F-box protein FBXL2 exerts human lung tumor suppressor-like activity by ubiquitin-mediated degradation of cyclin D3 resulting in cell cycle arrest

Dysregulated behavior of cell cycle proteins and their control by ubiquitin E3 ligases is an emerging theme in human lung cancer. Here, we identified and characterized the activity of a novel F-box protein, termed FBXL2, belonging to the SCF (Skip-Cullin1-F-box protein) E3 ligase family. Ectopically expressed FBXL2 triggered G2/M-phase arrest, induced chromosomal anomalies and increased apoptosis of transformed lung epithelia by mediating polyubiquitination and degradation of the mitotic regulator, cyclin D3. Unlike other F-box proteins that target phosphodegrons within substrates, FBXL2 uniquely recognizes a canonical calmodulin (CaM)-binding motif within cyclin D3 to facilitate its polyubiquitination. CaM bound and protected cyclin D3 from FBXL2 by direct intermolecular competition with the F-box protein for access within this motif. The chemotherapeutic agent vinorelbine increased apoptosis of human lung carcinoma cells by inducing FBXL2 expression and cyclin D3 degradation, an effect accentuated by CaM knockdown. Depletion of endogenous FBXL2 stabilized cyclin D3 levels, accelerated cancer cell growth and increased cell viability after vinorelbine treatment. Last, ectopic expression of FBXL2 significantly inhibited the growth and migration of tumorogenic cells and tumor formation in athymic nude mice. These observations implicate SCF(FBXL2) as an indispensible regulator of mitosis that serves as a tumor suppressor.     

Processing of cyclin E differs between normal and tumor breast cells

Cyclin E is a G1 cyclin essential for G1 to S-phase transition of the cell cycle with a profound role in oncogenesis. In tumor cells and tissues, cyclin E is overexpressed and present in its lower molecular weight (LMW) isoforms, and it can be used as a prognosticator for poor patient outcome. In this study, we have examined differences in the processing of cyclin E between normal mammary epithelial and breast cancer cell lines. Five NH2-terminally deleted epitope-tagged (FLAG) cyclin E vectors were constructed spanning the range of LMW forms observed in tumor cells. These constructs were transfected into normal and tumor cells and analyzed for the production of cyclin E-FLAG protein products by Western blot analysis with FLAG and cyclin E antibodies. Our results show that only tumor cells had the machinery to process these cyclin E-FLAG constructs to their LMW forms, whereas normal cells mainly expressed the full-length unprocessed form of each protein. Tumor and normal cells always process the cyclin E-FLAG protein in the same way as endogenously expressed cyclin E. This phenomenon is consistent with all of the cell lines used, regardless of transfection efficiency, time of processing posttransfection, or method of transfection. Furthermore, measurement of FLAG-associated kinase activity in the transfectants revealed that the protein products of the cyclin E-FLAG constructs are 10 times more active in tumor cells than in normal cells. These studies suggest that the LMW forms of cyclin E detected at a much higher level in tumor cells arise from posttranslational action of a protease.     

Inhibitory effect of p21 in MCF-7 cells is overcome by its coordinated stabilization with D-type cyclins

Coordinated accumulation of cyclin D1 and D3 is observed in 15% of primary breast cancers and in the breast cancer cell line MCF-7 this simultaneous overexpression is due to a defect in their ubiquitin-mediated proteolysis. The F-box protein Skp2 is a component of an SCF ubiquitin ligase complex and can associate with cyclin D1 and the cdk inhibitor p21 (Zhong-Kang et al., 1998). We extend this observation and show that cyclin D3 can also associate with Skp2 suggesting that cyclins D1, D3 and p21 may share the same SCF complex. In agreement with this hypothesis we report here that in primary breast cancers and in MCF-7 cells where cyclins D1 and D3 are elevated the level of p21 is also elevated. Further, we demonstrate that the turnover of p21 protein is reduced in MCF-7 cells. We show that p21 is active as a cdk inhibitor in this cell line but that the presence of elevated levels of cyclin D3 titrates p21 away from cyclin D1-cdk4/6 complexes and cdk2 complexes resulting in increased kinase activities. Our results suggest that a defect in the SCF complex may occur in 15-20% of breast cancers and that the resulting coordinated elevation of cyclins D1 and D3 overcomes the inhibition of cell cycle progression by p21. We propose that in the context of cyclins D1 and D3 overexpression, p21 may promote cell cycle progression.     

