胰腺癌细胞株和胰腺癌组织中miRNAs的表达检测

目的:探明miRNAs在胰腺癌细胞株及组织中的表达情况,证实miRNAs的差异表达与胰腺癌的发生有相关性.方法:对胰腺癌细胞株和胰腺癌组织进行总RNA的提取,通过Real-time PCR方法检测17种miRNAs(miR-16、miR-20a、miR-21、miR-26a、miR-142-3p、miR-155、miR-210、miR-181a、miR-181b、miR-196a、miR-939、miR-223、miR-1202、miR-1207-5p、miR-1825、miR-1228、miR-1915)在胰腺癌细胞株及胰腺癌组织中的表达,分析miRNAs的表达与胰腺癌患者临床表现之间有无相关性.结果:胰腺癌细胞株和胰腺癌组织中miRNAs的表达明显较正常胰腺组织高.癌组织及癌旁组织中miRNAs表达量在不同性别的胰腺癌患者中差异不大(P＞0.05),并且miRNAs的表达与患者年龄及其血清CA19-9关系不大.结论:经筛选的miRNAs可以作为胰腺癌的诊断标志.胰腺癌组织中的miRNAs表达并不是一成不变的,而是随着肿瘤的生长而不断发生变化.     

Comparative Proteomic Profiling of Pancreatic Ductal Adenocarcinoma Cell Lines

Pancreatic cancer is one of the most fatal cancers and is associated with limited diagnostic and therapeutic modalities. Currently, gemcitabine is the only effective drug and represents the preferred first-line treatment for chemotherapy. However, a high level of intrinsic or acquired resistance of pancreatic cancer to gemcitabine can contribute to the failure of gemcitabine treatment. To investigate the underlying molecular mechanisms for gemcitabine resistance in pancreatic cancer, we performed label-free quantification of protein expression in intrinsic gemcitabine-resistant and - sensitive human pancreatic adenocarcinoma cell lines using our improved proteomic strategy, combined with filter-aided sample preparation, single-shot liquid chromatography-mass spectrometry, enhanced spectral counting, and a statistical method based on a power law global error model. We identified 1931 proteins and quantified 787 differentially expressed proteins in the BxPC3, PANC-1, and HPDE cell lines. Bioinformatics analysis identified 15 epithelial to mesenchymal transition (EMT) markers and 13 EMT-related proteins that were closely associated with drug resistance were differentially expressed. Interestingly, 8 of these proteins were involved in glutathione and cysteine/methionine metabolism. These results suggest that proteins related to the EMT and glutathione metabolism play important roles in the development of intrinsic gemcitabine resistance by pancreatic cancer cell lines.     

Simple Monitoring of Gene Targeting Efficiency in Human Somatic Cell Lines Using the PIGA Gene

Gene targeting in most of human somatic cell lines has been labor-intensive because of low homologous recombination efficiency. The development of an experimental system that permits a facile evaluation of gene targeting efficiency in human somatic cell lines is the first step towards the improvement of this technology and its application to a broad range of cell lines. In this study, we utilized phosphatidylinositol glycan anchor biosynthesis class A (PIGA), a gene essential for the synthesis of glycosylphosphatidyl inositol (GPI) anchors, as a reporter of gene targeting events in human somatic cell lines. Targeted disruption of PIGA was quantitatively detected with FLAER, a reagent that specifically binds to GPI anchors. Using this PIGA-based reporter system, we successfully detected adeno-associated virus (AAV)-mediated gene targeting events both with and without promoter-trap enrichment of gene-targeted cell population. The PIGA-based reporter system was also capable of reproducing previous findings that an AAV-mediated gene targeting achieves a remarkably higher ratio of homologous versus random integration (H/R ratio) of targeting vectors than a plasmid-mediated gene targeting. The PIGA-based system also detected an approximately 2-fold increase in the H/R ratio achieved by a small negative selection cassette introduced at the end of the AAV-based targeting vector with a promoter-trap system. Thus, our PIGA-based system is useful for monitoring AAV-mediated gene targeting and will assist in improving gene targeting technology in human somatic cell lines.     

