61 results on '"Fantl WJ"'
Search Results
2. Multiparameter single-cell proteomic technologies give new insights into the biology of ovarian tumors.
- Author
-
Funingana IG, Bedia JS, Huang YW, Delgado Gonzalez A, Donoso K, Gonzalez VD, Brenton JD, Ashworth A, and Fantl WJ
- Subjects
- Female, Humans, Carboplatin therapeutic use, Tumor Microenvironment, Proteomics, Ovarian Neoplasms diagnosis, Ovarian Neoplasms etiology, Ovarian Neoplasms therapy
- Abstract
High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy. Its diagnosis at advanced stage compounded with its excessive genomic and cellular heterogeneity make curative treatment challenging. Two critical therapeutic challenges to overcome are carboplatin resistance and lack of response to immunotherapy. Carboplatin resistance results from diverse cell autonomous mechanisms which operate in different combinations within and across tumors. The lack of response to immunotherapy is highly likely to be related to an immunosuppressive HGSOC tumor microenvironment which overrides any clinical benefit. Results from a number of studies, mainly using transcriptomics, indicate that the immune tumor microenvironment (iTME) plays a role in carboplatin response. However, in patients receiving treatment, the exact mechanistic details are unclear. During the past decade, multiplex single-cell proteomic technologies have come to the forefront of biomedical research. Mass cytometry or cytometry by time-of-flight, measures up to 60 parameters in single cells that are in suspension. Multiplex cellular imaging technologies allow simultaneous measurement of up to 60 proteins in single cells with spatial resolution and interrogation of cell-cell interactions. This review suggests that functional interplay between cell autonomous responses to carboplatin and the HGSOC immune tumor microenvironment could be clarified through the application of multiplex single-cell proteomic technologies. We conclude that for better clinical care, multiplex single-cell proteomic technologies could be an integral component of multimodal biomarker development that also includes genomics and radiomics. Collection of matched samples from patients before and on treatment will be critical to the success of these efforts., (© 2023. The Author(s).)
- Published
- 2023
- Full Text
- View/download PDF
3. Elevated CD47 is a hallmark of dysfunctional aged muscle stem cells that can be targeted to augment regeneration.
- Author
-
Porpiglia E, Mai T, Kraft P, Holbrook CA, de Morree A, Gonzalez VD, Hilgendorf KI, Frésard L, Trejo A, Bhimaraju S, Jackson PK, Fantl WJ, and Blau HM
- Subjects
- Animals, Mice, Muscle, Skeletal, Aging, Disease Progression, CD47 Antigen, Myoblasts
- Abstract
In aging, skeletal muscle strength and regenerative capacity decline, due in part to functional impairment of muscle stem cells (MuSCs), yet the underlying mechanisms remain elusive. Here, we capitalize on mass cytometry to identify high CD47 expression as a hallmark of dysfunctional MuSCs (CD47
hi ) with impaired regenerative capacity that predominate with aging. The prevalent CD47hi MuSC subset suppresses the residual functional CD47lo MuSC subset through a paracrine signaling loop, leading to impaired proliferation. We uncover that elevated CD47 levels on aged MuSCs result from increased U1 snRNA expression, which disrupts alternative polyadenylation. The deficit in aged MuSC function in regeneration can be overcome either by morpholino-mediated blockade of CD47 alternative polyadenylation or antibody blockade of thrombospondin-1/CD47 signaling, leading to improved regeneration in aged mice, with therapeutic implications. Our findings highlight a previously unrecognized age-dependent alteration in CD47 levels and function in MuSCs, which underlies reduced muscle repair in aging., Competing Interests: Declaration of interests H.M.B. is cofounder of Rejuvenation Technologies Inc. and Epirium Bio. H.M.B. and E.P. are named inventors on patent application no. PCT/US2021/038549 held by Stanford University., (Copyright © 2022 Elsevier Inc. All rights reserved.)- Published
- 2022
- Full Text
- View/download PDF
4. Hybrid Fluorescent Mass-Tag Nanotrackers as Universal Reagents for Long-Term Live-Cell Barcoding.
- Author
-
Delgado-Gonzalez A, Laz-Ruiz JA, Cano-Cortes MV, Huang YW, Gonzalez VD, Diaz-Mochon JJ, Fantl WJ, and Sanchez-Martin RM
- Subjects
- Cell Line, Flow Cytometry methods, Proteomics, Electronic Data Processing, Fluorescent Dyes chemistry
- Abstract
Barcoding and pooling cells for processing as a composite sample are critical to minimize technical variability in multiplex technologies. Fluorescent cell barcoding has been established as a standard method for multiplexing in flow cytometry analysis. In parallel, mass-tag barcoding is routinely used to label cells for mass cytometry. Barcode reagents currently used label intracellular proteins in fixed and permeabilized cells and, therefore, are not suitable for studies with live cells in long-term culture prior to analysis. In this study, we report the development of fluorescent palladium-based hybrid-tag nanotrackers to barcode live cells for flow and mass cytometry dual-modal readout. We describe the preparation, physicochemical characterization, efficiency of cell internalization, and durability of these nanotrackers in live cells cultured over time. In addition, we demonstrate their compatibility with standardized cytometry reagents and protocols. Finally, we validated these nanotrackers for drug response assays during a long-term coculture experiment with two barcoded cell lines. This method represents a new and widely applicable advance for fluorescent and mass-tag barcoding that is independent of protein expression levels and can be used to label cells before long-term drug studies.
- Published
- 2022
- Full Text
- View/download PDF
5. Measuring trogocytosis between ovarian tumor and natural killer cells.
- Author
-
Delgado-Gonzalez A, Huang YW, Porpiglia E, Donoso K, Gonzalez VD, and Fantl WJ
- Subjects
- Cell Membrane metabolism, Female, Humans, Killer Cells, Natural metabolism, Ovarian Neoplasms metabolism, Trogocytosis
- Abstract
Trogocytosis is an active transport mechanism by which one cell extracts a plasma membrane fragment with embedded molecules from an adjacent cell in a contact-dependent process leading to the acquisition of a new function. Our protocol, which has general applicability, consolidates and optimizes existing protocols while highlighting key experimental variables to demonstrate that natural killer (NK) cells acquire the tetraspanin CD9 by trogocytosis from ovarian tumor cells. For complete details on the use and execution of this protocol, please refer to Gonzalez et al. (2021)., Competing Interests: The authors declare no competing interests. We have a published patent related to this study: WO2021/050200, PCT/US2020/046195., (© 2022 The Author(s).)
- Published
- 2022
- Full Text
- View/download PDF
6. CellSeg: a robust, pre-trained nucleus segmentation and pixel quantification software for highly multiplexed fluorescence images.
- Author
-
Lee MY, Bedia JS, Bhate SS, Barlow GL, Phillips D, Fantl WJ, Nolan GP, and Schürch CM
- Subjects
- Algorithms, Fluorescence, Software, Image Processing, Computer-Assisted, Neural Networks, Computer
- Abstract
Background: Algorithmic cellular segmentation is an essential step for the quantitative analysis of highly multiplexed tissue images. Current segmentation pipelines often require manual dataset annotation and additional training, significant parameter tuning, or a sophisticated understanding of programming to adapt the software to the researcher's need. Here, we present CellSeg, an open-source, pre-trained nucleus segmentation and signal quantification software based on the Mask region-convolutional neural network (R-CNN) architecture. CellSeg is accessible to users with a wide range of programming skills., Results: CellSeg performs at the level of top segmentation algorithms in the 2018 Kaggle Data Challenge both qualitatively and quantitatively and generalizes well to a diverse set of multiplexed imaged cancer tissues compared to established state-of-the-art segmentation algorithms. Automated segmentation post-processing steps in the CellSeg pipeline improve the resolution of immune cell populations for downstream single-cell analysis. Finally, an application of CellSeg to a highly multiplexed colorectal cancer dataset acquired on the CO-Detection by indEXing (CODEX) platform demonstrates that CellSeg can be integrated into a multiplexed tissue imaging pipeline and lead to accurate identification of validated cell populations., Conclusion: CellSeg is a robust cell segmentation software for analyzing highly multiplexed tissue images, accessible to biology researchers of any programming skill level., (© 2022. The Author(s).)
- Published
- 2022
- Full Text
- View/download PDF
7. Correction to: Mass Cytometry for the Characterization of Individual Cell Types in Ovarian Solid Tumors.
- Author
-
Gonzalez VD, Huang YW, and Fantl WJ
- Published
- 2022
- Full Text
- View/download PDF
8. Mass Cytometry for the Characterization of Individual Cell Types in Ovarian Solid Tumors.
- Author
-
Gonzalez VD, Huang YW, and Fantl WJ
- Subjects
- Antibodies, Female, Flow Cytometry, Humans, Isotopes, Mass Spectrometry, Single-Cell Analysis, Ovarian Neoplasms
- Abstract
Mass cytometry aka Cytometry by Time-Of-Flight (CyTOF) is one of several recently developed multiparametric single-cell technologies designed to address cellular heterogeneity within healthy and diseased tissue. Mass cytometry is an adaptation of flow cytometry in which antibodies are labeled with stable heavy metal isotopes and the readout is by time-of-flight mass spectrometry. With minimal spillover between channels, mass cytometry enables readouts of up to 60 parameters per single cell. Critically, mass cytometry can identify minority cell populations that are lost in bulk tissue analysis. Mass cytometry has been used to great effect for the study of immune cells. We have extended its use to examine single cells within disaggregated solid tissues, specifically freshly resected tubo-ovarian high-grade serous tumors. Here we detail our protocols designed to ensure the production of high-quality single-cell datasets. The methodology can be modified to accommodate the study of other solid tissues., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2022
- Full Text
- View/download PDF
9. High-grade serous ovarian tumor cells modulate NK cell function to create an immune-tolerant microenvironment.
- Author
-
Gonzalez VD, Huang YW, Delgado-Gonzalez A, Chen SY, Donoso K, Sachs K, Gentles AJ, Allard GM, Kolahi KS, Howitt BE, Porpiglia E, and Fantl WJ
- Subjects
- Antineoplastic Agents pharmacology, Carboplatin pharmacology, Cell Line, Tumor, Coculture Techniques, Cytokines metabolism, Cytotoxicity, Immunologic, Female, Humans, Killer Cells, Natural drug effects, Killer Cells, Natural metabolism, Lymphocytes, Tumor-Infiltrating drug effects, Lymphocytes, Tumor-Infiltrating metabolism, Neoplasms, Cystic, Mucinous, and Serous drug therapy, Neoplasms, Cystic, Mucinous, and Serous metabolism, Neoplasms, Cystic, Mucinous, and Serous pathology, Ovarian Neoplasms drug therapy, Ovarian Neoplasms metabolism, Ovarian Neoplasms pathology, Phenotype, Receptors, Natural Killer Cell metabolism, Tetraspanin 29 metabolism, Trogocytosis, Immune Tolerance drug effects, Killer Cells, Natural immunology, Lymphocytes, Tumor-Infiltrating immunology, Neoplasms, Cystic, Mucinous, and Serous immunology, Ovarian Neoplasms immunology, Tumor Escape drug effects, Tumor Microenvironment immunology
- Abstract
Tubo-ovarian high-grade serous carcinoma (HGSC) is unresponsive to immune checkpoint blockade despite significant frequencies of exhausted T cells. Here we apply mass cytometry and uncover decidual-like natural killer (dl-NK) cell subpopulations (CD56+CD9+CXCR3+KIR+CD3-CD16-) in newly diagnosed HGSC samples that correlate with both tumor and transitioning epithelial-mesenchymal cell abundance. We show different combinatorial expression patterns of ligands for activating and inhibitory NK receptors within three HGSC tumor compartments: epithelial (E), transitioning epithelial-mesenchymal (EV), and mesenchymal (vimentin expressing [V]), with a more inhibitory ligand phenotype in V cells. In cocultures, NK-92 natural killer cells acquire CD9 from HGSC tumor cells by trogocytosis, resulting in reduced anti-tumor cytokine production and cytotoxicity. Cytotoxicity in these cocultures is restored with a CD9-blocking antibody or CD9 CRISPR knockout, thereby identifying mechanisms of immune suppression in HGSC. CD9 is widely expressed in HGSC tumors and so represents an important new therapeutic target with immediate relevance for NK immunotherapy., Competing Interests: Declaration of interests The authors declare no competing interests. We have a published patent related to this study: WO2021/050200, PCT/US2020/046195., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
10. Profiling myelodysplastic syndromes by mass cytometry demonstrates abnormal progenitor cell phenotype and differentiation.
