László Homolya

HUN-REN Research Centre for Natural Sciences

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Balázs Sarkadi

HUN-REN Research Centre for Natural Sciences

ÀA

Àgota Apàti

HUN-REN Research Centre for Natural Sciences

Tamás I. Orbán

Eötvös Loránd University

János Réthelyi

Semmelweis University

György Várady

HUN-REN Research Centre for Natural Sciences

Zsuzsa Erdei

HUN-REN Research Centre for Natural Sciences

György Török

Semmelweis University

Zoltán Hegyi

University of Debrecen

Edit Hathy

Semmelweis University

András Váradi

Hungarian Research Network

Cooperative Institutions

Hungarian Academy of Sciences

164

Semmelweis University

138

HUN-REN Research Centre for Natural Sciences

Eötvös Loránd University

Institute of Molecular Life Sciences

Hungarian Research Network

University of Debrecen

University of Szeged

National Institute of Oncology

Magyar Agrár- és Élettudományi Egyetem

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Characterization of Human Neural Progenitor Cells and Neurons Differentiated from Induced Pluripotent Stem Cells

The FASEB Journal (2018)

Àgota Apàti Tünde Berecz Eszter Szabó Tamás I. Orbán László Homolya

Most studies focusing on neural development and the mechanism of neurodevelopmental disorders are based on animal (rodent or avian) models or post mortem human samples. Thus, the observations derived from these studies cannot be directly translated into mechanistic insights of human neural development and regeneration. Human pluripotent stem cells, which can potentially be differentiated into any cell types of the body, offer a new perspective to overcome this limitation. In the present study, we differentiated induced pluripotent stem cell lines toward the neural lineage. Using a multi‐step, directed differentiation protocol, specific human neural progenitor cells (NPCs) were generated which are committed to produce PROX‐1‐positive granule cells of dentate gyrus of the hippocampus. These NPCs were characterized in terms of morphology, expression of specific cellular markers, growth capacity, and capability of producing mature neurons. Next we investigated the calcium signals in NPCs and PROX1‐positive hippocampal neurons, expressing GCaMP6fast calcium indicator protein or loaded with Fluo4 calcium sensitive dye, in parallel experiments. We demonstrated the functional presence of several receptors, and also the lack of functioning sodium channels in NPCs, and spontaneous Ca 2+ gradients in neurons by both methods. We found that spontaneous calcium gradients and specific, ligand induced calcium signals were not significantly altered in the GCaMP6f expressing cells, suggesting the applicability of GCaMP6f for the investigation of fast calcium signals in neural cultures. We conclude that the characteristics of both the spontaneous and ligand‐induced Ca 2+ ‐signals, as well as their pharmacological modulation can be successfully examined in these human model cells by fluorescence imaging. Support or Funding Information This study is funded by the National Brain Research Program (NAP) of Hungary (grant numbers: KTIA_NAP_13‐1‐2013‐0001 to LH and KTIA_NAP_13‐2014‐0011 to JR), and the Momentum Program of the Hungarian Academy of Sciences (LP2012‐025 to LH). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Calcium imaging

Cell type

10.1096/fasebj.2018.32.1_supplement.615.5

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Citations (0)

Fluorescence Assay for Studying P-Glycoprotein Function at Single Cell Level

Springer eBooks (1996)

László Homolya Marianna Müller Zsolt Holló Balázs Sarkadi

Calcein

P-glycoprotein

Rhodamine 123

10.1007/978-1-4899-1866-6_36

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Interaction of bioactive hydrophobic peptides with the human multidrug transporter

The FASEB Journal (1994)

Balázs Sarkadi Marianna Müller László Homolya Zsolt Holló János Seprődi

In this report we demonstrate that various biologically active hydrophobic peptide derivatives, e.g., proteinase inhibitors, chemoattractants, ionophores, enkephalins, and immunosuppressants, stimulate a membrane ATPase activity associated with the human multidrug transporter (MDR1). The stimulation of the MDR1-ATPase by these agents does not correlate with their known biochemical or pharmacological activities but rather with their hydrophobicity. The peptides that show high-affinity interaction with the MDR1-ATPase also interfere strongly with fluorescent dye extrusion catalyzed by the multidrug transporter in intact cells and some have been shown to reverse drug resistance in cultured cells. These data suggest that several hydrophobic peptides behave as substrates of the multidrug transporter and may be used to modulate the chemotherapy resistance of tumor cells.—Sarkadi, B., Müller, M., Homolya, L., Holló, Z., Seprödi, J., Germann, U. A., Gottesman, M. M., Price, E. M., Boucher, R. C. Interaction of bioactive hydrophobic peptides with the human multidrug transporter. FASEB J. 8: 766-770; 1994.

