Publications by authors named "László Homolya"

60 Publications

Generation of Human Neural Progenitors from Blood Samples by Interrupted Reprogramming.

Methods Mol Biol 2021 Apr 8. Epub 2021 Apr 8.

Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary.

Human neuronal cell cultures are essential tools for biological and preclinical studies of our nervous system. Since we have very limited access to primary human neural samples, derivation of proliferative neural progenitor cells (NPCs) from cells harvested by minimally invasive sampling is a key issue. Here we describe a "shortcut" method to establish proliferative NPC cultures directly from peripheral blood mononuclear cells (PBMCs) via interrupted reprogramming. In addition, we provide procedures to characterize the NPC stage.
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http://dx.doi.org/10.1007/7651_2021_386DOI Listing
April 2021

The Plasma Membrane Ca Pump PMCA4b Regulates Melanoma Cell Migration through Remodeling of the Actin Cytoskeleton.

Cancers (Basel) 2021 Mar 17;13(6). Epub 2021 Mar 17.

Department of Transfusiology, Semmelweis University, H-1089 Budapest, Hungary.

We demonstrated that the plasma membrane Ca ATPase PMCA4b inhibits migration and metastatic activity of BRAF mutant melanoma cells. Actin dynamics are essential for cells to move, invade and metastasize, therefore, we hypothesized that PMCA4b affected cell migration through remodeling of the actin cytoskeleton. We found that expression of PMCA4b in A375 BRAF mutant melanoma cells induced a profound change in cell shape, cell culture morphology, and displayed a polarized migratory character. Along with these changes the cells became more rounded with increased cell-cell connections, lamellipodia and stress fiber formation. Silencing PMCA4b in MCF-7 breast cancer cells had a similar effect, resulting in a dramatic loss of stress fibers. In addition, the PMCA4b expressing A375 cells maintained front-to-rear Ca concentration gradient with the actin severing protein cofilin localizing to the lamellipodia, and preserved the integrity of the actin cytoskeleton from a destructive Ca overload. We showed that both PMCA4b activity and trafficking were essential for the observed morphology and motility changes. In conclusion, our data suggest that PMCA4b plays a critical role in adopting front-to-rear polarity in a normally spindle-shaped cell type through F-actin rearrangement resulting in a less aggressive melanoma cell phenotype.
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http://dx.doi.org/10.3390/cancers13061354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8002435PMC
March 2021

Medically Important Alterations in Transport Function and Trafficking of ABCG2.

Authors:
László Homolya

Int J Mol Sci 2021 Mar 10;22(6). Epub 2021 Mar 10.

Research Centre for Natural Sciences, Institute of Enzymology, H-1117 Budapest, Hungary.

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 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 , 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.
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http://dx.doi.org/10.3390/ijms22062786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001156PMC
March 2021

Identification of Specific Trafficking Defects of Naturally Occurring Variants of the Human ABCG2 Transporter.

Front Cell Dev Biol 2021 9;9:615729. Epub 2021 Feb 9.

Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences Centre of Excellence, Budapest, Hungary.

Proper targeting of the urate and xenobiotic transporter ATP-binding transporter subfamily G member 2 (ABCG2) to the plasma membrane (PM) is essential for its normal function. The naturally occurring Q141K and M71V polymorphisms in ABCG2, associated with gout and hyperuricemia, affect the cellular routing of the transporter, rather than its transport function. The cellular localization of ABCG2 variants was formerly studied by immunolabeling, which provides information only on the steady-state distribution of the protein, leaving the dynamics of its cellular routing unexplored. In the present study, we assessed in detail the trafficking of the wild-type, M71V-, and Q141K-ABCG2 variants from the endoplasmic reticulum (ER) to the cell surface using a dynamic approach, the so-called Retention Using Selective Hooks (RUSH) system. This method also allowed us to study the kinetics of glycosylation of these variants. We found that the fraction of Q141K- and M71V-ABCG2 that passes the ER quality control system is only partially targeted to the PM; a subfraction is immobile and retained in the ER. Surprisingly, the transit of these variants through the Golgi apparatus (either the appearance or the exit) was unaffected; however, their PM delivery beyond the Golgi was delayed. In addition to identifying the specific defects in the trafficking of these ABCG2 variants, our study provides a novel experimental tool for studying the effect of drugs that potentially promote the cell surface delivery of mutant or polymorphic ABCG2 variants with impaired trafficking.
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http://dx.doi.org/10.3389/fcell.2021.615729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900420PMC
February 2021

Identification of Neuronal Pentraxins as Synaptic Binding Partners of C1q and the Involvement of NP1 in Synaptic Pruning in Adult Mice.

