) via PI3K pathway.  相似文献   

8.
Cell adhesion to the extracellular matrix (ECM) is a requirement for proliferation that is typically lost in malignant cells. In the absence of adhesion, nontransformed cells arrest in G1 with increased levels of the cyclin-dependent kinase inhibitor p27. We have reported previously that the degradation of p27 requires its phosphorylation on Thr-187 and is mediated by Skp2, an F-box protein that associates with Skp1, Cul1, and Roc1/Rbx1 to form the SCF(Skp2) ubiquitin ligase complex. Here, we show that the accumulation of Skp2 protein is dependent on both cell adhesion and growth factors but that the induction of Skp2 mRNA is exclusively dependent on cell adhesion to the ECM. Conversely, the expression of the other three subunits of the SCF(Skp2) complex is independent of cell anchorage. Phosphorylation of p27 on Thr-187 is also not affected significantly by the loss of cell adhesion, demonstrating that increased p27 stability is not dependent on p27 dephosphorylation. Significantly, ectopic expression of Skp2 in nonadherent G1 cells resulted in p27 downregulation, entry into S phase, and cell division. The ability to induce adhesion-independent cell cycle progression was potentiated by coexpressing Skp2 with cyclin D1 but not with cyclin E, indicating that Skp2 and cyclin D1 cooperate to rescue proliferation in suspension cells. Our study shows that Skp2 is a key target of ECM signaling that controls cell proliferation.  相似文献   

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目的:研究p27^kip1蛋白、转录因子E2F-1在胃癌中的表达及其与胃癌病理参数乏问的关系,并探讨二者相关性。方法:应用免疫组化S-P法,对90例胃癌患者的癌组织、34例癌旁正常胃黏膜进行检测,分析p27^kip1、E2F-1蛋白表达及其与胃癌病理参数之间的关系。结果:p27^kip1在正常胃组织、胃癌组织中的阳性率分别为55.9%(19/34)、31.1%(28/90),二者差异有统计学意义(p〈0.05)。其阳性表达率与胃癌组织的浸润深度(p〈0.05)、组织学分型(p〈0.01)、分化程度(p〈0.01)、淋巴结转移(p〈0.05)均相关;E2F-1在正常胃组织、胃癌组织中的阳性率分别为17.6%(6/34)、36.7%(33/90),二者差异有统计学意义(p〈0.05),其阳性表达率与胃癌的组织学分型相关。而与胃癌的浸润深度(p〉0.05)、分化程度(p〉0.05)、淋巴结转移无关(p〉0.05)。结论:p27^kip1在胃癌组织中的表达明显低于在正常组织,相反,E2F-1蛋白在胃癌组织中的表达却明显增强;p27^kip1蛋白表达与胃癌的浸润深度、组织学分型、分化程度、淋巴结转移等病理参数相关;E2F-1表达与胃癌的组织学分型相关;p27^kip1、E2F-1在胃癌中的表迭呈负相关。  相似文献   

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p27Kip1 is a critical modulator of cell proliferation by controlling assembly, localization and activity of cyclin-dependent kinase (CDK). p27Kip1 also plays important roles in malignant transformation, modulating cell movement and interaction with the extracellular matrix. A critical p27Kip1 feature is the lack of a stable tertiary structure that enhances its “adaptability” to different interactors and explains the heterogeneity of its function. The absence of a well-defined folding underlines the importance of p27Kip1 post-translational modifications that might highly impact the protein functions. Here, we characterize the metabolism and CDK interaction of phosphoserine10-p27Kip1 (pS10- p27Kip1), the major phosphoisoform of p27Kip1. By an experimental strategy based on specific immunoprecipitation and bidimensional electrophoresis, we established that pS10-p27Kip1 is mainly bound to cyclin E/CDK2 rather than to cyclin A/CDK2. pS10- p27Kip1 is more stable than non-modified p27Kip1, since it is not (or scarcely) phosphorylated on T187, the post-translational modification required for p27Kip1 removal in the nucleus. pS10-p27Kip1 does not bind CDK1. The lack of this interaction might represent a mechanism for facilitating CDK1 activation and allowing mitosis completion. In conclusion, we suggest that nuclear p27Kip1 follows 2 almost independent pathways operating at different rates. One pathway involves threonine-187 and tyrosine phosphorylations and drives the protein toward its Skp2-dependent removal. The other involves serine-10 phosphorylation and results in the elongation of p27Kip1 half-life and specific CDK interactions. Thus, pS10-p27Kip1, due to its stability, might be thought as a major responsible for the p27Kip1-dependent arrest of cells in G1/G0 phase.  相似文献   