- Author
-
Behbehani GK, Finck R, Samusik N, Sridhar K, Fantl WJ, Greenberg PL, and Nolan GP
- Subjects
- Biopsy, Needle, Bone Marrow pathology, Bone Marrow Cells immunology, Bone Marrow Cells pathology, Bone Marrow Cells physiology, Cell Differentiation, Humans, Immunophenotyping methods, Myelodysplastic Syndromes pathology, Myelodysplastic Syndromes physiopathology, Phenotype, Pilot Projects, Flow Cytometry methods, Myelodysplastic Syndromes diagnosis, Stem Cells pathology, Stem Cells physiology
- Abstract
Background: We sought to enhance the cytometric analysis of myelodysplastic syndromes (MDS) by performing a pilot study of a single cell mass cytometry (MCM) assay to more comprehensively analyze patterns of surface marker expression in patients with MDS., Methods: Twenty-three MDS and five healthy donor bone marrow samples were studied using a 34-parameter mass cytometry panel utilizing barcoding and internal reference standards. The resulting data were analyzed by both traditional gating and high-dimensional clustering., Results: This high-dimensional assay provided three major benefits relative to traditional cytometry approaches: First, MCM enabled detection of aberrant surface maker at high resolution, detecting aberrancies in 27/31 surface markers, encompassing almost every previously reported MDS surface marker aberrancy. Additionally, three previously unrecognized aberrancies in MDS were detected in multiple samples at least one developmental stage: increased CD321 and CD99; and decreased CD47. Second, analysis of the stem and progenitor cell compartment (HSPCs), demonstrated aberrant expression in 21 of the 23 MDS samples, which were not detected in three samples from patients with idiopathic cytopenia of undetermined significance. These immunophenotypically abnormal HSPCs were also the single most significant distinguishing feature between clinical risk groups. Third, unsupervised clustering of high-parameter MCM data allowed identification of abnormal differentiation patterns associated with immunophenotypically aberrant myeloid cells similar to myeloid derived suppressor cells., Conclusions: These results demonstrate that high-parameter cytometry methods that enable simultaneous analysis of all bone marrow cell types could enhance the diagnostic utility of immunophenotypic analysis in MDS., (© 2020 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.)
- Published
- 2020
- Full Text
- View/download PDF
11. GateFinder: projection-based gating strategy optimization for flow and mass cytometry.
- Author
-
Aghaeepour N, Simonds EF, Knapp DJHF, Bruggner RV, Sachs K, Culos A, Gherardini PF, Samusik N, Fragiadakis GK, Bendall SC, Gaudilliere B, Angst MS, Eaves CJ, Weiss WA, Fantl WJ, and Nolan GP
- Subjects
- Algorithms, Biomarkers analysis, Flow Cytometry, Software
- Abstract
Motivation: High-parameter single-cell technologies can reveal novel cell populations of interest, but studying or validating these populations using lower-parameter methods remains challenging., Results: Here, we present GateFinder, an algorithm that enriches high-dimensional cell types with simple, stepwise polygon gates requiring only two markers at a time. A series of case studies of complex cell types illustrates how simplified enrichment strategies can enable more efficient assays, reveal novel biomarkers and clarify underlying biology., Availability and Implementation: The GateFinder algorithm is implemented as a free and open-source package for BioConductor: https://nalab.stanford.edu/gatefinder., Supplementary Information: Supplementary data are available at Bioinformatics online.
- Published
- 2018
- Full Text
- View/download PDF
12. Metal-isotope-tagged monoclonal antibodies for high-dimensional mass cytometry.
- Author
-
Han G, Spitzer MH, Bendall SC, Fantl WJ, and Nolan GP
- Subjects
- Animals, Antigens, CD analysis, Bismuth chemistry, Chelating Agents chemistry, Humans, Immunoassay methods, Immunoglobulin G chemistry, Indium chemistry, Isotopes chemistry, Jurkat Cells, Mice, Inbred C57BL, Palladium chemistry, Yttrium chemistry, Antibodies, Monoclonal chemistry, Flow Cytometry methods, Heterocyclic Compounds, 1-Ring chemistry, Immunoconjugates chemistry, Metals chemistry
- Abstract
Advances in single-cell mass cytometry have increasingly improved highly multidimensional characterization of immune cell heterogeneity. The immunoassay multiplexing capacity relies on monoclonal antibodies labeled with stable heavy-metal isotopes. To date, a variety of rare-earth elements and noble and post-transition metal isotopes have been used in mass cytometry; nevertheless, the methods used for antibody conjugation differ because of the individual metal coordination chemistries and distinct stabilities of various metal cations. Herein, we provide three optimized protocols for conjugating monoclonal IgG antibodies with 48 high-purity heavy-metal isotopes: (i) 38 isotopes of lanthanides, 2 isotopes of indium, and 1 isotope of yttrium; (ii) 6 isotopes of palladium; and (iii) 1 isotope of bismuth. Bifunctional chelating agents containing coordinative ligands of monomeric DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or polymeric pentetic acid (DTPA) were used to stably sequester isotopic cations in aqueous solutions and were subsequently coupled to IgG antibodies using site-specific biorthogonal reactions. Furthermore, quantification methods based on antibody inherent absorption at 280 nm and on extrinsic absorption at 562 nm after staining with bicinchoninic acid (BCA) are reported to determine metal-isotope-tagged antibodies. In addition, a freeze-drying procedure to prepare palladium isotopic mass tags is described. To demonstrate the utility, experiments using six palladium-tagged CD45 antibodies for barcoding assays of live immune cells in cytometry by time-of-flight (CyTOF) are described. Conjugation of pure isotopes of lanthanides, indium, or yttrium takes ~3.5 h. Conjugation of bismuth takes ~4 h. Preparation of palladium mass tags takes ~8 h. Conjugation of pure isotopes of palladium takes ~2.5 h. Antibody titration takes ~4 h.
- Published
- 2018
- Full Text
- View/download PDF
13. Publisher Correction: High-resolution myogenic lineage mapping by single-cell mass cytometry.
- Author
-
Porpiglia E, Samusik N, Ho ATV, Cosgrove BD, Mai T, Davis KL, Jager A, Nolan GP, Bendall SC, Fantl WJ, and Blau HM
- Abstract
In the version of this Article originally published, the name of author Andrew Tri Van Ho was coded wrongly, resulting in it being incorrect when exported to citation databases. This has been corrected, though no visible changes will be apparent.
- Published
- 2018
- Full Text
- View/download PDF
14. Single-cell developmental classification of B cell precursor acute lymphoblastic leukemia at diagnosis reveals predictors of relapse.
- Author
-
Good Z, Sarno J, Jager A, Samusik N, Aghaeepour N, Simonds EF, White L, Lacayo NJ, Fantl WJ, Fazio G, Gaipa G, Biondi A, Tibshirani R, Bendall SC, Nolan GP, and Davis KL
- Subjects
- Adult, B-Lymphocytes pathology, Female, Humans, Male, Middle Aged, Phenotype, Precursor B-Cell Lymphoblastic Leukemia-Lymphoma pathology, Recurrence, Risk Assessment, Signal Transduction, Survival Analysis, TOR Serine-Threonine Kinases metabolism, Young Adult, Precursor B-Cell Lymphoblastic Leukemia-Lymphoma classification, Precursor B-Cell Lymphoblastic Leukemia-Lymphoma diagnosis, Single-Cell Analysis
- Abstract
Insight into the cancer cell populations that are responsible for relapsed disease is needed to improve outcomes. Here we report a single-cell-based study of B cell precursor acute lymphoblastic leukemia at diagnosis that reveals hidden developmentally dependent cell signaling states that are uniquely associated with relapse. By using mass cytometry we simultaneously quantified 35 proteins involved in B cell development in 60 primary diagnostic samples. Each leukemia cell was then matched to its nearest healthy B cell population by a developmental classifier that operated at the single-cell level. Machine learning identified six features of expanded leukemic populations that were sufficient to predict patient relapse at diagnosis. These features implicated the pro-BII subpopulation of B cells with activated mTOR signaling, and the pre-BI subpopulation of B cells with activated and unresponsive pre-B cell receptor signaling, to be associated with relapse. This model, termed 'developmentally dependent predictor of relapse' (DDPR), significantly improves currently established risk stratification methods. DDPR features exist at diagnosis and persist at relapse. By leveraging a data-driven approach, we demonstrate the predictive value of single-cell 'omics' for patient stratification in a translational setting and provide a framework for its application to human cancer.
- Published
- 2018
- Full Text
- View/download PDF
15. Single-cell mass cytometry reveals distinct populations of brain myeloid cells in mouse neuroinflammation and neurodegeneration models.
- Author
-
Ajami B, Samusik N, Wieghofer P, Ho PP, Crotti A, Bjornson Z, Prinz M, Fantl WJ, Nolan GP, and Steinman L
- Subjects
- Animals, CREB-Binding Protein metabolism, CX3C Chemokine Receptor 1 genetics, CX3C Chemokine Receptor 1 metabolism, Disease Models, Animal, Huntingtin Protein genetics, Huntington Disease genetics, Luminescent Proteins genetics, Luminescent Proteins metabolism, Mice, Mice, Inbred C57BL, Mice, Mutant Strains, Mice, Transgenic, Monocytes, Mutation genetics, Myeloid Cells metabolism, Single-Cell Analysis methods, Superoxide Dismutase-1 genetics, Amyotrophic Lateral Sclerosis pathology, Brain pathology, Cytokines metabolism, Encephalomyelitis, Autoimmune, Experimental pathology, Huntington Disease pathology, Intracellular Signaling Peptides and Proteins metabolism, Myeloid Cells pathology
- Abstract
Neuroinflammation and neurodegeneration may represent two poles of brain pathology. Brain myeloid cells, particularly microglia, play key roles in these conditions. We employed single-cell mass cytometry (CyTOF) to compare myeloid cell populations in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, the R6/2 model of Huntington's disease (HD) and the mutant superoxide dismutase 1 (mSOD1) model of amyotrophic lateral sclerosis (ALS). We identified three myeloid cell populations exclusive to the CNS and present in each disease model. Blood-derived monocytes comprised five populations and migrated to the brain in EAE, but not in HD and ALS models. Single-cell analysis resolved differences in signaling and cytokine production within similar myeloid populations in EAE compared to HD and ALS models. Moreover, these analyses highlighted α5 integrin on myeloid cells as a potential therapeutic target for neuroinflammation. Together, these findings illustrate how neuropathology may differ between inflammatory and degenerative brain disease.
