Detection and destruction of HER2-positive cancer cells by Ultra Quenchbody-siRNA complex

Ultra Quenchbody (UQ-body) is a biosensor that utilizes the quenching behavior of the fluorescent dye linked to the antibody V region. When the corresponding antigen is bound to the UQ-body, the fluorescence is restored and allows the detection of target molecules easily and sensitively. In this paper, we constructed UQ-bodies to sensitively detect the human epidermal growth factor receptor 2 (HER2) cancer marker in solution or on cancer cells, which was further used to kill the cancer cells. A synthetic Fab fragment of anti-HER2 antibody Fab37 with many Trp residues at hypervariable region was prepared and labeled with fluorescent dyes to obtain the UQ-bodies. The UQ-body could detect HER2 in solution at concentrations as low as 20 pM with an EC₅₀ of 0.3 nM with a fourfold response. Fluorescence imaging of HER2-positive cells was successfully performed without any washing steps. To deliver small interfering RNA (siRNA) to cancer cells, a modified UQ-body with C-terminal 9R sequence was also prepared. HER2-positive cancer cells were effectively killed by polo-like kinase 1 siRNA intracellularly delivered by the UQ-body-9R. The novel approach employing siRNA-empowered UQ-body could detect and image the HER2 antigen easily and sensitively, and effectively kill the HER2-positive cancer cells.     

Automated live cell imaging of green fluorescent protein degradation in individual fibroblasts.

To accurately interpret the data from fluorescent proteins as reporters of gene activation within living cells, it is important to understand the kinetics of the degradation of the reporter proteins. We examined the degradation kinetics over a large number (>1,000) of single, living cells from a clonal population of NIH3T3 fibroblasts that were stably transfected with a destabilized, enhanced green fluorescent protein (eGFP) reporter driven by the tenascin-C promoter. Data collection and quantification of the fluorescence protein within a statistically significant number of individual cells over long times (14 h) by automated microscopy was facilitated by culturing cells on micropatterned arrays that confined their migration and allowed them to be segmented using phase contrast images. To measure GFP degradation rates unambiguously, protein synthesis was inhibited with cycloheximide. Results from automated live cell microscopy and image analysis indicated a wide range of cell-to-cell variability in the GFP fluorescence within individual cells. Degradation for this reporter was analyzed as a first order rate process with a degradation half-life of 2.8 h. We found that GFP degradation rates were independent of the initial intensity of GFP fluorescence within cells. This result indicates that higher GFP abundance in some cells is likely due to higher rates of gene expression, because it is not due to systematically lower rates of protein degradation. The approach described in this study will assist the quantification and understanding of gene activity within live cells using fluorescent protein reporters.     

Protein aggregates are transported to vacuoles by a macroautophagic mechanism in nutrient-starved plant cells

When a fusion protein of cytochrome b5 (Cyt b5) and the red fluorescent protein (RFP) are expressed in tobacco BY-2 cells, the expressed protein forms intracellular aggregates that emit red fluorescence. When such cells are grown to the stationary phase or incubated in nutrient limited medium, RFP fluorescence can be detected in the vacuolar lumen. We investigated this transport mechanism using a limited-nitrogen model. E-64 and 3-methyladenine, which inhibit autophagic processes, blocked the transport of the RFP signal to the vacuole. We next traced the autophagic process in tobacco cells using YFP fused with the tobacco Atg8 homologue (YFP-NtAtg8) and analyzed the contribution of autophagy to the vacuolar transport of the aggregates. Under limited-nitrogen conditions, the aggregates were degraded in preference to other organelles, and the autophagosomes colocalized with the aggregates at a higher frequency than with mitochondria. This is the first demonstration that selective macroautophagic degradation can occur in plant cells.     