11.
Uncontrolled cell proliferation, genomic instability and cancer are closely related to the abnormal activation of the cell cycle. Therefore, blocking the cell cycle of cancer cells has become one of the key goals for treating malignancies. Unfortunately, the factors affecting cell cycle progression remain largely unknown. In this study, we have explored the effects of Notch3 on the cell cycle in breast cancer cell lines by 3 methods: overexpressing the intra-cellular domain of Notch3 (N3ICD), knocking-down Notch3 by RNA interference, and using X-ray radiation exposure. The results revealed that overexpression of Notch3 arrested the cell cycle at the G0/G1 phase, and inhibited the proliferation and colony-formation rate in the breast cancer cell line, MDA-MB-231. Furthermore, overexpressing N3ICD upregulated Cdh1 expression and resulted in p27Kip accumulation by accelerating Skp2 degradation. Conversely, silencing of Notch3 in the breast cancer cell line, MCF-7, caused a decrease in expression levels of Cdh1 and p27Kip at both the protein and mRNA levels, while the expression of Skp2 only increased at the protein level. Correspondingly, there was an increase in the percentage of cells in the G0/G1 phase and an elevated proliferative ability and colony-formation rate, which may be caused by alterations of the Cdh1/Skp2/p27 axis. These results were also supported by exposing MDA-MB-231 cells or MCF-7 treated with siN3 to X-irradiation at various doses. Overall, our data showed that overexpression of N3ICD upregulated the expression of Cdh1 and caused p27Kip accumulation by accelerating Skp2 degradation, which in turn led to cell cycle arrest at the G0/G1 phase, in the context of proliferating breast cancer cell lines. These findings help to illuminate the precision therapy targeted to cell cycle progression, required for cancer treatment.  相似文献   

12.
Oxidative stress-induced apoptosis is mediated by ERK1/2 phosphorylation   总被引:7,自引:0,他引:7  
Oxidative stress is known to induce apoptosis in a wide variety of cell types, apparently by modulating intracellular signaling pathways. High concentrations of H2O2 have been found to induce apoptosis in L929 mouse fibroblast cells. To elucidate the mechanisms of H2O2-mediated apoptosis, ERK1/2, p38-MAPK, and JNK1/2 phosphorylation was examined, and ERK1/2 and JNK1/2 were found to be activated by H2O2. Inhibition of ERK1/2 activation by treatment of L929 cells with PD98059 or dominant-negative ERK2 transfection blocked H2O2-induced apoptosis, while inhibition of JNK1/2 by dominant-negative JNK1 or JNK2 or MKK4 or MKK7 transfection did not affect H2O2-mediated apoptosis. H2O2-mediated ERK1/2 activation was not only Ras-Raf dependent, but also both tyrosine kinase (PDGFbeta receptor and Src) and PKCdelta dependent. H2O2-mediated PKCdelta-dependent and tyrosine kinase-dependent ERK1/2 activations were independent from each other. Based on the above results, we suggest for the first time that oxidative damage-induced apoptosis is mediated by ERK1/2 phosphorylation which is not only Ras-Raf dependent, but also both tyrosine kinase and PKCdelta dependent.  相似文献   