- Published
- 2018
- Full Text
- View/download PDF
16. Commonly Occurring Cell Subsets in High-Grade Serous Ovarian Tumors Identified by Single-Cell Mass Cytometry.
- Author
-
Gonzalez VD, Samusik N, Chen TJ, Savig ES, Aghaeepour N, Quigley DA, Huang YW, Giangarrà V, Borowsky AD, Hubbard NE, Chen SY, Han G, Ashworth A, Kipps TJ, Berek JS, Nolan GP, and Fantl WJ
- Subjects
- Aged, Aged, 80 and over, Antibodies, Neoplasm metabolism, Carboplatin pharmacology, Cell Line, Tumor, Cluster Analysis, Cystadenocarcinoma, Serous metabolism, Cystadenocarcinoma, Serous pathology, Drug Resistance, Neoplasm drug effects, Female, Humans, Middle Aged, Neoplasm Proteins metabolism, Neoplasm Recurrence, Local pathology, Ovarian Neoplasms metabolism, Ovarian Neoplasms pathology, Phenotype, Prognosis, Flow Cytometry methods
- Abstract
We have performed an in-depth single-cell phenotypic characterization of high-grade serous ovarian cancer (HGSOC) by multiparametric mass cytometry (CyTOF). Using a CyTOF antibody panel to interrogate features of HGSOC biology, combined with unsupervised computational analysis, we identified noteworthy cell types co-occurring across the tumors. In addition to a dominant cell subset, each tumor harbored rarer cell phenotypes. One such group co-expressed E-cadherin and vimentin (EV), suggesting their potential role in epithelial mesenchymal transition, which was substantiated by pairwise correlation analyses. Furthermore, tumors from patients with poorer outcome had an increased frequency of another rare cell type that co-expressed vimentin, HE4, and cMyc. These poorer-outcome tumors also populated more cell phenotypes, as quantified by Simpson's diversity index. Thus, despite the recognized genomic complexity of the disease, the specific cell phenotypes uncovered here offer a focus for therapeutic intervention and disease monitoring., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
17. Atomic mass tag of bismuth-209 for increasing the immunoassay multiplexing capacity of mass cytometry.
- Author
-
Han G, Chen SY, Gonzalez VD, Zunder ER, Fantl WJ, and Nolan GP
- Subjects
- Antibodies, Monoclonal chemistry, Humans, Immunoassay, Immunoconjugates chemistry, Bismuth chemistry, Flow Cytometry methods, Killer Cells, Natural, Mass Spectrometry methods, Single-Cell Analysis methods
- Abstract
Mass cytometry (or CyTOF) is an atomic mass spectrometry-based single-cell immunoassay technology, which has provided an increasingly systematic and sophisticated view in basic biological and clinical studies. Using elemental reporters composed of stable heavy metal isotopes, more than 50 cellular parameters are measured simultaneously. However, this current multiplexing does not meet the theoretical capability of CyTOF instrumentation with 135 detectable channels, primarily due to the limitation of available chemistries for conjugating elemental mass tags to affinity reagents. To address this issue, we develop herein additional metallic mass tag based on bismuth-209 (
209 Bi) for efficient conjugation to monoclonal antibody. This enables the use of an addtional channel m/z = 209 of CyTOF for single-cell immunoassays. Bismuth has nearly the same charge-to-radius ratio as lanthanide elements; thus, bismuth(III) cations (209 Bi3+ ) could coordinate with DTPA chelators in the same geometry of O- and N-donor groups as that of lanthanide. In this report, the coordination chemistry of209 Bi3+ with DTPA chelators and Maxpar® X8 polymers were investigated in details. Accordingly, the protocols of conjugating antibody with bismuth mass tag were provided. A method based on UV-Vis absorbance at 280 nm of209 Bi3+ -labeling DTPA complexes was developed to evaluate the stoichiometric ratio of209 Bi3+ cations to the conjugated antibody. Side-by-side single-cell analysis experiments with bismuth- and lanthanide-tagged antibodies were carried out to compare the analytical sensitivities. The measurement accuracy of bismuth-tagged antibody was validated within in vitro assay using primary human natural killer cells. Furthermore, bismuth-tagged antibodies were successfully employed in cell cycle measurements and high-dimensional phenotyping immunoassays. © 2017 International Society for Advancement of Cytometry., (© 2017 International Society for Advancement of Cytometry.)- Published
- 2017
- Full Text
- View/download PDF
18. Upregulation of Human Endogenous Retrovirus-K Is Linked to Immunity and Inflammation in Pulmonary Arterial Hypertension.
- Author
-
Saito T, Miyagawa K, Chen SY, Tamosiuniene R, Wang L, Sharpe O, Samayoa E, Harada D, Moonen JAJ, Cao A, Chen PI, Hennigs JK, Gu M, Li CG, Leib RD, Li D, Adams CM, Del Rosario PA, Bill M, Haddad F, Montoya JG, Robinson WH, Fantl WJ, Nolan GP, Zamanian RT, Nicolls MR, Chiu CY, Ariza ME, and Rabinovitch M
- Subjects
- Adolescent, Adult, Animals, Antigen-Antibody Complex biosynthesis, Antigen-Antibody Complex immunology, Cells, Cultured, Child, Coculture Techniques, Female, Humans, Hypertension, Pulmonary metabolism, Hypertension, Pulmonary pathology, Infant, Inflammation immunology, Inflammation metabolism, Inflammation pathology, Inflammation Mediators metabolism, Leukocytes, Mononuclear immunology, Leukocytes, Mononuclear metabolism, Leukocytes, Mononuclear pathology, Male, Middle Aged, Rats, Rats, Sprague-Dawley, SAM Domain and HD Domain-Containing Protein 1 biosynthesis, SAM Domain and HD Domain-Containing Protein 1 immunology, Young Adult, Hypertension, Pulmonary immunology, Inflammation Mediators immunology, Up-Regulation physiology, Viral Proteins biosynthesis, Viral Proteins immunology
- Abstract
Background: Immune dysregulation has been linked to occlusive vascular remodeling in pulmonary arterial hypertension (PAH) that is hereditary, idiopathic, or associated with other conditions. Circulating autoantibodies, lung perivascular lymphoid tissue, and elevated cytokines have been related to PAH pathogenesis but without a clear understanding of how these abnormalities are initiated, perpetuated, and connected in the progression of disease. We therefore set out to identify specific target antigens in PAH lung immune complexes as a starting point toward resolving these issues to better inform future application of immunomodulatory therapies., Methods: Lung immune complexes were isolated and PAH target antigens were identified by liquid chromatography tandem mass spectrometry, confirmed by enzyme-linked immunosorbent assay, and localized by confocal microscopy. One PAH antigen linked to immunity and inflammation was pursued and a link to PAH pathophysiology was investigated by next-generation sequencing, functional studies in cultured monocytes and endothelial cells, and hemodynamic and lung studies in a rat., Results: SAM domain and HD domain-containing protein 1 (SAMHD1), an innate immune factor that suppresses HIV replication, was identified and confirmed as highly expressed in immune complexes from 16 hereditary and idiopathic PAH versus 12 control lungs. Elevated SAMHD1 was localized to endothelial cells, perivascular dendritic cells, and macrophages, and SAMHD1 antibodies were prevalent in tertiary lymphoid tissue. An unbiased screen using metagenomic sequencing related SAMHD1 to increased expression of human endogenous retrovirus K (HERV-K) in PAH versus control lungs (n=4). HERV-K envelope and deoxyuridine triphosphate nucleotidohydrolase mRNAs were elevated in PAH versus control lungs (n=10), and proteins were localized to macrophages. HERV-K deoxyuridine triphosphate nucleotidohydrolase induced SAMHD1 and proinflammatory cytokines (eg, interleukin 6, interleukin 1β, and tumor necrosis factor α) in circulating monocytes, pulmonary arterial endothelial cells, and also activated B cells. Vulnerability of pulmonary arterial endothelial cells (PAEC) to apoptosis was increased by HERV-K deoxyuridine triphosphate nucleotidohydrolase in an interleukin 6-independent manner. Furthermore, 3 weekly injections of HERV-K deoxyuridine triphosphate nucleotidohydrolase induced hemodynamic and vascular changes of pulmonary hypertension in rats (n=8) and elevated interleukin 6., Conclusions: Our study reveals that upregulation of the endogenous retrovirus HERV-K could both initiate and sustain activation of the immune system and cause vascular changes associated with PAH., (© 2017 American Heart Association, Inc.)
- Published
- 2017
- Full Text
- View/download PDF
19. High-resolution myogenic lineage mapping by single-cell mass cytometry.
- Author
-
Porpiglia E, Samusik N, Ho ATV, Cosgrove BD, Mai T, Davis KL, Jager A, Nolan GP, Bendall SC, Fantl WJ, and Blau HM
- Subjects
- Animals, Biomarkers metabolism, Cell Proliferation, Cells, Cultured, Elapid Venoms toxicity, Fusion Regulatory Protein-1 metabolism, Genes, Reporter, Genotype, High-Throughput Screening Assays, Hyaluronan Receptors metabolism, Integrin beta4 metabolism, Luminescent Proteins genetics, Luminescent Proteins metabolism, Mice, Inbred C57BL, Mice, Transgenic, Muscle, Skeletal drug effects, Muscle, Skeletal injuries, Muscle, Skeletal pathology, MyoD Protein metabolism, Myoblasts, Skeletal drug effects, Myoblasts, Skeletal pathology, PAX7 Transcription Factor deficiency, PAX7 Transcription Factor genetics, Phenotype, Stem Cells drug effects, Stem Cells pathology, Tetraspanin 29 metabolism, Time Factors, Cell Lineage, Cell Separation methods, Flow Cytometry methods, Muscle Development drug effects, Muscle, Skeletal metabolism, Myoblasts, Skeletal metabolism, Regeneration drug effects, Single-Cell Analysis methods, Stem Cells metabolism
- Abstract
Muscle regeneration is a dynamic process during which cell state and identity change over time. A major roadblock has been a lack of tools to resolve a myogenic progression in vivo. Here we capitalize on a transformative technology, single-cell mass cytometry (CyTOF), to identify in vivo skeletal muscle stem cell and previously unrecognized progenitor populations that precede differentiation. We discovered two cell surface markers, CD9 and CD104, whose combined expression enabled in vivo identification and prospective isolation of stem and progenitor cells. Data analysis using the X-shift algorithm paired with single-cell force-directed layout visualization defined a molecular signature of the activated stem cell state (CD44
+ /CD98+ /MyoD+ ) and delineated a myogenic trajectory during recovery from acute muscle injury. Our studies uncover the dynamics of skeletal muscle regeneration in vivo and pave the way for the elucidation of the regulatory networks that underlie cell-state transitions in muscle diseases and ageing.- Published
- 2017
- Full Text
- View/download PDF
20. High-throughput precision measurement of subcellular localization in single cells.
- Author
-
Burns TJ, Frei AP, Gherardini PF, Bava FA, Batchelder JE, Yoshiyasu Y, Yu JM, Groziak AR, Kimmey SC, Gonzalez VD, Fantl WJ, and Nolan GP
- Subjects
- Cell Nucleus metabolism, Cytoplasm metabolism, Cytoplasm ultrastructure, DNA Damage genetics, Humans, Protein Transport genetics, Subcellular Fractions ultrastructure, Flow Cytometry methods, High-Throughput Screening Assays methods, Single-Cell Analysis methods
- Abstract
To quantify visual and spatial information in single cells with a throughput of thousands of cells per second, we developed Subcellular Localization Assay (SLA). This adaptation of Proximity Ligation Assay expands the capabilities of flow cytometry to include data relating to localization of proteins to and within organelles. We used SLA to detect the nuclear import of transcription factors across cell subsets in complex samples. We further measured intranuclear re-localization of target proteins across the cell cycle and upon DNA damage induction. SLA combines multiple single-cell methods to bring about a new dimension of inquiry and analysis in complex cell populations. © 2017 International Society for Advancement of Cytometry., (© 2017 International Society for Advancement of Cytometry.)
- Published
- 2017
- Full Text
- View/download PDF
21. Single-cell systems-level analysis of human Toll-like receptor activation defines a chemokine signature in patients with systemic lupus erythematosus.