Front Immunol 2020 8;11:599771. Epub 2021 Feb 8.

ELTE NAP Neuroimmunology Research Group, Department of Biochemistry, Institute of Biology, ELTE Eötvös Loránd University, Budapest, Hungary.

Elements of the immune system particularly that of innate immunity, play important roles beyond their traditional tasks in host defense, including manifold roles in the nervous system. Complement-mediated synaptic pruning is essential in the developing and healthy functioning brain and becomes aberrant in neurodegenerative disorders. C1q, component of the classical complement pathway, plays a central role in tagging synapses for elimination; however, the underlying molecular mechanisms and interaction partners are mostly unknown. Neuronal pentraxins (NPs) are involved in synapse formation and plasticity, moreover, NP1 contributes to cell death and neurodegeneration under adverse conditions. Here, we investigated the potential interaction between C1q and NPs, and its role in microglial phagocytosis of synapses in adult mice. We verified that NPs interact with C1q, as well as activate the complement system. Flow cytometry, immunostaining and co-immunoprecipitation showed that synapse-bound C1q colocalizes and interacts with NPs. High-resolution confocal microscopy revealed that microglia-surrounded C1q-tagged synapses are NP1 positive. We have also observed the synaptic occurrence of C4 suggesting that activation of the classical pathway cannot be ruled out in synaptic plasticity in healthy adult animals. In summary, our results indicate that NPs play a regulatory role in the synaptic function of C1q. Whether this role can be intensified upon pathological conditions, such as in Alzheimer's disease, is to be disclosed.
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http://dx.doi.org/10.3389/fimmu.2020.599771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897678PMC
February 2021

Generation of multiple iPSC clones from a male schizophrenia patient carrying de novo mutations in genes KHSRP, LRRC7, and KIR2DL1, and his parents.

Stem Cell Res 2021 Mar 25;51:102140. Epub 2020 Dec 25.

Molecular Psychiatry Research Group, National Brain Research Program (NAP), Hungarian Academy of Sciences and Semmelweis University, Hungary; Department of Psychiatry and Psychotherapy, Semmelweis University, Budapest, Hungary. Electronic address:

Here we describe the generation of induced pluripotent stem cell lines from each member - male proband, mother, father - of a schizophrenia case-parent trio that participated in an exome sequencing study, and 3 de novo mutations were identified in the proband. Peripheral blood mononuclear cells were obtained from all three individuals and reprogrammed using Sendai virus particles carrying the Yamanaka transgenes. These 3 iPSC lines (iPSC-SZ-HU-MO 1, iPSC-SZ-HU-FA 1, and iPSC-SZ-HU-PROB 1) represent a resource for examining the functional significance of the identified de novo mutations in the molecular pathophysiology of schizophrenia.
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http://dx.doi.org/10.1016/j.scr.2020.102140DOI Listing
March 2021

Large Stokes-shift bioorthogonal probes for STED, 2P-STED and multi-color STED nanoscopy.

Methods Appl Fluoresc 2021 Jan 9;9(1):015006. Epub 2021 Jan 9.

Chemical Biology Research Group, Institute of Organic Chemistry, Research Centre for Natural Sciences, Magyar tudósok krt. 2., H-1117 Budapest, Hungary. Department of Biophysics and Radiation Biology, Semmelweis University, Tűzoltó u. 37-47., H-1094 Budapest, Hungary. Laboratory of Molecular Cell Biology, Institute of Enzymology, Research Centre for Natural Sciences, Magyar tudósok krt. 2., H-1117 Budapest, Hungary.

Synthesis and multiple STED imaging applications of four, red-emitting (610-670 nm), tetrazine-functionalized fluorescent probes (CBRD = Chemical Biology Research group Dye 1-4) with large Stokes-shift is presented. Present studies revealed the super-resolution microscopy applicability of the probes as demonstrated through bioorthogonal labeling scheme of cytoskeletal proteins actin and keratin-19, and mitochondrial protein TOMM20. Furthermore, super-resolved images of insulin receptors in live-cell bioorthogonal labeling schemes through a genetically encoded cyclooctynylated non-canonical amino acid are also presented. The large Stokes-shifts and the wide spectral bands of the probes enabled the use of two common depletion lasers (660 nm and 775 nm). The probes were also found suitable for super-resolution microscopy in combination with two-photon excitation (2P-STED) resulting in improved spatial resolution. One of the dyes was also used together with two commercial dyes in the three-color STED imaging of intracellular structures.
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http://dx.doi.org/10.1088/2050-6120/abb363DOI Listing
January 2021

Functional Comparison of Blood-Derived Human Neural Progenitor Cells.