Protein Aggregates are Transported to Vacuoles by Macroautophagic Mechanism in Nutrient-Starved Plant Cells

When a fusion protein of cytochrome b5 (Cyt b5) and the red fluorescent protein (RFP) are expressed in tobacco BY-2 cells, the expressed protein forms intracellular aggregates that emit red fluorescence. When such cells are grown to the stationary phase or incubated with nutrient limit medium, RFP fluorescence can be detected in the vacuolar lumen. We investigated this transport mechanism using a limited-nitrogen model. E-64 and 3-methyladenine, which inhibit authophagic processes, inhibited the transport of RFP signal to the vacuole. We next traced the autophagic process in tobacco cells using YFP fused with the tobacco Atg8 homologue (YFP–NtAtg8) and analyzed the contribution of autophagy to the vacuolar transport of the aggregates. Under limited-nitrogen conditions, the aggregates were degraded in preference to otherorganelles, and the autophagosomes colocalized with the aggregates at a higher frequency than with mitochondria. This is the first demonstration that selective macroautophagic degradation occurs in plant cells.     

珊瑚和海葵来源红荧光蛋白的研究和应用

绿色荧光蛋白作为标记蛋白和报告蛋白在生物学研究中应用越来越广。但在荧光共振能量转移(fluorescenceresonanceenergytransfer,FRET)等技术中存在一些缺陷,需要更大波长范围的荧光蛋白。最近研究发现了多种来源于珊瑚和海葵的红荧光蛋白,这些长波长的荧光蛋白对绿色荧光蛋白是一种很好的代替和补充,可以实现细胞内多荧光标记,提供更理想的FRET荧光对。经随机突变和定点突变等方法改建获得的红荧光蛋白变种显示出更高的荧光强度,成熟时间也更短。目前应用较多的是来源于香菇珊瑚(Discosomasp.)的红荧光蛋白DsRed。     

Development and investigation of recombinant immunotoxin protein 4D5scFv-mCherry-PE(40)

Development of agents for theranostics implies combining the targeting module, the effector module, and the detection module within the same complex or recombinant protein. We have constructed, isolated, and characterized the 4D5scFv-mCherry-PE(40) protein, which exhibits fluorescent properties and specifically binds to cancer cells expressing the HER2 receptor and reduces their viability. The ability of the obtained targeted antitumor agent 4D5scFv-mCherry-PE(40) to selectively stain the HER2-positive cells and its highly selective cytotoxicity against these cells make the obtained targeted recombinant protein 4D5scFv-mCherry-PE(40) a promising theranostic agent for the diagnostics and therapy of HER2-positive human tumors.     

In vivo imaging and quantitative monitoring of autophagic flux in tobacco BY-2 cells

Autophagy has been shown to play essential roles in the growth, development and survival of eukaryotic cells. However, simple methods for quantification and visualization of autophagic flux remain to be developed in living plant cells. Here, we analyzed the autophagic flux in transgenic tobacco BY-2 cell lines expressing fluorescence-tagged NtATG8a as a marker for autophagosome formation. Under sucrose-starved conditions, the number of punctate signals of YFP-NtATG8a increased, and the fluorescence intensity of the cytoplasm and nucleoplasm decreased. Conversely, these changes were not observed in BY-2 cells expressing a C-terminal glycine deletion mutant of the NtATG8a protein (NtATG8aΔG). To monitor the autophagic flux more easily, we generated a transgenic BY-2 cell line expressing NtATG8a fused to a pH-sensitive fluorescent tag, a tandem fusion of the acid-insensitive RFP and the acid-sensitive YFP. In sucrose-rich conditions, both fluorescent signals were detected in the cytoplasm and only weakly in the vacuole. In contrast, under sucrose-starved conditions, the fluorescence intensity of the cytoplasm decreased, and the RFP signal clearly increased in the vacuole, corresponding to the fusion of the autophagosome to the vacuole and translocation of ATG8 from the cytoplasm to the vacuole. Moreover, we introduce a novel simple easy way to monitor the autophagic flux non-invasively by only measuring the ratio of fluorescence of RFP and YFP in the cell suspension using a fluorescent image analyzer without microscopy. The present in vivo quantitative monitoring system for the autophagic flux offers a powerful tool for determining the physiological functions and molecular mechanisms of plant autophagy induced by environmental stimuli.     

