Lattice Structure of Cytoplasmic Microtubules in a Cultured Mammalian Cell

Tubulin can polymerize in two distinct arrangements: “B-lattices,” in which the α-tubulins of one protofilament lie next to α-tubulins in the neighboring protofilaments, or the “A” configuration, where α-tubulins lie beside β-tubulins. Microtubules (MTs) in flagellar axonemes and those assembled from pure tubulin in vitro display only B-lattices, but recent work shows that A-lattices are found when tubulin co-polymerizes in vitro with an allele of end-binding protein 1 that lacks C-terminal sequences. This observation suggests that cytoplasmic MTs, which form in the presence of this “tip-associating protein,” may have A-lattices. To test this hypothesis, we have decorated interphase MTs in 3T3 cells with monomeric motor domains from the kinesin-like protein Eg5. These MTs show only B-lattices, as confirmed by visual inspection of electron cryo-tomograms and power spectra of single projection views, imaged at higher electron dose. This result is significant because 13 protofilament MTs with B-lattices must include a “seam,” one lateral domain where adjacent dimers are in the A-configuration. It follows that cytoplasmic MTs are not cylindrically symmetric; they have two distinct faces, which may influence the binding patterns of functionally significant MT-interacting proteins.     

Apoptotic action of estrogen

Estrogen as a mitogen stimulates cell proliferation and prevents cell death in many cell types. In patients, estrogen is known to stimulate breast and uterus cancer development. Ironically, high doses of estrogen can induce regression of hormone-dependent breast cancer in postmenopausal women. The comprehensive mechanism by which estrogen induces tumor recession in breast cancer is still unknown, but activation of the Fas/FasL pathways plays a key role in this process. Laboratory studies show that the apoptotic action of estrogen is the major factor leading to cell number decreases in several cell types. The effects of estrogen are estrogen-receptor dependent. In this mini review, we will focus on the latest findings regarding estrogen apoptotic effects in several cell models, including breast cancer cells, and summarize the possible mechanisms involved in these estrogen mediated processes. New potential implications for the pharmacological control of breast cancer with estrogen in post-menopausal women are also discussed.     

糖鞘脂GM1调控生长密度相关的乳腺细胞体外增殖抑制

细胞在体内增殖受到制约,以确保器官的正常大小和组织稳态的维持,体外培养的细胞也存在接触抑制生长现象.分布于细胞膜上的糖鞘脂具有调控细胞增殖的作用.本研究探讨了糖鞘脂GM1对人乳腺细胞MCF-10A、人乳腺癌细胞BT-549和SK-BR3增殖的影响.通过对细胞不同接种量研究细胞增殖的变化;利用流式细胞术检测细胞在不同密度生长时GM1的表达差异;探索细胞在不同密度生长时外源添加GM1对细胞增殖的影响;构建GM1干扰和过表达细胞并检测转染细胞株的增殖差异.结果显示,相较于常规密度,在低密度和高密度生长时,细胞增殖受到抑制,GM1表达量提高;GM1处理抑制低密度和高密度生长时细胞增殖,对常规密度生长细胞没有显著影响.在低密度和高密度生长时,GM1干扰细胞增殖能力提高,而GM1过表达细胞增殖能力下降.综上,本实验研究证实GM1抑制乳腺细胞MCF-10A、乳腺癌细胞BT-549和SKBR-3在体外低密度和高密度生长时的细胞增殖,为研究GM1抑制细胞增殖分子机理提供了工作基础.     