Int J Mol Sci 2020 Nov 30;21(23). Epub 2020 Nov 30.

Institute of Enzymology, Research Centre for Natural Sciences, 1117 Budapest, Hungary.

Induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) are promising tools to model complex neurological or psychiatric diseases, including schizophrenia. Multiple studies have compared patient-derived and healthy control NPCs derived from iPSCs in order to investigate cellular phenotypes of this disease, although the establishment, stabilization, and directed differentiation of iPSC lines are rather expensive and time-demanding. However, interrupted reprogramming by omitting the stabilization of iPSCs may allow for the generation of a plastic stage of the cells and thus provide a shortcut to derive NPSCs directly from tissue samples. Here, we demonstrate a method to generate shortcut NPCs (sNPCs) from blood mononuclear cells and present a detailed comparison of these sNPCs with NPCs obtained from the same blood samples through stable iPSC clones and a subsequent neural differentiation (classical NPCs-cNPCs). Peripheral blood cells were obtained from a schizophrenia patient and his two healthy parents (a case-parent trio), while a further umbilical cord blood sample was obtained from the cord of a healthy new-born. The expression of stage-specific markers in sNPCs and cNPCs were compared both at the protein and RNA levels. We also performed functional tests to investigate Wnt and glutamate signaling and the oxidative stress, as these pathways have been suggested to play important roles in the pathophysiology of schizophrenia. We found similar responses in the two types of NPCs, suggesting that the shortcut procedure provides sNPCs, allowing an efficient screening of disease-related phenotypes.
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http://dx.doi.org/10.3390/ijms21239118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730078PMC
November 2020

Investigation of de novo mutations in a schizophrenia case-parent trio by induced pluripotent stem cell-based in vitro disease modeling: convergence of schizophrenia- and autism-related cellular phenotypes.

Stem Cell Res Ther 2020 11 27;11(1):504. Epub 2020 Nov 27.

National Brain Research Project (NAP) Molecular Psychiatry Research Group, Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary.

Background: De novo mutations (DNMs) have been implicated in the etiology of schizophrenia (SZ), a chronic debilitating psychiatric disorder characterized by hallucinations, delusions, cognitive dysfunction, and decreased community functioning. Several DNMs have been identified by examining SZ cases and their unaffected parents; however, in most cases, the biological significance of these mutations remains elusive. To overcome this limitation, we have developed an approach of using induced pluripotent stem cell (iPSC) lines from each member of a SZ case-parent trio, in order to investigate the effects of DNMs in cellular progenies of interest, particularly in dentate gyrus neuronal progenitors.

Methods: We identified a male SZ patient characterized by early disease onset and negative symptoms, who is a carrier of 3 non-synonymous DNMs in genes LRRC7, KHSRP, and KIR2DL1. iPSC lines were generated from his and his parents' peripheral blood mononuclear cells using Sendai virus-based reprogramming and differentiated into neuronal progenitor cells (NPCs) and hippocampal dentate gyrus granule cells. We used RNASeq to explore transcriptomic differences and calcium (Ca) imaging, cell proliferation, migration, oxidative stress, and mitochondrial assays to characterize the investigated NPC lines.

Results: NPCs derived from the SZ patient exhibited transcriptomic differences related to Wnt signaling, neuronal differentiation, axonal guidance and synaptic function, and decreased Ca reactivity to glutamate. Moreover, we could observe increased cellular proliferation and alterations in mitochondrial quantity and morphology.

Conclusions: The approach of reprograming case-parent trios represents an opportunity for investigating the molecular effects of disease-causing mutations and comparing these in cell lines with reduced variation in genetic background. Our results are indicative of a partial overlap between schizophrenia and autism-related phenotypes in the investigated family.

