HSP90高表达细胞株的建立及细胞增殖性状的改变

目的:建立稳定高表达热休克蛋白90(HSP90)细胞株,研究其对细胞增殖的影响.方法:含人HSP90 13全长基因的重组质粒pSmycHSP经亚克隆、纯化、酶切鉴定后,用电穿孔法转染到小鼠成纤维细胞系NIH-3T3细胞内.经G418筛选、克隆分离培养,用免疫细胞化学、免疫印迹鉴定阳性克隆.以转染空质粒的NIH-3T3细胞为对照,用MTT法、流式细胞术测定,分析HSP90高表达对细胞增殖和细胞周期的影响.结果:转染pSmycHSP的NIH-3T3细胞HSP90染色增强,生长速度减慢,S期DNA含量降低.结论:己建立稳定高表达热休克蛋白90(HSP90)NIH-3T3细胞株;转染pSmycHSP的NIH-3T3细胞能够有效地表达HSP90,影响细胞周期,使细胞增殖迟滞.     

慢病毒介导的GFP-Hsp90αE47A 基因在HepG2 细胞表达及对 细胞增殖性影响的研究

目的:建立真核细胞表达的GFP-Hsp90αE47A基因重组慢病毒载体三质粒包装细胞系统,并检测其对细胞增殖性的影响,为进一步研究HSP90分子伴侣功能奠定基础。方法:制备完整的重组慢病毒载体三质粒系统:转移质粒(Hsp90αE47A/psin-GFP),包装质粒(ΔNRF)及包膜蛋白质粒(VSV-G)。磷酸钙法将三质粒共转染293T包装细胞,48h后收集病毒上清。将制备好的慢病毒颗粒感染HepG2细胞,在荧光显微镜下观察报告基因GFP的表达情况,Westernblot检测HepG2细胞GFP-Hsp90α表达。MTT法检测细胞增殖情况。结果:转染后的293T和感染后的HepG2细胞能观察到较强的绿色荧光,培养液上清病毒滴度约为3.0×103ifu/μl,HepG2细胞中有GFP-Hsp90α蛋白的表达。内源性Hsp90α表达无明显上升(为对照组的1.05±0.15倍,P<0.05,t检验),有明显外源性GFP-Hsp90αE47A蛋白的表达,为对照组内源性Hsp90α的0.68±0.12倍。外源性GFP-Hsp90αE47A蛋白的表达HepG2细胞增殖活性于第4d有明显抑制。(1.051±0.03vs1.349±0.05,P<0.05,t检验)。结论:成功建立重组慢病毒载体的三质粒包装细胞系统,并将GFP-Hsp90αE47A基因在HepG2细胞中稳定表达,且并未引起细胞明显的热休克反应而导致的内源性Hsp90α增高;且能明显抑制细胞增殖,为后期Hsp90α分子伴侣功能进行研究奠定基础。     

慢病毒介导的GFP-Hsp90αE47A基因在HepG2细胞表达及对细胞增殖性影响的研究

目的：建立真核细胞表达的GFP-Hsp90αE47A基因重组慢病毒载体三质粒包装细胞系统,并检测其对细胞增殖性的影响,为进一步研究HSP90分子伴侣功能奠定基础。方法：制备完整的重组慢病毒载体三质粒系统：转移质粒（Hsp90αE47A/psin-GFP）,包装质粒（ΔNRF）及包膜蛋白质粒（VSV-G）。磷酸钙法将三质粒共转染293T包装细胞,48h后收集病毒上清。将制备好的慢病毒颗粒感染HepG2细胞,在荧光显微镜下观察报告基因GFP的表达情况,Westernblot检测HepG2细胞GFP-Hsp90α表达。MTT法检测细胞增殖情况。结果：转染后的293T和感染后的HepG2细胞能观察到较强的绿色荧光,培养液上清病毒滴度约为3.0×103ifu/μl,HepG2细胞中有GFP-Hsp90α蛋白的表达。内源性Hsp90α表达无明显上升（为对照组的1.05±0.15倍,P〈0.05,t检验）,有明显外源性GFP-Hsp90αE47A蛋白的表达,为对照组内源性Hsp90α的0.68±0.12倍。外源性GFP-Hsp90αE47A蛋白的表达HepG2细胞增殖活性于第4d有明显抑制。（1.051±0.03vs1.349±0.05,P〈0.05,t检验）。结论：成功建立重组慢病毒载体的三质粒包装细胞系统,并将GFP-Hsp90αE47A基因在HepG2细胞中稳定表达,且并未引起细胞明显的热休克反应而导致的内源性Hsp90α增高;且能明显抑制细胞增殖,为后期Hsp90α分子伴侣功能进行研究奠定基础。     

