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1.
Sci Rep ; 14(1): 13227, 2024 06 09.
Article in English | MEDLINE | ID: mdl-38851782

ABSTRACT

There are hundreds of genes typically overexpressed in breast cancer cells and it's often assumed that their overexpression contributes to cancer progression. However, the precise proportion of these overexpressed genes contributing to tumorigenicity remains unclear. To address this gap, we undertook a comprehensive screening of a diverse set of seventy-two genes overexpressed in breast cancer. This systematic screening evaluated their potential for inducing malignant transformation and, concurrently, assessed their impact on breast cancer cell proliferation and viability. Select genes including ALDH3B1, CEACAM5, IL8, PYGO2, and WWTR1, exhibited pronounced activity in promoting tumor formation and establishing gene dependencies critical for tumorigenicity. Subsequent investigations revealed that CEACAM5 overexpression triggered the activation of signaling pathways involving ß-catenin, Cdk4, and mTOR. Additionally, it conferred a growth advantage independent of exogenous insulin in defined medium and facilitated spheroid expansion by inducing multiple layers of epithelial cells while preserving a hollow lumen. Furthermore, the silencing of CEACAM5 expression synergized with tamoxifen-induced growth inhibition in breast cancer cells. These findings underscore the potential of screening overexpressed genes for both oncogenic drivers and tumor dependencies to expand the repertoire of therapeutic targets for breast cancer treatment.


Subject(s)
Breast Neoplasms , Cell Proliferation , Gene Expression Regulation, Neoplastic , Humans , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Female , Cell Proliferation/genetics , Cell Line, Tumor , Signal Transduction , Oncogenes , beta Catenin/metabolism , beta Catenin/genetics , Tamoxifen/pharmacology , Animals , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , TOR Serine-Threonine Kinases/metabolism , TOR Serine-Threonine Kinases/genetics , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 4/metabolism , Cell Transformation, Neoplastic/genetics
2.
Nat Protoc ; 17(9): 1903-1925, 2022 09.
Article in English | MEDLINE | ID: mdl-35840661

ABSTRACT

Here we provide a detailed tutorial on CRISPR in vivo screening. Using the mouse as the model organism, we introduce a range of CRISPR tools and applications, delineate general considerations for 'transplantation-based' or 'direct in vivo' screening design, and provide details on technical execution, sequencing readouts, computational analyses and data interpretation. In vivo screens face unique pitfalls and limitations, such as delivery issues or library bottlenecking, which must be counteracted to avoid screening failure or flawed conclusions. A broad variety of in vivo phenotypes can be interrogated such as organ development, hematopoietic lineage decision and evolutionary licensing in oncogenesis. We describe experimental strategies to address various biological questions and provide an outlook on emerging CRISPR applications, such as genetic interaction screening. These technological advances create potent new opportunities to dissect the molecular underpinnings of complex organismal phenotypes.


Subject(s)
CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Animals , Gene Library , Genetic Testing , Mice , Phenotype
3.
JCI Insight ; 7(10)2022 05 23.
Article in English | MEDLINE | ID: mdl-35439169

ABSTRACT

Systemic therapies for pancreatic ductal adenocarcinoma (PDAC) remain unsatisfactory. Clinical prognosis is particularly poor for tumor subtypes with activating aberrations in the MYC pathway, creating an urgent need for novel therapeutic targets. To unbiasedly find MYC-associated epigenetic dependencies, we conducted a drug screen in pancreatic cancer cell lines. Here, we found that protein arginine N-methyltransferase 5 (PRMT5) inhibitors triggered an MYC-associated dependency. In human and murine PDACs, a robust connection of MYC and PRMT5 was detected. By the use of gain- and loss-of-function models, we confirmed the increased efficacy of PRMT5 inhibitors in MYC-deregulated PDACs. Although inhibition of PRMT5 was inducing DNA damage and arresting PDAC cells in the G2/M phase of the cell cycle, apoptotic cell death was executed predominantly in cells with high MYC expression. Experiments in primary patient-derived PDAC models demonstrated the existence of a highly PRMT5 inhibitor-sensitive subtype. Our work suggests developing PRMT5 inhibitor-based therapies for PDAC.


Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Animals , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Drug Evaluation, Preclinical , Early Detection of Cancer , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Epigenesis, Genetic , Humans , Mice , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Protein-Arginine N-Methyltransferases/genetics , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Pancreatic Neoplasms
4.
Nat Protoc ; 17(4): 1142-1188, 2022 04.
Article in English | MEDLINE | ID: mdl-35288718

ABSTRACT

Genetically engineered mouse models (GEMMs) transformed the study of organismal disease phenotypes but are limited by their lengthy generation in embryonic stem cells. Here, we describe methods for rapid and scalable genome engineering in somatic cells of the liver and pancreas through delivery of CRISPR components into living mice. We introduce the spectrum of genetic tools, delineate viral and nonviral CRISPR delivery strategies and describe a series of applications, ranging from gene editing and cancer modeling to chromosome engineering or CRISPR multiplexing and its spatio-temporal control. Beyond experimental design and execution, the protocol describes quantification of genetic and functional editing outcomes, including sequencing approaches, data analysis and interpretation. Compared to traditional knockout mice, somatic GEMMs face an increased risk for mouse-to-mouse variability because of the higher experimental demands of the procedures. The robust protocols described here will help unleash the full potential of somatic genome manipulation. Depending on the delivery method and envisaged application, the protocol takes 3-5 weeks.


Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Neoplasms , Animals , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Editing/methods , Liver , Mice , Mice, Knockout , Neoplasms/genetics , Pancreas
6.
Nat Rev Cancer ; 20(10): 573-593, 2020 10.
Article in English | MEDLINE | ID: mdl-32636489

ABSTRACT

With the genetic portraits of all major human malignancies now available, we next face the challenge of characterizing the function of mutated genes, their downstream targets, interactions and molecular networks. Moreover, poorly understood at the functional level are also non-mutated but dysregulated genomes, epigenomes or transcriptomes. Breakthroughs in manipulative mouse genetics offer new opportunities to probe the interplay of molecules, cells and systemic signals underlying disease pathogenesis in higher organisms. Herein, we review functional screening strategies in mice using genetic perturbation and chemical mutagenesis. We outline the spectrum of genetic tools that exist, such as transposons, CRISPR and RNAi and describe discoveries emerging from their use. Genome-wide or targeted screens are being used to uncover genomic and regulatory landscapes in oncogenesis, metastasis or drug resistance. Versatile screening systems support experimentation in diverse genetic and spatio-temporal settings to integrate molecular, cellular or environmental context-dependencies. We also review the combination of in vivo screening and barcoding strategies to study genetic interactions and quantitative cancer dynamics during tumour evolution. These scalable functional genomics approaches are transforming our ability to interrogate complex biological systems.


Subject(s)
Genetic Association Studies , Genetic Predisposition to Disease , Genomics , Neoplasms/diagnosis , Neoplasms/genetics , Animals , CRISPR-Cas Systems , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Viral , DNA Transposable Elements , Early Detection of Cancer , Genetic Association Studies/methods , Genetic Testing/methods , Genomics/methods , Humans , Mutagenesis/drug effects , Mutagenesis/radiation effects , Neoplasms/therapy , Translational Research, Biomedical
7.
Nucleic Acids Res ; 48(13): e78, 2020 07 27.
Article in English | MEDLINE | ID: mdl-32479629

ABSTRACT

The systematic perturbation of genomes using CRISPR/Cas9 deciphers gene function at an unprecedented rate, depth and ease. Commercially available sgRNA libraries typically contain tens of thousands of pre-defined constructs, resulting in a complexity challenging to handle. In contrast, custom sgRNA libraries comprise gene sets of self-defined content and size, facilitating experiments under complex conditions such as in vivo systems. To streamline and upscale cloning of custom libraries, we present CLUE, a bioinformatic and wet-lab pipeline for the multiplexed generation of pooled sgRNA libraries. CLUE starts from lists of genes or pasted sequences provided by the user and designs a single synthetic oligonucleotide pool containing various libraries. At the core of the approach, a barcoding strategy for unique primer binding sites allows amplifying different user-defined libraries from one single oligonucleotide pool. We prove the approach to be straightforward, versatile and specific, yielding uniform sgRNA distributions in all resulting libraries, virtually devoid of cross-contaminations. For in silico library multiplexing and design, we established an easy-to-use online platform at www.crispr-clue.de. All in all, CLUE represents a resource-saving approach to produce numerous high quality custom sgRNA libraries in parallel, which will foster their broad use across molecular biosciences.