- Author
-
O'Gorman WE, Hsieh EW, Savig ES, Gherardini PF, Hernandez JD, Hansmann L, Balboni IM, Utz PJ, Bendall SC, Fantl WJ, Lewis DB, Nolan GP, and Davis MM
- Subjects
- Cells, Cultured, Chemokines genetics, Chemokines metabolism, Humans, Lipopolysaccharide Receptors metabolism, Lupus Erythematosus, Systemic genetics, Lymphocyte Activation, NF-kappa B metabolism, Organ Specificity, Signal Transduction, Single-Cell Analysis methods, Transcriptome, Killer Cells, Natural immunology, Lupus Erythematosus, Systemic immunology, Monocytes immunology, T-Lymphocytes immunology, Toll-Like Receptors metabolism
- Abstract
Background: Activation of Toll-like receptors (TLRs) induces inflammatory responses involved in immunity to pathogens and autoimmune pathogenesis, such as in patients with systemic lupus erythematosus (SLE). Although TLRs are differentially expressed across the immune system, a comprehensive analysis of how multiple immune cell subsets respond in a system-wide manner has not been described., Objective: We sought to characterize TLR activation across multiple immune cell subsets and subjects, with the goal of establishing a reference framework against which to compare pathologic processes., Methods: Peripheral whole-blood samples were stimulated with TLR ligands and analyzed by means of mass cytometry simultaneously for surface marker expression, activation states of intracellular signaling proteins, and cytokine production. We developed a novel data visualization tool to provide an integrated view of TLR signaling networks with single-cell resolution. We studied 17 healthy volunteer donors and 8 patients with newly diagnosed and untreated SLE., Results: Our data revealed the diversity of TLR-induced responses within cell types, with TLR ligand specificity. Subsets of natural killer cells and T cells selectively induced nuclear factor κ light chain enhancer of activated B cells in response to TLR2 ligands. CD14(hi) monocytes exhibited the most polyfunctional cytokine expression patterns, with more than 80 distinct cytokine combinations. Monocytic TLR-induced cytokine patterns were shared among a group of healthy donors, with minimal intraindividual and interindividual variability. Furthermore, autoimmune disease altered baseline cytokine production; newly diagnosed untreated SLE patients shared a distinct monocytic chemokine signature, despite clinical heterogeneity., Conclusion: Mass cytometry defined a systems-level reference framework for human TLR activation, which can be applied to study perturbations in patients with inflammatory diseases, such as SLE., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
22. Mass Cytometric Functional Profiling of Acute Myeloid Leukemia Defines Cell-Cycle and Immunophenotypic Properties That Correlate with Known Responses to Therapy.
- Author
-
Behbehani GK, Samusik N, Bjornson ZB, Fantl WJ, Medeiros BC, and Nolan GP
- Subjects
- Antineoplastic Combined Chemotherapy Protocols therapeutic use, Biomarkers, Bone Marrow Cells metabolism, Bone Marrow Examination, Cluster Analysis, Flow Cytometry, Genotype, Humans, Immunophenotyping, Karyotype, Leukemia, Myeloid, Acute diagnosis, Leukemia, Myeloid, Acute drug therapy, Leukemia, Myeloid, Acute genetics, Neoplastic Stem Cells metabolism, Neoplastic Stem Cells pathology, Reproducibility of Results, Treatment Outcome, Cell Cycle genetics, Leukemia, Myeloid, Acute metabolism, Phenotype
- Abstract
Unlabelled: Acute myeloid leukemia (AML) is characterized by a high relapse rate that has been attributed to the quiescence of leukemia stem cells (LSC), which renders them resistant to chemotherapy. However, this hypothesis is largely supported by indirect evidence and fails to explain the large differences in relapse rates across AML subtypes. To address this, bone marrow aspirates from 41 AML patients and five healthy donors were analyzed by high-dimensional mass cytometry. All patients displayed immunophenotypic and intracellular signaling abnormalities within CD34(+)CD38(lo) populations, and several karyotype- and genotype-specific surface marker patterns were identified. The immunophenotypic stem and early progenitor cell populations from patients with clinically favorable core-binding factor AML demonstrated a 5-fold higher fraction of cells in S-phase compared with other AML samples. Conversely, LSCs in less clinically favorable FLT3-ITD AML exhibited dramatic reductions in S-phase fraction. Mass cytometry also allowed direct observation of the in vivo effects of cytotoxic chemotherapy., Significance: The mechanisms underlying differences in relapse rates across AML subtypes are poorly understood. This study suggests that known chemotherapy sensitivities of common AML subsets are mediated by cell-cycle differences among LSCs and provides a basis for using in vivo functional characterization of AML cells to inform therapy selection., (©2015 American Association for Cancer Research.)
- Published
- 2015
- Full Text
- View/download PDF
23. IMMUNOLOGY. An interactive reference framework for modeling a dynamic immune system.
- Author
-
Spitzer MH, Gherardini PF, Fragiadakis GK, Bhattacharya N, Yuan RT, Hotson AN, Finck R, Carmi Y, Zunder ER, Fantl WJ, Bendall SC, Engleman EG, and Nolan GP
- Subjects
- Animals, Bone Marrow immunology, Circadian Rhythm immunology, Flow Cytometry, Genetic Variation, Humans, Mice, Mice, Inbred C57BL, Models, Biological, Phenotype, Reference Standards, Immune System cytology, Immune System immunology
- Abstract
Immune cells function in an interacting hierarchy that coordinates the activities of various cell types according to genetic and environmental contexts. We developed graphical approaches to construct an extensible immune reference map from mass cytometry data of cells from different organs, incorporating landmark cell populations as flags on the map to compare cells from distinct samples. The maps recapitulated canonical cellular phenotypes and revealed reproducible, tissue-specific deviations. The approach revealed influences of genetic variation and circadian rhythms on immune system structure, enabled direct comparisons of murine and human blood cell phenotypes, and even enabled archival fluorescence-based flow cytometry data to be mapped onto the reference framework. This foundational reference map provides a working definition of systemic immune organization to which new data can be integrated to reveal deviations driven by genetics, environment, or pathology., (Copyright © 2015, American Association for the Advancement of Science.)
- Published
- 2015
- Full Text
- View/download PDF
24. Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm.
- Author
-
Zunder ER, Finck R, Behbehani GK, Amir el-AD, Krishnaswamy S, Gonzalez VD, Lorang CG, Bjornson Z, Spitzer MH, Bodenmiller B, Fantl WJ, Pe'er D, and Nolan GP
- Subjects
- Software, Staining and Labeling methods, Algorithms, Flow Cytometry methods, Palladium, Single-Cell Analysis methods
- Abstract
Mass-tag cell barcoding (MCB) labels individual cell samples with unique combinatorial barcodes, after which they are pooled for processing and measurement as a single multiplexed sample. The MCB method eliminates variability between samples in antibody staining and instrument sensitivity, reduces antibody consumption and shortens instrument measurement time. Here we present an optimized MCB protocol. The use of palladium-based labeling reagents expands the number of measurement channels available for mass cytometry and reduces interference with lanthanide-based antibody measurement. An error-detecting combinatorial barcoding scheme allows cell doublets to be identified and removed from the analysis. A debarcoding algorithm that is single cell-based rather than population-based improves the accuracy and efficiency of sample deconvolution. This debarcoding algorithm has been packaged into software that allows rapid and unbiased sample deconvolution. The MCB procedure takes 3-4 h, not including sample acquisition time of ∼1 h per million cells.
- Published
- 2015
- Full Text
- View/download PDF
25. Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining.
- Author
-
Behbehani GK, Thom C, Zunder ER, Finck R, Gaudilliere B, Fragiadakis GK, Fantl WJ, and Nolan GP
- Subjects
- Humans, Jurkat Cells, U937 Cells, Cytophotometry methods, High-Throughput Screening Assays methods, Optical Imaging methods, Saponins
- Abstract
Fluorescent cellular barcoding and mass-tag cellular barcoding are cytometric methods that enable high sample throughput, minimize inter-sample variation, and reduce reagent consumption. Previously employed barcoding protocols require that barcoding be performed after surface marker staining, complicating combining the technique with measurement of alcohol-sensitive surface epitopes. This report describes a method of barcoding fixed cells after a transient partial permeabilization with 0.02% saponin that results in efficient and consistent barcode staining with fluorescent or mass-tagged reagents while preserving surface marker staining. This approach simplifies barcoding protocols and allows direct comparison of surface marker staining of multiple samples without concern for variations in the antibody cocktail volume, antigen-antibody ratio, or machine sensitivity. Using this protocol, cellular barcoding can be used to reliably detect subtle differences in surface marker expression., (© 2014 International Society for Advancement of Cytometry.)
- Published
- 2014
- Full Text
- View/download PDF
26. Clinical recovery from surgery correlates with single-cell immune signatures.
- Author
-
Gaudillière B, Fragiadakis GK, Bruggner RV, Nicolau M, Finck R, Tingle M, Silva J, Ganio EA, Yeh CG, Maloney WJ, Huddleston JI, Goodman SB, Davis MM, Bendall SC, Fantl WJ, Angst MS, and Nolan GP
- Subjects
- Aged, Biomarkers blood, CREB-Binding Protein blood, Female, Hip Joint physiopathology, Humans, Lipopolysaccharide Receptors blood, Male, Middle Aged, Monocytes metabolism, NF-kappa B blood, Phenotype, Phosphorylation, Postoperative Complications blood, Postoperative Complications physiopathology, Predictive Value of Tests, Protein Interaction Maps, Recovery of Function, STAT3 Transcription Factor blood, Time Factors, Treatment Outcome, Arthroplasty, Replacement, Hip adverse effects, Flow Cytometry, Hip Joint surgery, Immunophenotyping methods, Monocytes immunology, Postoperative Complications immunology, Signal Transduction immunology
- Abstract
Delayed recovery from surgery causes personal suffering and substantial societal and economic costs. Whether immune mechanisms determine recovery after surgical trauma remains ill-defined. Single-cell mass cytometry was applied to serial whole-blood samples from 32 patients undergoing hip replacement to comprehensively characterize the phenotypic and functional immune response to surgical trauma. The simultaneous analysis of 14,000 phosphorylation events in precisely phenotyped immune cell subsets revealed uniform signaling responses among patients, demarcating a surgical immune signature. When regressed against clinical parameters of surgical recovery, including functional impairment and pain, strong correlations were found with STAT3 (signal transducer and activator of transcription), CREB (adenosine 3',5'-monophosphate response element-binding protein), and NF-κB (nuclear factor κB) signaling responses in subsets of CD14(+) monocytes (R = 0.7 to 0.8, false discovery rate <0.01). These sentinel results demonstrate the capacity of mass cytometry to survey the human immune system in a relevant clinical context. The mechanistically derived immune correlates point to diagnostic signatures, and potential therapeutic targets, that could postoperatively improve patient recovery., (Copyright © 2014, American Association for the Advancement of Science.)
- Published
- 2014
- Full Text
- View/download PDF
27. Single-cell mass cytometry for analysis of immune system functional states.
- Author
-
Bjornson ZB, Nolan GP, and Fantl WJ
- Subjects
- Cell Cycle, Cell Survival, Humans, Proteomics, Flow Cytometry methods, Immune System
- Abstract
Mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on cell populations at single-cell resolution. Datasets are generated with panels of up to 45 antibodies. Each antibody is conjugated to a polymer chelated with a stable metal isotope, usually in the lanthanide series of the periodic table. Antibody panels recognize surface markers to delineate cell types simultaneously with intracellular signaling molecules to measure biological functions, such as metabolism, survival, DNA damage, cell cycle and apoptosis, to provide an overall determination of the network state of an individual cell. This review will cover the basics of mass cytometry as well as outline assays developed for the platform that enhance the immunologist's analytical arsenal., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
28. Single-cell mass cytometry adapted to measurements of the cell cycle.
- Author
-
Behbehani GK, Bendall SC, Clutter MR, Fantl WJ, and Nolan GP
- Subjects
- Animals, Antibodies chemistry, Bone Marrow Cells metabolism, Bone Marrow Cells physiology, Cell Differentiation, Cell Line, Cell Proliferation, Cell Separation, Cyclin A metabolism, Cyclin B1 metabolism, DNA Replication, Flow Cytometry, Hematopoiesis, Histones metabolism, Humans, Immunophenotyping, Membrane Proteins metabolism, Mice, Retinoblastoma Protein metabolism, Staining and Labeling, T-Lymphocytes metabolism, T-Lymphocytes physiology, Transition Elements chemistry, Cell Cycle Checkpoints, Single-Cell Analysis methods
- Abstract
Mass cytometry is a recently introduced technology that utilizes transition element isotope-tagged antibodies for protein detection on a single-cell basis. By circumventing the limitations of emission spectral overlap associated with fluorochromes utilized in traditional flow cytometry, mass cytometry currently allows measurement of up to 40 parameters per cell. Recently, a comprehensive mass cytometry analysis was described for the hematopoietic differentiation program in human bone marrow from a healthy donor. The current study describes approaches to delineate cell cycle stages utilizing 5-iodo-2-deoxyuridine (IdU) to mark cells in S phase, simultaneously with antibodies against cyclin B1, cyclin A, and phosphorylated histone H3 (S28) that characterize the other cell cycle phases. Protocols were developed in which an antibody against phosphorylated retinoblastoma protein (Rb) at serines 807 and 811 was used to separate cells in G0 and G1 phases of the cell cycle. This mass cytometry method yielded cell cycle distributions of both normal and cancer cell populations that were equivalent to those obtained by traditional fluorescence cytometry techniques. We applied this to map the cell cycle phases of cells spanning the hematopoietic hierarchy in healthy human bone marrow as a prelude to later studies with cancers and other disorders of this lineage., (Copyright © 2012 International Society for Advancement of Cytometry.)