丙型肝炎病毒核心蛋白基因真核表达质粒的构建和表达

目的:构建丙型肝炎病毒(HCV)核心蛋白真核表达质粒,并在HepG2细胞中稳定表达.方法:采用RT-RCR方法从丙型肝炎患者血清中得到HCVC区的cDNA序列,然后克隆入pcDNA3.0载体,构建真核表达质粒pcDNA-HCV,并进行酶切鉴定和测序鉴定;采用脂质体转染技术将pcDNA-HCV稳定转染于HepG2细胞系,并用G-418进行筛选;采用Western Blotting方法和免疫细胞化学方法检测HepG2细胞中HCV核心蛋白表达情况.结果:从HCV感染者血清中扩增得到的503bpHCV cDNA序列.属于HCV 1型基因C区,并成功构建了真核表达质粒pcDNA-HCV.经Western blotting和免疫细胞化学检测,稳定转染的HepG2细胞中有HCV核心蛋白的表达.结论:成功构建HCV核心蛋白真核表达质粒pcDNA-HCV,并可以在真核细胞HepG2中稳定表达.     

Anti-HIF-1α-pEGFP重组质粒的构建及表达

目的构建EGFP-HIF-1α反义重组质粒,转染人宫颈癌Hela细胞,用以探讨HIF-1α蛋白在宫颈癌的生长、转移中的作用。方法应用基因重组技术构建EGFP-HIF-1α反义重组质粒,通过脂质体介导将其转染入Hela细胞,倒置荧光显微镜观察转染效果,Western blot检测HIF-1α蛋白的表达。结果酶切鉴定结果显示含EGFP-HIF-1α反义重组质粒构建成功,并能在Hela细胞中封闭HIF-1α蛋白的表达。结果 EGFP-HIF-1α反义重组质粒构建及转染成功,封闭Hela细胞中HIF-1α蛋白的表达,为进一步研究HIF-1α蛋白在宫颈癌的生长、转移中的作用提供试验基础。     

重组人凝血因子Ⅷ表达质粒的构建及其在HepG2细胞中的表达

目的:构建含有B区缺失型(△760aa-1639aa)人凝血因子Ⅷ(B domain-deleted human FⅧ,BDDhFⅧ)的真核表达质粒,转染HepG2细胞使其稳定表达人凝血因子Ⅷ。方法:将BDDhFVIII基因片段插入pcDNA4/v5-his空载体中构建重组真核表达质粒,测序正确后电转入HepG2细胞,经Ni-NTA纯化,利用Western blot检测凝血因子Ⅷ在HepG2细胞中的表达,持续培养获得稳定表达BDDhFⅧ蛋白的细胞株。结果:经限制性酶切和测序鉴定均证实重组真核表达质粒pcDNA4/v5-his-BDDhFⅧ成功构建,在转染HepG2细胞后,Western blot检测证实人凝血因子Ⅷ可以在HepG2细胞中正确表达。结论:成功构建了人凝血因子Ⅷ的稳定细胞株,并能在HepG2细胞表达目的蛋白。     

pcDNA3.1-Canstatin-3Flag真核表达载体的构建及转染HepG2细胞中的表达

目的:构建pcDNA3.1-Canstatin-3Flag载体并稳定转染肝癌HepG2细胞,检测canstatin在mRNA水平的表达。方法:胎盘中提取总RNA,RT-PCR法获得canstatinDNA,克隆至pcDNA3.1(-)载体中,并测序,重组质粒pcDNA3.1-Canstatin-3Flag转染肝癌HepG2细胞,G418筛选出稳定转染细胞,RT-PCR检测canstatin mRNA表达。结果:1.成功构建出pcDNA3.1-Canstatin-3Flag重组质粒;2.获得稳定转染pcDNA3.1-Canstatin-3Flag的肝癌HepG2细胞;3.发现转染后的肝癌HepG2细胞canstatin在mRNA水平比未转染细胞有明显的增强。结论:获得了稳定转染pcDNA3.1-Canstatin-3Flag的肝癌HepG2细胞,为后期canstatin在肝癌中的研究提供了支持。     

朱砂叶螨HSP90基因克隆及原核表达

采用RT-PCR及RACE技术克隆朱砂叶螨Tetranychus cinnabarinus的热激蛋白90(HSP90)基因, 并进行序列分析, 得到一条长2 595 bp的cDNA序列, 该序列开放阅读框（open reading frame, ORF）为2 169 bp, 编码722个氨基酸, 分子量约为83.45 kDa, 理论等电点为4.81, 3′非编码区（untranslated region, UTR）为249 bp, 5′UTR为177 bp。通过Antheprot分析发现5个HSP90家族的签名序列及胞质HSP90特征序列MEEVD。同源性分析表明, 朱砂叶螨HSP90编码区核苷酸序列和其他已知的HSP90, 尤其是节肢动物昆虫的HSP90, 具有很高的相似性。将鉴定正确的原核重组表达质粒pET43a-TcHSP90, 转化大肠杆菌Escherichia coli BL21(origami) 进行原核表达, 应用SDS-PAGE和Western blotting技术分离并检测融合蛋白, 结果表明构建的原核表达质粒可以在宿主菌中稳定、正确表达。朱砂叶螨TcHSP90基因的克隆、原核表达, 为进一步研究HSP90的性质和功能的研究提供有用的实验材料。     