Subject(s)
Cloning, Molecular , Gene Library , RNA, Guide, Kinetoplastida/genetics , Animals , CRISPR-Cas Systems/genetics , Genome , Humans , Mice , Oligonucleotides/genetics
9.
Curr Opin Genet Dev ; 54: 105-109, 2019 02.
Article in English | MEDLINE | ID: mdl-31121413

ABSTRACT

RNA molecules are subject to a complex co-transcriptional and post-transcriptional life cycle, controlled at all stages by RNA binding proteins (RBPs) and non-coding RNAs that influence mRNA stability, splicing, localization, and decay. Together with mechanisms regulating the process of transcription itself, non-coding RNAs and RBPs contribute to a model of para-transcriptional coordination of gene expression, which is utilized during normal tissue physiology and cancer development in order to execute complex gene expression programs. Several key regulators of RNA biology, such as certain splice factors, represent bona fide cancer vulnerabilities, but our understanding of these processes is still far away from being comprehensive. Genetic forward screens utilizing technologies such as transposons, RNAi and CRISPR aid the field in rapidly establishing functional phenotypes and genetic cancer cell addictions. This review focuses on four individual regulatory gene expression processes governed by regulators of the RNA life cycle, the impact of functional genomics on streamlining the discovery process and the role of such mechanisms in tumor biology.


Subject(s)
CRISPR-Cas Systems/genetics , Neoplasms/genetics , RNA Stability/genetics , RNA, Untranslated/genetics , Animals , Disease Models, Animal , Gene Expression Regulation, Neoplastic/genetics , Humans , Mice , RNA/genetics , RNA-Binding Proteins/genetics
10.
Mol Cancer Ther ; 17(8): 1773-1780, 2018 08.
Article in English | MEDLINE | ID: mdl-29720559

ABSTRACT

Mutated in approximately 30% of human cancers, Ras GTPases are the most common drivers of oncogenesis and render tumors unresponsive to many standard therapies. Despite decades of research, no drugs directly targeting Ras are currently available. We have previously characterized a small protein antagonist of K-Ras, R11.1.6, and demonstrated its direct competition with Raf for Ras binding. Here we evaluate the effects of R11.1.6 on Ras signaling and cellular proliferation in a panel of human cancer cell lines. Through lentiviral transduction, we generated cell lines that constitutively or through induction with doxycycline express R11.1.6 or a control protein YW1 and show specific binding by R11.1.6 to endogenous Ras through microscopy and co-immunoprecipitation experiments. Genetically encoded intracellular expression of this high-affinity Ras antagonist, however, fails to measurably disrupt signaling through either the MAPK or PI3K pathway. Consistently, cellular proliferation was unaffected as well. To understand this lack of signaling inhibition, we quantified the number of molecules of R11.1.6 expressed by the inducible cell lines and developed a simple mathematical model describing the competitive binding of Ras by R11.1.6 and Raf. This model supports a potential mechanism for the lack of biological effects that we observed, suggesting stoichiometric and thermodynamic barriers that should be overcome in pharmacologic efforts to directly compete with downstream effector proteins localized to membranes at very high effective concentrations. Mol Cancer Ther; 17(8); 1773-80. ©2018 AACR.


Subject(s)
Neoplasms/genetics , Proto-Oncogene Proteins c-raf/metabolism , ras Proteins/metabolism , Cell Line, Tumor , Humans , Neoplasms/pathology , Signal Transduction
11.
Cancer Cell ; 32(4): 411-426.e11, 2017 10 09.
Article in English | MEDLINE | ID: mdl-28966034

ABSTRACT

Glioblastoma (GBM) is a devastating malignancy with few therapeutic options. We identify PRMT5 in an in vivo GBM shRNA screen and show that PRMT5 knockdown or inhibition potently suppresses in vivo GBM tumors, including patient-derived xenografts. Pathway analysis implicates splicing in cellular PRMT5 dependency, and we identify a biomarker that predicts sensitivity to PRMT5 inhibition. We find that PRMT5 deficiency primarily disrupts the removal of detained introns (DIs). This impaired DI splicing affects proliferation genes, whose downregulation coincides with cell cycle defects, senescence and/or apoptosis. We further show that DI programs are evolutionarily conserved and operate during neurogenesis, suggesting that they represent a physiological regulatory mechanism. Collectively, these findings reveal a PRMT5-regulated DI-splicing program as an exploitable cancer vulnerability.