A splice variant of Skp2 is retained in the cytoplasm and fails to direct cyclin D1 ubiquitination in the uterine cancer cell line SK-UT.

Cyclin D1 is an important regulator of the transition from G1 into S phase of the cell cycle. The level to which cyclin D1 accumulates is tightly regulated. One mechanism contributing to the control of cyclin D1 levels is the regulation of its ubiquitination. SK-UT-1B cells are deficient in the degradation of D-type cyclins. We show here that p27, a substrate of the SCF(Skp2) ubiquitin ligase complex, is coordinately stabilized in SK-UT-1B cells. Further, we show that expression of Skp2 in SK-UT-1B cells rescues the cyclin D1 and p27 degradation defect observed in this cell line. These results therefore indicate that the SCF(Skp2) ubiquitin ligase complex affects the ubiquitination of cyclin D1. In addition, we show that SK-UT-1B cells express a novel splice variant of Skp2 that localizes to the cytoplasm and that cyclin D1 ubiquitination takes place in the nucleus. We propose that the translocation of Skp2 into the nucleus is required for the ubiquitination of cyclin D1 and that the absence of the SCF(Skp2) complex in the nucleus of SK-UT-1B cells is the mechanism underlying the ubiquitination defect observed in this cell line. Finally, our data indicates that differential splicing of F-box proteins may represent an additional level of regulation of the F-box mediated ubiquitination pathway.     

一种新的黑色素瘤相关抗原MAGE-C2的基因克隆及表达

目的:克隆人黑色素瘤相关抗原MAGE-C2的cDNA,构建真核、原核表达载体并转入相应细胞获得表达.方法:采用RT-PCR技术,从直肠癌细胞系SW480的总RNA中克隆了MAGE-C2 cDNA序列,并将开放读框分别插入质粒pEGFP-C1和pGEX-4T-3中,获得pEGFP-MAGE-C2绿色荧光蛋白(GFP)融合表达载体和pGEX-MAGE-C2谷胱甘肽转移酶(GST)融合蛋白表达载体,将pEGFP-MAGE-C2转染293T细胞,观察绿色荧光融合蛋白在细胞中的定位;将pGEX-MAGE-C2转化大肠杆菌BL21株,经IPTG诱导表达GST融合蛋白,并用谷胱甘肽亲和层析法纯化重组蛋白.结果:获得MAGE-C2开放读框并构建表达载体,测序结果与GenBank收录的序列相一致.真核表达载体转染293T细胞后显示MAGE-C2蛋白定位于细胞核;原核重组蛋白大部分以可溶性形式表达,亲和层析后,获得了较高纯度的MAGE-C2-GST融合蛋白.分析显示原核表达GST融合蛋白的相对分子质量为70 000,使用抗GST单克隆抗体进行Western blot分析证实为目的蛋白.结论:成功的获得MAGE-C2基因,构建了其表达载体并获得表达,为进一步制备MAGE-C2特异性单抗及深入探讨MAGE-C2可能参与肿瘤发生发展的机制提供了重要条件.     

Expression of G1 phase regulators in MG-63 osteosarcoma cell line.

