Mdm2、PTEN表达和细菌L型感染与卵巢肿瘤的相关性研究

目的探讨Mdm2癌基因、PTEN抑癌基因和细菌L型感染在卵巢肿瘤中的表达及临床意义,并分析它们之间的相互影响和协同作用。方法应用免疫组化、原位杂交和革兰染色等方法检测97例卵巢乳头状癌及23例卵巢乳头状瘤组织中Mdm2、PTEN的表达及细菌L型检出率,并用χ^2检验进行统计学处理。结果卵巢恶性肿瘤中Mdm2蛋白和基因的表达率均明显高于良性肿瘤（P〈0．0001-0．0005）。Mdm2蛋白和基因的表达在卵巢乳头状癌中临床分期Ⅲ、Ⅳ期的表达率明显高于Ⅰ、Ⅱ期（P〈0．005）;随着病理分级增高而显著增加（P〈0．005）;腹腔淋巴结有转移、有腹水比无转移者、无腹水者差异有显著性（P〈0．01～0．05）,呈正相关。PTEN蛋白及基因的表达在卵巢良性肿瘤均明显高于恶性肿瘤,病理分级Ⅰ、Ⅱ级的阳性表达率明显高于Ⅲ级;临床分期Ⅰ、Ⅱ期中的阳性表达率明显高于Ⅲ、Ⅳ期（P〈0．001-0．005）;腹腔淋巴结无转移者、无腹水者比有转移、有腹水差异有显著性（P〈0．01～0．05）,呈负相关。细菌L型检出阳性率与病理分级、临床分期差异有显著性（P〈0．05）,腹腔淋巴结有转移、有腹水比无转移者、无腹水者差异有非常显著性（P〈0．01）。结论Mdm2、PTEN基因在卵巢肿瘤中有不同程度的异常表达,两种基因均可作为判断卵巢肿瘤生物学行为及患者预后参考指标。L型感染极有可能导致基因的变异或过表达,因此L型感染可能成为诱发肿瘤形成的原因之一。细菌L型感染可能诱导PTEN抑癌基因、Mdm2癌基区的变异和异常表达以及它们相互协同在卵巢肿瘤发生和发展过程中起重要作用。     

Simultaneous loss of the DLC1 and PTEN tumor suppressors enhances breast cancer cell migration

The phosphatase and tensin homolog (PTEN) gene is a tumor suppressor frequently deleted or mutated in sporadic tumors of the breast, prostate, endometrium and brain. The protein acts as a dual specificity phosphatase for lipids and proteins. PTEN loss confers a growth advantage to cells, protects from apoptosis and favors cell migration. The deleted in liver cancer 1 (DLC1) gene has emerged as a novel tumor suppressor downregulated in a variety of tumor types including those of the breast. DLC1 contains a Rho GTPase activating domain that is involved in the inhibition of cell proliferation, migration and invasion. To investigate how simultaneous loss of PTEN and DLC1 contributes to cell transformation, we downregulated both proteins by RNA interference in the non-invasive MCF7 breast carcinoma cell line. Joint depletion of PTEN and DLC1 resulted in enhanced cell migration in wounding and chemotactic transwell assays. Interestingly, both proteins were found to colocalize at the plasma membrane and interacted physically in biochemical pulldowns and coimmunoprecipitations. We therefore postulate that the concerted local inactivation of signaling pathways downstream of PTEN and DLC1, respectively, is required for the tight control of cell migration.     

Cell cycle arrest by the PTEN tumor suppressor is target cell specific and may require protein phosphatase activity

PTEN, a tumor suppressor commonly targeted in human cancer, possesses phosphatase activities toward both protein and lipid substrates. While PTEN suppresses gliomas through cell cycle inhibition which requires its lipid phosphatase activity, PTEN's effects on other tumor types and the role of its protein phosphatase activity are controversial or unknown. Here we show that exogenous wild-type PTEN arrests some, but not all human breast cancer cell lines in G1, in a manner independent of endogenous PTEN. Unexpectedly, the G129E mutant of PTEN selectively deficient in the lipid phosphatase activity still blocked the cell cycle of MCF-7 cells, while the G129R and H123Y mutants lacking both phosphatase activities were ineffective. These results suggest that PTEN's protein phosphatase activity likely contributes to its tumor suppressor function in subsets of tumors and that elucidation of downstream targets which dictate cellular responses to PTEN may have important implications for future cancer treatment strategies.     