Subject(s)
Brain Neoplasms/pathology , Glioma/pathology , Introns , Protein-Arginine N-Methyltransferases/physiology , Animals , Cell Cycle/drug effects , Cell Differentiation , Cell Line, Tumor , Cell Proliferation/drug effects , Glioma/drug therapy , Glioma/genetics , High-Throughput Screening Assays , Humans , Isoquinolines/pharmacology , Mice , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Pyrimidines/pharmacology , RNA Splicing
12.
Expert Rev Anticancer Ther ; 16(9): 977-87, 2016 Sep.
Article in English | MEDLINE | ID: mdl-27410491

ABSTRACT

INTRODUCTION: Whereas tumorigenic processes have traditionally been attributed to gene amplification, deletion, or mutation, it is now clear that epigenetic changes represent an additional hallmark of cancer. This review explains the basic principles of epigenetic regulation and therapy, provides an overview of clinically approved drugs, introduces novel targets and compounds, and discusses the potential reasons behind treatment success and failure. AREAS COVERED: We provide a brief introduction to the concept of epigenetic regulation in general and explain how epigenetic pathways are altered in cancer. Based on this, we go on to explore the rational behind epigenetic cancer therapy, provide an overview of clinical success and failure of specific drugs, describe novel pharmaceutical targets, review epigenetic combination treatment, and finally discuss biological concepts influencing treatment success. Expert commentary: Even though many early epigenetic therapy trials had disappointing results, lessons learned from these studies have heavily influenced the design of modern trials leading to improved therapeutic outcomes. Better preclinical model systems may help to reduce the risk of clinical failure and to identify high-confidence targets for clinical follow-up.


Subject(s)
Antineoplastic Agents/pharmacology , Epigenesis, Genetic , Neoplasms/pathology , Animals , Genome, Human , Humans , Molecular Targeted Therapy , Neoplasms/drug therapy , Neoplasms/genetics , Treatment Failure , Treatment Outcome
13.
Proc Natl Acad Sci U S A ; 113(27): E3892-900, 2016 07 05.
Article in English | MEDLINE | ID: mdl-27325776

ABSTRACT

Targeted transcriptional regulation is a powerful tool to study genetic mediators of cellular behavior. Here, we show that catalytically dead Cas9 (dCas9) targeted to genomic regions upstream or downstream of the transcription start site allows for specific and sustainable gene-expression level alterations in tumor cells in vitro and in syngeneic immune-competent mouse models. We used this approach for a high-coverage pooled gene-activation screen in vivo and discovered previously unidentified modulators of tumor growth and therapeutic response. Moreover, by using dCas9 linked to an activation domain, we can either enhance or suppress target gene expression simply by changing the genetic location of dCas9 binding relative to the transcription start site. We demonstrate that these directed changes in gene-transcription levels occur with minimal off-target effects. Our findings highlight the use of dCas9-mediated transcriptional regulation as a versatile tool to reproducibly interrogate tumor phenotypes in vivo.


Subject(s)
Bacterial Proteins/genetics , Endonucleases/genetics , Gene Expression Regulation, Leukemic , Genetic Techniques , Leukemia, Experimental , Animals , CRISPR-Associated Protein 9 , DNA Damage , DNA Modification Methylases/metabolism , DNA Repair Enzymes/metabolism , Dacarbazine/analogs & derivatives , Drug Resistance, Neoplasm , Mice , Sequence Analysis, RNA , Temozolomide , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/metabolism
14.
Cancer Cell ; 28(5): 623-637, 2015 Nov 09.
Article in English | MEDLINE | ID: mdl-26602816

ABSTRACT

In normal cells, p53 is activated by DNA damage checkpoint kinases to simultaneously control the G1/S and G2/M cell cycle checkpoints through transcriptional induction of p21(cip1) and Gadd45α. In p53-mutant tumors, cell cycle checkpoints are rewired, leading to dependency on the p38/MK2 pathway to survive DNA-damaging chemotherapy. Here we show that the RNA binding protein hnRNPA0 is the "successor" to p53 for checkpoint control. Like p53, hnRNPA0 is activated by a checkpoint kinase (MK2) and simultaneously controls both cell cycle checkpoints through distinct target mRNAs, but unlike p53, this is through the post-transcriptional stabilization of p27(Kip1) and Gadd45α mRNAs. This pathway drives cisplatin resistance in lung cancer, demonstrating the importance of post-transcriptional RNA control to chemotherapy response.