- Published
- 2012
- Full Text
- View/download PDF
29. A platinum-based covalent viability reagent for single-cell mass cytometry.
- Author
-
Fienberg HG, Simonds EF, Fantl WJ, Nolan GP, and Bodenmiller B
- Subjects
- Cell Adhesion, Cell Membrane Permeability, Cell Survival, DNA analysis, DNA chemistry, Female, Fluorescent Dyes, Humans, Single-Cell Analysis, Tumor Cells, Cultured, Cisplatin chemistry, Flow Cytometry methods, Mass Spectrometry methods, Platinum chemistry, Staining and Labeling methods
- Abstract
In fluorescence-based flow cytometry, cellular viability is determined with membrane-impermeable fluorescent reagents that specifically enter and label plasma membrane-compromised nonviable cells. A recent technological advance in flow cytometry uses antibodies conjugated to elemental metal isotopes, rather than to fluorophores, to allow signal detection by atomic mass spectrometry. Unhampered by the limitations of overlapping emission fluorescence, mass cytometry increases the number of parameters that can be measured in single cells. However, mass cytometry is unable to take advantage of current fluorescent viability dyes. An alternative methodology was therefore developed here in which the platinum-containing chemotherapy drug cisplatin was used to resolve live and dead cells by mass cytometry. In a 1-min incubation step, cisplatin preferentially labeled nonviable cells from both adherent and suspension cultures, resulting in a platinum signal quantifiable by mass cytometry. This protocol was compatible with established sample processing steps for intracellular cytometry. Furthermore, the live/dead ratios were comparable between mass- and fluorescence-based cytometry. Importantly, although cisplatin is a known DNA-damaging agent, a 1-min "pulse" of cisplatin did not induce observable DNA damage or apoptotic responses even within 6-h post-exposure. Cisplatin can therefore be used as a viability reagent for a wide range of mass cytometry protocols., (Copyright © 2012 International Society for Advancement of Cytometry.)
- Published
- 2012
- Full Text
- View/download PDF
30. Signaling changes in the stem cell factor-AKT-S6 pathway in diagnostic AML samples are associated with disease relapse.
- Author
-
Kornblau SM, Covey T, Putta S, Cohen A, Woronicz J, Fantl WJ, Gayko U, and Cesano A
- Published
- 2011
- Full Text
- View/download PDF
31. Association of reactive oxygen species-mediated signal transduction with in vitro apoptosis sensitivity in chronic lymphocytic leukemia B cells.
- Author
-
Palazzo AL, Evensen E, Huang YW, Cesano A, Nolan GP, and Fantl WJ
- Subjects
- Adult, Aged, Aged, 80 and over, Apoptosis immunology, B-Lymphocytes immunology, B-Lymphocytes metabolism, Biomarkers, Tumor metabolism, Cell Line, Tumor, Female, Humans, Hydrogen Peroxide pharmacology, Leukemia, Lymphocytic, Chronic, B-Cell immunology, Leukemia, Lymphocytic, Chronic, B-Cell metabolism, Male, Middle Aged, Phosphorylation drug effects, Proteome drug effects, Proteome immunology, Proteome metabolism, Signal Transduction immunology, Vidarabine analogs & derivatives, Vidarabine pharmacology, Apoptosis drug effects, B-Lymphocytes drug effects, B-Lymphocytes pathology, Leukemia, Lymphocytic, Chronic, B-Cell pathology, Reactive Oxygen Species pharmacology, Signal Transduction drug effects, Single-Cell Analysis
- Abstract
Background: Chronic lymphocytic leukemia (CLL) is a B cell malignancy with a variable clinical course and unpredictable response to therapeutic agents. Single cell network profiling (SCNP) utilizing flow cytometry measures alterations in signaling biology in the context of molecular changes occurring in malignancies. In this study SCNP was used to identify proteomic profiles associated with in vitro apoptotic responsiveness of CLL B cells to fludarabine, as a basis for ultimately linking these with clinical outcome., Methodology/principal Finding: SCNP was used to quantify modulated-signaling of B cell receptor (BCR) network proteins and in vitro F-ara-A mediated apoptosis in 23 CLL samples. Of the modulators studied the reactive oxygen species, hydrogen peroxide (H₂O₂), a known intracellular second messenger and a general tyrosine phosphatase inhibitor stratified CLL samples into two sub-groups based on the percentage of B cells in a CLL sample with increased phosphorylation of BCR network proteins. Separately, in the same patient samples, in vitro exposure to F-ara-A also identified two sub-groups with B cells showing competence or refractoriness to apoptotic induction. Statistical analysis showed that in vitro F-ara-A apoptotic proficiency was highly associated with the proficiency of CLL B cells to undergo H₂O₂-augmented signaling., Conclusions/significance: This linkage in CLL B cells among the mechanisms governing chemotherapy-induced apoptosis increased signaling of BCR network proteins and a likely role of phosphatase activity suggests a means of stratifying patients for their response to F-ara-A based regimens. Future studies will examine the clinical applicability of these findings and also the utility of this approach in relating mechanism to function of therapeutic agents.
- Published
- 2011
- Full Text
- View/download PDF
32. Functional characterization of FLT3 receptor signaling deregulation in acute myeloid leukemia by single cell network profiling (SCNP).
- Author
-
Rosen DB, Minden MD, Kornblau SM, Cohen A, Gayko U, Putta S, Woronicz J, Evensen E, Fantl WJ, and Cesano A
- Subjects
- Adult, Aged, Aged, 80 and over, Female, Flow Cytometry, Humans, Leukemia, Myeloid, Acute pathology, Male, Middle Aged, Tumor Cells, Cultured, Leukemia, Myeloid, Acute metabolism, Signal Transduction, fms-Like Tyrosine Kinase 3 metabolism
- Abstract
Background: Molecular characterization of the FMS-like tyrosine kinase 3 receptor (FLT3) in cytogenetically normal acute myeloid leukemia (AML) has recently been incorporated into clinical guidelines based on correlations between FLT3 internal tandem duplications (FLT3-ITD) and decreased disease-free and overall survival. These mutations result in constitutive activation of FLT3, and FLT3 inhibitors are currently undergoing trials in AML patients selected on FLT3 molecular status. However, the transient and partial responses observed suggest that FLT3 mutational status alone does not provide complete information on FLT3 biological activity at the individual patient level. Examination of variation in cellular responsiveness to signaling modulation may be more informative., Methodology/principal Findings: Using single cell network profiling (SCNP), cells were treated with extracellular modulators and their functional responses were quantified by multiparametric flow cytometry. Intracellular signaling responses were compared between healthy bone marrow myeloblasts (BMMb) and AML leukemic blasts characterized as FLT3 wild type (FLT3-WT) or FLT3-ITD. Compared to healthy BMMb, FLT3-WT leukemic blasts demonstrated a wide range of signaling responses to FLT3 ligand (FLT3L), including elevated and sustained PI3K and Ras/Raf/Erk signaling. Distinct signaling and apoptosis profiles were observed in FLT3-WT and FLT3-ITD AML samples, with more uniform signaling observed in FLT3-ITD AML samples. Specifically, increased basal p-Stat5 levels, decreased FLT3L induced activation of the PI3K and Ras/Raf/Erk pathways, decreased IL-27 induced activation of the Jak/Stat pathway, and heightened apoptotic responses to agents inducing DNA damage were observed in FLT3-ITD AML samples. Preliminary analysis correlating these findings with clinical outcomes suggests that classification of patient samples based on signaling profiles may more accurately reflect FLT3 signaling deregulation and provide additional information for disease characterization and management., Conclusions/significance: These studies show the feasibility of SCNP to assess modulated intracellular signaling pathways and characterize the biology of individual AML samples in the context of genetic alterations.
- Published
- 2010
- Full Text
- View/download PDF
33. Distinct patterns of DNA damage response and apoptosis correlate with Jak/Stat and PI3kinase response profiles in human acute myelogenous leukemia.
- Author
-
Rosen DB, Putta S, Covey T, Huang YW, Nolan GP, Cesano A, Minden MD, and Fantl WJ
- Subjects
- Etoposide pharmacology, Humans, Intracellular Space drug effects, Intracellular Space metabolism, Signal Transduction drug effects, Staurosporine pharmacology, Apoptosis drug effects, DNA Damage, Janus Kinases metabolism, Leukemia, Myeloid, Acute genetics, Leukemia, Myeloid, Acute pathology, Phosphatidylinositol 3-Kinases metabolism, STAT Transcription Factors metabolism
- Abstract
Background: Single cell network profiling (SCNP) utilizing flow cytometry measures alterations in intracellular signaling responses. Here SCNP was used to characterize Acute Myeloid Leukemia (AML) disease subtypes based on survival, DNA damage response and apoptosis pathways., Methodology and Principal Findings: Thirty four diagnostic non-M3 AML samples from patients with known clinical outcome were treated with a panel of myeloid growth factors and cytokines, as well as with apoptosis-inducing agents. Analysis of induced Jak/Stat and PI3K pathway responses in blasts from individual patient samples identified subgroups with distinct signaling profiles that were not seen in the absence of a modulator. In vitro exposure of patient samples to etoposide, a DNA damaging agent, revealed three distinct "DNA damage response (DDR)/apoptosis" profiles: 1) AML blasts with a defective DDR and failure to undergo apoptosis; 2) AML blasts with proficient DDR and failure to undergo apoptosis; 3) AML blasts with proficiency in both DDR and apoptosis pathways. Notably, AML samples from clinical responders fell within the "DDR/apoptosis" proficient profile and, as well, had low PI3K and Jak/Stat signaling responses. In contrast, samples from clinical non responders had variable signaling profiles often with in vitro apoptotic failure and elevated PI3K pathway activity. Individual patient samples often harbored multiple, distinct, leukemia-associated cell populations identifiable by their surface marker expression, functional performance of signaling pathway in the face of cytokine or growth factor stimulation, as well as their response to apoptosis-inducing agents., Conclusions and Significance: Characterizing and tracking changes in intracellular pathway profiles in cell subpopulations both at baseline and under therapeutic pressure will likely have important clinical applications, potentially informing the selection of beneficial targeted agents, used either alone or in combination with chemotherapy.
- Published
- 2010
- Full Text
- View/download PDF
34. Dynamic single-cell network profiles in acute myelogenous leukemia are associated with patient response to standard induction therapy.
- Author
-
Kornblau SM, Minden MD, Rosen DB, Putta S, Cohen A, Covey T, Spellmeyer DC, Fantl WJ, Gayko U, and Cesano A
- Subjects
- Adult, Aged, Apoptosis drug effects, Cohort Studies, Female, Flow Cytometry, Humans, Leukemia, Myeloid, Acute diagnosis, Leukemia, Myeloid, Acute genetics, Male, Middle Aged, Mutation, Predictive Value of Tests, Prospective Studies, Reproducibility of Results, Signal Transduction drug effects, Single-Cell Analysis, Young Adult, fms-Like Tyrosine Kinase 3 metabolism, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Leukemia, Myeloid, Acute drug therapy, Leukemia, Myeloid, Acute metabolism
- Abstract
Purpose: Complete response to induction chemotherapy is observed in approximately 60% of patients with newly diagnosed non-M3 acute myelogenous leukemia (AML). However, no methods exist to predict with high accuracy at the individual patient level the response to standard AML induction therapy., Experimental Design: We applied single-cell network profiling (SCNP) using flow cytometry, a tool that allows a comprehensive functional assessment of intracellular signaling pathways in heterogeneous tissues, to two training cohorts of AML samples (n = 34 and 88) to predict the likelihood of response to induction chemotherapy., Results: In the first study, univariate analysis identified multiple signaling "nodes" (readouts of modulated intracellular signaling proteins) that correlated with response (i.e., AUC(ROC) > or = 0.66; P < or = 0.05) at a level greater than age. After accounting for age, similar findings were observed in the second study. For patients <60 years old, complete response was associated with the presence of intact apoptotic pathways. In patients > or =60 years old, nonresponse was associated with FLT3 ligand-mediated increase in phosphorylated Akt and phosphorylated extracellular signal-regulated kinase. Results were independent of cytogenetics, FLT3 mutational status, and diagnosis of secondary AML., Conclusions: These data emphasize the value of performing quantitative SCNP under modulated conditions as a basis for the development of tests highly predictive for response to induction chemotherapy. SCNP provides information distinct from other known prognostic factors such as age, secondary AML, cytogenetics, and molecular alterations and is potentially combinable with the latter to improve clinical decision making. Independent validation studies are warranted., (Copyright 2010 AACR.)