Generation of Recombinant Chinese Hamster Ovary Cell Lines by Microinjection

Microinjection is a gene transfer technique enabling partial control of plasmid delivery into the nucleus or cytoplasm of cultured animal cells. Here this method was used to establish various recombinant mammalian cell lines. The injection volume was estimated by fluorescence quantification of injected fluorescein isothyocynate (FITC)-dextran. The DNA concentration and injection pressure were then optimized for microinjection into the nucleus or cytoplasm using a reporter plasmid encoding the green fluorescent protein (GFP). Nuclear microinjection was more sensitive to changes in these two parameters than was cytoplasmic microinjection. Under optimal conditions, 80–90% of the cells were GFP-positive 1 day after microinjection into the nucleus or the cytoplasm. Recombinant cell lines were recovered following microinjection or calcium phosphate transfection and analyzed for the level and stability of recombinant protein production. In general, the efficiency of recovery of recombinant cell lines and the stability of reporter protein expression over time were higher following microinjection as compared to CaPi transfection. The results demonstrate the feasibility of using microinjection as a method to generate recombinant cell lines. Revisions requested 27 October 2005; Revisions received 12 December 2005     

Monitoring of Dynamic Microbiological Processes Using Real-Time Flow Cytometry

We describe a straightforward approach to continuously monitor a variety of highly dynamic microbiological processes in millisecond resolution with flow cytometry, using standard bench-top instrumentation. Four main experimental examples are provided, namely: (1) green fluorescent protein expression by antibiotic-stressed Escherichia coli, (2) fluorescent labeling of heat-induced membrane damage in an autochthonous freshwater bacterial community, (3) the initial growth response of late stationary E. coli cells inoculated into fresh growth media, and (4) oxidative disinfection of a mixed culture of auto-fluorescent microorganisms. These examples demonstrate the broad applicability of the method to diverse biological experiments, showing that it allows the collection of detailed, time-resolved information on complex processes.     

Real-Time Monitoring of the Secretory Function of Cultured Adrenal Chromaffin Cells

A system to discriminate the real-time dynamics of the secretory function in cultured adrenal chromaffin cells, using a cell bed perfusion technique and an amperometric detector, was established. Examination of basal conditions revealed that the electrode potential and flow rate are crucial factors for monitoring precise dynamics of the secretory process. Stimulation of the cells either with acetylcholine (ACh) or with high K+ concentration caused a transient current response. The current responses showed concentration dependence for both stimuli, and also showed a high correlation with the amount of catecholamines (CA) in the respective peak fraction of perfusate. Either prolonged cholinergic stimulation or maintained depolarization produced a transient response, which is not attributable to a depletion of releasable storage of CA as indicated by double-stimulation experiments. Stimulation with high K+ concentration evoked an additional release of CA even after the cellular response to prolonged ACh was inactivated, whereas maintained depolarization with high K+ produced both facilitatory and inhibitory effects on the cell responsiveness to ACh. Most probably the transient natures of the secretory responses to ACh and to high K+ are mediated by different mechanisms. All the results suggest that the direct monitoring is profitable for studies on the regulatory mechanisms of the secretory function.     

Real-Time Monitoring of Protein Conformational Changes Using a Nano-Mechanical Sensor

Proteins can switch between different conformations in response to stimuli, such as pH or temperature variations, or to the binding of ligands. Such plasticity and its kinetics can have a crucial functional role, and their characterization has taken center stage in protein research. As an example, Topoisomerases are particularly interesting enzymes capable of managing tangled and supercoiled double-stranded DNA, thus facilitating many physiological processes. In this work, we describe the use of a cantilever-based nanomotion sensor to characterize the dynamics of human topoisomerase II (Topo II) enzymes and their response to different kinds of ligands, such as ATP, which enhance the conformational dynamics. The sensitivity and time resolution of this sensor allow determining quantitatively the correlation between the ATP concentration and the rate of Topo II conformational changes. Furthermore, we show how to rationalize the experimental results in a comprehensive model that takes into account both the physics of the cantilever and the dynamics of the ATPase cycle of the enzyme, shedding light on the kinetics of the process. Finally, we study the effect of aclarubicin, an anticancer drug, demonstrating that it affects directly the Topo II molecule inhibiting its conformational changes. These results pave the way to a new way of studying the intrinsic dynamics of proteins and of protein complexes allowing new applications ranging from fundamental proteomics to drug discovery and development and possibly to clinical practice.     

Mass Spectrometry-based Quantitative Proteomic Profiling of Human Pancreatic and Hepatic Stellate Cell Lines

The functions of the liver and the pancreas differ; however, chronic inflammation in both organs is associated with fibrosis. Evidence suggests that fibrosis in both organs is partially regulated by organ-specific stellate cells. We explore the proteome of human hepatic stellate cells (hHSC) and human pancreatic stellate cells (hPaSC) using mass spectrometry (MS)-based quantitative proteomics to investigate pathophysiologic mechanisms. Proteins were isolated from whole cell lysates of immortalized hHSC and hPaSC. These proteins were tryptically digested, labeled with tandem mass tags (TMT), fractionated by OFFGEL, and subjected to MS. Proteins significantly different in abundance (P < 0.05) were classified via gene ontology (GO) analysis. We identified 1223 proteins and among them, 1222 proteins were quantifiable. Statistical analysis determined that 177 proteins were of higher abundance in hHSC, while 157 were of higher abundance in hPaSC. GO classification revealed that proteins of relatively higher abundance in hHSC were associated with protein production, while those of relatively higher abundance in hPaSC were involved in cell structure. Future studies using the methodologies established herein, but with further upstream fractionation and/or use of enhanced MS instrumentation will allow greater proteome coverage, achieving a comprehensive proteomic analysis of hHSC and hPaSC.     