构建稳定表达RFP及嘌呤霉素抗性的K562细胞株

目的构建稳定表达红色荧光蛋白（red fluorescent protein,RFP）和嘌呤霉素（puromycin）抗性的K562．PM．RFP细胞株,便于慢性粒细胞性白血病研究中K562细胞的观察和筛选。方法采用PCR法获得RFP片段,将其插入到慢病毒pGC-FU-3FLAG-IRES—Puromycin载体中获得pGC—PM—RFP重组质粒,经脂质体转染到293T细胞中获得慢病毒LV—PM—RFP,有限稀释法检测慢病毒在293T细胞中的转染效率,用包装获得的慢病毒感染K562细胞,经嘌呤霉素筛选获得RFP阳性的K562-PM—RFP细胞株。结果PCR及测序结果证实目的基因RFP正确克隆至慢病毒质粒中,经慢病毒LV—PM-RFP感染的K562细胞能在嘌呤霉素抗性培养基中存活,并稳定表达RFP。结论成功构建了慢病毒重组质粒pGC—PM-RFP,并获得了携带RFP及嘌呤霉素抗性基因的K562-PM—RFP细胞株。     

A brilliant monomeric red fluorescent protein to visualize cytoskeleton dynamics in Dictyostelium

Red fluorescent proteins (RFPs) combined with GFP are attractive probes for double-fluorescence labeling of proteins in live cells. However, the application of these proteins is restrained by stable oligomer formation and by their weak fluorescence in vivo. Previous attempts to eliminate these problems by mutagenesis of RFP from Discosoma (DsRed) resulted in the monomeric mRFP1 and in the tetrameric RedStar RFP, which is distinguished by its enhanced fluorescence in vivo. Based on these mutations, we have generated an enhanced monomeric RFP, mRFPmars, and report its spectral properties. Together with green fluorescent labels, we used mRFPmars to visualize filamentous actin structures and microtubules in Dictyostelium cells. This enhanced RFP proved to be suitable to monitor the dynamics of cytoskeletal proteins in cell motility, mitosis, and endocytosis using dual-wavelength fluorescence microscopy.     

In vitro differentiation and maturation of mouse embryonic stem cells into hepatocytes

It is difficult to induce the maturation of embryonic stem (ES) cells into hepatocytes in vitro. We previously reported that Thy1-positive mesenchymal cells derived from the mouse fetal liver promote the maturation of hepatic progenitor cells. Here, we isolated alpha-fetoprotein (AFP)-producing cells from mouse ES cells for subsequent differentiation into hepatocytes in vitro by coculture with Thy1-positive cells. ES cells expressing green fluorescent protein (GFP) under the control of an AFP promoter were cultured under serum- and feeder layer-free culture conditions. The proportion of GFP-positive cells plateaued at 41.6 +/- 12.2% (means +/- SD) by day 7. GFP-positive cells, isolated by flow cytometry, were cultured in the presence or absence of Thy1-positive cells as a feeder layer. Isolated GFP-positive cells were stained for AFP, Foxa2, and albumin. The expression of mRNAs encoding tyrosine amino transferase, tryptophan 2,3-dioxygenase, and glucose-6-phosphatase were only detected following coculture with Thy1-positive cells. Following coculture with Thy1-positive cells, the isolated cells produced and stored glycogen. Ammonia clearance activity was also enhanced following coculture. Electron microscopic analysis indicated that the cocultured cells exhibited the morphologic features of mature hepatocytes. In conclusion, coculture with Thy1-positive cells in vitro induced the maturation of AFP-producing cells isolated from ES cell cultures into hepatocytes.     