Cyclins and cyclin-dependent kinases (cdks) form complexes that govern transitions during cell cycle phases. In this study we characterized a human osteosarcoma cell line, MG-63, for the expression level of cyclin D1, cyclin E, cdk4, cdk2, and cell cycle inhibitors pRb and p21. To investigate the role of these proteins we treated MG-63 cells with tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Cell proliferation analysis demonstrated an increased proliferation of MG-63 cells with IL-6, while TNF-alpha acted as an anti-proliferative agent. Immunoblotting revealed an increased expression of p21 with TNF-alpha and its complex with cdk2. TNF-alpha reduced the expression of the cyclin E-cdk2 complex. TNF-alpha did not affect the amount of cyclin D1, cyclin E, cdk4, cdk2, and of cyclin D1-cdk4 complex. IL-6 decreased p21 expression and its complex with cdk2, while it increased the cyclin E-cdk2 complex. Cyclin D1 and cdk4 expression and their complex did not change after IL-6 treatment, nor did cyclin E and cdk2 protein expression. Hyperphosphorylated/dephosphorylated Rb protein ratio was reduced with TNF-alpha whereas it increased with IL-6. These results may suggest an important role of p21 and of cyclin E-cdk2 complex in the G1 phase regulation through pRb phosphorylation in MG-63 cells.     

HuR contributes to cyclin E1 deregulation in MCF-7 breast cancer cells

Many cancers overexpress cyclin E1 and its tumor-specific low molecular weight (LMW) isoforms. However, the mechanism of cyclin E1 deregulation in cancers is still not well understood. We show here that the mRNA-binding protein HuR increases cyclin E1 mRNA stability in MCF-7 breast carcinoma cells. Thus, mRNA stabilization may be a key event in the deregulation of cyclin E1 in MCF-7 cells. Compared with MCF10A immortalized breast epithelial cells, MCF-7 cells overexpress full-length cyclin E1 and its LMW isoforms and exhibit increased cyclin E1 mRNA stability. Increased mRNA stability is associated with a stable adenylation state and an increased ratio of cytoplasmic versus nuclear HuR. UV cross-link competition and UV cross-link immunoprecipitation assays verified that HuR specifically bound to the cyclin E1 3'-untranslated region. Knockdown of HuR with small interfering RNA (siRNA) in MCF-7 cells decreased cyclin E1 mRNA half-life (t(1/2)) and its protein level: a 22% decrease for the full-length isoforms and 80% decrease for the LMW isoforms. HuR siRNA also delayed G(1)-S phase transition and inhibited MCF-7 cell proliferation, which was partially recovered by overexpression of a LMW isoform of cyclin E1. Overexpression of HuR in MCF10A cells increased cyclin E1 mRNA t(1/2) and its protein level. Taken together, our data show that HuR critically contributes to cyclin E1 overexpression and its growth-promoting function, at least in part by increasing cyclin E1 mRNA stability, which provides a new mechanism of cyclin E1 deregulation in breast cancer.     

非小细胞肺癌中PAX6表达及其对肿瘤细胞增殖与侵袭作用机制研究

背景与目的：转录因子PAX6主要表达于胚胎期,不同肿瘤中PAX6呈现高表达,通过不同的信号通路发挥肿瘤抑制或促进作用。检测PAX6在非小细胞肺癌(non-small cell lung cancer,NSCLC)中的表达,通过RNAi下调其表达,研究PAX6对肿瘤细胞侵袭和增殖力以及cyclin E、p38表达水平的影响。方法：应用蛋白印记检测86例NSCLC肿瘤组织及癌旁配对组织以及2例细胞系中PAX6的表达情况。应用PAX6特异性siRNA序列,转染NSCLC细胞系A549,MTT法、Transwell小室、划痕实验检测转染前后细胞增殖、侵袭和迁移能力的对比。蛋白质印记法(Western blot)检测转染前后cyclin E及p38表达情况。结果：对比癌旁组织及正常人支气管上皮细胞16HBE,PAX6在NSCLC组织及A549细胞系中显著高表达。选取高效siRNA序列转染细胞系后,PAX6表达的下调抑制了肿瘤细胞的增殖及集落形成,G1期细胞比例增加,细胞侵袭及迁移力均下降。PAX6基因敲低细胞cyclin E及p38活性受到抑制,表达下调。结论：PAX6通过调控MAPK通路及cyclin E的表达加速肿瘤细胞的增殖、侵袭及迁移,是潜在的NSCLC诊疗靶点。     