Limitations: Our study investigated only one family; therefore, the generalizability of findings is limited. We could not derive iPSCs from two other siblings to test for possible genetic effects in the family that are not driven by DNMs. The transcriptomic and functional assays were limited to the NPC stage, although these variables should also be investigated at the mature neuronal stage.
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http://dx.doi.org/10.1186/s13287-020-01980-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694414PMC
November 2020

Syndecan-4 Modulates Cell Polarity and Migration by Influencing Centrosome Positioning and Intracellular Calcium Distribution.

Front Cell Dev Biol 2020 15;8:575227. Epub 2020 Oct 15.

Department of Biochemistry, Faculty of Medicine, University of Szeged, Szeged, Hungary.

Efficient cell migration requires cellular polarization, which is characterized by the formation of leading and trailing edges, appropriate positioning of the nucleus and reorientation of the Golgi apparatus and centrosomes toward the leading edge. Migration also requires the development of an asymmetrical front-to-rear calcium (Ca) gradient to regulate focal adhesion assembly and actomyosin contractility. Here we demonstrate that silencing of syndecan-4, a transmembrane heparan sulfate proteoglycan, interferes with the correct polarization of migrating mammalian myoblasts (i.e., activated satellite stem cells). In particular, syndecan-4 knockdown completely abolished the intracellular Ca gradient, abrogated centrosome reorientation and thus decreased cell motility, demonstrating the role of syndecan-4 in cell polarity. Additionally, syndecan-4 exhibited a polarized distribution during migration. Syndecan-4 knockdown cells exhibited decreases in the total movement distance during directional migration, maximum and vectorial distances from the starting point, as well as average and maximum cell speeds. Super-resolution direct stochastic optical reconstruction microscopy images of syndecan-4 knockdown cells revealed nanoscale changes in the actin cytoskeletal architecture, such as decreases in the numbers of branches and individual branch lengths in the lamellipodia of the migrating cells. Given the crucial importance of myoblast migration during embryonic development and postnatal muscle regeneration, we conclude that our results could facilitate an understanding of these processes and the general role of syndecan-4 during cell migration.
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http://dx.doi.org/10.3389/fcell.2020.575227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593626PMC
October 2020

The ABCG2/BCRP transporter and its variants - from structure to pathology.

FEBS Lett 2020 Dec 16;594(23):4012-4034. Epub 2020 Oct 16.

Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary.

The ABCG2 protein has a key role in the transport of a wide range of structurally dissimilar endo- and xenobiotics in the human body, especially in the tissue barriers and the metabolizing or secreting organs. The human ABCG2 gene harbors a high number of polymorphisms and mutations, which may significantly modulate its expression and function. Recent high-resolution structural data, complemented with molecular dynamic simulations, may significantly help to understand intramolecular movements and substrate handling, as well as the effects of mutations on the membrane transporter function of ABCG2. As reviewed here, structural alterations may result not only in direct alterations in drug binding and transporter activity, but also in improper folding or problems in the carefully regulated process of trafficking, including vesicular transport, endocytosis, recycling, and degradation. Here, we also review the clinical importance of altered ABCG2 expression and function in general drug metabolism, cancer multidrug resistance, and impaired uric acid excretion, leading to gout.
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http://dx.doi.org/10.1002/1873-3468.13947DOI Listing
December 2020

Anionic food color tartrazine enhances antibacterial efficacy of histatin-derived peptide DHVAR4 by fine-tuning its membrane activity.

Q Rev Biophys 2020 03 2;53:e5. Epub 2020 Mar 2.

Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, H-1117, Hungary.

Here it is demonstrated how some anionic food additives commonly used in our diet, such as tartrazine (TZ), bind to DHVAR4, an antimicrobial peptide (AMP) derived from oral host defense peptides, resulting in significantly fostered toxic activity against both Gram-positive and Gram-negative bacteria, but not against mammalian cells. Biophysical studies on the DHVAR4-TZ interaction indicate that initially large, positively charged aggregates are formed, but in the presence of lipid bilayers, they rather associate with the membrane surface. In contrast to synergistic effects observed for mixed antibacterial compounds, this is a principally different mechanism, where TZ directly acts on the membrane-associated AMP promoting its biologically active helical conformation. Model vesicle studies show that compared to dye-free DHVAR4, peptide-TZ complexes are more prone to form H-bonds with the phosphate ester moiety of the bilayer head-group region resulting in more controlled bilayer fusion mechanism and concerted severe cell damage. AMPs are considered as promising compounds to combat formidable antibiotic-resistant bacterial infections; however, we know very little on their in vivo actions, especially on how they interact with other chemical agents. The current example illustrates how food dyes can modulate AMP activity, which is hoped to inspire improved therapies against microbial infections in the alimentary tract. Results also imply that the structure and function of natural AMPs could be manipulated by small compounds, which may also offer a new strategic concept for the future design of peptide-based antimicrobials.
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http://dx.doi.org/10.1017/S0033583520000013DOI Listing
March 2020

Synaptic mitochondrial dysfunction and septin accumulation are linked to complement-mediated synapse loss in an Alzheimer's disease animal model.

