在大鼠肝癌发生过程中GABA-BR1的表达变化及意义

为探讨γ-氨基丁酸B型受体1(γ-aminobutyric acid type B1 receptor,GABA-BR1)与肝癌发生的关系,利用二乙基亚硝胺(DEN)制备诱发型大鼠肝癌模型,模拟人类肝癌发生发展过程,采用免疫组织化学技术对GABA-BR1和细胞周期素E(Cyclin E)蛋白在大鼠肝脏中的表达进行定性和定量分析.结果显示,在正常肝组织中有少量散在分布的GABA-BR1表达阳性的细胞;在变性或坏死灶周围GABA-BR1表达阳性的肝细胞较多;增生结节中部分肝细胞呈过表达;在肝癌细胞中广泛表达.Cyclin E与GABA-BR1阳性表达的细胞分布特点相似.实验结果提示,在肝癌发生发展过程GABA-BR1过表达,可能对肝细胞的异常增生或癌变过程起到抑制作用.     

ELV对青春期大鼠睾丸中VEGF与VEGFR2蛋白表达的影响

目的研究血管内皮生长因子(VEGF)及其受体2(VEGFR2)在实验性左侧精索静脉曲张大鼠睾丸中的表达和定位,探讨精索静脉曲张中VEGF和VEGFR2的可能作用。方法通过部分结扎左肾静脉建立大鼠实验性左侧精索静脉曲张模型,于术后2周和4周取材,采用免疫组化法检测VEGF、VEGFR2在睾丸上的表达变化。结果 ELV2周与4周组大鼠两侧睾丸中VEGF蛋白表达均上调,但ELV组间VEGF蛋白表达没有明显变化;ELV2周组大鼠睾丸中VEGFR2蛋白的表达与对照组比较增强,而4周组比对照组和2周组均显著增强。结论实验性左侧精索静脉曲张对VEGF、VEGFR2蛋白的表达有影响,说明它们与男性不育可能有一定的关系。     

葡萄糖6-磷酸脱氢酶调控细胞周期蛋白E1和细胞周期蛋白依赖性激酶2促进细胞增殖及其在肾透明细胞癌中的预后价值

肾透明细胞癌(clear cell renal cell carcinoma,ccRCC)是一种转移率高、预后差的细胞代谢性疾病,对其有效诊疗及预后分子标志物的研究十分重要。葡萄糖6-磷酸脱氢酶(glucose 6-phosphatedehydrogenase, G6PD)在ccRCC中高表达,并提示患者不良预后,其促进ccRCC细胞增殖的分子机制有待进一步揭示。本研究发现,降低G6PD可抑制细胞周期G₁/S期转化并显著抑制ccRCC细胞增殖。G6PD可在细胞水平调控G₁/S期转化及增殖相关因子Cyclin D1,CDK4,CDK6,Cyclin E1和CDK2基因表达。TCGA数据库分析结果表明,ccRCC 中Cyclin D1,Cyclin E1 和 CDK2的mRNA 水平显著升高,而CDK4表达无明显差异,CDK6表达却显著降低。相关性分析结果显示,G6PD与Cyclin D1呈显著负相关(P<0.0001),G6PD与CDK4,CDK6之间无显著相关性(P>0.05),G6PD与Cyclin E1(P<0.0001)以及CDK2(P<0.05)显著正相关。进一步免疫组化检测结果表明,Cyclin E1和 CDK2在ccRCC肿瘤组织中表达显著升高。生存预后分析结果显示,Cyclin D1高表达提示ccRCC患者整体预后更为良好,CDK4和CDK6表达水平在ccRCC患者总生存率预测中无意义;而Cyclin E1和CDK2高表达均可提示ccRCC患者预后不良。进一步细胞水平检测发现,Cyclin E1、CDK2表达降低可显著逆转G6PD促进ccRCC细胞增殖的能力。综上,与增殖相关因子Cyclin D1,CDK4和CDK6相比,G6PD有可能通过促进Cyclin E1和CDK2表达升高而发挥促进 ccRCC肿瘤细胞增殖的作用,并且这3者的异常高表达有望成为ccRCC患者不良预后的独立生存预测因素。     

Cyclin D1与细胞周期调控

细胞周期是细胞生命活动中一个最重要的过程,其关键是G1 期的启动.细胞周期蛋白(Cyclin)、细胞周期蛋白依赖性激酶(CDKs)和CDK抑制因子(CKIs)是参与钿胞周期调控的主要因子.Cyclin D1是调控细胞周期G1期的关键蛋白,是一个比其他Cyclins更加敏感的指标,对细胞周期调控至关重要.综述Cyclin D1的结构和功能及其在肿瘤组织中的表达特征,初步分析Cyclin D在昆虫细胞周期调控的研究.     

