Vorinostat Eliminates Multicellular Resistance of Mesothelioma 3D Spheroids via Restoration of Noxa Expression

When grown in 3D cultures as spheroids, mesothelioma cells acquire a multicellular resistance to apoptosis that resembles that of solid tumors. We have previously found that resistance to the proteasome inhibitor bortezomib in 3D can be explained by a lack of upregulation of Noxa, the pro-apoptotic BH3 sensitizer that acts via displacement of the Bak/Bax-activator BH3-only protein, Bim. We hypothesized that the histone deacetylase inhibitor vorinostat might reverse this block to Noxa upregulation in 3D. Indeed, we found that vorinostat effectively restored upregulation of Noxa protein and message and abolished multicellular resistance to bortezomib in the 3D spheroids. The ability of vorinostat to reverse resistance was ablated by knockdown of Noxa or Bim, confirming the essential role of the Noxa/Bim axis in the response to vorinostat. Addition of vorinostat similarly increased the apoptotic response to bortezomib in another 3D model, the tumor fragment spheroid, which is grown from human mesothelioma ex vivo. In addition to its benefit when used with bortezomib, vorinostat also enhanced the response to cisplatin plus pemetrexed, as shown in both 3D models. Our results using clinically relevant 3D models show that the manipulation of the core apoptotic repertoire may improve the chemosensitivity of mesothelioma. Whereas neither vorinostat nor bortezomib alone has been clinically effective in mesothelioma, vorinostat may undermine chemoresistance to bortezomib and to other therapies thereby providing a rationale for combinatorial strategies.     

口蹄疫病毒3D基因的克隆表达及功能分析

利用RT-PCR技术扩增了口蹄疫病毒(FMDV)编码RNA依赖的RNA聚合酶的3D基因,并将其克隆到原核表达质粒载体pET-28a( )中。3D基因经测序确认后在大肠杆菌BL-21中表达,表达产物纯化的目的蛋白进行Western-blotting检测,获得分子量约55KDa的单一3D基因表达产物。利用RNA体外复制体系和荧光定量PCR技术,证明纯化的3D基因表达产物RNA依赖的RNA聚合酶具有较高的酶活性,可以在体外从头合成FMDVRNA,且主要以引物依赖的方式合成病毒基因组。     

口蹄疫病毒3D基因的克隆表达及功能分析

利用RT-PCR技术扩增了口蹄疫病毒(FMDV)编码RNA依赖的RNA聚合酶的3D基因,并将其克隆到原核表达质粒载体pET-28a(+)中.3D基因经测序确认后在大肠杆菌BL-21中表达,表达产物纯化的目的蛋白进行Western-blotting检测,获得分子量约55KDa的单一3D基因表达产物.利用RNA体外复制体系和荧光定量PCR技术,证明纯化的3D基因表达产物RNA依赖的RNA聚合酶具有较高的酶活性,可以在体外从头合成FMDV RNA,且主要以引物依赖的方式合成病毒基因组.     

TRAIL-Mediated Apoptosis in Breast Cancer Cells Cultured as 3D Spheroids

TNF-alpha-related-apoptosis-inducing-ligand (TRAIL) has been explored as a therapeutic drug to kill cancer cells. Cancer cells in the circulation are subjected to apoptosis-inducing factors. Despite the presence of these factors, cells are able to extravasate and metastasize. The homotypic and heterotypic cell-cell interactions in a tumor are known to play a crucial role in bestowing important characteristics to cancer cells that leave the primary site. Spheroid cell culture has been extensively used to mimic these physiologically relevant interactions. In this work, we show that the breast cancer cell lines BT20 and MCF7, cultured as 3D tumor spheroids, are more resistant to TRAIL-mediated apoptosis by downregulating the expression of death receptors (DR4 and DR5) that initiate TRAIL-mediated apoptosis. For comparison, we also investigated the effect of TRAIL on cells cultured as a 2D monolayer. Our results indicate that tumor spheroids are enriched for CD44^hiCD24^loALDH1^hi cells, a phenotype that is predominantly known to be a marker for breast cancer stem cells. Furthermore, we attribute the TRAIL-resistance and cancer stem cell phenotype observed in tumor spheroids to the upregulation of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE₂) pathway. We show that inhibition of the COX-2/PGE₂ pathway by treating tumor spheroids with NS-398, a selective COX-2 inhibitor, reverses the TRAIL-resistance and decreases the incidence of a CD44^hiCD24^lo population. Additionally, we show that siRNA mediated knockdown of COX-2 expression in MCF7 cells render them sensitive to TRAIL by increasing the expression of DR4 and DR5. Collectively, our results show the effect of the third-dimension on the response of breast cancer cells to TRAIL and suggest a therapeutic target to overcome TRAIL-resistance.     