Generation and Characterisation of Stable Cell Lines Expressing Recombinant Human N-Methyl-d-Aspartate Receptor Subtypes

Abstract: Transfection of mouse L(tk-) cells with human N -methyl- d -aspartate (NMDA) receptor subunit cDNAs under the control of a dexamethasone-inducible promoter has been used to generate two stable cell lines expressing NR1a/NR2A receptors and a stable cell line expressing NR1a/NR2B receptors. The cell lines have been characterised by northern and western blot analyses, and the pharmacology of the recombinant receptors determined by radioligand binding techniques. Pharmacological differences were identified between the two NMDA receptor subtypes. The glutamate site antagonist d,l -(ε)-2-[³H]amino-4-propyl-5-phosphono-3-pentanoic acid ([³H]CGP 39653) had high affinity for NR1a/NR2A receptors ( K _D = 3.93 n M ) but did not bind to NR1a/NR2B receptors. Glycine site agonists showed a 2.6–5.4-fold higher affinity for NR1a/NR2B receptors. Data from radioligand binding studies indicated that one of the cell lines, NR1a/NR2A-I, expressed a stoichiometric excess of the NR1a subunit, which may exist as homomeric assemblies. This observation has implications when interpreting data from pharmacological analysis of recombinant receptors, as well as understanding the assembly and control of expression of native NMDA receptors.     

Bmi1 Enhances Tumorigenicity and Cancer Stem Cell Function in Pancreatic Adenocarcinoma

Background

Bmi1 is an integral component of the Polycomb Repressive Complex 1 (PRC1) and is involved in the pathogenesis of multiple cancers. It also plays a key role in the functioning of endogenous stem cells and cancer stem cells. Previous work implicated a role for cancer stem cells in the pathogenesis of pancreatic cancer. We hypothesized that Bmi1 plays an integral role in enhancing pancreatic tumorigenicity and the function of cancer stem cells in pancreatic ductal adenocarcinoma.

Methods

We measured endogenous Bmi1 levels in primary human pancreatic ductal adenocarcinomas, pancreatic intraepithelial neoplasias (PanINs) and normal pancreas by immunohistochemistry and Western blotting. The function of Bmi1 in pancreatic cancer was assessed by alteration of Bmi1 expression in several cell model systems by measuring cell proliferation, cell apoptosis, in vitro invasion, chemotherapy resistance, and in vivo growth and metastasis in an orthotopic model of pancreatic cancer. We also assessed the cancer stem cell frequency, tumorsphere formation, and in vivo growth of human pancreatic cancer xenografts after Bmi1 silencing.

Results

Bmi1 was overexpressed in human PanINs, pancreatic cancers, and in several pancreatic cancer cell lines. Overexpression of Bmi1 in MiaPaCa2 cells resulted in increased proliferation, in vitro invasion, larger in vivo tumors, more metastases, and gemcitabine resistance while opposite results were seen when Bmi1 was silenced in Panc-1 cells. Bmi1 was overexpressed in the cancer stem cell compartment of primary human pancreatic cancer xenografts. Pancreatic tumorspheres also demonstrated high levels of Bmi1. Silencing of Bmi1 inhibited secondary and tertiary tumorsphere formation, decreased primary pancreatic xenograft growth, and lowered the proportion of cancer stem cells in the xenograft tissue.

Conclusions

Our results implicate Bmi1 in the invasiveness and growth of pancreatic cancer and demonstrate its key role in the regulation of pancreatic cancer stem cells.     

Liraglutide Improves Pancreatic Beta Cell Mass and Function in Alloxan-Induced Diabetic Mice

Glucagon-like peptide-1 (GLP-1) receptor agonists potentiate glucose-induced insulin secretion. In addition, they have been reported to increase pancreatic beta cell mass in diabetic rodents. However, the precise mode of action of GLP-1 receptor agonists still needs to be elucidated. Here we clarify the effects of the human GLP-1 analog liraglutide on beta cell fate and function by using an inducible Cre/loxP-based pancreatic beta cell tracing system and alloxan-induced diabetic mice. Liraglutide was subcutaneously administered once daily for 30 days. The changes in beta cell mass were examined as well as glucose tolerance and insulin secretion. We found that chronic liraglutide treatment improved glucose tolerance and insulin response to oral glucose load. Thirty-day treatment with liraglutide resulted in a 2-fold higher mass of pancreatic beta cells than that in vehicle group. Liraglutide increased proliferation rate of pancreatic beta cells and prevented beta cells from apoptotic cells death. However, the relative abundance of YFP-labeled beta cells to total beta cells was no different before and after liraglutide treatment, suggesting no or little contribution of neogenesis to the increase in beta cell mass. Liraglutide reduced oxidative stress in pancreatic islet cells of alloxan-induced diabetic mice. Furthermore, the beneficial effects of liraglutide in these mice were maintained two weeks after drug withdrawal. In conclusion, chronic liraglutide treatment improves hyperglycemia by ameliorating beta cell mass and function in alloxan-induced diabetic mice.     