人乳头瘤病毒E7蛋白对细胞周期G1/S检查点的影响

高危型人乳头瘤病毒(HPV)感染与宫颈癌的发生关系已经明确,HPV编码的早期蛋白E7是HPV致宫颈癌的主要相关蛋白之一。G_1/S检查点是细胞周期中不可逆的关键点,决定了细胞进入细胞周期与否,与肿瘤的发生发展关系密切。pRb蛋白是调控细胞周期G_1/S检查点中的限速底物,是起调控作用的主要因子之一。E7通过影响pRb蛋白、E2F家族、Cyclin/CDK2复合物、p27蛋白、Dyrk1B等细胞周期相关蛋白来影响G_1/S检查点的正常工作,形成失控的细胞增殖。高危型HPV E7蛋白对细胞周期检查点的影响,既是HPV致癌机制的重要组成部分,也可能成为攻克此类癌症的突破口。本文综述了高危型HPV E7蛋白对细胞周期G_1/S检查点的影响。     

VEGF、VEGFR2在青春期大鼠睾丸、附睾及附睾精子上的表达

目的通过对血管内皮生长因子(VEGF)及其受体VEGFR2在青春期大鼠睾丸及附睾表达的研究,探讨其在雄性生殖器官中的作用。方法采用免疫组化法检测VEGF、VEGFR2在SD大鼠睾丸和附睾的表达定位,用免疫荧光法检测它们在大鼠附睾精子上的表达定位。结果VEGF及VEGFR2在青春期大鼠睾丸和附睾组织中均有表达。在睾丸中,VEGF主要表达于精原细胞胞质、精子细胞发育中的顶体、Sertoli细胞胞质及精子残余体内,Leydig细胞胞质也有阳性表达;VEGFR2主要表达于精子细胞发育中的顶体和间质细胞胞质。在附睾中,VEGF表达于附睾管上皮所有主细胞胞质内;而VEGFR2表达于附睾管头段和尾段上皮主细胞胞质内,体段免疫染色阴性。免疫荧光显示,VEGF与VEGFR2都与精子头部顶体、尾部颈段、中段和主段相结合,末段未见阳性荧光。结论VEGF及VEGFR2在大鼠的睾丸和附睾中均有表达,其表达定位具有细胞特异性和区域特异性,提示其可能在大鼠睾丸精子发生和附睾精子成熟中发挥重要作用。     

血小板衍生生长因子受体β对骨肉瘤细胞株HOS增殖的影响

血管形成是肿瘤生长过程中不可或缺的因素。血小板衍生生长因子受体(platelet-derived growth factor receptor, PDGFR)通过与其配体结合刺激新生血管形成,与多种肿瘤的发生发展密切相关。研究表明其亚型PDGFRβ高表达于大多数骨肉瘤病人标本及细胞株中,促进肿瘤增殖,但具体机制仍未明确。在本研究中,分别对HOS骨肉瘤细胞株进行特异性配体PDGFBB刺激处理及小干扰RNA (siRNA)敲降PDGFRβ处理,然后检测其增殖、细胞周期及相关蛋白的表达。结果表明:经过PDGFBB刺激后细胞增殖能力增强;细胞周期结果显示处于G1期的HOS细胞数量增多,S期和G2/M期细胞数量减少,G2期相关蛋白Cyclin B1和CDK1以及G1期相关蛋白CyclinE2和CDK2表达增高,提示PDGFBB可能促进了G2/M期转化。而经siRNA干扰后,PDGFRβ在mRNA和蛋白水平均显示表达量下降;细胞增殖受到抑制。检测细胞周期结果发现在敲降PDGFRβ后,进入S期的细胞数量增加了约9.71%,而G2/M期的细胞数量约减少了6.78%,S期相关蛋白Cyclin A1和CDK2明显降低,提示发生了S期阻滞。因此我们推测,PDGFRβ通过调控细胞周期进程影响骨肉瘤细胞的增殖。本研究为PDGFRβ影响HOS细胞增殖及其潜在机制提供了理论依据,为PDGFRβ可以作为治疗骨肉瘤的潜在治疗靶点提供了理论依据。     