慢病毒介导的RNA干涉对乳腺癌SKBR3细胞HER2受体的下调及生长抑制

大约30％的乳腺癌中有表皮生长因子受体家族蛋白HER2的过表达,此类癌症的预后差,恶性程度高。RNA干涉（RNAi）是最近发展起来能特异性抑制哺乳动物细胞中基因表达的新技术。本文在以往获得的能够产生良好基因沉默效应的小干涉RNA（siRNA）的基础上,构建了U6和H1双启动子siRNA表达载体,并转染HER2高表达乳腺癌SKBR3细胞定量测定了其HER2下调效应。随后,siRNA表达盒经LR重组反应被克隆入慢病毒载体中,在成功包装成病毒后,感染SKBR3并经荧光定量PCR、蛋白印迹杂交和流式细胞仪一系列实验证明慢病毒介导的RNAi确实能有效地下调肿瘤抗原HER2的表达。细胞长期增殖实验表明经慢病毒处理后细胞生长得到抑制。我们的研究为进一步阐明HER2与癌症恶化的关系以及发展新的基因治疗药物提供了工具和可能。     

Interaction of fluorescent gonadotropin-releasing hormone with receptors in cultured pituitary cells

A fluorescent derivative of the gonadotropin-releasing hormone (GnRH) agonist analog, [D-Lys6]GnRH, was synthesized for receptor studies and shown to be biologically active. The rhodamine-derivatized peptide (Rh-GnRH) retained 40% of the receptor binding activity of [D-Lys6]GnRH, and 50% of the luteinizing hormone-releasing activity assayed in cultured pituitary cells. The fluorescent analog was employed to visualize the distribution of GnRH receptors in cultured pituitary cells, using the technique of video-intensified fluorescence microscopy. The binding of Rh-GnRH was confined to the large gonadotrophs which comprised 15% of the cell population. The specificity of the binding was shown by the absence of significant fluorescence in the presence of a 100-fold excess of [D-Lys6]GnRH, or when Rh-GnRH was incubated with choriocarcinoma, neuroblastoma, or 3T3 cell lines devoid of GnRH receptors. The interaction of Rh-GnRH with living pituitary cells was characterized by an initial diffuse distribution, followed by the formation of polar aggregates that later appeared to be internalized. These observations emphasize the value of fluorescent derivatives of GnRH for elucidating the course of the interaction with specific receptors on pituitary gonadotrophs. The initial results indicate that GnRH-receptor complexes undergo aggregation during stimulation of luteinizing hormone release, and are later internalized for subsequent degradation and/ or intracellular actions.     

Generation of Two-color Transgenic Zebrafish Using the Green and Red Fluorescent Protein Reporter Genes gfp and rfp

Two tissue-specific promoters were used to express both green fluorescent protein (GFP) and red fluorescent protein (RFP) in transgenic zebrafish embryos. One promoter (CK), derived from a cytokeratin gene, is active specifically in skin epithelia in embryos, and the other promoter (MLC) from a muscle-specific gene encodes a myosin light chain 2 polypeptide. When the 2 promoters drove the 2 reporter genes to express in the same embryos, both genes were faithfully expressed in the respective tissues, skin or muscle. When the 2 fluorescent proteins were expressed in the same skin or muscle cells under the same promoter, GFP fluorescence appeared earlier than RFP fluorescence in both skin and muscle tissues, probably owing to a higher detection sensitivity of GFP. However, RFP appeared to be more stable as its fluorescence steadily increased during development. Finally, F₁ transgenic offspring were obtained expressing GFP in skin cells under the CK promoter and RFP in muscle cells under the MLC promoter. Our study demonstrates the feasibility of monitoring expression of multiple genes in different tissues in the same transgenic organism.     

Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis

BACKGROUND: To demonstrate that senescent fibroblasts stimulate the proliferation and neoplastic transformation of premalignant epithelial cells (Krtolica et al.: Proc Natl Acad Sci USA 98:12072-12077, 2001), we developed methods to quantify the proliferation of epithelial cells cocultured with fibroblasts. METHODS: We stained epithelial-fibroblast cocultures with the fluorescent DNA-intercalating dye 4,6-diamidino-2-phenylindole (DAPI), or expressed green fluorescent protein (GFP) in the epithelial cells, and then cultured them with fibroblasts. The cocultures were photographed under an inverted microscope with appropriate filters, and the fluorescent images were captured with a digital camera. We modified an image analysis program to selectively recognize the smaller, more intensely fluorescent epithelial cell nuclei in DAPI-stained cultures and used the program to quantify areas with DAPI fluorescence generated by epithelial nuclei or GFP fluorescence generated by epithelial cells in each field. RESULTS: Analysis of the image areas with DAPI and GFP fluorescences produced nearly identical quantification of epithelial cells in coculture with fibroblasts. We confirmed these results by manual counting. In addition, GFP labeling permitted kinetic studies of the same coculture over multiple time points. CONCLUSIONS: The image analysis-based quantification method we describe here is an easy and reliable way to monitor cells in coculture and should be useful for a variety of cell biological studies.     

In vivo method to monitor changes in HER2 expression using near-infrared fluorescence imaging

Human epidermal growth factor receptor type 2 (HER2) is a well-known biomarker that is overexpressed in many breast carcinomas. HER2 expression level is an important factor to optimize the therapeutic strategy and monitor the treatment. We used albumin binding domain-fused HER2-specific Affibody molecules, labeled with Alexa Fluor750 dye, to characterize HER2 expression in vivo. Near-infrared optical imaging studies were carried out using mice with subcutaneous HER2-positive tumors. Animals were divided into groups of five: no treatment and 12 hours and 1 week after treatment of the tumors with the Hsp90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG). The compartmental ligands-receptor model, describing binding kinetics, was used to evaluate HER2 expression from the time sequence of the fluorescence images after the intravenous probe injection. The normalized rate of accumulation of the specific fluorescent biomarkers, estimated from this time sequence, linearly correlates with the conventional ex vivo enzyme-linked immunosorbent assay (ELISA) readings for the same tumor. Such correspondence makes properly arranged fluorescence imaging an excellent candidate for estimating HER2 overexpression in tumors, complementing ELISA and other ex vivo assays. Application of this method to the fluorescence data from HER2-positive xenografts reveals that the 17-DMAG treatment results in downregulation of HER2. Application of the AngioSense 750 probe confirmed the antiangiogenic effect of 17-DMAG found with Affibody-Alexa Fluor 750 conjugate.     

A Fluorescent Protein Scaffold for Presenting Structurally Constrained Peptides Provides an Effective Screening System to Identify High Affinity Target-Binding Peptides

Peptides that have high affinity for target molecules on the surface of cancer cells are crucial for the development of targeted cancer therapies. However, unstructured peptides often fail to bind their target molecules with high affinity. To efficiently identify high-affinity target-binding peptides, we have constructed a fluorescent protein scaffold, designated gFPS, in which structurally constrained peptides are integrated at residues K131–L137 of superfolder green fluorescent protein. Molecular dynamics simulation supported the suitability of this site for presentation of exogenous peptides with a constrained structure. gFPS can present 4 to 12 exogenous amino acids without a loss of fluorescence. When gFPSs presenting human epidermal growth factor receptor type 2 (HER2)-targeting peptides were added to the culture medium of HER2-expressing cells, we could easily identify the peptides with high HER2-affinity and -specificity based on gFPS fluorescence. In addition, gFPS could be expressed on the yeast cell surface and applied for a high-throughput screening. These results demonstrate that gFPS has the potential to serve as a powerful tool to improve screening of structurally constrained peptides that have a high target affinity, and suggest that it could expedite the one-step identification of clinically applicable cancer cell-binding peptides.