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels¹or in biomaterial scaffolds². Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.Download video file.^{(44M, mov)}     

基于微流控芯片构建三维基质支架中的多细胞球体模型

抗癌药物在进行动物和临床试验以前,需要用体外肿瘤组织模型评估药效.由于三维(3D)多细胞球体(multicellular tumor spheroids MCTSs)在抗药性和组织结构等方面与体内肿瘤组织相似,常被用作体外肿瘤组织模型.为监测MCTSs在形成过程中,肿瘤细胞之间和肿瘤细胞与基质之间的相互作用,基于微流控技术基础上自行设计和构建MCTSs模型.该肿瘤MCTSs模型实验结果表明,在3D微环境下,血清能够诱导MDA-MB-231形成直径为289μm的MCTSs,肿瘤细胞MCTSs之间有相互靠近的趋势,并且发现凋亡细胞多分布在MCTSs之间.肿瘤坏死因子(tumor necrosis factor-α,TNF-α)诱导MDA-MB-231形成MCTSs之间没有相互靠近的趋势,并且MCTSs直径的长度很难达到100μm.以上结果表明,该模型有望为研究肿瘤形成MCTSs机制和药物筛选提供有用的体外肿瘤模型.     

Regulation of Adipogenesis and Key Adipogenic Gene Expression by 1, 25-Dihydroxyvitamin D in 3T3-L1 Cells

The functions of 1, 25-dihydroxyvitamin D (1, 25-(OH)₂D₃) in regulating adipogenesis, adipocyte differentiation and key adipogenic gene expression were studied in 3T3-L1 preadipocytes. Five concentrations (0.01, 0.1, 1, 10, 100nM) of 1, 25-(OH)₂D₃ were studied and lipid accumulation measured by Oil Red O staining and expression of adipogenic genes quantified using quantitative real-time PCR. Adipogenic responses to 1, 25-(OH)₂D₃ were determined on 6, and 12 h, and days 1-10 after induction of adipogenesis by a hormonal cocktail with or without 1, 25-(OH)₂D₃. In response to 1, 25-(OH)₂D₃ (1, 10, and 100 nM), lipid accumulation and the expression of PPARγ, C/EBPα, FABP4 and SCD-1 were inhibited through day 10, and vitamin D receptor expression was inhibited in the early time points. The greatest inhibitory effect was upon expression of FABP4. Expression of SREBP-1c was only affected on day 2. The lowest concentrations of 1, 25-(OH)₂D₃ tested did not affect adipocyte differentiation or adipogenic gene expression. The C/EBPα promoter activity response to 1, 25-(OH)₂D₃ was also tested, with no effect detected. These results indicate that 1, 25-(OH)₂D₃ inhibited adipogenesis via suppressing adipogenic-specific genes, and is invoked either during PPARγ activation or immediately up-stream thereof. Gene expression down-stream of PPARγ especially FABP4 was strongly inhibited, and we suggest that the role of 1, 25-(OH)₂D₃ in regulating adipogenesis will be informed by further studies of adipogenic-specific gene promoter activity.     

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.     

