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1.
Sci Signal ; 11(540)2018 07 24.
Article in English | MEDLINE | ID: mdl-30042127

ABSTRACT

Cells respond to DNA damage by activating complex signaling networks that decide cell fate, promoting not only DNA damage repair and survival but also cell death. We have developed a multiscale computational model that quantitatively links chemotherapy-induced DNA damage response signaling to cell fate. The computational model was trained and calibrated on extensive data from U2OS osteosarcoma cells, including the cell cycle distribution of the initial cell population, signaling data measured by Western blotting, and cell fate data in response to chemotherapy treatment measured by time-lapse microscopy. The resulting mechanistic model predicted the cellular responses to chemotherapy alone and in combination with targeted inhibitors of the DNA damage response pathway, which we confirmed experimentally. Computational models such as the one presented here can be used to understand the molecular basis that defines the complex interplay between cell survival and cell death and to rationally identify chemotherapy-potentiating drug combinations.


Subject(s)
Antineoplastic Agents/pharmacology , Bone Neoplasms/pathology , DNA Damage , Osteosarcoma/pathology , Ovarian Neoplasms/pathology , Small Molecule Libraries/pharmacology , Animals , Apoptosis , Bone Neoplasms/drug therapy , Bone Neoplasms/genetics , Cell Cycle , Cell Proliferation , DNA Repair , Drug Therapy, Combination , Female , Humans , Mice , Osteosarcoma/drug therapy , Osteosarcoma/genetics , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/genetics , Signal Transduction , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
2.
Proc Natl Acad Sci U S A ; 112(2): 512-7, 2015 Jan 13.
Article in English | MEDLINE | ID: mdl-25548173

ABSTRACT

The differentiation of effector CD8(+) T cells is critical for the development of protective responses to pathogens and for effective vaccines. In the first few hours after activation, naive CD8(+) T cells initiate a transcriptional program that leads to the formation of effector and memory T cells, but the regulation of this process is poorly understood. Investigating the role of specific transcription factors (TFs) in determining CD8(+) effector T-cell fate by gene knockdown with RNAi is challenging because naive T cells are refractory to transduction with viral vectors without extensive ex vivo stimulation, which obscures the earliest events in effector differentiation. To overcome this obstacle, we developed a novel strategy to test the function of genes in naive CD8(+) T cells in vivo by creating bone marrow chimera from hematopoietic progenitors transduced with an inducible shRNA construct. Following hematopoietic reconstitution, this approach allowed inducible in vivo gene knockdown in any cell type that developed from this transduced progenitor pool. We demonstrated that lentivirus-transduced progenitor cells could reconstitute normal hematopoiesis and develop into naive CD8(+) T cells that were indistinguishable from wild-type naive T cells. This experimental system enabled induction of efficient gene knockdown in vivo without subsequent manipulation. We applied this strategy to show that the TF BATF is essential for initial commitment of naive CD8(+) T cells to effector development but becomes dispensable by 72h. This approach makes possible the study of gene function in vivo in unperturbed cells of hematopoietic origin that are refractory to viral transduction.


Subject(s)
Basic-Leucine Zipper Transcription Factors/antagonists & inhibitors , Basic-Leucine Zipper Transcription Factors/immunology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , RNA Interference , Animals , Basic-Leucine Zipper Transcription Factors/genetics , CD8-Positive T-Lymphocytes/cytology , Cell Differentiation , Gene Knockdown Techniques , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/metabolism , Lentivirus/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , RNA, Small Interfering/genetics , Transduction, Genetic , Transplantation Chimera
3.
Biol Psychiatry ; 73(7): 683-90, 2013 Apr 01.
Article in English | MEDLINE | ID: mdl-23237312

ABSTRACT

BACKGROUND: Ankyrin 3 (ANK3) has been strongly implicated as a risk gene for bipolar disorder (BD) by recent genome-wide association studies of patient populations. However, the genetic variants of ANK3 contributing to BD risk and their pathological function are unknown. METHODS: To gain insight into the potential disease relevance of ANK3, we examined the function of mouse Ank3 in the regulation of psychiatric-related behaviors using genetic, neurobiological, pharmacological, and gene-environment interaction (G×E) approaches. Ank3 expression was reduced in mouse brain either by viral-mediated RNA interference or through disruption of brain-specific Ank3 in a heterozygous knockout mouse. RESULTS: RNA interference of Ank3 in hippocampus dentate gyrus induced a highly specific and consistent phenotype marked by decreased anxiety-related behaviors and increased activity during the light phase, which were attenuated by chronic treatment with the mood stabilizer lithium. Similar behavioral alterations of reduced anxiety and increased motivation for reward were also exhibited by Ank3+/- heterozygous mice compared with wild-type Ank3+/+ mice. Remarkably, the behavioral traits of Ank3+/- mice transitioned to depression-related features after chronic stress, a trigger of mood episodes in BD. Ank3+/- mice also exhibited elevated serum corticosterone, suggesting that reduced Ank3 expression is associated with elevated stress reactivity. CONCLUSIONS: This study defines a new role for Ank3 in the regulation of psychiatric-related behaviors and stress reactivity that lends support for its involvement in BD and establishes a general framework for determining the disease relevance of genes implicated by patient genome-wide association studies.