siRNA干扰Mad2蛋白的表达对乳腺癌MCF-7细胞紫杉醇药物敏感性的影响

目的研究纺锤体检测点蛋白Mad2对乳腺癌细胞MCF-7紫杉醇敏感性的影响。方法运用小片段干扰RNA技术下调MCF-7细胞中Mad2蛋白的表达,经不同浓度的紫杉醇药物浓度处理,通过细胞术检测细胞周期分布,MTY法检测细胞存活率的变化,RT—PCR及Western印迹法检测相关蛋白的表达改变情况。结果siRNA可以特异性干扰MCF-7细胞中Mad2蛋白水平的表达,同时显著降低紫杉醇诱导细胞发生有丝分裂阻滞的比例,导致CyclinB1的表达下调,凋亡率降低;并且Mad2表达下调后,MCF-7细胞中黏附分子E-cadherin的表达量也降低。结论Mad2在紫杉醇诱导MCF-7细胞发生有丝分裂周期阻滞中发挥重要作用,进而影响细胞对紫杉醇的敏感性,同时细胞黏附分子E—cadherin可能参与这一过程的发生。     

The G-protein-coupled estrogen receptor agonist G-1 suppresses proliferation of ovarian cancer cells by blocking tubulin polymerization

The G-protein-coupled estrogen receptor 1 (GPER) has recently been reported to mediate the non-genomic action of estrogen in different types of cells and tissues. G-1 (1-[4-(6-bromobenzo[1,3] dioxol-5yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone) was developed as a potent and selective agonist for GPER. G-1 has been shown to induce the expression of genes and activate pathways that facilitate cancer cell proliferation by activating GPER. Here we demonstrate that G-1 has an anticancer potential with a mechanism similar to vinca alkaloids, the commonly used chemotherapy drugs. We found that G-1 blocks tubulin polymerization and thereby interrupts microtubule assembly in ovarian cancer cells leading to the arrest of cell cycle in the prophase of mitosis and the suppression of ovarian cancer cell proliferation. G-1 treatment also induces apoptosis of ovarian cancer cells. The ability of G-1 to target microtubules to suppress ovarian cancer cell proliferation makes it a promising candidate drug for treatment of ovarian cancer.     

SH3GL1在紫杉醇耐药乳腺癌中的表达及临床意义

膜结合蛋白SH3GL1参与调控某些肿瘤细胞生物学行为,而其对紫杉醇耐药乳腺癌细胞的恶性生物学行为影响尚未见报道.为阐明 SH3GL1对紫杉醇耐药敏感性的影响以及潜在的分子机制,本研究首先采用免疫组化法证实SH3GL1在紫杉醇耐药乳腺癌组织高表达(P<0.001).Western印迹法证实,SH3GL1在紫杉醇耐药细胞株MCF-7/PTX高表达(P<0.01);随后,采用MTT分别检测（0,50,100 nmol/L）紫杉醇处理后MCF-7细胞株增殖情况,同时Western印迹法检测SH3GL1表达,发现100 nmol/L紫杉醇能够抑制MCF-7细胞增殖(P<0.05);同时抑制SH3GL1表达(P<0.01); 敲减SH3GL1表达后,紫杉醇耐药细胞株MCF-7/PTX和MCF-7增殖速率降低(P<0.05),耐药基因MDR1表达降低(P<0.05),p-AKT和p-gp水平下降(P<0.05).上述结果表明,降低SH3GL1表达可以减弱紫杉醇耐药性,增加乳腺癌对紫杉醇的敏感性,这为临床上紫杉醇耐药乳腺癌患者的治疗提供了新的靶点.     

EB病毒潜伏膜蛋白1通过Survivin介导细胞增殖和抑制细胞凋亡

利用间接免疫荧光、基因转染、抗体剔除 (Ab knock out)、细胞平板集落形成、流式细胞术以及半胱氨酸天冬酰胺酶 (caspase3)活性检测等方法 ,从survivin核移位、Rb磷酸化、细胞周期演进、细胞克隆形成和细胞凋亡等方面 ,探讨EB病毒潜伏膜蛋白 1(LMP1)调控细胞增殖和细胞凋亡双重效应的分子机制 .结果发现 ,LMP1表达介导survivin核移位 ,促进细胞Rb磷酸化增加 ,S期细胞数显著增加 ;LMP1通过survivin促进细胞克隆形成 .用Ab knock out阻断survivin核移位和survivin反义核酸抑制survivin表达时 ,Rb磷酸化水平降低 ,S期细胞减少 ,抑制LMP1介导的细胞增殖 ,活化细胞caspase 3,诱导细胞凋亡 .结果提示 ,EB病毒LMP1通过survivin促进细胞增殖和抑制细胞凋亡     