Cell Mol Life Sci 2020 Dec 7;77(24):5243-5258. Epub 2020 Feb 7.

ELTE NAP Neuroimmunology Research Group, Department of Biochemistry, Institute of Biology, ELTE Eötvös Loránd University, Budapest, Hungary.

Synaptic functional disturbances with concomitant synapse loss represent central pathological hallmarks of Alzheimer's disease. Excessive accumulation of cytotoxic amyloid oligomers is widely recognized as a key event that underlies neurodegeneration. Certain complement components are crucial instruments of widespread synapse loss because they can tag synapses with functional impairments leading to their engulfment by microglia. However, an exact understanding of the affected synaptic functions that predispose to complement-mediated synapse elimination is lacking. Therefore, we conducted systematic proteomic examinations on synaptosomes prepared from an amyloidogenic mouse model of Alzheimer's disease (APP/PS1). Synaptic fractions were separated according to the presence of the C1q-tag using fluorescence-activated synaptosome sorting and subjected to proteomic comparisons. The results raised the decline of mitochondrial functions in the C1q-tagged synapses of APP/PS1 mice based on enrichment analyses, which was verified using flow cytometry. Additionally, proteomics results revealed extensive alterations in the level of septin protein family members, which are known to dynamically form highly organized pre- and postsynaptic supramolecular structures, thereby affecting synaptic transmission. High-resolution microscopy investigations demonstrated that synapses with considerable amounts of septin-3 and septin-5 show increased accumulation of C1q in APP/PS1 mice compared to the wild-type ones. Moreover, a strong positive correlation was apparent between synaptic septin-3 levels and C1q deposition as revealed via flow cytometry and confocal microscopy examinations. In sum, our results imply that deterioration of synaptic mitochondrial functions and alterations in the organization of synaptic septins are associated with complement-dependent synapse loss in Alzheimer's disease.
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http://dx.doi.org/10.1007/s00018-020-03468-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7671981PMC
December 2020

The importance of transporters and cell polarization for the evaluation of human stem cell-derived hepatic cells.

PLoS One 2020 23;15(1):e0227751. Epub 2020 Jan 23.

Molecular Cell Biology Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences Centre of Excellence, Budapest, Hungary.

One of the most promising applications of human pluripotent stem cells is their utilization for human-based pharmacological models. Despite the fact that membrane transporters expressed in the liver play pivotal role in various hepatic functions, thus far only little attention was devoted to the membrane transporter composition of the stem cell-derived liver models. In the present work, we have differentiated HUES9, a human embryonic stem cell line, toward the hepatic lineage, and monitored the expression levels of numerous differentiation marker and liver transporter genes with special focus on ABC transporters. In addition, the effect of bile acid treatment and polarizing culturing conditions on hepatic maturation has been assessed. We found that most transporter genes crucial for hepatic functions are markedly induced during hepatic differentiation; however, as regards the transporter composition the end-stage cells still exhibited dual, hepatocyte and cholangiocyte character. Although the bile acid treatment and sandwich culturing only slightly influenced the gene expressions, the stimulated cell polarization resulted in formation of bile canaliculi and proper localization of transporters. Our results point to the importance of membrane transporters in human stem cell-derived hepatic models and demonstrate the relevance of cell polarization in generation of applicable cellular models with correctly localized transporters. On the basis of our observations we suggest that conventional criteria for the evaluation of the quality of stem cell-derived hepatocyte-like cells ought to be augmented with additional elements, such as polarized and functional expression of hepatic transporters.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0227751PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6977753PMC
April 2020

Cellular Processing of the ABCG2 Transporter-Potential Effects on Gout and Drug Metabolism.

Cells 2019 10 8;8(10). Epub 2019 Oct 8.

Institute of Enzymology, Research Centre for Natural Sciences, Magyar Tudosok krt. 2, 1117 Budapest, Hungary.