细胞周期相关因子与乳腺细胞的癌变及生物学特性的相关性研究

目的：研究正常乳腺上皮细胞与乳腺癌细胞之间以及不同恶性程度的乳腺癌细胞之间的细胞周期相关因子的表达异同。方法：采用Western印迹法检测正常乳腺细胞株AG11132A、ER阳性和乳腺癌细胞株MCF－7及ER阴性的乳腺癌细胞株MDA－MB－231之间细胞周期蛋白D1、E及P21蛋白表达的异同及与其生物学特性的关系。结果：（1）正常乳腺上皮细胞株高表达P21蛋白,低表达周期蛋白E。与正常细胞相比,乳腺癌细胞株MCF－7、MDA－MB－231细胞高表达周期蛋白E,其中表达的周期蛋白E中存在异常的你分子量周期蛋白E成分,而正常乳腺上皮细胞不表达这种异常 的周期蛋白E。（2）在乳腺癌细胞株之间,相对ER阳性的MCF－7细胞,ER阴性的MDA－MB－231细胞则高表达周期蛋白E,基本无表达P21蛋白。结论L（1）正常细胞与这间、相对低恶性程度的民相对高恶性和蔼的癌细胞之间的差别是多环节的、质和量异常导致的结果;（2）周期蛋白E及P21均可反映乳腺癌细胞的增殖活性和恶性程度,可能是乳腺癌的临床预后指标。     

MAGE-B5, MAGE-B6, MAGE-C2, and MAGE-C3: four new members of the MAGE family with tumor-specific expression

A number of genes of the MAGE-A, B, and C families have been shown to code for antigens that are recognized on many human tumors by autologous cytolytic T lymphocytes. These antigens ought to be strictly tumor specific because the encoding MAGE genes are not expressed in normal adult cells except for male germline cells, which lack HLA expression. To identify new genes of this type, we performed representational difference analysis on a melanoma cell line by subtraction with a normal skin sample. This led to the identification of MAGE-C2, a new member of the MAGE-C family. A search for nucleotide sequences encoding MAGE-like proteins in public databases led to the identification of three additional MAGE genes, which were named MAGE-B5, MAGE-B6, and MAGE-C3. The four new MAGE genes are not expressed in normal tissues, except for testis, and are expressed in tumors of different histological origins. Therefore, like other MAGE genes expressed specifically in tumors, MAGE-B5, MAGE-B6, MAGE-C2, and MAGE-C3 ought to encode antigens that could be targets for cancer immunotherapy.     

Detection of cyclin b1 expression in g(1)-phase cancer cell lines and cancer tissues by postsorting Western blot analysis

Protein complex of cyclin B1 and cyclin-dependent protein kinase 1 induces phosphorylation of key substrates that mediate cell cycle transition during the G(2)-M phase. It is believed that cyclin B1 accumulates in the S phase of the cell cycle and reaches the maximal level at mitosis but is absent in G(1)-phase cells. In the present study, we demonstrated that cyclin B1 was expressed in the arrested G(1)-phase MOLT-4 lymphocyte leukemia cells and in G(1) phase T-7 transitional tumor cells, as determined by flow cytometry. In addition, we showed that cyclin B1 was detected in the G(1) phase in breast cancer cells from patient tissues and in lymphocytes from patients with leukemia. These findings were confirmed for the first time by postsorting Western blot analysis and by confocal microscopy. Furthermore, by using postsorting Western blotting, we found that cyclin B1 was expressed in different time-window sections of the G(1) phase under different conditions. For the asynchronously growing T-7 cells, cyclin B1 was detected in early G(1) phase, whereas in MOLT-4 cells arrested in G(1)-S phase, cyclin B1 was mainly detected in late G(1) phase. We propose that the cyclin B1 expressed in the G(1) phase may differ from that expressed in the G(2)-M phase, and that this unscheduled type of cyclin B1 may play an important role in tumorigenesis and apoptosis.     