Paclitaxel administration and its effects on clinically relevant human cancer and non cancer cell lines

Comparisons of the effects of clinically relevant concentrations of the anticancer agent paclitaxel on growth, viability, and apoptosis were determined using in vitro human cell cultures. Growth of the cervical cancer cell line, HeLa-S3, was significantly reduced, and apoptotic index was significantly increased, after 24 h in cultures treated with 12 nM paclitaxel. In contrast, hepatic carcinoma (HEpG2) cells capable of detoxifying paclitaxel were only affected at paclitaxel concentrations ge120 nM. The previously uncharacterized non-cancerous human microvessel endothelial cell line HMEC-1, was more sensitive to paclitaxel treatment than both HeLa-S3 and HEpG2 cells, demonstrating decreased growth and increased apoptosis with 1.2 nM paclitaxel. These results are significant in the design of in vitro cell culture systems to study drug metabolism and toxicity.     

siRNA沉默SIRT1基因对宫颈癌细胞HeLa增殖和凋亡的影响

化学合成靶向SIRT1基因的小干扰RNA,脂质体法转染人宫颈癌细胞株HeLa,观察小干扰RNA沉默SIRT1基因对HeLa增殖及细胞凋亡的影响。在优化siRNA SIRT1转染条件的基础上,应用RT-PCR和Western blot分别检测各组SIRT1 mRNA、SIRT1蛋白及凋亡相关蛋白的表达;CCK-8法检测细胞增殖抑制率;Hoechst荧光染色法和流式细胞仪检测细胞凋亡。结果表明,siRNA SIRT1转染细胞组SIRT1 mRNA水平和蛋白表达量明显低于对照组;siRNA SIRT1转染组细胞增殖受抑制,细胞凋亡率明显增加;凋亡相关蛋白P53、P21表达上调,Survivin表达下调。上述结果表明:siRNA SIRT1诱导的HeLa细胞凋亡与P53、P21、Survivin通路关系密切,但siRNA SIRT1诱导HeLa细胞凋亡的详尽机制有待进一步研究。     

靶向IRE1a干扰质粒构建及对ERS时细胞增殖及凋亡的影响

目的构建靶向人IRE1a的shRNA干扰质粒（pSUPER-IRE1a）并观察其对人HeLa细胞和HepG2细胞增殖及凋亡的影响。方法设计并合成靶向IRE1a基因的两条shRNA,分别克隆至真核表达载体pSUPER构建重组质粒pS1、pS2,依次转染入HeLa细胞和HepG2细胞中。采用RT—PCR检测pS1、pS2转染前后IRE1a在HeLa细胞和HepG2细胞中的mRNA水平,免疫印迹检测pS1、pS2转染前后IRE1a蛋白的表达;MTT比色法、BrdU／DAPI双免疫荧光法及流式细胞仪分别检测各重组质粒对HeLa细胞和HepG2细胞增殖及凋亡的影响。结果干扰质粒（pSUPER-IRE1a）能有效抑制HeLa细胞和HepG2细胞中IRE1a基因的表达;成功转染后,细胞处于内质网应激（ER stress）状态时,各实验组细胞增殖率及凋亡率与对照组比较,差异均具有统计学意义P〈0．05）。结论成功构建靶向人IRE1a的shRNA真核表达载体pS1、pS2,有效抑制了HeLa细胞和HepG2细胞中IRE1a的表达;细胞处于ERS状态时,IRE1a-shRNA有效促进HeLa、HepG2两种肿瘤细胞的增殖;抑制HeLa和HepG2细胞的凋亡。