A novel model to identify interaction partners of the PTEN tumor suppressor gene in human bladder cancer

Phosphatase and tensin homolog (PTEN), deleted on chromosome 10, is a potent tumor suppressor. PTEN expression is reduced in advanced bladder cancer and reduction correlates with disease stage. To gain insights into the function of PTEN in human bladder cancer by identifying its binding partners, we developed a novel IPTG inducible PTEN expression system and evaluated this system in the PTEN null UMUC-3 human bladder cancer xenograft model. In this model, induction of PTEN in vivo resulted in reduced tumor growth. We used mass spectrometry to identify PTEN interaction partners in these cells, which identified known interaction partners major vault protein (MVP) and paxillin as well as a novel interaction partner, TRK fused gene (TFG). In conclusion, using a biologically relevant model system to dissect PTEN tumor suppressor function in human bladder cancer, we identified three molecules important for many cellular functions in complex with PTEN.     

PTEN在心肌肥厚中的作用初探

目的：观察肿瘤抑制因子PTEN mRNA在腹主动脉狭窄大鼠及卡托普利处理的腹主动脉狭窄大鼠左心室肌中的表达,以探讨PTEN在心肌肥厚发生发展中的可能作用。方法：采用腹主动脉狭窄术制备压力超负荷心肌肥厚动物模型,于术后4周应用逆转录—聚合酶链式反应(RT—PCR)方法,分别检测和观察对照组、心肌肥厚组和卡托普利组大鼠左心室PTEN mRNA表达的变化。结果：①与对照组相比,心肌肥厚组大鼠左心室肌PTEN mRNA表达减少;②与心肌肥厚组相比,卡托普利组大鼠左心室肌PTEN mRNA表达增加,接近对照组。结论：PTEN在心肌肥厚发生发展中可能起负调控作用,该作用与肾素—血管紧张素系统密切相关。     

Thioredoxin-1 binds to the C2 domain of PTEN inhibiting PTEN's lipid phosphatase activity and membrane binding: a mechanism for the functional loss of PTEN's tumor suppressor activity

Thioredoxin-1 (Trx-1) is a 12 kDa redox protein that is overexpressed in a large number of human tumors. Elevated Trx-1 is associated with increased tumor cell proliferation, inhibited apoptosis, aggressive tumor growth, and decreased patient survival. The molecular mechanisms for the promotion of tumorigenesis by Trx-1 are not known. PTEN is a major tumor suppressor of human cancer that acts by hydrolyzing membrane phosphatidylinositol (PtdIns)-3-phosphates, thus, preventing the activation of the survival signaling kinase Akt by PtdIns-3-kinase. We show that Trx-1 binds in a redox dependent manner to PTEN to inhibit its PtdIns-3-phosphatase activity which results in increased Akt activation in cells. Molecular docking and site-specific mutation studies show that the binding of Trx-1 to PTEN occurs through a disulfide bond between the active site Cys(32) of Trx-1 and Cys(212) of the C2 domain of PTEN leading to steric interference by bound Trx-1 of the catalytic site of PTEN and of the C2 lipid membrane-binding domain. The results of the study suggest that the increased levels of Trx-1 in human tumors could lead to functional inhibition of PTEN tumor suppressor activity providing an additional mechanism for tumorigenesis with loss of PTEN activity.     

丹皮酚对肝癌MHCC97-H细胞PTEN、AKT表达的影响

目的:探讨丹皮酚(Paeonol,Pae)在体外对人肝癌MHCC97-H细胞PTEN、AKT表达的影响。方法:体外培养人肝癌MHCC97-H细胞,MTT法检测丹皮酚对MHCC97-H细胞的增殖抑制作用,RT-PCR法检测PTEN、Akt1、Akt2mRNA表达,West- ern Blot法检测PTEN、p-AKT蛋白的表达。结果:丹皮酚呈时间剂量依赖性抑制人肝癌MHCC97-H细胞的增殖;肝癌MHCC97-H细胞低表达PTEN,高表达AKT,丹皮酚能显著上调MHCC97-H细胞PTEN表达,下调AKT表达。结论:丹皮酚可上调抑癌基因PTEN的表达,下调致癌基因AKT的表达,抑制MHCC97-H细胞的增殖。     

Proteome profile changes that are differentially regulated by lipid and protein phosphatase activities of tumor suppressor PTEN in PTEN-expressing U-87 MG human glioblastoma cells