13.
Syndecan-2 was found to detach from RACK1 and associate with caveolin-2 and Ras in cells transformed with oncogenic ras. Most of syndecan-2 from transformed cells was revealed with negligible phosphorylations at tyrosine residues. We experimented with HeLa cells transfected with plasmids encoding syndecan-2 and its mutants (syndecan-2(Y180F), syndecan-2(Y192F), and syndecan-2(Y180,192F)) to provide evidences that PY180 of syndecan-2 is a binding site for RACK1 and is deprived in cells transfected with oncogenic ras. However, in HeLa cells transfected with syndecan-2(Y180F), RACK1 was found to sustain its reactions with syndecan-2 independent of phosphorylation. The finding of syndecan-2 reactive with caveolin-2/Ras suggests the molecular complex most likely to obstruct RACK1 for functional attachment at syndecan-2, as revealed in cells transfected with oncogenic ras. We provided evidences to reinforce the view that molecular rearrangements upon transformation are specific and interesting.  相似文献   

14.
HeLa细胞中Skp2的表达调控p21WAFCIP1稳定性   总被引:2,自引:0,他引:2       下载免费PDF全文
泛素 蛋白酶体抑制剂MG 132处理的HeLa和SaoS 2细胞中 ,低表达的p2 1WAF CIP1受泛素 蛋白酶体通路调控 .为探讨肿瘤细胞中高表达的E3连接酶底物结合亚基 Skp2和低表达的p2 1WAF CIP1之间的关系 ,在HeLa细胞中转染Skp2的反义寡核苷酸后 ,p2 1表达水平明显升高 .同时 ,相对于转染空载体和Skp2ΔF(Skp2的F box缺失突变体 )的真核表达载体 ,转染全长Skp2的HeLa细胞中 ,p2 1表达水平显著下降 ,说明肿瘤细胞中Skp2调节p2 1的稳定性 ,并且这种调控作用依赖于Skp2蛋白中的F box结构 .免疫荧光结果表明 ,Skp2和p2 1共定位于细胞核 ,这为两者间的相互作用提供了前提 ;体内相互免疫共沉淀结果表明 ,p2 1和其它SCFSkp2 的底物一样与Skp2直接结合 ,并且它们之间的结合区不在F box结构域 ,这一结果在体外的GST pulldown实验中得到验证  相似文献   

15.
Skp2, the substrate-binding subunit of an SCF ubiquitin ligase complex, is a key regulator of cell cycle progression that targets substrates for degradation by the 26S proteasome. We have now shown that ablation of Skp2 in primary mouse embryonic fibroblasts (MEFs) results both in impairment of adipocyte differentiation and in the accumulation of the cyclin-dependent kinase inhibitor p27Kip1, a principal target of the SCFSkp2 complex. Genetic ablation of p27Kip1 in MEFs promoted both lipid accumulation and adipocyte-specific gene expression. However, depletion of p27Kip1 by adenovirus-mediated RNA interference failed to correct the impairment of adipocyte differentiation in Skp2-/- MEFs. In contrast, troglitazone, a high-affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ), largely restored lipid accumulation and PPARγ gene expression in Skp2−/− MEFs. Our data suggest that Skp2 plays an essential role in adipogenesis in MEFs in a manner that is at least in part independent of regulation of p27Kip1 expression.  相似文献   