Oligopeptide

10.1096/fasebj.8.10.7914178

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Citations (90)

Nucleotide binding is the critical regulator of ABCG2 conformational transitions

bioRxiv (Cold Spring Harbor Laboratory) (2022)

Zsuzsanna Gyöngy Gábor Mocsár Éva Hegedüs Thomas Stockner Zsuzsanna Ritter

Abstract ABCG2 is an exporter type ABC protein that can expel numerous chemically unrelated xeno- and endobiotics from cells. When expressed in tumor cells or tumor stem cells, ABCG2 confers multidrug resistance, contributing to the failure of chemotherapy. Molecular details orchestrating substrate translocation and ATP hydrolysis remain elusive. Here we present methods to concomitantly investigate substrate and nucleotide binding by ABCG2 in cells. Using the conformation-sensitive antibody 5D3, we show that the switch from the inward-facing (IF) to the outward-facing (OF) conformation of ABCG2 is induced by nucleotide binding. IF-OF transition is facilitated by substrates, and hindered by the inhibitor Ko143. Direct measurements of 5D3 and substrate binding to ABCG2 indicate that the high-to-low affinity switch of the drug binding site coincides with the transition from the IF to the OF conformation. Low substrate binding persists in the post-hydrolysis state, supporting that dissociation of the ATP hydrolysis products is required to reset the high substrate affinity IF conformation of ABCG2.

Abcg2

Conformational change

10.1101/2022.10.19.512890

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Effect of Loss of P2Y 2 Receptor Gene Expression on Nucleotide Regulation of Murine Epithelial Cl − Transport

Carolina Digital Repository (University of North Carolina at Chapel Hill) (1999)

Victoria L. Cressman Eduardo R. Lazarowski László Homolya Richard C. Boucher Beverly H. Koller

P2Y receptor

10.17615/1qrc-3z19

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Application of a Human Multidrug Transporter (ABCG2) Variant as Selectable Marker in Gene Transfer to Progenitor Cells

Human Gene Therapy (2003)

Olga Ujhelly Csilla Özvegy György Várady Judit Cervenak László Homolya

Abcg2

Efflux

George (robot)

10.1089/104303403321209005

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Citations (44)

Fluorescent cellular indicators are extruded by the multidrug resistance protein.

Journal of Biological Chemistry (1993)

László Homolya Zsolt Holló Ursula A. Germann I Pastan M M Gottesman

In this report we show that NIH-3T3 mouse fibroblasts stably expressing the human multidrug transporter (MDR1 or P-glycoprotein), in contrast to the control NIH-3T3 cells, actively extrude the hydrophobic acetoxymethyl ester (AM) derivatives used for cellular loading of various fluorescent calcium and pH indicators. This dye extrusion is blocked by competing substrates and inhibitors of the multidrug transporters, e.g. by verapamil, vincristine, sodium orthovanadate, oligomycin, and a monoclonal anti-MDR1 antibody. The hydrophilic free acid forms of the indicators are not exported by MDR1. We also demonstrate that in isolated cell membranes the MDR1-ATPase, similar to that by known substrates of the transporter, is stimulated by the AM derivatives of fluorescent dyes whereas the free acid forms of the dyes are without effect. Since (i) the AM derivatives of the fluorescent indicators rapidly permeate the cell membrane and are readily cleaved by high activity and large capacity cytoplasmic esterases and (ii) the free acid forms are not substrates for export by MDR1, the observations above suggest that dye extrusion by MDR1 may occur without a cytoplasmic appearance of the AM compounds. These data also call attention to the possible interaction of widely used hydrophobic fluorescent indicators with MDR1 and offer an efficient detection of MDR1-expressing tumor cells as well as a screening method for examining drug interactions with the multidrug transporter.