- Published
- 2010
- Full Text
- View/download PDF
35. beta-Catenin regulates vascular endothelial growth factor expression in colon cancer.
- Author
-
Easwaran V, Lee SH, Inge L, Guo L, Goldbeck C, Garrett E, Wiesmann M, Garcia PD, Fuller JH, Chan V, Randazzo F, Gundel R, Warren RS, Escobedo J, Aukerman SL, Taylor RN, and Fantl WJ
- Subjects
- Adenocarcinoma blood supply, Adenocarcinoma etiology, Adenocarcinoma metabolism, Adenocarcinoma pathology, Adenomatous Polyposis Coli genetics, Adenomatous Polyposis Coli metabolism, Animals, Binding Sites, Colon metabolism, Colonic Neoplasms blood supply, Colonic Neoplasms etiology, Colonic Neoplasms metabolism, Colonic Neoplasms pathology, Cytoskeletal Proteins genetics, Endothelial Growth Factors genetics, Fibroblast Growth Factor 2 analysis, Gene Expression Regulation, Neoplastic drug effects, Genes, APC, Genes, ras, Growth Substances genetics, Humans, Intercellular Signaling Peptides and Proteins genetics, Intestinal Mucosa metabolism, Lymphokines genetics, Mice, Mice, Inbred C57BL, Mice, Mutant Strains, Neoplasm Proteins genetics, Oligodeoxyribonucleotides, Antisense pharmacology, RNA, Messenger biosynthesis, RNA, Messenger genetics, RNA, Neoplasm biosynthesis, RNA, Neoplasm genetics, Recombinant Fusion Proteins physiology, Signal Transduction, Subcellular Fractions chemistry, Trans-Activators genetics, Transfection, Tumor Cells, Cultured drug effects, Tumor Cells, Cultured metabolism, Vascular Endothelial Growth Factor A, Vascular Endothelial Growth Factors, beta Catenin, Adenocarcinoma genetics, Colonic Neoplasms genetics, Cytoskeletal Proteins physiology, Endothelial Growth Factors biosynthesis, Gene Expression Regulation, Neoplastic physiology, Intercellular Signaling Peptides and Proteins biosynthesis, Lymphokines biosynthesis, Neoplasm Proteins biosynthesis, Promoter Regions, Genetic genetics, Trans-Activators physiology
- Abstract
To evaluate whether beta-catenin signaling has a role in the regulation of angiogenesis in colon cancer, a series of angiogenesis-related gene promoters was analyzed for beta-catenin/TCF binding sites. Strikingly, the gene promoter of human vascular endothelial growth factor (VEGF, or VEGF-A) contains seven consensus binding sites for beta-catenin/TCF. Analysis of laser capture microdissected human colon cancer tissue indicated a direct correlation between up-regulation of VEGF-A expression and adenomatous polyposis coli (APC) mutational status (activation of beta-catenin signaling) in primary tumors. In metastases, this correlation was not observed. Analysis by immunohistochemistry of intestinal polyps in mice heterozygous for the multiple intestinal neoplasia gene (Min/+) at 5 months revealed an increase and redistribution of VEGF-A in proximity to those cells expressing nuclear beta-catenin with a corresponding increase in vessel density. Transfection of normal colon epithelial cells with activated beta-catenin up-regulated levels of VEGF-A mRNA and protein by 250-300%. When colon cancer cells with elevated beta-catenin levels were treated with beta-catenin antisense oligodeoxynucleotides, VEGF-A expression was reduced by more than 50%. Taken together, our observations indicate a close link between beta-catenin signaling and the regulation of VEGF-A expression in colon cancer.
- Published
- 2003
36. c-Src/Lyn kinases activate Helicobacter pylori CagA through tyrosine phosphorylation of the EPIYA motifs.
- Author
-
Stein M, Bagnoli F, Halenbeck R, Rappuoli R, Fantl WJ, and Covacci A
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Bacterial Proteins genetics, Binding Sites, CSK Tyrosine-Protein Kinase, Helicobacter pylori genetics, Molecular Sequence Data, Phosphorylation, Pyrazoles pharmacology, Pyrimidines pharmacology, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods, Tyrosine genetics, src-Family Kinases antagonists & inhibitors, Antigens, Bacterial, Bacterial Proteins metabolism, Helicobacter pylori metabolism, Protein-Tyrosine Kinases metabolism, Tyrosine metabolism, src-Family Kinases metabolism
- Abstract
The human pathogen Helicobacter pylori colonizes the mucous layer of the stomach. During parasitic infection, freely swimming bacteria adhere to the gastric epithelial cells and trigger intracellular signalling pathways. This process requires the translocation of the effector protein CagA into the host cell through a specialized type IV secretion system encoded in the cag pathogenicity island. Following transfer, CagA is phosphorylated on tyrosine residues by a host cell kinase. Here, we describe how the tyrosine phosphorylation of CagA is restricted to a previously identified repeated sequence called D1. This sequence is located in the C-terminal half of the protein and contains the five-amino-acid motif EPIYA, which is amplified by duplications in a large fraction of clinical isolates. Tyrosine phosphorylation of CagA is essential for the activation process that leads to dramatic changes in the morphology of cells growing in culture. In addition, we observed that two members of the src kinases family, c-Src and Lyn, account for most of the CagA-specific kinase activity in host cell lysates. Thus, CagA translocation followed by tyrosine phosphorylation at the EPIYA motifs promotes a growth factor-like response with intense cytoskeletal rearrangements, cell elongation effects and increased cellular motility.
- Published
- 2002
- Full Text
- View/download PDF
37. Elevated expression of axin2 and hnkd mRNA provides evidence that Wnt/beta -catenin signaling is activated in human colon tumors.
- Author
-
Yan D, Wiesmann M, Rohan M, Chan V, Jefferson AB, Guo L, Sakamoto D, Caothien RH, Fuller JH, Reinhard C, Garcia PD, Randazzo FM, Escobedo J, Fantl WJ, and Williams LT
- Subjects
- Axin Protein, Base Sequence, Cell Line, Colonic Neoplasms genetics, Colonic Neoplasms pathology, DNA Primers, DNA, Complementary, Humans, Molecular Sequence Data, Oligonucleotide Array Sequence Analysis, Promoter Regions, Genetic, RNA, Messenger metabolism, Wnt Proteins, beta Catenin, Colonic Neoplasms metabolism, Cytoskeletal Proteins metabolism, Proteins genetics, Proto-Oncogene Proteins metabolism, RNA, Messenger genetics, Repressor Proteins, Signal Transduction, Trans-Activators, Zebrafish Proteins
- Abstract
Genetic studies have identified mutations in key regulators of the Wnt/beta-catenin pathway in a variety of cancers, most frequently in colon cancers. However, whether the pathway is activated in clinical cancer samples is not easily determined, and therefore it is useful to find markers that could be surrogates to show activation of the Wnt/beta-catenin pathway. Gene expression profiles were analyzed in SW620, a colon cancer cell line in which beta-catenin levels are stabilized as a consequence of truncated adenomatous polyposis coli and were compared with profiles of the same cells transfected with antisense oligodeoxynucleotides. Treatment of cells with beta-catenin antisense oligodeoxynucleotides resulted in a decrease in the levels of axin2 and human naked cuticle (hnkd) mRNAs. Interestingly, the proteins encoded by both of these mRNAs are known inhibitors of the beta-catenin pathway. In 30 human cell lines derived from different origins, axin2 and hnkd were expressed only in human colon cancer cell lines that are known to have activating mutations in the Wnt/beta-catenin pathway. Further, levels of both axin2 and hnkd mRNA were also found to be elevated in about 65% of laser microdissected cells from human colon tumors compared with laser microdissected cells of normal morphology from the same patient samples. The increased expression of axin2 and hnkd correlated with truncations in adenomatous polyposis coli in the same patient samples. These results reveal that it is possible to detect activation of a carcinogenic pathway in human cancer samples with specific markers.
- Published
- 2001
- Full Text
- View/download PDF
38. PAR-1 is a Dishevelled-associated kinase and a positive regulator of Wnt signalling.
- Author
-
Sun TQ, Lu B, Feng JJ, Reinhard C, Jan YN, Fantl WJ, and Williams LT
- Subjects
- Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, CHO Cells, Cricetinae, Cytoskeletal Proteins drug effects, Dishevelled Proteins, Drosophila embryology, Drosophila Proteins, Humans, JNK Mitogen-Activated Protein Kinases, Mitogen-Activated Protein Kinases drug effects, Mitogens physiology, Molecular Sequence Data, Phosphorylation, Protein Binding, Protein Serine-Threonine Kinases metabolism, Protein Serine-Threonine Kinases pharmacology, Proto-Oncogene Proteins drug effects, Proto-Oncogene Proteins pharmacology, Sequence Alignment, Signal Transduction drug effects, Transcriptional Activation drug effects, Tumor Cells, Cultured, Wnt Proteins, Xenopus, Xenopus Proteins, beta Catenin, Caenorhabditis elegans Proteins, Phosphoproteins metabolism, Protein Serine-Threonine Kinases physiology, Proto-Oncogene Proteins physiology, Trans-Activators, Zebrafish Proteins
- Abstract
Wnt signalling regulates beta-catenin-dependent developmental processes through the Dishevelled protein (Dsh). Dsh regulates two distinct pathways, one mediated by beta-catenin and the other by Jun kinase (JNK). We have purified a Dsh-associated kinase from Drosophila that encodes a homologue of Caenorhabditis elegans PAR-1, a known determinant of polarity during asymmetric cell divisions. Treating cells with Wnt increases endogenous PAR-1 activity coincident with Dsh phosphorylation. PAR-1 potentiates Wnt activation of the beta-catenin pathway but blocks the JNK pathway. Suppressing endogenous PAR-1 function inhibits Wnt signalling through beta-catenin in mammalian cells, and Xenopus and Drosophila embryos. PAR-1 seems to be a positive regulator of the beta-catenin pathway and an inhibitor of the JNK pathway. These findings show that PAR-1, a regulator of polarity, is also a modulator of Wnt-beta-catenin signalling, indicating a link between two important developmental pathways.