大鼠发育过程中海马细胞周期相关蛋白的表达与分布

目的观察正常SD大鼠发育过程中海马细胞周期相关蛋白表达及分布特点,探讨其与脑老化的关系.方法采用免疫组织化学方法观察不同发育时期(1周,2月,4月,10月,15月)Cyclin D1、CDK4、P16、NeuN表达的规律.结果在各年龄组Cyclin D1、CDK4、P16、NeuN阳性细胞层的厚度随着增龄而逐渐变薄.各阳性细胞的排列逐渐由紧密变得松散,胞体逐渐增大,各阳性细胞逐渐伸出轴突进入分子层.P16随月龄的增长在海马各区染色增强,但P16阳性细胞数目减少.结论 Cyclin D1、CDK4、P16、NeuN在海马发育的各个时期均有表达,老龄大鼠海马内Cyclin D1、CDK4、P16表达的下调提示细胞增殖活性受限,这可能与脑老化有关.     

细胞周期蛋白依赖性激酶2的功能及其抑制剂的研究

细胞周期蛋白依赖性激酶2(cyclin-dependent kinase 2,CDK2)是CDK家族中的重要成员之一。CDK2的表达或功能异常与多种疾病(如肿瘤、病毒复制与感染、免疫缺陷性疾病和雄性不育等)发生机制密切相关。CDK2抑制剂已成为抗肿瘤药物研发中的一个重要靶点。该文对CDK2在细胞周期调控、细胞增殖、细胞分化、细胞凋亡中的作用机制以及CDK2抑制剂的研发进行综述。     

HBXIP基因对乙肝病毒X蛋白诱导细胞凋亡的影响

探讨乙型肝炎病毒X蛋白结合蛋白(hepatitisBXinteractingprotein ,HBXIP)基因在乙型肝炎病毒X蛋白(HBX)诱导肝癌细胞凋亡时对细胞周期的影响.构建HBXIP基因真核表达载体pcDNA3 hbxip ,进行瞬时基因转染,将克隆有HBx基因的pCMV X (分别为1μg、2 μg和3μg)和pcDNA3 hbxip质粒分别和共转染至人H74 0 2肝癌细胞中(总体积分别为5 0 μl) .发现瞬时转染3μgpCMV X质粒后,肝癌细胞凋亡发生率为34 4 % ,肝癌细胞的细胞周期相关蛋白p2 7表达水平发生明显上调;与对照组相比,瞬时转染1μg、2 μg和3μg时,细胞周期蛋白D和细胞周期蛋白E的表达水平均发生明显上调,但随着HBX水平的增加细胞周期蛋白D和细胞周期蛋白E的表达水平发生明显下降;在稳定转染pCMV X质粒的H74 0 2 X肝癌细胞中无明显的细胞凋亡发生,研究发现p2 7的表达水平发生了明显下调,而细胞周期蛋白D和细胞周期蛋白E的表达水平发生了明显上调;当pcDNA3 hbxip质粒与pCMV X质粒进行共瞬时转染时,细胞凋亡发生率由pcDNA3质粒与pCMV X质粒共转染时的2 9 2 %下降为13 3% ,p2 7的表达水平发生了下调,但细胞周期蛋白D和细胞周期蛋白E的表达水平无明显变化.研究结果表明,瞬时转染一定剂量的x基因可导致肝癌细胞发生凋亡,细胞周期相关蛋白p2 7、细胞周期蛋白D和     

Ran GTPase protein promotes human pancreatic cancer proliferation by deregulating the expression of Survivin and cell cycle proteins

Ran, a member of the Ras GTPase family, has important roles in nucleocytoplasmic transport. Herein, we detected Ran expression in pancreatic cancer and explored its potential role on tumour progression. Overexpressed Ran in pancreatic cancer tissues was found highly correlated with the histological grade. Downregulation of Ran led to significant suppression of cell proliferation, cell cycle arrest at the G1/S phase and induction of apoptosis. In vivo studies also validated that result. Further studies revealed that those effects were at least partly mediated by the downregulation of Cyclin A, Cyclin D1, Cyclin E, CDK2, CDK4, phospho-Rb and Survivin proteins and up regulation of cleaved Caspase-3.     