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目的探讨维甲酸对A549细胞增殖和凋亡及相关基因表达的影响。方法MTT法观察ATRA对A549细胞增殖的抑制作用;流式细胞仪、AO/EB荧光双染法检测细胞凋亡;免疫细胞化学检测ATRA处理前后A549细胞Skp2、p27^kip1蛋白表达的情况。结果ATRA处理后①MTT法结果显示ATRA对A549细胞具有增殖抑制作用,在一定范围内呈时间-剂量依赖性。②AO/EB荧光双染色法观察到ATRA 25μmol/L作用A549细胞48h后,即可发现典型的凋亡形态学改变。③流式细胞仪结果出现凋亡峰,与对照组细胞相比,实验组细胞周期延长,主要表现为G0/G1期细胞比例增加,同时S期细胞比例减少。④免疫细胞化学结果显示,ATRA 25μmol/L处理细胞48h后,维甲酸处理组Skp2有明显下调,p27^kip1则明显上调。结论ATRA具有抑制肺腺癌A549细胞增殖,诱导细胞凋亡的作用,其机制可能与下调Skp2,上调p27^kip1蛋白的表达水平有关。  相似文献   

17.
Epidermal growth factor receptor variant III (EGFRvIII), the most common EGFR mutation, is associated with cell migration of glioblastoma multiforme (GBM) cases; however, the mechanism has not been elucidated. In this study, we found that the EGFRvIII-promoted glioma cell migration was closely linked to high levels of tyrosine phosphorylation in focal adhesion kinase (FAK) Y397. We also demonstrated that EGFRvIII formed a complex with FAK, resulting in enhanced tyrosine phosphorylation levels of FAK Y397 and EGFR Y1068. After knockdown of FAK expression via anti-FAK shRNA, the U87ΔEGFR cell migration was significantly inhibited, accompanying with the reduced phosphorylation levels of extracellular signal-regulated kinase (ERK1/2). Furthermore, the role of ERK1/2 in FAK-regulated cell migration was confirmed. Taken together, our results suggest that FAK and its downstream molecule ERK were involved in EGFRvIII-promoted glioma cell migration in U87ΔEGFR cells.  相似文献   

18.
Fyn is a member of the Src-family protein tyrosine kinases and plays important roles in both neurons and oligodendrocytes. Here we report association of Fyn with p250GAP, a RhoGAP protein that is expressed predominantly in brain. p250GAP interacts with Fyn both in vitro and in vivo. p250GAP is tyrosine phosphorylated by Fyn when co-expressed in HEK293T cells. This phosphorylation appears to enhance the interaction between p250GAP and Fyn. Furthermore, the level of tyrosine phosphorylation of p250GAP increases upon differentiation of the oligodendrocyte cell line CG4. Given that Fyn activity is up-regulated during oligodendrocyte maturation, the results argue that p250GAP is phosphorylated by Fyn in oligodendrocytes. Tyrosine phosphorylation of p250GAP by Fyn would regulate its RhoGAP activity, subcellular localization, or interactions with other proteins, leading to morphological and phenotypic changes of oligodendrocytes.  相似文献   

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为了探讨p27Kip1蛋白和CyclinD1蛋白在非小细胞肺癌(NSCLC)中的表达及意义,收集临床手术切除的非小细胞肺癌组织蜡块64例及正常肺组织10例,应用免疫组化(S-P法)检测组织中p27Kip1蛋白和CyclinD1蛋白的表达,结合临床病理资料和随访资料进行回顾性研究。实验发现NSCLC组织中p27Kip1蛋白表达和CyclinD1蛋白表达均明显不同于正常肺组织(P<0.01)。p27Kip1蛋白表达降低与NSCLC肿瘤大小、病理分级、分期增加、淋巴结转移之间有相关性(P<0.05),但与肿瘤组织学分型无相关性(P>0.05)。CyclinD1蛋白过表达与组织学分型、肿瘤大小、病理分级、临床分期、淋巴结转移无相关性(P>0.05)。p27Kip1蛋白表达与CyclinD1蛋白表达之间呈显著负相关(P<0.01)。cox单因素及多因素分析,p27Kip1蛋白低表达及CyclinD1过表达是影响NSCLC患者预后的主要因素。实验结果显示,NSCLC组织中,p27Kip1蛋白表达降低,而CyclinD1过表达,二者与NSCLC的发生发展机制有关,可作为预后指标,有利于NSCLC患者预后判断及个体化治疗。  相似文献   

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