Subject(s)
Ankyrins/genetics , Anxiety Disorders/genetics , Anxiety Disorders/physiopathology , Bipolar Disorder/genetics , Lithium Chloride/pharmacology , Stress, Psychological/genetics , Stress, Psychological/physiopathology , Animals , Ankyrins/physiology , Anxiety Disorders/blood , Anxiety Disorders/drug therapy , Corticosterone/blood , Dentate Gyrus/physiology , Male , Mice , Mice, Knockout , Mice, Transgenic , Stress, Psychological/blood , Stress, Psychological/drug therapy
4.
Nat Methods ; 8(8): 659-61, 2011 Jun 26.
Article in English | MEDLINE | ID: mdl-21706014

ABSTRACT

Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss-of-function and gain-of-function experiments. We describe the production of a sequence-confirmed, clonal collection of over 16,100 human open-reading frames (ORFs) encoded in a versatile Gateway vector system. Using this ORFeome resource, we created a genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.


Subject(s)
Cloning, Molecular/methods , Genetic Vectors/genetics , Genomic Library , Lentivirus/genetics , Humans , Open Reading Frames
5.
Nature ; 468(7326): 968-72, 2010 Dec 16.
Article in English | MEDLINE | ID: mdl-21107320

ABSTRACT

Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed ∼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.


Subject(s)
Drug Resistance, Neoplasm , MAP Kinase Kinase Kinases/metabolism , MAP Kinase Signaling System , Mitogen-Activated Protein Kinases/metabolism , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Allosteric Regulation , Cell Line, Tumor , Clinical Trials as Topic , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Enzyme Activation/drug effects , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Library , Humans , Indoles/pharmacology , Indoles/therapeutic use , MAP Kinase Kinase Kinases/genetics , Melanoma/drug therapy , Melanoma/enzymology , Melanoma/genetics , Melanoma/metabolism , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinase Kinases/metabolism , Open Reading Frames/genetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins B-raf/chemistry , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins c-raf/genetics , Proto-Oncogene Proteins c-raf/metabolism , Sulfonamides/pharmacology , Sulfonamides/therapeutic use , Vemurafenib
6.
Nat Methods ; 7(4): 307-9, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20208532

ABSTRACT

We describe a Toxoplasma gondii strain that will permit the use of site-specific recombination to study the host-parasite interactions of this organism. This Toxoplasma strain efficiently injects a Cre fusion protein into host cells. In a Cre-reporter cell line, a single parasite invasion induced Cre-mediated recombination in 95% of infected host cells. By infecting Cre-reporter mice with these parasites, we also monitored host-cell infection in vivo.


Subject(s)
Integrases/metabolism , Toxoplasma/enzymology , Toxoplasmosis/parasitology , Animals , Host-Parasite Interactions , Integrases/genetics , Integrases/immunology , Mice , Mice, Transgenic , Microscopy, Fluorescence , Plasmids/genetics , Recombination, Genetic , Toxoplasma/genetics , Transduction, Genetic
7.
FASEB J ; 23(5): 1431-40, 2009 May.
Article in English | MEDLINE | ID: mdl-19141533

ABSTRACT

An understanding of nuclear reprogramming is fundamental to the use of cells in regenerative medicine. Due to technological obstacles, the time course and extent of reprogramming of cells following fusion has not been assessed to date. Here, we show that hundreds of genes are activated or repressed within hours of fusion of human keratinocytes and mouse muscle cells in heterokaryons, and extensive changes are observed within 4 days. This study was made possible by the development of a broadly applicable approach, species-specific transcriptome amplification (SSTA), which enables global resolution of transcripts derived from the nuclei of two species, even when the proportions of species-specific transcripts are highly skewed. Remarkably, either phenotype can be dominant; an excess of primary keratinocytes leads to activation of the keratinocyte program in muscle cells and the converse is true when muscle cells are in excess. We conclude that nuclear reprogramming in heterokaryons is rapid, extensive, bidirectional, and dictated by the balance of regulators contributed by the cell types.


Subject(s)
Cellular Reprogramming , Gene Expression Profiling/methods , Hybrid Cells/physiology , Animals , Cell Differentiation , Cell Fusion , Humans , Hybrid Cells/metabolism , Keratinocytes/physiology , Mice , Oligonucleotide Array Sequence Analysis
8.
J Cell Biol ; 180(5): 1005-19, 2008 Mar 10.
Article in English | MEDLINE | ID: mdl-18332221

ABSTRACT

Cell-cell fusion is critical to the normal development of certain tissues, yet the nature and degree of conservation of the underlying molecular components remains largely unknown. Here we show that the two guanine-nucleotide exchange factors Brag2 and Dock180 have evolutionarily conserved functions in the fusion of mammalian myoblasts. Their effects on muscle cell formation are distinct and are a result of the activation of the GTPases ARF6 and Rac, respectively. Inhibition of ARF6 activity results in a lack of physical association between paxillin and beta(1)-integrin, and disruption of paxillin transport to sites of focal adhesion. We show that fusion machinery is conserved among distinct cell types because Dock180 deficiency prevented fusion of macrophages and the formation of multinucleated giant cells. Our results are the first to demonstrate a role for a single protein in the fusion of two different cell types, and provide novel mechanistic insight into the function of GEFs in the morphological maturation of multinucleated cells.