The phosphatase and tensin homolog tumor suppressor (PTEN) belongs to a class of "gatekeeper" tumor suppressors together with p53, retinoblastoma and adenomatous polyposis. It is considered one of the most important tumor suppressors in the post p53 era. Previously to identify the molecules involved in the signaling network regulated by PTEN using proteomic tools, we reported global proteome profiles at different time points using the PTEN inducible NIH3T3 cells (Kim, S.-y., Kim, Y. S., Bahk, Y. Y., Mol. Cells 2003, 15, 396-405). However, the system had a critical limitation that NIH3T3 cell has endogenous wild-type PTEN and, thus to be exact, the induced PTEN could not give the answer about the real physiological roles of this tumor suppressor. Here, to find out PTEN-related protein network we have established various PTEN (wild-type, an activity inert C124G, and a lipid phosphatase deficient G129E)-expressing cell clones in U-87 MG human glioblastoma cells lacking detectable PTEN as a result of genetic lesions. In this biological context, we compared their morphological and expression patterns, and proteome images of each PTEN-expressing cell clone by 2-DE followed by identification with MALDI-TOF MS. We obtained some pieces of evidence that morphological change by PTEN expression is mediated by its protein phosphatase activity and their growth rate by the lipid phosphatase activity. The proteomic approaches showed that 30 proteins possibly correlated with PTEN's protein phosphatase activity (13 down-regulated and 17 up-regulated) and 20 with the lipid phosphatase activity (14 down-regulated and 6 up-regulated) were identified. Taken together, we conclude that the comparative analysis of proteome from various PTEN-expressing cells has yielded interpretable data to elucidate the protein network directly and/or indirectly caused by individual phosphatase activities of PTEN in vivo.     

槐定碱联合顺铂对卵巢癌中FHIT，Survivin 及PTEN表达的影响

目的:探讨槐定碱联合顺铂对卵巢癌中脆性组氨酸三联(FHIT)、凋亡抑制基因survivin、抑癌基因PTEN的表达影响研究。方法:收集我院卵巢癌患者60例,随机分为实验组和对照组,每组30例,所有患者在给予纠正电解质与酸碱平衡等常规治疗后,对照组患者给予顺铂注射液进行治疗,实验组患者在对照组的基础上给予槐定碱注射液进行治疗。观察并比较治疗前后两组患者卵巢癌组织中FHIT,survivin及PTEN表达水平的变化情况以及不良反应的发生率。结果:与治疗前相比,治疗后两组患者FHIT及PTEN表达水平均升高,survivin表达水平均降低(P0.05),治疗结束后与对照组相比,实验组患者FHIT及PTEN表达水平较高(P0.05);与对照组相比,实验组患者survivin表达水平较低(P0.05);且两组患者不良反应率相当(P0.05)。结论:槐定碱联合顺铂可以有效降低卵巢癌的恶性程度,减慢肿瘤细胞的增殖发展,延缓病情,其机制可能与升高抑癌基因FHIT和PTEN的表达水平,以及降低凋亡抑制基因survivin的表达有关。     

Structural and functional characterization of the Wnt inhibitor APC membrane recruitment 1 (Amer1)

Amer1/WTX binds to the tumor suppressor adenomatous polyposis coli and acts as an inhibitor of Wnt signaling by inducing β-catenin degradation. We show here that Amer1 directly interacts with the armadillo repeats of β-catenin via a domain consisting of repeated arginine-glutamic acid-alanine (REA) motifs, and that Amer1 assembles the β-catenin destruction complex at the plasma membrane by recruiting β-catenin, adenomatous polyposis coli, and Axin/Conductin. Deletion or specific mutations of the membrane binding domain of Amer1 abolish its membrane localization and abrogate negative control of Wnt signaling, which can be restored by artificial targeting of Amer1 to the plasma membrane. In line, a natural splice variant of Amer1 lacking the plasma membrane localization domain is deficient for Wnt inhibition. Knockdown of Amer1 leads to the activation of Wnt target genes, preferentially in dense compared with sparse cell cultures, suggesting that Amer1 function is regulated by cell contacts. Amer1 stabilizes Axin and counteracts Wnt-induced degradation of Axin, which requires membrane localization of Amer1. The data suggest that Amer1 exerts its negative regulatory role in Wnt signaling by acting as a scaffold protein for the β-catenin destruction complex and promoting stabilization of Axin at the plasma membrane.     