Fluorescent protein

10.1016/s0021-9258(20)80566-3

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Citations (498)

Nucleotide binding is the critical regulator of ABCG2 conformational transitions

eLife (2023)

Zsuzsanna Gyöngy Gábor Mocsár Éva Hegedüs Thomas Stockner Zsuzsanna Ritter

ABCG2 is an exporter-type ABC protein that can expel numerous chemically unrelated xeno- and endobiotics from cells. When expressed in tumor cells or tumor stem cells, ABCG2 confers multidrug resistance, contributing to the failure of chemotherapy. Molecular details orchestrating substrate translocation and ATP hydrolysis remain elusive. Here, we present methods to concomitantly investigate substrate and nucleotide binding by ABCG2 in cells. Using the conformation-sensitive antibody 5D3, we show that the switch from the inward-facing (IF) to the outward-facing (OF) conformation of ABCG2 is induced by nucleotide binding. IF-OF transition is facilitated by substrates, and hindered by the inhibitor Ko143. Direct measurements of 5D3 and substrate binding to ABCG2 indicate that the high-to-low affinity switch of the drug binding site coincides with the transition from the IF to the OF conformation. Low substrate binding persists in the post-hydrolysis state, supporting that dissociation of the ATP hydrolysis products is required to reset the high substrate affinity IF conformation of ABCG2.

Abcg2

Conformational change

10.7554/elife.83976

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Citations (10)

Medically Important Alterations in Transport Function and Trafficking of ABCG2

International Journal of Molecular Sciences (2021)

László Homolya

Several polymorphisms and mutations in the human ABCG2 multidrug transporter result in reduced plasma membrane expression and/or diminished transport function. Since ABCG2 plays a pivotal role in uric acid clearance, its malfunction may lead to hyperuricemia and gout. On the other hand, ABCG2 residing in various barrier tissues is involved in the innate defense mechanisms of the body; thus, genetic alterations in ABCG2 may modify the absorption, distribution, excretion of potentially toxic endo- and exogenous substances. In turn, this can lead either to altered therapy responses or to drug-related toxic reactions. This paper reviews the various types of mutations and polymorphisms in ABCG2, as well as the ways how altered cellular processing, trafficking, and transport activity of the protein can contribute to phenotypic manifestations. In addition, the various methods used for the identification of the impairments in ABCG2 variants and the different approaches to correct these defects are overviewed.

Abcg2

10.3390/ijms22062786

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Citations (21)

Characterization of NF-κB Reporter U937 Cells and Their Application for the Detection of Inflammatory Immune-Complexes

PLoS ONE (2016)

Csilla Kecse-Nagy Zoltán Szittner Krisztián Papp Zoltán Hegyi Paolo Rovero

Our study tested the hypothesis that immunoglobulins differ in their ability to activate the nuclear factor-κB pathway mediated cellular responses. These responses are modulated by several properties of the immune complex, including the ratio of antibody isotypes binding to antigen. Immunoassays allow the measurement of antigen specific antibodies belonging to distinct immunoglobulin classes and subclasses but not the net biological effect of the combination of these antibodies. We set out to develop a biosensor that is suitable for the detection and characterization of antigen specific serum antibodies. We genetically modified the monocytoid U937 cell line carrying Fc receptors with a plasmid encoding NF-κB promoter-driven GFP. This clone, U937-NF-κB, was characterized with respect to FcR expression and response to solid-phase immunoglobulins. Human IgG3, IgG4 and IgG1 induced GFP production in a time- and dose-dependent manner, in this order of efficacy, while IgG2 triggered no activation at the concentrations tested. IgA elicited no response alone but showed significant synergism with IgG3 and IgG4. We confirmed the importance of activation via FcγRI by direct stimulation with monoclonal antibody and by competition assays. We used citrullinated peptides and serum from rheumatoid arthritis patients to generate immune complexes and to study the activation of U937-NF-κB, observing again a synergistic effect between IgG and IgA. Our results show that immunoglobulins have distinct pro-inflammatory potential, and that U937-NF-κB is suitable for the estimation of biological effects of immune-complexes, offering insight into monocyte activation and pathogenesis of antibody mediated diseases.

U937 cell

Monocyte

10.1371/journal.pone.0156328

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Citations (15)