Insufficient effect of p27(KIP1) to inhibit cyclin D1 in human esophageal cancer in vitro

The cell cycle is controlled by protein complexes composed of cyclins and cyclin-dependent kinases. p27KIP1 (p27) is one of the Kip/Cip family cyclin-dependent kinase inhibitory proteins which negatively regulate cell cycle progression, and have been proposed as candidate tumor suppressor genes. To examine the role of p27 in the development of human esophageal squamous cell carcinoma (ESCC), we performed Western blot and immunoprecipitation analyses of the levels of expression of p27 protein in a series of ESCC cell lines. This protein was expressed at various levels in these cell lines during exponential growth. p27 level was significantly associated with that of cyclin D1, but not of cyclin E. Further cell cycle synchronization studies demonstrated that p27 was free or bound with affinity to cyclin E-CDK2 more than to cyclin D1-CDK4 or cyclin D1-CDK6. It is known that overexpression of cyclin D1 rather than cyclin E is involved in the pathogenesis of ESCC. Our findings indicated that high expression of p27 throughout the G1 to S phase may inhibit more likely cyclin E, than cyclin D1, which promotes tumor growth of esophageal squamous cell carcinoma.     

Altered complex formation between p21waf, p27kip and their partner G1 cyclins determines the stimulatory or inhibitory transforming growth factor-beta1 growth response of human fibroblasts

TGF-beta1 stimulates proliferation of WI38 human embryo fibroblasts but inhibits that of their SV40-transformed counterparts, VA13 cells. Protein expression levels of cyclins A, D1, E and that of cdk2 and cdk4 were not affected by TGF-beta1 in either of these cells. However, TGF-beta1-treatment increases cdk2 kinase activity in WI38 cells and reduces it in VA13 cells. The same treatment reduces the amount of p21waf present in complexes with cyclins D1 and E in growth-stimulated WI38 cells, but the reverse applies in growth-inhibited VA13 cells. Mitogenic stimulation of WI38 fibroblasts correlated with decreased expression of p27kip protein and reduced amounts of it in complex with cyclin E. In contrast, proliferative inhibition of VA13 fibroblasts by TGF-beta1 caused a reduction of p27kip in complexes with cyclin D1, but increased it in complexes with cyclin E, without affecting the overall level of p27kip protein expression. Thus, in this human fibroblast model, TGF-beta1-mediated stimulation or inhibition of proliferation depends on modulation in the amounts of p21waf and p27kip in complexes with cyclins D1 and E.     

细胞周期蛋白E小干扰RNA靶向调控乳腺癌细胞的生长

目的 研究乳腺癌细胞中细胞周期蛋白E(cyelin E)对乳腺癌细胞肿瘤生物学特征的影响.方法 用带有cyclin E小干扰RNA(siRNA)的真核表达载体pEGFP/CCNE2转染乳腺痛细胞系MCF-7.采用逆转录聚合酶链反应(RT-PCR)和Western blot方法分别在RNA水平和蛋白水平检测siRNA对细胞内cyclin E表达水平的影响,CCK-8方法检测转染后细胞株生长增殖能力以及对化疗药物的敏感性,流式细胞仪检测细胞周期的变化,裸鼠移植瘤成瘤实验检测转染后细胞成瘤能力的变化.结果 pEGFP/CCNE2转染后,MCF-7细胞内cyclin E的表达受到抑制,cyclin E mRNA相对表达水平为0.23±0.05,蛋白相对表达水平为0.24±0.05;细胞的生长增殖能力下降,抑制率为68.56%±0.08%,对化疗药物的敏感性增加;细胞被大量阻滞在G1期,G1期细胞占77.38%.细胞的成瘤能力降低,移植瘤体积缩小.结论 抑制乳腺癌细胞中cyclin E的表达能够抑制乳腺癌细胞的生长、分化和增殖,提高肿瘤对化疗药物的敏感性.     