PVB与PF方案对局部晚期宫颈癌PTEN、VEGF和CD105表达的影响

目的:比较PVB与PF方案对局部晚期宫颈癌患者PTEN、VEGF和CD105表达的影响。方法:选取我院妇科收治的局部晚期宫颈癌患者150例,随机分为两组,其中PVB组75例予顺铂、长春新碱、博来霉素化疗;PF组75例予博来霉素、顺铂配合5-氟尿嘧啶治疗。观察并对比治疗前后患者临床疗效及患者癌组织PTEN、VEGF表达阳性率、CD105抗体标记的肿瘤MVD值等指标。结果:1治疗后两组PTEN、VEGF表达阳性率均有所改善,与PVB组(76.3%)比较,PF组PTEN表达阳性率(88.3%)明显升高,差异有统计学意义(P0.05);与PVB组(15.3%)比较,PF组VEGF表达阳性率(6.7%)明显降低,差异有统计学意义(P0.05);2治疗后两组MVD值均有所改善,与PVB组(38.93±9.89)比较,PF组MVD值(35.61±10.33)明显降低,差异有统计学意义(P0.05);3治疗后,与PVB组(76.67%)比较,PF组的有效率(86.67%)明显较高,差异有统计学意义(P0.05)。结论:与PVB方案相比,PF方案能够使局部晚期宫颈癌患者PTEN表达水平升高,降低VEGF和CD105表达水平,临床疗效更确切。     

Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C–Cdc20 complex

The Anaphase Promoting Complex/Cyclosome (APC/C) in complex with its co‐activator Cdc20 is responsible for targeting proteins for ubiquitin‐mediated degradation during mitosis. The activity of APC/C–Cdc20 is inhibited during prometaphase by the Spindle Assembly Checkpoint (SAC) yet certain substrates escape this inhibition. Nek2A degradation during prometaphase depends on direct binding of Nek2A to the APC/C via a C‐terminal MR dipeptide but whether this motif alone is sufficient is not clear. Here, we identify Kif18A as a novel APC/C–Cdc20 substrate and show that Kif18A degradation depends on a C‐terminal LR motif. However in contrast to Nek2A, Kif18A is not degraded until anaphase showing that additional mechanisms contribute to Nek2A degradation. We find that dimerization via the leucine zipper, in combination with the MR motif, is required for stable Nek2A binding to and ubiquitination by the APC/C. Nek2A and the mitotic checkpoint complex (MCC) have an overlap in APC/C subunit requirements for binding and we propose that Nek2A binds with high affinity to apo‐APC/C and is degraded by the pool of Cdc20 that avoids inhibition by the SAC.     

宫颈癌组织中的PTEN, VEGF和CD105的表达及临床意义

目的:探讨宫颈癌组织中的人第10号染色体缺失的磷酸酶及张力蛋白同源基因(PTEN)、血管内皮生长因子(VEGF)和内皮因子(CD105)的表达及临床意义。方法:收集2010年1月至2016年2月我院收治的宫颈癌患者组织标本320例和同期正常宫颈组织标本100例,采用SP免疫组化法检测宫颈癌和正常宫颈组织中的PTEN、VEGF和CD105,分析各指标在宫颈癌、正常宫颈组织及不同类型宫颈癌组织中的表达,并采用Sperman分析三者相关关系。结果:宫颈癌组织VEGF阳性率、CD105-MVD高于正常宫颈组织,而PTEN阳性率低于正常宫颈组织,差异均有统计学意义(P0.05);宫颈癌组织FIGO分期Ⅰ~Ⅱ期的VEGF阳性率、CD105-MVD低于Ⅲ~Ⅳ期,而PTEN阳性率高于Ⅲ~Ⅳ期;宫颈癌组织伴有淋巴结转移的VEGF阳性率、CD105-MVD高于无转移,而PTEN阳性率低于无转移,差异均有统计学意义(P0.05);Sperman相关性显示VEGF与PTEN,CD105-MVD与PTEN呈现明显的负相关关系(rs=-0.564,-0.893,P0.05),VEGF与CD105-MVD呈现正相关关系(rs=0.825,P0.05)。结论:PTEN在宫颈癌组织表达降低,CD105和VEGF升高,三个指标在宫颈癌的发生、发展、转移及预后中具有重要作用。     

TSPYL5 is involved in cell growth and the resistance to radiation in A549 cells via the regulation of p21 and PTEN/AKT pathway

TSPYL5, encoding testis-specific Y-like protein, has been postulated to be a tumor suppressor gene, and its hypermethylation is often associated with human disease, especially cancer. In this study, we report that the TSPYL5 gene was less methylated (30%) in A549 lung adenocarcinoma cells, which are relatively resistant to γ-radiation, than in H460 lung cancer cells, in which the TSPYL5 gene was hypermethylated (95%); thus, the expression level of TSPYL5 is much higher in A549 cells than in H460 cells. We showed that TSPYL5 suppression with silencing RNA in A549 cells up-regulated cellular PTEN, followed by down-regulation of AKT activation. Therefore, blockage of TSPYL5 sensitized A549 cells to cytotoxic agents such as γ-radiation. In addition, TSPYL5 suppression also showed an increased level of p21^WAF1/Cip1 and subsequently induced inhibition of cell growth in A549 cells. The overexpression of TSPYL5 in H460 cells showed the opposite effects. This study provides the first demonstration that TSPYL5 modulates cell growth and sensitization of cells to the detrimental effects of damaging agents via regulation of p21^WAF1/Cip1 and PTEN/AKT pathway.     