Subject(s)
Giant Cells/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Macrophages/metabolism , Myoblasts/metabolism , ADP-Ribosylation Factor 6 , ADP-Ribosylation Factors/genetics , ADP-Ribosylation Factors/metabolism , Animals , Cell Adhesion/physiology , Cell Communication/physiology , Cell Fusion , Cell Line , Focal Adhesions/metabolism , Focal Adhesions/ultrastructure , Giant Cells/ultrastructure , Guanine Nucleotide Exchange Factors/genetics , Integrin beta1/metabolism , Macrophages/ultrastructure , Mice , Myoblasts/ultrastructure , Neuropeptides/genetics , Neuropeptides/metabolism , Paxillin/metabolism , RNA, Small Interfering , rac GTP-Binding Proteins/genetics , rac GTP-Binding Proteins/metabolism , rac1 GTP-Binding Protein
9.
Mol Ther ; 11(4): 591-9, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15771961

ABSTRACT

beta-Globin transgenes regulated by the locus control region (LCR) are dominantly silenced by linked bacterial reporter genes in transgenic mice. Enhanced green fluorescent protein (eGFP) from jellyfish is an alternative reporter used in retrovirus vectors to transfer LCRbeta-globin genes into bone marrow. We show here that the eGFP coding sequence silences LCRbeta-globin in transgenic mice, but the PGK promoter did not provoke such silencing. As eGFP contains 60 CpG dinucleotides, which are targets of DNA methylation, we synthesized a novel CpG-free variant called dmGFP. Its utility was demonstrated in MSCV retrovirus vectors transcriptionally controlled by the viral 5'LTR or internal PGK or EF1alpha promoter. Specific fluorescence was detected from eGFP, and at lower levels from dmGFP, in transduced mouse CFU-S and embryonic stem cells. While eGFP was rarely silenced in CFU-S, dmGFP was not silenced in these progenitors. Moreover, the dmGFP coding sequence did not silence LCRbeta-globin in transgenic mice, showing that the eGFP silencing mechanism acts primarily via CpG dinucleotides. However, LCRbeta-globin expression remained suboptimal, indicating that other silencing pathways recognize dmGFP in the absence of CpG dinucleotides. We conclude that dmGFP ameliorates silencing, but optimal LCRbeta-globin expression is obtained in the absence of nonmammalian reporters.


Subject(s)
CpG Islands/physiology , Gene Silencing , Globins/genetics , Green Fluorescent Proteins/genetics , Locus Control Region/genetics , Animals , Base Sequence , CpG Islands/genetics , Dinucleotide Repeats/genetics , Dinucleotide Repeats/physiology , Genes, Reporter/genetics , Mice , Mice, Transgenic , Molecular Sequence Data , Mutation , Transgenes
10.
J Cell Sci ; 117(Pt 10): 1979-88, 2004 Apr 15.
Article in English | MEDLINE | ID: mdl-15054107

ABSTRACT

The multipotent nature of skeletal muscle-derived side population cells is demonstrated by their myogenic and hematopoietic potential in vivo. However, whether muscle side population cells are derived from the bone marrow is unclear. To study the long-term contribution of the hematopoietic system to muscle side population, whole bone marrow cells from Ly5.1 males or from e-GFP transgenic male mice were transplanted into lethally irradiated Ly5.2 females. Long-term cell trafficking of donor bone marrow cells to muscle side population was monitored 17 times in a 34-week study. Fluorescence-activated cell sorter analyses were used to detect Ly5.1 and GFP(+) donor cells, which were confirmed by fluorescence in situ hybridization of the Y-chromosome. Analyses post-transplantation indicated that whereas cells of donor origin could be found in the muscle, donor bone marrow cells had contributed little to the muscle side population. Attempts to increase cell trafficking by induced muscle damage again confirmed that more than 90% of side population cells present in the muscle were derived from the host. These results demonstrate that muscle side population cells are not replenished by the bone marrow and suggest a non-hematopoietic origin for this cell population.


Subject(s)
Bone Marrow Cells/metabolism , Bone Marrow Transplantation/methods , Muscle, Skeletal/metabolism , Animals , Benzimidazoles/pharmacology , Cell Separation , Cobra Cardiotoxin Proteins/metabolism , Female , Flow Cytometry , Green Fluorescent Proteins/metabolism , Immunohistochemistry , In Situ Hybridization, Fluorescence , Leukocyte Common Antigens/biosynthesis , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Muscles/cytology , Phenotype , Sex Factors , Time Factors , Y Chromosome/ultrastructure
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