E2F3a在U251胶质瘤细胞中的功能研究

背景与目的：E2F3a是一种重要的转录激活因子，在多种肿瘤组织中均呈现表达上调。E2F3a在恶性化程度较高的神经胶质瘤中表达较高，目前它在胶质瘤细胞中的功能尚不清楚。本文的目的是研究E2F3。对U251胶质瘤细胞周期和凋亡的影响。方法：通过脂质体介导质粒转染在U251细胞中过表达E2F3a：用Western blotting检测细胞内E2F3a蛋白质的表达水平：用PT-核DNA染色结合流式细胞术分析细胞周期分布：用Annexin V—PE和7-AAD染色结合流式细胞术分析细胞凋亡比例；用实时荧光定量PCR方法测定细胞内A2、B1、D1和E细胞周期素mRNA的相对表达水平。结果：与空载体转染对照组相比．E2F3a过表达可将位于S期的细胞比例提高28％（P〈0．01），将位于G0/G1期和G2/M的细胞比例分别降低15％和13％（P〈0．01）。E2F3a过表达对细胞凋亡无明显影响。进一步研究发现，E2F3a可显著上调细胞周期素D1和E的mRNA表达．而不影响周期素A2和B1的mRNA表达。结论：E2F3a是胶质瘤细胞增殖的促进因子，它可通过上调细胞周期素D1和E的表达加速细胞由G，期进入S期。     

Role of the ubiquitin ligase Fbw7 in cancer progression

Fbw7 is a member of F-box family proteins, which constitute one subunit of Skp1, Cul1, and F-box protein (SCF) ubiquitin ligase complex. SCF(Fbw7) targets a set of well-known oncoproteins, including c-Myc, cyclin E, Notch, c-Jun, and Mcl-1, for ubiquitylation and degradation. Fbw7 provides specificity of the ubiquitylation of these substrate proteins via recognition of a consensus phosphorylated degron. Through regulation of several important proteins, Fbw7 controls diverse cellular processes, including cell-cycle progression, cell proliferation, differentiation, DNA damage response, maintenance of genomic stability, and neural cell stemness. As reduced Fbw7 expression level and loss-of-function mutations are found in a wide range of human cancers, Fbw7 is generally considered as a tumor suppressor. However, the exact mechanisms underlying Fbw7-induced tumor suppression is unclear. This review focuses on regulation network, biological functions, and genetic alteration of Fbw7 in connection with its role in cancer development.     

The low molecular weight cyclin E isoforms augment angiogenesis and metastasis of human melanoma cells in vivo

Immunohistochemical analysis has consistently shown that cyclin E is up-regulated in human malignant melanomas in vivo. Here we analyzed such expression in more detail and show that cyclin E is overexpressed and present in low molecular weight (LMW) isoforms in metastatic melanoma and in a subset of primary invasive melanoma tumor tissues, but not in benign nevi. Human metastatic melanoma cell lines, but not normal melanocytes, also expressed the LMW cyclin E forms. The biological significance of these findings was established by showing that overexpression of two LMW cyclin E forms named cyclin E truncated 1 [cyclinE(T1)] and cyclin E truncated 2 [cyclinE(T2)] in a low tumorigenic and non-metastatic primary cutaneous melanoma cell line generated angiogenic tumors with prominent perineural invasion compared with full-length cyclin E. In addition, cyclin E(T1)- and cyclin E(T2)-expressing melanoma cells displayed a dramatic increase in the incidence and number of metastases in an experimental lung metastasis assay. Together, these results indicate that the LMW cyclin E forms are functional and likely act as regulators of human melanoma tumor progression and invasion.