Purinergic Receptor-mediated Rapid Depletion of Nuclear Phosphorylated Akt Depends on Pleckstrin Homology Domain Leucine-rich Repeat Phosphatase,Calcineurin, Protein Phosphatase 2A,and PTEN Phosphatases

Akt is an important oncoprotein, and data suggest a critical role for nuclear Akt in cancer development. We have previously described a rapid (3–5 min) and P2X7-dependent depletion of nuclear phosphorylated Akt (pAkt) and effects on downstream targets, and here we studied mechanisms behind the pAkt depletion. We show that cholesterol-lowering drugs, statins, or extracellular ATP, induced a complex and coordinated response in insulin-stimulated A549 cells leading to depletion of nuclear pAkt. It involved protein/lipid phosphatases PTEN, pleckstrin homology domain leucine-rich repeat phosphatase (PHLPP1 and -2), protein phosphatase 2A (PP2A), and calcineurin. We employed immunocytology, immunoprecipitation, and proximity ligation assay techniques and show that PHLPP and calcineurin translocated to the nucleus and formed complexes with Akt within 3 min. Also PTEN translocated to the nucleus and then co-localized with pAkt close to the nuclear membrane. An inhibitor of the scaffolding immunophilin FK506-binding protein 51 (FKBP51) and calcineurin, FK506, prevented depletion of nuclear pAkt. Furthermore, okadaic acid, an inhibitor of PP2A, prevented the nuclear pAkt depletion. Chemical inhibition and siRNA indicated that PHLPP, PP2A, and PTEN were required for a robust depletion of nuclear pAkt, and in prostate cancer cells lacking PTEN, transfection of PTEN restored the statin-induced pAkt depletion. The activation of protein and lipid phosphatases was paralleled by a rapid proliferating cell nuclear antigen (PCNA) translocation to the nucleus, a PCNA-p21^cip1 complex formation, and cyclin D1 degradation. We conclude that these effects reflect a signaling pathway for rapid depletion of pAkt that may stop the cell cycle.     

Colocalization of APC and DLG at the tips of cellular protrusions in cultured epithelial cells and its dependency on cytoskeletons

Adenomatous polyposis coli gene product (APC) is a tumor suppressor linked to familial adenomatous polyposis and is thought to be involved in cellular polarization and migration in moving epithelial cells. APC interacts with the mammalian homolog of Discs large (DLG). DLG is a member of the membrane-associated guanylate kinase superfamily and is thought to function as a scaffolding protein that coordinates the assembly of a lateral plasma membrane-localized protein complex in epithelial cells. We confirmed the suitability of several anti-APC antibodies for immunocytochemical analysis. Using these antibodies, we showed that APC clusters were colocalized with DLG protein at cellular protrusions of subconfluent MDCK cells. A portion of the clusters was found at the tips of microtubules extending into the cellular protrusions. In addition, actin stress fibers converged near the clusters. When microtubules were disrupted by nocodazole, the colocalization of APC and DLG was lost due to the disappearance of APC clusters. However, the coclusters remained after depolymerization of actin filaments with latrunculin A. This is the first report showing colocalization of APC and DLG in non-polarized epithelial cells. This colocalization suggests that DLG functions not only at the lateral cell–cell contact sites of polarized epithelial cells but also at the protrusions of non-polarized epithelial cells through the interaction with APC protein.     

Redox regulation of the tumor suppressor PTEN by glutaredoxin 5 and Ycp4

Human PTEN (phosphatase and tensin homolog deleted on chromosome 10; a phosphatidylinositol 3-phosphatase) expressed in Saccharomyces cerevisiae was oxidized in a time- and H₂O₂-concentration-dependent manner. Oxidized hPTEN was reduced by cellular reductants as in human cells. The reduction rate of oxidized hPTEN was monitored in S. cerevisiae mutants in which the genes involved in redox homeostasis had been disrupted. Reduction of hPTEN was delayed in each of S. cerevisiae grx5Δ and ycp4Δ mutants. Expression of Grx5 and Ycp4 in each of the mutants rescued the reduction rate of oxidized hPTEN. Furthermore, an in vitro assay revealed that the human Grx5/GSH system efficiently catalyzed the reduction of oxidized hPTEN. These results suggest that the reduction of oxidized hPTEN is regulated by Grx5 and Ycp4.