The human ABCG2 is an important plasma membrane multidrug transporter, involved in uric acid secretion, modulation of absorption of drugs, and in drug resistance of cancer cells. Variants of the ABCG2 transporter, affecting cellular processing and trafficking, have been shown to cause gout and increased drug toxicity. In this paper, we overview the key cellular pathways involved in the processing and trafficking of large membrane proteins, focusing on ABC transporters. We discuss the information available for disease-causing polymorphic variants and selected mutations of ABCG2, causing increased degradation and impaired travelling of the transporter to the plasma membrane. In addition, we provide a detailed in silico analysis of an as yet unrecognized loop region of the ABCG2 protein, in which a recently discovered mutation may actually promote ABCG2 membrane expression. We suggest that post-translational modifications in this unstructured loop at the cytoplasmic surface of the protein may have special influence on ABCG2 processing and trafficking.
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http://dx.doi.org/10.3390/cells8101215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6830335PMC
October 2019

Cellular expression and function of naturally occurring variants of the human ABCG2 multidrug transporter.

Cell Mol Life Sci 2020 Jan 28;77(2):365-378. Epub 2019 Jun 28.

Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar Tudosok krt. 2, Budapest, 1117, Hungary.

The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics; thus the relatively frequent polymorphic and mutant ABCG2 variants in the population may significantly alter disease conditions and pharmacological effects. Low-level or non-functional ABCG2 expression may increase individual drug toxicity, reduce cancer drug resistance, and result in hyperuricemia and gout. In the present work we have studied the cellular expression, trafficking, and function of nine naturally occurring polymorphic and mutant variants of ABCG2. A comprehensive analysis of the membrane localization, transport, and ATPase activity, as well as retention and degradation in intracellular compartments was performed. Among the examined variants, R147W and R383C showed expression and/or protein folding defects, indicating that they could indeed contribute to ABCG2 functional deficiency. These studies and the applied methods should significantly promote the exploration of the medical effects of these personal variants, promote potential therapies, and help to elucidate the specific role of the affected regions in the folding and function of the ABCG2 protein.
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http://dx.doi.org/10.1007/s00018-019-03186-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6971004PMC
January 2020

release of MVB-like small extracellular vesicle clusters by colorectal carcinoma cells.

J Extracell Vesicles 2019 8;8(1):1596668. Epub 2019 Apr 8.

Molecular Medicine Research Group, Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary.

Small extracellular vesicles (EVs) are membrane enclosed structures that are usually released from cells upon exocytosis of multivesicular bodies (MVBs) as a collection of separate, free EVs. In this study, we analysed paraffin embedded sections of archived human colorectal cancer samples. We studied 3D reconstructions of confocal microscopic images complemented by HyVolution and STED imaging. Unexpectedly, we found evidence that large, MVB-like aggregates of ALIX/CD63 positive EV clusters were released by migrating tumour cells. These structures were often captured with partial or complete extra-cytoplasmic localization at the interface of the plasma membrane of the tumour cell and the stroma. Their diameter ranged between 0.62 and 1.94 μm (mean±S.D.: 1.17 ± 0.34 μm). High-resolution 3D reconstruction showed that these extracellular MVB-like EV clusters were composed of distinguishable internal particles of small EV size (mean±S.D.: 128.96 ± 16.73 nm). , HT29 colorectal cancer cells also showed the release of similar structures as confirmed by immunohistochemistry and immune electron microscopy. Our results provide evidence for an transmission of MVB-like EV clusters through the plasma membrane. Immunofluorescent-based detection of the MVB like small EV clusters in archived pathological samples may represent a novel and unique opportunity which enables analysis of EV release in human tissues.
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http://dx.doi.org/10.1080/20013078.2019.1596668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461071PMC
April 2019

Enhancement of chicken primordial germ cell in vitro maintenance using an automated cell image analyser.

Acta Vet Hung 2018 12;66(4):518-529

2 National Agricultural Research and Innovation Center, Agricultural Biotechnology Institute, Animal Biotechnology Department , Szent-Györgyi Albert u. 4, H-2100 Gödöllő , Hungary.