Subject(s)
Cell Cycle Checkpoints/genetics , Drug Resistance, Neoplasm/genetics , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Mutation , Neoplasms/genetics , Tumor Suppressor Protein p53/genetics , Aged , Animals , Antineoplastic Agents/therapeutic use , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cisplatin/therapeutic use , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cyclin-Dependent Kinase Inhibitor p27/genetics , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Female , Gene Expression Regulation, Neoplastic , Genetic Pleiotropy , HEK293 Cells , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , Male , Mice, Inbred C57BL , Middle Aged , Neoplasms/drug therapy , Neoplasms/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Survival Analysis
15.
Cancer Res ; 75(15): 3127-38, 2015 Aug 01.
Article in English | MEDLINE | ID: mdl-26025730

ABSTRACT

Glioblastoma (GBM) is often treated with the cytotoxic drug temozolomide, but the disease inevitably recurs in a drug-resistant form after initial treatment. Here, we report that in GBM cells, even a modest decrease in the mismatch repair (MMR) components MSH2 and MSH6 have profound effects on temozolomide sensitivity. RNAi-mediated attenuation of MSH2 and MSH6 showed that such modest decreases provided an unexpectedly strong mechanism of temozolomide resistance. In a mouse xenograft model of human GBM, small changes in MSH2 were sufficient to suppress temozolomide-induced tumor regression. Using The Cancer Genome Atlas to analyze mRNA expression patterns in tumors from temozolomide-treated GBM patients, we found that MSH2 transcripts in primary GBM could predict patient responses to initial temozolomide therapy. In recurrent disease, the absence of microsatellite instability (the standard marker for MMR deficiency) suggests a lack of involvement of MMR in the resistant phenotype of recurrent disease. However, more recent studies reveal that decreased MMR protein levels occur often in recurrent GBM. In accordance with our findings, these reported decreases may constitute a mechanism by which GBM evades temozolomide sensitivity while maintaining microsatellite stability. Overall, our results highlight the powerful effects of MSH2 attenuation as a potent mediator of temozolomide resistance and argue that MMR activity offers a predictive marker for initial therapeutic response to temozolomide treatment.


Subject(s)
Dacarbazine/analogs & derivatives , Glioblastoma/drug therapy , MutS Homolog 2 Protein/metabolism , Animals , Antineoplastic Agents, Alkylating/pharmacology , Carmustine/pharmacology , Cell Line, Tumor/drug effects , Cell Line, Tumor/radiation effects , DNA Modification Methylases/metabolism , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dacarbazine/pharmacology , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm/genetics , Gene Knockdown Techniques , Genes, p53 , Glioblastoma/metabolism , Glioblastoma/mortality , Glioblastoma/pathology , Humans , Mice, Inbred C57BL , MutS Homolog 2 Protein/genetics , Radiation, Ionizing , Survival Analysis , Temozolomide , Tumor Suppressor Proteins/metabolism , Xenograft Model Antitumor Assays
17.
J Neurosci Methods ; 233: 13-7, 2014 Aug 15.
Article in English | MEDLINE | ID: mdl-24938397

ABSTRACT

BACKGROUND: A functional reconstitution of channel proteins in planar lipid bilayers is still very versatile to study structure/function correlates under well-defined conditions at the single protein level. NEW METHOD: In this study we present an improved planar lipid bilayer technique in which an air bubble is used for stabilizing unstable/leaky bilayers or for removing excess lipids. The bubble can also be used as a tool for reducing the number of channels in the bilayer with the goal of having only one active channel in the membrane. RESULTS: Stable planar lipid bilayers are formed within seconds to minutes. In the case of multiple channel insertion the air bubble can be used to reduce the number of channels within minutes. COMPARISON WITH EXISTING METHOD(S): The simple improvement of the classical folding technique guarantees a very fast creation of stable bilayers even with difficult phospholipids in a conventional vertical bilayer set-up; it requires no modifications of the existing set-up. CONCLUSIONS: This technique is very easy to handle and guarantees successful single channel recordings for any kind of planar lipid bilayer experiment.