核输入蛋白α/β真核表达载体的构建及细胞内定位

目的:构建带有myc标签的核输入蛋白(importin)α/β的真核表达载体,转染人胚肾293T细胞并分析其表达。方法:以乳腺癌文库为模板PCR扩增核输入蛋白α/β基因,扩增产物插入真核表达载体p XJ-40-myc,经双酶切和测序鉴定;将空载体与重组质粒分别转染人胚肾293T细胞,通过Western印迹和免疫荧光技术检测核输入蛋白α/β的表达。结果:酶切鉴定与测序结果表明构建的myc-Importinα/β真核表达载体正确;Western印迹检测到重组质粒在293T细胞中的表达;免疫荧光检测到核输入蛋白α定位于细胞质和细胞核,而核输入蛋白β定位于细胞核。结论:构建了核输入蛋白α/β的真核表达载体,并确定了其在细胞中的表达定位。     

小鼠DLL1真核表达载体的构建及其在肿瘤细胞中的表达

目的:构建带绿色荧光蛋白的小鼠DLL1全长基因真核表达载体,并在肿瘤细胞中表达。方法:利用PCR特异性引物扩增出DLL1基因全长,将克隆的基因片段插入带绿色荧光蛋白的真核表达载体pIRES2-EGFP质粒中。然后利用脂质体将重组质粒pIRES2-EGFP-DLL1转染进小鼠B16黑色素瘤细胞中,并通过G418筛选后选取生长良好、荧光强度高的三株单克隆进行mRNA水平DLL1表达的鉴定。结果:成功扩增小鼠DLL1的全长基因。克隆入质粒载体后,通过DNA序列测定证实其序列正确。将构建的pIRES2-EGFP-DLL1质粒转染小鼠B16黑色素瘤细胞,经过G418筛选和荧光显微镜观察后,挑选得到GFP阳性率90%以上的稳定转染细胞株。RT-PCR检测稳定转染细胞的mDLL1的表达显著增加,进一步证实了pIRES2-EGFP-DLL1的表达效能。结论:成功构建了小鼠DLL1基因的真核表达质粒,证实其在真核细胞B16中可以表达。     

The anti-myeloma activity of a novel purine scaffold HSP90 inhibitor PU-H71 is via inhibition of both HSP90A and HSP90B1

Background

The ribonucleotide reductase M1 (RRM1) gene encodes the regulatory subunit of ribonucleotide reductase, the molecular target of gemcitabine. The overexpression of RRM1 mRNA in tumor tissues is reported to be associated with gemcitabine resistance. Thus, single nucleotide polymorphisms (SNPs) of the RRM1 gene are potential biomarkers of the response to gemcitabine chemotherapy. We investigated whether RRM1 expression in peripheral blood mononuclear cells (PBMCs) or SNPs were associated with clinical outcome after gemcitabine-based chemotherapy in advanced non-small cell lung cancer (NSCLC) patients.

Methods

PBMC samples were obtained from 62 stage IIIB and IV patients treated with gemcitabine-based chemotherapy. RRM1 mRNA expression levels were assessed by real-time PCR. Three RRM1 SNPs, -37C→A, 2455A→G and 2464G→A, were assessed by direct sequencing.

Results

RRM1 expression was detectable in 57 PBMC samples, and SNPs were sequenced in 56 samples. The overall response rate to gemcitabine was 18%; there was no significant association between RRM1 mRNA expression and response rate (P = 0.560). The median progression-free survival (PFS) was 23.3 weeks in the lower expression group and 26.9 weeks in the higher expression group (P = 0.659). For the -37C→A polymorphism, the median PFS was 30.7 weeks in the C(-)37A group, 24.7 weeks in the A(-)37A group, and 23.3 weeks in the C(-)37C group (P = 0.043). No significant difference in PFS was observed for the SNP 2455A→G or 2464G→A.

Conclusions

The RRM1 polymorphism -37C→A correlated with PFS in NSCLC patients treated with gemcitabine-based chemotherapy. No significant correlation was found between PBMC RRM1 mRNA expression and the efficacy of gemcitabine.     