Activation of cyclin-dependent kinase 2 by full length and low molecular weight forms of cyclin E in breast cancer cells

Cyclin E, a positive regulator of the cell cycle, controls the transition of cells from G(1) to S phase. Deregulation of the G(1)-S checkpoint contributes to uncontrolled cell division, a hallmark of cancer. We have reported previously that cyclin E is overexpressed in breast cancer and such overexpression is usually accompanied by the appearance of low molecular weight isoforms of cyclin E protein, which are not present in normal cells. Furthermore, we have shown that the expression of cyclin E low molecular weight isoforms can be used as a reliable prognostic marker for breast cancer to predict patient outcome. In this study we examined the role of cyclin E in directly activating cyclin-dependent kinase (CDK) 2. For this purpose, a series of N-terminal deleted forms of cyclin E corresponding to the low molecular weight forms detected only in cancer cells were translated in vitro and mixed with cell extracts. These tumor-specific N-terminal deleted forms of cyclin E are able to activate CDK2. Addition of cyclin E into both normal and tumor cell extracts was shown to increase the levels of CDK2 activity, along with an increase in the amount of phosphorylated CDK2. The increase in CDK2 activity was because of cyclin E binding to endogenous CDK2 in complex with endogenous cyclin E, cyclin A, or unbound CDK2. The increase in CDK2 phosphorylation was through a pathway involving cyclin-activating kinase, but addition of cyclin E to an extract containing unphosphorylated CDK2 can still lead to increase in CDK2 activity. Our data suggest that the ability of high levels of full-length and low molecular weight forms of cyclin E to activate CDK2 may be one mechanism that leads to the constitutive activation of cyclin E.CDK2 complexes leading to G(1)/S deregulation and tumor progression.     

人细胞周期蛋白D1/CDK4基因的真核表达及生物活性鉴定

通过生物工程获得人重组细胞周期蛋白 (cyclinD1 )及细胞周期蛋白激酶CDK4蛋白 ,作为抗癌药物筛选的分子靶点 .从人HL 6 0细胞中获得细胞周期蛋白D1 CDK4基因的cDNA ,先克隆至pGEMT Easy载体上 ,再经重组构建供体质粒pFastBac D1和pFastBac CDK4 .重组供体质粒转化感受态DH1 0Bac细胞 ,挑取确证为白色克隆的菌落振荡培养 ,分离制备高纯度杆粒DNA .以重组病毒适量感染昆虫细胞Tn 5B1 4 ,利用Bac to Bac杆状病毒表达系统在昆虫细胞Tn 5B1 4 (Hi5 )中表达相应的重组蛋白 .应用昆虫杆状病毒表达系统 (Bac to Bac)在昆虫细胞Tn 5B1 4中分别高效表达了人细胞周期蛋白D1和CDK4蛋白 .SDS PAGE分析表明 ,表达量占细胞可溶性蛋白质的 2 0 %左右 ,表达产物经Ni2 + NTA亲和层析纯化后纯度达 85 %以上 .研究表明 ,昆虫细胞表达的细胞周期蛋白D1和CDK4蛋白能促进Rb蛋白的磷酸化 ,具有生物活性 .成功构建了细胞周期蛋白D1及CDK4真核杆状病毒表达载体 ,并且在昆虫细胞中正确表达了具有生物活性的细胞周期蛋白D1及CDK4融合蛋白 .     

Cyclin T1 overexpression induces malignant transformation and tumor growth

Human PTEFb is a protein kinase composed by CDK9 and Cyclin T that controls the elongation phase of RNA Pol II. This complex also affects the activation and differentiation program of lymphoid cells. In this study we found that several head and neck tumor cell lines overexpress PTEFb. We also established that Cyclin T1 is able to induce transformation in vitro, as we determined by foci and colony formation assays. Nu/nu mice s.c. injected with stable transfected Cyclin T1 cells (NIH 3T3 Cyclin T1) developed tumors faster than animals injected with control cells (NIH 3T3 β-gal). In vitro, NIH 3T3 Cyclin T1 cells show increased proliferation and CDK4-Rb phosphorylation. Even more, silencing E2F1 expression (shRNA E2F1) in NIH 3T3 cells resulted in a dramatic inhibition of Cyclin T1-induced foci. All these data demonstrate for the first time the Cyclin T1 oncogenic function and suggest a role for this protein in controlling cell cycle probably via Rb/E2F1 pathway.Key words: cyclin T1, CDK9, PTEFb     

Oxidative stress regulates expression of VEGFR1 in myeloid cells: link to tumor-induced immune suppression in renal cell carcinoma