Primordial germ cells (PGCs) were isolated from blood samples of chicken embryos. We established four PGC lines: two males (FS-ZZ-101, GFP-ZZ-4ZP) and two females (FS-ZW-111, GFP-ZW-5ZP). We could not detect a significant difference in the marker expression profile, but there was a remarkable difference between the proliferation rates of these PGC lines. We monitored the number of PGCs throughout a three-day period using a high-content screening cell imaging and analysing system (HCS). We compared three different initial cell concentrations in the wells: ~1000 cells (1×, ~4000 (4× and ~8000 (8×. For the GFPZW- 5ZP, FS-ZZ-101 and FS-ZW-111 PGC lines the lowest doubling time was observed at 4× concentration, while for GFP-ZZ-4ZP we found the lowest doubling time at 1× concentration. At 8× initial concentration, the growth rate was high during the first two days for all cell lines, but this was followed by the appearance of cell aggregates decreasing the cell growth rate. We could conclude that the difference in proliferation rate could mainly be attributed to genotypic variation in the established PGC lines, but external factors such as cell concentration and quality of the culture medium also affect the growth rate of PGCs.
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http://dx.doi.org/10.1556/004.2018.046DOI Listing
December 2018

Application of human pluripotent stem cells and pluripotent stem cell-derived cellular models for assessing drug toxicity.

Expert Opin Drug Metab Toxicol 2019 Jan 17;15(1):61-75. Epub 2018 Dec 17.

a Institute of Enzymology , Research Centre for Natural Sciences , Budapest , Hungary.

: Human pluripotent stem cells (hPSCs) are capable of differentiating into all types of cells in the body and so provide suitable toxicology screening systems even for hard-to-obtain human tissues. Since hPSCs can also be generated from differentiated cells and current gene editing technologies allow targeted genome modifications, hPSCs can be applied for drug toxicity screening both in normal and disease-specific models. Targeted hPSC differentiation is still a challenge but cardiac, neuronal or liver cells, and complex cellular models are already available for practical applications. : The authors review new gene-editing and cell-biology technologies to generate sensitive toxicity screening systems based on hPSCs. Then the authors present the use of undifferentiated hPSCs for examining embryonic toxicity and discuss drug screening possibilities in hPSC-derived models. The authors focus on the application of human cardiomyocytes, hepatocytes, and neural cultures in toxicity testing, and discuss the recent possibilities for drug screening in a 'body-on-a-chip' model system. : hPSCs and their genetically engineered derivatives provide new possibilities to investigate drug toxicity in human tissues. The key issues in this regard are still the selection and generation of proper model systems, and the interpretation of the results in understanding drug effects.
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http://dx.doi.org/10.1080/17425255.2019.1558207DOI Listing
January 2019

Clinically relevant mutations in the ABCG2 transporter uncovered by genetic analysis linked to erythrocyte membrane protein expression.

Sci Rep 2018 05 10;8(1):7487. Epub 2018 May 10.

Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar Tudosok krt. 2, Budapest, 1117, Hungary.

The ABCG2 membrane protein is a key xeno- and endobiotic transporter, modulating the absorption and metabolism of pharmacological agents and causing multidrug resistance in cancer. ABCG2 is also involved in uric acid elimination and its impaired function is causative in gout. Analysis of ABCG2 expression in the erythrocyte membranes of healthy volunteers and gout patients showed an enrichment of lower expression levels in the patients. By genetic screening based on protein expression, we found a relatively frequent, novel ABCG2 mutation (ABCG2-M71V), which, according to cellular expression studies, causes reduced protein expression, although with preserved transporter capability. Molecular dynamics simulations indicated a stumbled dynamics of the mutant protein, while ABCG2-M71V expression in vitro could be corrected by therapeutically relevant small molecules. These results suggest that personalized medicine should consider this newly discovered ABCG2 mutation, and genetic analysis linked to protein expression provides a new tool to uncover clinically important mutations in membrane proteins.
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http://dx.doi.org/10.1038/s41598-018-25695-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5945641PMC
May 2018

Generation of multidrug resistant human tissues by overexpression of the ABCG2 multidrug transporter in embryonic stem cells.

PLoS One 2018 12;13(4):e0194925. Epub 2018 Apr 12.

Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.