Novel Pancreatic Cancer Cell Lines Derived from Genetically Engineered Mouse Models of Spontaneous Pancreatic Adenocarcinoma: Applications in Diagnosis and Therapy

Pancreatic cancer (PC) remains one of the most lethal human malignancies with poor prognosis. Despite all advances in preclinical research, there have not been significant translation of novel therapies into the clinics. The development of genetically engineered mouse (GEM) models that produce spontaneous pancreatic adenocarcinoma (PDAC) have increased our understanding of the pathogenesis of the disease. Although these PDAC mouse models are ideal for studying potential therapies and specific genetic mutations, there is a need for developing syngeneic cell lines from these models. In this study, we describe the successful establishment and characterization of three cell lines derived from two (PDAC) mouse models. The cell line UN-KC-6141 was derived from a pancreatic tumor of a Kras^G12D;Pdx1-Cre (KC) mouse at 50 weeks of age, whereas UN-KPC-960 and UN-KPC-961 cell lines were derived from pancreatic tumors of Kras^G12D;Trp53^R172H;Pdx1-Cre (KPC) mice at 17 weeks of age. The cancer mutations of these parent mice carried over to the daughter cell lines (i.e. Kras^G12D mutation was observed in all three cell lines while Trp53 mutation was observed only in KPC cell lines). The cell lines showed typical cobblestone epithelial morphology in culture, and unlike the previously established mouse PDAC cell line Panc02, expressed the ductal marker CK19. Furthermore, these cell lines expressed the epithelial-mesenchymal markers E-cadherin and N-cadherin, and also, Muc1 and Muc4 mucins. In addition, these cell lines were resistant to the chemotherapeutic drug Gemcitabine. Their implantation in vivo produced subcutaneous as well as tumors in the pancreas (orthotopic). The genetic mutations in these cell lines mimic the genetic compendium of human PDAC, which make them valuable models with a high potential of translational relevance for examining diagnostic markers and therapeutic drugs.     

Generation and Characterisation of Novel Pancreatic Adenocarcinoma Xenograft Models and Corresponding Primary Cell Lines

Pancreatic adenocarcinoma is one of the most lethal cancer types, currently lacking efficient treatment. The heterogeneous nature of these tumours are poorly represented by the classical pancreatic cell lines, which have been through strong clonal selection in vitro, and are often derived from metastases. Here, we describe the establishment of novel pancreatic adenocarcinoma models, xenografts and corresponding in vitro cell lines, from primary pancreatic tumours. The morphology, differentiation grade and gene expression pattern of the xenografts resemble the original tumours well. The cell lines were analysed for colony forming capacity, tumourigenicity and expression of known cancer cell surface markers and cancer stem-like characteristics. These primary cell models will be valuable tools for biological and preclinical studies for this devastating disease.     

Human Fucci Pancreatic Beta Cell Lines: New Tools to Study Beta Cell Cycle and Terminal Differentiation

Regulation of cell cycle in beta cells is poorly understood, especially in humans. We exploited here the recently described human pancreatic beta cell line EndoC-βH2 to set up experimental systems for cell cycle studies. We derived 2 populations from EndoC-βH2 cells that stably harbor the 2 genes encoding the Fucci fluorescent indicators of cell cycle, either from two vectors, or from a unique bicistronic vector. In proliferating non-synchronized cells, the 2 Fucci indicators revealed cells in the expected phases of cell cycle, with orange and green cells being in G1 and S/G2/M cells, respectively, and allowed the sorting of cells in different substeps of G1. The Fucci indicators also faithfully red out alterations in human beta cell proliferative activity since a mitogen-rich medium decreased the proportion of orange cells and inflated the green population, while reciprocal changes were observed when cells were induced to cease proliferation and increased expression of some beta cell genes. In the last situation, acquisition of a more differentiated beta cell phenotype correlates with an increased intensity in orange fluorescence. Hence Fucci beta cell lines provide new tools to address important questions regarding human beta cell cycle and differentiation.     

Improved Genome Editing in Human Cell Lines Using the CRISPR Method

The Cas9/CRISPR system has become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-introduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1.