小麦胁迫相关基因W1的克隆及表达模式分析

应用噬菌体原位杂交技术从干旱胁迫诱导的小麦cDNA文库中克隆到一个胁迫诱导的基因片段别。删全长cDNA为901bp,其中,编码区长498bp,编码166个氨基酸。Southern杂交表明,W1是一个低拷贝基因。RT—PCR结果表明,W1受干旱、低温的诱导,但不受高盐的诱导。氨基酸序列分析发现W1有一个USP保守区（pfam00582）。同源性分析发现W1与一个水稻胁迫诱导蛋白（NM_001061239）的同源性为83％,但该类蛋白的功能尚无报道。肼是小麦第1个被克隆的胁迫相关蛋白基因,该基因的克隆有助于阐明小麦的抗逆机制,并为今后培育抗逆性小麦品种提供候选基因。     

磷脂酶D(PLD)基因的结构、表达及其表达产物在信号转导中的作用

磷脂酶D(PLDEC 3 .1 .4.4)水解磷脂 (PL) ,磷脂构成生物膜的骨架 ,磷脂酶的激活不仅对细胞的结构和稳定性有很重要的作用 ,而且调控许多重要的细胞生理功能 ,例如PLD在信号转导、小泡运输、有丝分裂、激素作用的发挥、细胞骨架组装、防御反应以及种子萌发和衰老过程中都起重要作用。近年来它在跨膜信号转导中的重要作用 ,越来越引起人们的重视 ,成为新的研究热点。介绍了磷脂酶基因的结构特点、亚细胞定位、表达的激活抑制以及其表达产物作为胞内信号分子在植物信号转导中的重要作用。     

磷脂酶D（PLD）基因的结构、表达及其表达产物在信号转导中的作用

磷脂酶D(PLD EC 3.1.4.4)水解磷脂(PL),磷脂构成生物膜的骨架, 磷脂酶的激活不仅对细胞的结构和稳定性有很重要的作用,而且调控许多重要的细胞生理功能,例如PLD在信号转导、小泡运输、有丝分裂 、激素作用的发挥、细胞骨架组装、防御反应以及种子萌发和衰老过程中都起重要作用。近年来它在跨膜信号转导中的重要作用，越来越引起人们的重视，成为新的研究热点。介绍了磷脂酶基因的结构特点、亚细胞定位、表达的激活抑制以及其表达产物作为胞内信号分子在植物信号转导中的重要作用。     

马铃薯StAP1基因的分子克隆与表达分析

采用RT-PCR方法从马铃薯品种‘Désirée’中克隆了StAP1cDNA序列（GenBank登录号GU220568）。该基因cDNA开放阅读框长度为735bp,编码一个由244个氨基酸残基组成的蛋白,该蛋白分子量为28.57kDa,理论等电点为8.32。StAP1蛋白含有1个高度保守的MADS结构域,与烟草NAP1具有较高一致性。组织表达分析显示,StAP1在马铃薯植株的顶芽、花和叶中有较高水平的转录表达,在块茎中有微量表达。利用反义StCOL转基因马铃薯植株进一步分析表明,StAP1的表达受StCOL的调控。     

结核杆菌 D29 噬菌体几丁质酶基因的表达及功能研究

噬菌体一般通过表达内溶素来降解宿主菌细胞壁上的肽聚糖 . 用 PCR 技术从结核杆菌 D29 噬菌体基因组中克隆了 gene10 ,并使其在大肠杆菌中得到了高效表达,蛋白质 C 端带有 6×His. 用镍柱亲和纯化了大肠杆菌表达的 gp10 蛋白可溶性部分 . 活性测定表明, gp10 不但具有几丁质酶活性,还具有溶菌酶活性,是一种双功能的酶 . 耻垢杆菌经 gp10 作用后,其生长受到抑制,扫描电镜观察发现部分耻垢杆菌被降解 . 说明与其他种类噬菌体降解细胞壁的方式不同, D29 噬菌体可能利用 gp10 的溶菌酶活性使结核杆菌细胞壁降解 . 这有助于揭示结核杆菌噬菌体与其宿主的相互作用机制,是关于噬菌体几丁质酶的首次报道 .