Metastatic renal cell carcinoma (RCC) associates with overproduction of vascular endothelial growth factor (VEGF) due to the mutation/inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. Herein we demonstrate that implantation of human RCC tumor cells into athymic nude mice promotes the appearance of VEGF receptor 1 (VEGFR1)/CD11b double-positive myeloid cells in peripheral blood. Avastin-mediated VEGF neutralization was capable of significantly reducing the numbers of circulating VEGFR1+ myeloid cells. Conversely, up-regulation of VEGFR1 by myeloid cells could also be achieved in vitro by coculturing bone marrow cells with RCC-conditioned medium or by short-term exposure of naive myeloid cells to oxidative stress. Treatment of myeloid cells with H2O2, lipid peroxidation product 4-hydroxy-2(E)-nonenal, or an inhibitor of thioredoxin reductase all resulted in increased expression of VEGFR1. Furthermore, after exposure to oxidative stress, myeloid cells acquire immunosuppressive features and become capable of inhibiting T cell proliferation. Data suggest that tumor-induced oxidative stress may promote both VEGFR1 up-regulation and immunosuppressive function in bone marrow-derived myeloid cells. Analysis of tumor tissue and peripheral blood from patients with metastatic RCC revealed that VEGFR1+ cells can be also found in cancer patients. Restoration of immunocompetence in metastatic RCC patients by pharmacological elimination of VEGFR1+ cells may have a significant impact on the therapeutic efficacy of cancer vaccines or other immune-based therapies.     

Phosphorylation of mammalian CDC6 by cyclin A/CDK2 regulates its subcellular localization.

Cyclin-dependent kinases (CDKs) are essential for regulating key transitions in the cell cycle, including initiation of DNA replication, mitosis and prevention of re-replication. Here we demonstrate that mammalian CDC6, an essential regulator of initiation of DNA replication, is phosphorylated by CDKs. CDC6 interacts specifically with the active Cyclin A/CDK2 complex in vitro and in vivo, but not with Cyclin E or Cyclin B kinase complexes. The cyclin binding domain of CDC6 was mapped to an N-terminal Cy-motif that is similar to the cyclin binding regions in p21(WAF1/SDI1) and E2F-1. The in vivo phosphorylation of CDC6 was dependent on three N-terminal CDK consensus sites, and the phosphorylation of these sites was shown to regulate the subcellular localization of CDC6. Consistent with this notion, we found that the subcellular localization of CDC6 is cell cycle regulated. In G1, CDC6 is nuclear and it relocalizes to the cytoplasm when Cyclin A/CDK2 is activated. In agreement with CDC6 phosphorylation being specifically mediated by Cyclin A/CDK2, we show that ectopic expression of Cyclin A, but not of Cyclin E, leads to rapid relocalization of CDC6 from the nucleus to the cytoplasm. Based on our data we suggest that the phosphorylation of CDC6 by Cyclin A/CDK2 is a negative regulatory event that could be implicated in preventing re-replication during S phase and G2.     

In vivo study on the effects of curcumin on the expression profiles of anti-tumour genes (VEGF, CyclinD1 and CDK4) in liver of rats injected with DEN

In this study we investigated the effects of curcumin, derived from plant Curcuma longa, on oxidative toxicity, and the possible molecular mechanism of antitumour of curcumin in liver cancer rats. Results showed that blood levels of Gamma-glutamyltransferase, aspartate aminotransferase, alanine aminotransferase, glutathione S-transferase, and liver level of MD were significantly decreased after curcumin feeding. Levels of the liver malondialdehyde MDA, nitric oxide and antioxidant enzymes were significantly increased. Moreover, RT-PCR and Western blot analysis results showed that curcumin treatment significantly decreased liver vascular endothelial growth factor (VEGF), CyclinD1 and CDK4 mRNA expression levels and CyclinD1 and CDK4 proteins levels in liver cancer rats. These findings were confirmed by histopathology. It is concluded that curcumin can protect the liver from the damage caused by N-nitrosodiethylamine. Moreover, curcumin has the potential to be used in a therapy for liver cancer. The present data provide evidence to support the presence of free radicals and VEGF, CyclinD1 and CDK4 mRNA in rat tumour cells. Studies are in progress in order to further characterize the role of VEGF, CyclinD1 and CDK4 mRNA in liver cancer cells and in hepatic therapeutics.