The HSP90 complex of plants

Heat shock protein 90 (HSP90) is a highly conserved and essential molecular chaperone involved in maturation and activation of signaling proteins in eukaryotes. HSP90 operates as a dimer in a conformational cycle driven by ATP binding and hydrolysis. HSP90 often functions together with co-chaperones that regulate the conformational cycle and/or load a substrate "client" protein onto HSP90. In plants, immune sensing NLR (nucleotide-binding domain and leucine-rich repeat containing) proteins are among the few known client proteins of HSP90. In the process of chaperoning NLR proteins, co-chaperones, RAR1 and SGT1 function together with HSP90. Recent structural and functional analyses indicate that RAR1 dynamically controls conformational changes of the HSP90 dimer, allowing SGT1 to bridge the interaction between NLR proteins and HSP90. Here, we discuss the regulation of NLR proteins by HSP90 upon interaction with RAR1 and SGT1, emphasizing the recent progress in our understanding of the structure and function of the complex. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

HSP90 modulates actin dynamics: Inhibition of HSP90 leads to decreased cell motility and impairs invasion

HSP90, a major molecular chaperone, plays an essential role in the maintenance of several signaling molecules. Inhibition of HSP90 by inhibitors such as 17-allylamino-demethoxy-geldanamycin (17AAG) is known to induce apoptosis in various cancer cells by decreasing the activation or expression of pro-survival molecules such as protein kinase B (Akt). While we did not observe either decrease in expression or activation of pro-survival signaling molecules in human breast cancer cells upon inhibiting HSP90 with 17AAG, we did observe a decrease in cell motility of transformed cells, and cell motility and invasion of cancer cells. We found a significant decrease in the number of filopodia and lamellipodia, and in the F-actin bundles upon HSP90 inhibition. Our results show no change in the active forms or total levels of FAK and Pax, or in the activation of Rac-1 and Cdc-42; however increased levels of HSP90, HSP90α and HSP70 were observed upon HSP90 inhibition. Co-immuno-precipitation of HSP90 reveals interaction of HSP90 with G-actin, which increases upon HSP90 inhibition. FRET results show a significant decrease in interaction between actin monomers, leading to decreased actin polymerization upon HSP90 inhibition. We observed a decrease in the invasion of human breast cancer cells in the matrigel assay upon HSP90 inhibition. Over-expression of αB-crystallin, known to be involved in actin dynamics, did not abrogate the effect of HSP90 inhibition. Our work provides the molecular mechanism by which HSP90 inhibition delays cell migration and should be useful in developing cancer treatment strategies with known anti-cancer drugs such as cisplatin in combination with HSP90 inhibitors.     

Mitochondrial HSP90s and tumor cell metabolism

The control of protein homeostasis, or proteostasis, has been traditionally viewed through the lenses of a general housekeeping function that all cells need, regardless of pathway specification or link to defined cellular responses. A more updated perspective considers proteostasis as an essential adaptive mechanism, taking place in specialized subcellular organelles, and maintaining the functionality of defined cellular networks. Fresh experimental evidence now identifies heat shock protein 90 (HSP90) chaperones as pivotal regulators of proteostasis in mitochondria, selectively in tumor cells. This function connects to a global network of cellular compensation, linking autophagy, endoplasmic reticulum (ER) stress and metabolic reprogramming in a single adaptive continuum, and offers prime opportunities for novel cancer therapeutics.     

N-aryl-benzimidazolones as novel small molecule HSP90 inhibitors

We describe the development of a novel series of N-aryl-benzimidazolone HSP90 inhibitors (9) targeting the N-terminal ATP-ase site. SAR development was influenced by structure-based design based around X-ray structures of ligand bound HSP90 complexes. Lead compounds exhibited high binding affinities, ATP-ase inhibition and cellular client protein degradation.     

HSP90 controls SIR2 mediated gene silencing

In recent years, Hsp90 is found to interact with several telomeric proteins at various phases of cell cycle. The Hsp90 chaperone system controls assembly and disassembly of telomere structures and thus maintains the dynamic state of telomere. Here, for the first time we report that the activity of another telomeric protein Sir2p is modulated by Hsp82, the ortholog of Hsp90 from budding yeast (Saccharomyces cerevisiae). In a temperature sensitive Hsp90 deficient yeast strain (iG170Dhsp82), less abundant Sir2p is observed, resulting in de-repression of telomere silencing and a complete loss of mating type silencing. Intriguingly, over expression of Hsp90, either by exposing cells to heat shock or by introducing HSP82 overexpression plasmid also yields reduced level of Sir2p, with a consequential loss of telomere silencing. Thus, Hsp90 homeostasis maintains the cellular pool of Sir2p and thereby controls the reversible nature of telomere silencing. Interestingly, such regulation is independent of one of its major co-chaperones Sba1 (human ortholog of p23).