- Published
- 2001
- Full Text
- View/download PDF
39. Cell autonomous regulation of multiple Dishevelled-dependent pathways by mammalian Nkd.
- Author
-
Yan D, Wallingford JB, Sun TQ, Nelson AM, Sakanaka C, Reinhard C, Harland RM, Fantl WJ, and Williams LT
- Subjects
- Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Calcium-Binding Proteins, Carrier Proteins genetics, Cloning, Molecular, Dishevelled Proteins, Enzyme Activation, Insect Proteins chemistry, Insect Proteins genetics, JNK Mitogen-Activated Protein Kinases, Mice, Mice, Inbred BALB C, Mitogen-Activated Protein Kinases metabolism, Molecular Sequence Data, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins pharmacology, RNA, Messenger biosynthesis, RNA, Messenger drug effects, Sequence Homology, Amino Acid, Signal Transduction, Wnt Proteins, Carrier Proteins metabolism, Drosophila Proteins, Phosphoproteins metabolism, Zebrafish Proteins
- Abstract
Genetic studies have identified Drosophila Naked Cuticle (Nkd) as an antagonist of the canonical Wnt/beta-catenin signaling pathway, but its mechanism of action remains obscure [Zeng, W., Wharton, K. A., Jr., Mack, J. A., Wang, K., Gadbaw, M., et al. (2000) Nature (London) 403, 789--795]. Here we have cloned a cDNA encoding a mammalian homolog of Drosophila Nkd, mNkd, and demonstrated that mNkd interacts directly with Dishevelled. Dishevelled is an intracellular mediator of both the canonical Wnt pathway and planar cell polarity (PCP) pathway. Activation of the c-Jun-N-terminal kinase has been implicated in the PCP pathway. We showed that mNkd acts in a cell-autonomous manner not only to inhibit the canonical Wnt pathway but also to stimulate c-Jun-N-terminal kinase activity. Expression of mNkd disrupted convergent extension in Xenopus, consistent with a role for mNkd in the PCP pathway. These data suggest that mNkd may act as a switch to direct Dishevelled activity toward the PCP pathway, and away from the canonical Wnt pathway.
- Published
- 2001
- Full Text
- View/download PDF
40. Regulation of cyclooxygenase-2 and periostin by Wnt-3 in mouse mammary epithelial cells.
- Author
-
Haertel-Wiesmann M, Liang Y, Fantl WJ, and Williams LT
- Subjects
- Animals, Calcium-Calmodulin-Dependent Protein Kinases antagonists & inhibitors, Calcium-Calmodulin-Dependent Protein Kinases metabolism, Cell Line, Cyclooxygenase 2, Cytoskeletal Proteins genetics, Cytoskeletal Proteins metabolism, Dinoprostone metabolism, Epithelial Cells drug effects, Epithelial Cells enzymology, Epithelial Cells metabolism, Genes, Reporter genetics, Glycogen Synthase Kinase 3, Glycogen Synthase Kinases, Isoenzymes metabolism, Lithium Chloride pharmacology, Mammary Glands, Animal cytology, Mammary Glands, Animal drug effects, Mammary Glands, Animal enzymology, Mice, Mutation, Oligonucleotide Array Sequence Analysis, Oligonucleotides, Antisense genetics, Oligonucleotides, Antisense metabolism, Prostaglandin-Endoperoxide Synthases metabolism, Proteins genetics, RNA, Messenger genetics, RNA, Messenger metabolism, Signal Transduction, Wnt Proteins, Wnt3 Protein, beta Catenin, Cell Adhesion Molecules genetics, Gene Expression Regulation drug effects, Isoenzymes genetics, Mammary Glands, Animal metabolism, Prostaglandin-Endoperoxide Synthases genetics, Proteins metabolism, Trans-Activators
- Abstract
Wnt family members are critical in developmental processes and have been shown to promote carcinogenesis when ectopically expressed in the mouse mammary gland. The gene expression pattern mediated by Wnt is pivotal for these diverse responses. The Wnt pathway has been conserved among different species. Genetic studies have shown that Wnt effects are mediated, at least in part, by beta-catenin, which regulates transcription of "downstream genes." Wnt stimulation inactivates glycogen-synthase kinase-3beta (GSK-3) with subsequent stabilization of beta-catenin, which after heterodimerizing with lymphocyte enhancer factor-1/T-cell factor cofactors stimulates transcription. To establish whether Wnt-stimulated transcription is mediated solely by beta-catenin, a comparison was made of gene expression profiles in response to Wnt-3, overexpression of beta-catenin, and inhibition of GSK-3. Infection of cells with Wnt-3 and inhibition of GSK-3 regulate a set of genes that include cyclooxygenase-2 and periostin. Interestingly, overexpression of beta-catenin or reducing beta-catenin levels with antisense oligonucleotide transfection did not have any effect on cyclooxygenase-2 or periostin expression, thereby defining a Wnt pathway, which cannot be mimicked by beta-catenin overexpression.
- Published
- 2000
- Full Text
- View/download PDF
41. 16K human prolactin inhibits vascular endothelial growth factor-induced activation of Ras in capillary endothelial cells.
- Author
-
D'Angelo G, Martini JF, Iiri T, Fantl WJ, Martial J, and Weiner RI
- Subjects
- Biological Transport, Cyclic AMP metabolism, Cyclic AMP-Dependent Protein Kinases metabolism, Endothelial Growth Factors pharmacology, Endothelium, Vascular drug effects, Enzyme Activation drug effects, GRB10 Adaptor Protein, GTPase-Activating Proteins, Humans, Lymphokines pharmacology, Membrane Proteins metabolism, Peptide Fragments metabolism, Peptide Fragments pharmacology, Prolactin pharmacology, Proteins metabolism, Proto-Oncogene Proteins c-raf drug effects, Proto-Oncogene Proteins c-raf metabolism, Receptor Protein-Tyrosine Kinases drug effects, Receptor Protein-Tyrosine Kinases metabolism, Receptors, Growth Factor drug effects, Receptors, Growth Factor metabolism, Receptors, Vascular Endothelial Growth Factor, Signal Transduction, Son of Sevenless Proteins, Vascular Endothelial Growth Factor A, Vascular Endothelial Growth Factors, ras GTPase-Activating Proteins, ras Proteins metabolism, Endothelial Growth Factors metabolism, Endothelium, Vascular metabolism, Genes, ras, Lymphokines metabolism, Prolactin metabolism
- Abstract
Signaling pathways mediating the antiangiogenic action of 16K human (h)PRL include inhibition of vascular endothelial growth factor (VEGF)-induced activation of the mitogen-activated protein kinases (MAPK). To determine at which step 16K hPRL acts to inhibit VEGF-induced MAPK activation, we assessed more proximal events in the signaling cascade. 16K hPRL treatment blocked VEGF-induced Raf-1 activation as well as its translocation to the plasma membrane. 16K hPRL indirectly increased cAMP levels; however, the blockade of Raf-1 activation was not dependent on the stimulation of cAMP-dependent protein kinase (PKA), but rather on the inhibition of the GTP-bound Ras. The VEGF-induced tyrosine phosphorylation of the VEGF receptor, Flk-1, and its association with the Shc/Grb2/Ras-GAP (guanosine triphosphatase-activating protein) complex were unaffected by 16K hPRL treatment. In contrast, 16K hPRL prevented the VEGF-induced phosphorylation and dissociation of Sos from Grb2 at 5 min, consistent with inhibition by 16K hPRL of the MEK/MAPK feedback on Sos. The inhibition of Ras activation was paralleled by the increased phosphorylation of 120 kDa proteins comigrating with Ras-GAP. Taken together, these findings show that 16K hPRL inhibits the VEGF-induced Ras activation; this antagonism represents a novel and potentially important mechanism for the control of angiogenesis.
- Published
- 1999
- Full Text
- View/download PDF
42. Regulation of the MAP kinase pathway by mammalian Ksr through direct interaction with MEK and ERK.
- Author
-
Yu W, Fantl WJ, Harrowe G, and Williams LT
- Subjects
- Amino Acid Sequence, Animals, COS Cells, Calcium-Calmodulin-Dependent Protein Kinases metabolism, DNA Replication drug effects, Fibroblasts drug effects, Fibroblasts metabolism, MAP Kinase Kinase 1, Mice, Microinjections, Molecular Sequence Data, Protein Binding, Protein Kinases metabolism, Rats, ras Proteins metabolism, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase Kinases, Protein Kinases physiology, Protein Serine-Threonine Kinases metabolism, Protein-Tyrosine Kinases metabolism, Signal Transduction
- Abstract
Background: Genetic screens in Drosophila melanogaster and Caenorhabditis elegans identified the kinase suppressor of Ras, Ksr, as a new component in the Ras intracellular signaling pathway. In these organisms, mutations in Ksr resulted in attenuation of Ras-mediated signaling. Homologs of Ksr have also been isolated from mice and humans; their precise role in Ras signaling is not well defined. Here, we present data showing interactions between the murine form of Ksr (mKsr-1) and other components of the Ras pathway., Results: To gain insight into the biological function of Ksr, we used a yeast two-hybrid screen and found an interaction between the carboxy-terminal region of mKsr-1 and mitogen-activated protein (MAP) kinase kinase 1 (MAPKK-1 or MEK-1). An interaction was also detected between MAP kinase (also called extracellular signal-regulated kinase; ERK), and the amino-terminal region of mKsr-1. These interactions were recapitulated in COS-7 cells. Further, when COS-7 cells were transfected with either full-length mKsr-1 or only its carboxy-terminal region, an inhibition of serum-stimulated MAP kinase activation was observed. Microinjection of full-length mKsr-1 or its carboxy-terminal, but not its amino-terminal region, blocked serum-induced DNA synthesis in rat embryo fibroblasts. Co-injection of mKsr-1 with MEK-1 reversed the blockade., Conclusions: Together with the data from genetic analyses, our findings lead us to propose that mKsr-1 may control MAP kinase signaling by serving as a scaffold protein that links MEK and its substrate ERK.
- Published
- 1998
- Full Text
- View/download PDF
43. Reduction of food intake and weight gain by the ob protein requires a specific secondary structure and is reversible.
- Author
-
Giese K, Fantl WJ, Vitt C, Stephans JC, Cousens L, Wachowicz M, and Williams LT
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cloning, Molecular, DNA Primers chemistry, Diet, Disulfides chemistry, Dithiothreitol pharmacology, Electrophoresis, Polyacrylamide Gel, Immunoblotting, Leptin, Mice, Mice, Obese genetics, Mice, Obese metabolism, Molecular Sequence Data, Protein Processing, Post-Translational, Protein Structure, Secondary, Proteins genetics, Proteins pharmacology, Rats, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Recombinant Proteins pharmacology, Sequence Analysis, Body Weight, Obesity genetics, Proteins chemistry
- Abstract
Background: Obesity, the condition of excessive accumulation of fat is a poorly understood disorder and is a risk factor for type II diabetes, hypertension, and hyperlipidaemia. Recently, a putative mouse obese gene was cloned and its product, termed ob protein, was shown to be involved in the regulation of body weight., Materials and Methods: Bacterial and insect cells were used for expression of recombinant mouse ob protein. Amino-terminal sequence analysis and site-directed mutagenesis were used to identify and characterize the mature form of ob protein. Genetically obese mice and wild-type rats were used to determine the biological activity of ob protein., Results: Mouse ob protein is synthesized as a precursor molecule, the mature form of which was found in mouse serum. Biochemical analysis identified the processing site in the ob precursor molecule and an intramolecular disulfide bond in the mature form that is necessary for activity. Reduction of food intake and weight gain after administration of ob protein to genetically obese mice and wild-type rats is reversible., Discussion: This study demonstrates that ob protein is a secreted satiety factor which regulates body weight and reduces food intake even in animals with no genetic body weight abnormalities. The failure of ob protein to effect these parameters in db/db mice supports the hypothesis that these mice are deficient in a signaling molecule that normally responds to the ob protein.
- Published
- 1996
44. Ras-dependent induction of cellular responses by constitutively active phosphatidylinositol-3 kinase.
- Author
-
Hu Q, Klippel A, Muslin AJ, Fantl WJ, and Williams LT
- Subjects
- 3T3 Cells, Animals, Genes, fos genetics, Mice, Phosphatidylinositol 3-Kinases, Phosphotransferases (Alcohol Group Acceptor) biosynthesis, Promoter Regions, Genetic physiology, Recombinant Fusion Proteins biosynthesis, Transcription, Genetic physiology, Xenopus laevis, Phosphotransferases (Alcohol Group Acceptor) physiology, Signal Transduction physiology, ras Proteins physiology
- Abstract
Phosphatidylinositol (Pl)-3 kinase is one of many enzymes stimulated by growth factors. A constitutively activated mutant, p110, that functions independently of growth factor stimulation was constructed to determine the specific responses regulated by Pl-3 kinase. The p110 protein exhibited high specific activity as a Pl-3 kinase and as a protein kinase. Expression of p110 in NIH 3T3 cells induced transcription from the fos promoter. Co-expression of dominant negative Ras blocked this response. When expressed in Xenopus laevis oocytes, p110 increased the amount of guanosine 5'-triphosphate-bound Ras, caused activation of the Ras effector Raf-1, and induced Ras-dependent oocyte maturation. These findings show that Pl-3 kinase can stimulate diverse Ras-dependent cellular processes, including oocyte maturation and fos transcription.