Subject(s)
Lipid Bilayers/chemical synthesis , Air , Dimyristoylphosphatidylcholine/chemistry , Membrane Potentials , Microelectrodes , Phosphatidylcholines/chemistry , Phospholipids/chemistry , Potassium Channels/chemistry , Time Factors
18.
Cell ; 156(3): 590-602, 2014 Jan 30.
Article in English | MEDLINE | ID: mdl-24485462

ABSTRACT

Therapy-resistant microenvironments represent a major barrier toward effective elimination of disseminated malignancies. Here, we show that select microenvironments can underlie resistance to antibody-based therapy. Using a humanized model of treatment refractory B cell leukemia, we find that infiltration of leukemia cells into the bone marrow rewires the tumor microenvironment to inhibit engulfment of antibody-targeted tumor cells. Resistance to macrophage-mediated killing can be overcome by combination regimens involving therapeutic antibodies and chemotherapy. Specifically, the nitrogen mustard cyclophosphamide induces an acute secretory activating phenotype (ASAP), releasing CCL4, IL8, VEGF, and TNFα from treated tumor cells. These factors induce macrophage infiltration and phagocytic activity in the bone marrow. Thus, the acute induction of stress-related cytokines can effectively target cancer cells for removal by the innate immune system. This synergistic chemoimmunotherapeutic regimen represents a potent strategy for using conventional anticancer agents to alter the tumor microenvironment and promote the efficacy of targeted therapeutics.


Subject(s)
Disease Models, Animal , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Tumor Microenvironment , Animals , Cyclophosphamide/therapeutic use , Cytokines/immunology , Drug Resistance, Neoplasm , Heterografts , Humans , Immunity, Innate , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Macrophages/immunology , Mice , Neoplasm Transplantation
19.
Biochim Biophys Acta ; 1838(4): 1096-103, 2014 Apr.
Article in English | MEDLINE | ID: mdl-23791706

ABSTRACT

The viral channel KcvNTS belongs to the smallest K(+) channels known so far. A monomer of a functional homotetramer contains only 82 amino acids. As a consequence of the small size the protein is almost fully submerged into the membrane. This suggests that the channel is presumably sensitive to its lipid environment. Here we perform a comparative analysis for the function of the channel protein embedded in three different membrane environments. 1. Single-channel currents of KcvNTS were recorded with the patch clamp method on the plasma membrane of HEK293 cells. 2. They were also measured after reconstitution of recombinant channel protein into classical planar lipid bilayers and 3. into horizontal bilayers derived from giant unilamellar vesicles (GUVs). The recombinant channel protein was either expressed and purified from Pichia pastoris or from a cell-free expression system; for the latter a new approach with nanolipoprotein particles was used. The data show that single-channel activity can be recorded under all experimental conditions. The main functional features of the channel like a large single-channel conductance (80pS), high open-probability (>50%) and the approximate duration of open and closed dwell times are maintained in all experimental systems. An apparent difference between the approaches was only observed with respect to the unitary conductance, which was ca. 35% lower in HEK293 cells than in the other systems. The reason for this might be explained by the fact that the channel is tagged by GFP when expressed in HEK293 cells. Collectively the data demonstrate that the small viral channel exhibits a robust function in different experimental systems. This justifies an extrapolation of functional data from these systems to the potential performance of the channel in the virus/host interaction. This article is part of a Special Issue entitled: Viral Membrane Proteins-Channels for Cellular Networking.


Subject(s)
Potassium Channels/chemistry , Viral Matrix Proteins/chemistry , Amino Acid Sequence , HEK293 Cells , Humans , Lipid Bilayers/chemistry , Models, Biological , Molecular Sequence Data , Potassium Channels/physiology , Viral Matrix Proteins/physiology
20.
Cell Stem Cell ; 12(6): 639-41, 2013 Jun 06.
Article in English | MEDLINE | ID: mdl-23746971

ABSTRACT

BMI1 is a known oncogenic transcriptional repressor in glioblastoma stem-like cells, but its downstream mediators are poorly understood. Recently, in Cancer Cell, Gargiulo et al. (2013) designed a rational in vivo RNAi screen based on BMI1 ChIP-seq from neural progenitors and identified functional tumor suppressor targets, including Atf3 and Cbx7.


Subject(s)
Bone Morphogenetic Proteins/metabolism , Endoplasmic Reticulum Stress , Glioblastoma/genetics , Neoplastic Stem Cells/metabolism , Polycomb Repressive Complex 1/genetics , Polycomb Repressive Complex 1/metabolism , Proto-Oncogene Proteins/metabolism , Transforming Growth Factor beta/metabolism , Animals , Humans
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