- Published
- 1995
- Full Text
- View/download PDF
45. Activation of Na+/H+ exchange by platelet-derived growth factor involves phosphatidylinositol 3'-kinase and phospholipase C gamma.
- Author
-
Ma YH, Reusch HP, Wilson E, Escobedo JA, Fantl WJ, Williams LT, and Ives HE
- Subjects
- Animals, Binding Sites, CHO Cells, Calcium metabolism, Cricetinae, Epithelium drug effects, Epithelium metabolism, Female, Hydrogen-Ion Concentration, Isoenzymes metabolism, Mammary Glands, Animal metabolism, Mice, Mutagenesis, Site-Directed, Phosphatidylinositol 3-Kinases, Point Mutation, Receptor, Platelet-Derived Growth Factor beta, Receptors, Platelet-Derived Growth Factor biosynthesis, Receptors, Platelet-Derived Growth Factor drug effects, Recombinant Proteins biosynthesis, Recombinant Proteins drug effects, Recombinant Proteins metabolism, Transfection, Phosphotransferases (Alcohol Group Acceptor) metabolism, Platelet-Derived Growth Factor pharmacology, Receptors, Platelet-Derived Growth Factor physiology, Sodium-Hydrogen Exchangers metabolism, Type C Phospholipases metabolism
- Abstract
The effect of site-specific mutations in the mouse platelet-derived growth factor (PDGF) beta-receptor on activation of the Na+/H+ exchanger was examined in normal murine mammary gland epithelial (NMuMG) and Chinese hamster ovary (CHO) cells. These cells, which do not normally express PDGF receptors, were stably transfected with PDGF beta-receptor cDNA. Intracellular pH and Ca2+ were monitored using fluorescent probes. In both NMuMG and CHO cells expressing wild-type PDGF beta-receptors, PDGF B/B activated the amiloride-sensitive Na+/H+ exchanger. In both cell types, cell alkalinization was reduced by approximately 50% with a receptor mutant Y708F,Y719F which cannot bind phosphatidylinositol (PI) 3'-kinase. An inhibitor of PI 3'-kinase, LY294002, also inhibited alkalinization by 43% in cells with wild-type, but not Y708F,Y719F receptors. PDGF-induced intracellular Ca2+ release was not affected by this mutation. Both alkalinization and Ca2+ release were reduced by nearly 100% with the mutant Y977F,Y989F, which cannot bind phospholipase C gamma (PLC gamma). Y739F, a mutant that fails to bind the GTPase-activating protein did not affect PDGF-induced alkalinization. In protein kinase C (PKC) down-regulated NMuMG cells (wild-type receptor), PDGF no longer activated the Na+/H+ exchanger. In contrast, in PKC down-regulated CHO cells (wild-type receptor), PDGF-induced alkalinization was attenuated by only 37%. This residual activity was unaffected by the Y708F,Y719F mutation, but was completely eliminated by removal of medium Ca2+. These findings indicate that phospholipase C gamma is essential for activation of Na+/H+ exchange. PI 3'-kinase participates in PKC-dependent activation of Na+/H+ exchange by PDGF. In CHO cells, there is a second, Ca(2+)-dependent mechanism for activation of the exchanger.
- Published
- 1994
46. Activation of Raf-1 by 14-3-3 proteins.
- Author
-
Fantl WJ, Muslin AJ, Kikuchi A, Martin JA, MacNicol AM, Gross RW, and Williams LT
- Subjects
- 14-3-3 Proteins, Animals, CDC2 Protein Kinase metabolism, Cells, Cultured, Enzyme Activation, Oocytes, Oogenesis genetics, Oogenesis physiology, Proto-Oncogene Proteins c-raf, Recombinant Proteins metabolism, Saccharomyces cerevisiae genetics, Xenopus, Nerve Tissue Proteins metabolism, Protein Serine-Threonine Kinases metabolism, Proto-Oncogene Proteins metabolism, Tyrosine 3-Monooxygenase
- Abstract
The protein Raf-1, a key mediator of mitogenesis and differentiation, associates with p21ras (refs 1-3). However, the regulation of the serine/threonine kinase activity of Raf-1 is still not understood. Using the yeast two-hybrid system, we identified two structurally related proteins that interact with the aminoterminal region of Raf-1. These proteins, 14-3-3 zeta (PLA2) and 14-3-3 beta (HS1), are members of the 14-3-3 family of proteins. Expression of 14-3-3 proteins in Xenopus oocytes enhanced Raf-1 activity and promoted Raf-1-dependent oocyte maturation. A dominant negative mutant of Raf-1 blocked the effects of 14-3-3 protein.
- Published
- 1994
- Full Text
- View/download PDF
47. HIV-1 Nef leads to inhibition or activation of T cells depending on its intracellular localization.
- Author
-
Baur AS, Sawai ET, Dazin P, Fantl WJ, Cheng-Mayer C, and Peterlin BM
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, CD8-Positive T-Lymphocytes chemistry, Gene Products, nef analysis, Humans, Hybrid Cells physiology, Intracellular Fluid chemistry, Lymphocyte Activation, Molecular Sequence Data, NF-kappa B metabolism, Recombinant Fusion Proteins analysis, Tumor Cells, Cultured chemistry, Tumor Cells, Cultured metabolism, Viral Fusion Proteins analysis, nef Gene Products, Human Immunodeficiency Virus, Genes, nef immunology, HIV-1 genetics, T-Lymphocytes immunology
- Abstract
Nef of primate lentiviruses is required for viremia and progression to AIDS in monkeys. Negative, positive, and no effects of Nef have also been reported on viral replication in cells. To reconcile these observations, we expressed a hybrid CD8-Nef protein in Jurkat cells. Two opposite phenotypes were found, which depended on the intracellular localization of Nef. Expressed in the cytoplasm or on the cell surface, the chimera inhibited or activated early signaling events from the T cell antigen receptor. Activated Jurkat cells died by apoptosis, and only cells with mutated nef genes expressing truncated Nefs survived, which rendered Nef nonfunctional. These mutations paralleled those in other viral strains passaged in vitro. Not only do these positional effects of Nef reconcile diverse phenotypes of Nef and suggest a role for its N-terminal myristylation, but they also explain effects of Nef in HIV infection and progression to AIDS.
- Published
- 1994
- Full Text
- View/download PDF
48. Platelet-derived growth factor receptor mediates activation of ras through different signaling pathways in different cell types.
- Author
-
Satoh T, Fantl WJ, Escobedo JA, Williams LT, and Kaziro Y
- Subjects
- Animals, B-Lymphocytes, CHO Cells, Calcimycin pharmacology, Cell Line, Cricetinae, GTPase-Activating Proteins, Guanosine Diphosphate metabolism, Guanosine Triphosphate metabolism, Humans, Interleukin-3 pharmacology, Kinetics, Mice, Phosphatidylinositol 3-Kinases, Receptors, Platelet-Derived Growth Factor genetics, Recombinant Proteins metabolism, Recombinant Proteins pharmacology, Tetradecanoylphorbol Acetate pharmacology, Transfection, ras GTPase-Activating Proteins, Phosphotransferases metabolism, Platelet-Derived Growth Factor pharmacology, Proteins metabolism, Proto-Oncogene Proteins p21(ras) metabolism, Receptors, Platelet-Derived Growth Factor physiology, Signal Transduction, Type C Phospholipases metabolism
- Abstract
A series of pieces of evidence have shown that Ras protein acts as a transducer of the platelet-derived growth factor (PDGF) receptor-mediated signaling pathway: (i) formation of Ras.GTP is detected immediately on PDGF stimulation, and (ii) a dominant inhibitory mutant Ras, as well as a neutralizing anti-Ras antibody, can interfere with PDGF-induced responses. On the other hand, several signal transducing molecules including phosphatidylinositol 3-kinase (PI3-K), GTPase-activating protein (GAP), and phospholipase C gamma (PLC gamma) bind directly to the PDGF receptor and become tyrosine phosphorylated. Recently, it was shown that specific phosphorylated tyrosines of the PDGF receptor are responsible for interaction between the receptor and each signaling molecule. However, the roles of these signaling molecules have not been elucidated, and it remains unclear which molecules are implicated in the Ras pathway. In this study, we measured Ras activation in cell lines expressing mutant PDGF receptors that are deficient in coupling with specific molecules. In fibroblast CHO cells, a mutant receptor (Y708F/Y719F [PI3-K-binding sites]) was unable to stimulate Ras, whereas another mutant (Y739F [the GAP-binding site]) could do so, suggesting an indispensable role of PI3-K or a protein that binds to the same sites as PI3-K for PDGF-stimulated Ras activation. By contrast, both of the above mutants were capable of stimulating Ras protein in a pro-B-cell line, BaF3. Furthermore, a mutant receptor (Y977F/Y989F [PLC gamma-binding sites]) could fully activate Ras, and the direct activation of protein kinase C and calcium mobilization had almost no effect on the GDP/GTP state of Ras in this cell line. These results suggest that, in the pro-B-cell transfectants, each of the above pathways (PI3-K, GAP, and PLC gamma) can be eliminated without a loss of Ras activation. It remains unclear whether another unknown essential pathway which regulates Ras protein exists within BaF3 cells. Therefore, it is likely that several different PDGF receptor-mediated signaling pathways function upstream of Ras, and the extent of the contribution of each pathway for the regulation of Ras may differ among different cell types.
- Published
- 1993
- Full Text
- View/download PDF
49. Signalling by receptor tyrosine kinases.
- Author
-
Fantl WJ, Johnson DE, and Williams LT
- Subjects
- Animals, Growth Substances metabolism, Mutagenesis, Phosphotyrosine, Receptors, Cell Surface genetics, Receptors, Cell Surface physiology, Signal Transduction genetics, Tyrosine analogs & derivatives, Tyrosine metabolism, Protein-Tyrosine Kinases physiology, Signal Transduction physiology
- Published
- 1993
- Full Text
- View/download PDF
50. Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways.
- Author
-
Fantl WJ, Escobedo JA, Martin GA, Turck CW, del Rosario M, McCormick F, and Williams LT
- Subjects
- 3T3 Cells, Amino Acid Sequence, Animals, Binding Sites, CHO Cells, Cell Division, Cells, Cultured, Cricetinae, DNA Mutational Analysis, GTP Phosphohydrolases metabolism, GTPase-Activating Proteins, In Vitro Techniques, Mice, Molecular Sequence Data, Peptide Mapping, Peptides chemistry, Peptides metabolism, Phosphatidylinositol 3-Kinases, Phosphotyrosine, Receptors, Cell Surface chemistry, Receptors, Platelet-Derived Growth Factor, Signal Transduction, Structure-Activity Relationship, Tyrosine metabolism, Phosphotransferases metabolism, Platelet-Derived Growth Factor physiology, Proteins metabolism, Receptors, Cell Surface metabolism, Tyrosine analogs & derivatives
- Abstract
The receptor for platelet-derived growth factor (PDGF) binds two proteins containing SH2 domains, GTPase activating protein (GAP) and phosphatidylinositol 3-kinase (PI3-kinase). The sites on the receptor that mediate this interaction were identified by using phosphotyrosine-containing peptides representing receptor sequences to block specifically binding of either PI3-kinase or GAP. These results suggested that PI3-kinase binds two phosphotyrosine residues, each located in a 5 aa motif with an essential methionine at the fourth position C-terminal to the tyrosine. Point mutations at these sites caused a selective elimination of PI3-kinase binding and loss of PDGF-stimulated DNA synthesis. Mutation of the binding site for GAP prevented the receptor from associating with or phosphorylating GAP, but had no effect on PI3-kinase binding and little effect on DNA synthesis. Therefore, GAP and PI3-kinase interact with the receptor by binding to different phosphotyrosine-containing sequence motifs.
- Published
- 1992
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.