The ABCG2 multidrug transporter provides resistance against various endo- and xenobiotics, and protects the stem cells against toxins and stress conditions. We have shown earlier that a GFP-tagged version of ABCG2 is fully functional and may be used to follow the expression, localization and function of this transporter in living cells. In the present work we have overexpressed GFP-ABCG2, driven by a constitutive (CAG) promoter, in HUES9 human embryonic stem cells. Stem cell clones were generated to express the wild-type and a substrate-mutant (R482G) GFP-ABCG2 variant, by using the Sleeping Beauty transposon system. We found that the stable overexpression of these transgenes did not change the pluripotency and growth properties of the stem cells, nor their differentiation capacity to hepatocytes or cardiomyocytes. ABCG2 overexpression provided increased toxin resistance in the stem cells, and protected the derived cardiomyocytes against doxorubicin toxicity. These studies document the potential of a stable ABCG2 expression for engineering toxin-resistant human pluripotent stem cells and selected stem cell derived tissues.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194925PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5896897PMC
July 2018

Characterization of calcium signals in human induced pluripotent stem cell-derived dentate gyrus neuronal progenitors and mature neurons, stably expressing an advanced calcium indicator protein.

Mol Cell Neurosci 2018 04 6;88:222-230. Epub 2018 Feb 6.

Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary. Electronic address:

Pluripotent stem cell derived human neuronal progenitor cells (hPSC-NPCs) and their mature neuronal cell culture derivatives may efficiently be used for central nervous system (CNS) drug screening, including the investigation of ligand-induced calcium signalization. We have established hippocampal NPC cultures derived from human induced PSCs, which were previously generated by non-integrating Sendai virus reprogramming. Using established protocols these NPCs were differentiated into hippocampal dentate gyrus neurons. In order to study calcium signaling without the need of dye loading, we have stably expressed an advanced calcium indicator protein (GCaMP6fast) in the NPCs using the Sleeping Beauty transposon system. We observed no significant effects of the long-term GCaMP6 expression on NPC morphology, gene expression pattern or neural differentiation capacity. In order to compare the functional properties of GCaMP6-expressing neural cells and the corresponding parental cells loaded with calcium indicator dye Fluo-4, a detailed characterization of calcium signals was performed. We found that the calcium signals induced by ATP, glutamate, LPA, or proteases - were similar in these two systems. Moreover, the presence of the calcium indicator protein allowed for a sensitive, repeatable detection of changes in calcium signaling during the process of neurogenesis and neuronal maturation.
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http://dx.doi.org/10.1016/j.mcn.2018.02.003DOI Listing
April 2018

A new fluorescent dye accumulation assay for parallel measurements of the ABCG2, ABCB1 and ABCC1 multidrug transporter functions.

PLoS One 2018 17;13(1):e0190629. Epub 2018 Jan 17.

Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.

ABC multidrug transporters are key players in cancer multidrug resistance and in general xenobiotic elimination, thus their functional assays provide important tools for research and diagnostic applications. In this study we have examined the potential interactions of three key human ABC multidrug transporters with PhenGreen diacetate (PGD), a cell permeable fluorescent metal ion indicator. The non-fluorescent, hydrophobic PGD rapidly enters the cells and, after cleavage by cellular esterases, in the absence of quenching metal ions, PhenGreen (PG) becomes highly fluorescent. We found that in cells expressing functional ABCG2, ABCB1, or ABCC1 transporters, cellular PG fluorescence is strongly reduced. This fluorescence signal in the presence of specific transporter inhibitors is increased to the fluorescence levels in the control cells. Thus the PG accumulation assay is a new, unique tool for the parallel determination of the function of the ABCG2, ABCB1, and ABCC1 multidrug transporters. Since PG has very low cellular toxicity, the PG accumulation assay also allows the selection, separation and culturing of selected cell populations expressing either of these transporters.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0190629PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5771559PMC
February 2018

Functional shift with maintained regenerative potential following portal vein ligation.

Sci Rep 2017 12 22;7(1):18065. Epub 2017 Dec 22.

Hepato-Pancreatico-Biliary Surgery Research Center Hungary, 1st Department of Surgery, Semmelweis University, Budapest, Hungary.

Selective portal vein ligation (PVL) allows the two-stage surgical resection of primarily unresectable liver tumours by generating the atrophy and hypertrophy of portally ligated (LL) and non-ligated lobes (NLL), respectively. To evaluate critically important underlying functional alterations, present study characterised in vitro and vivo liver function in male Wistar rats (n = 106; 210-250 g) before, and 24/48/72/168/336 h after PVL. Lobe weights and volumes by magnetic resonance imaging confirmed the atrophy-hypertrophy complex. Proper expression and localization of key liver transporters (Ntcp, Bsep) and tight junction protein ZO-1 in isolated hepatocytes demonstrated constantly present viable and well-polarised cells in both lobes. In vitro taurocholate and bilirubin transport, as well as in vivo immunohistochemical Ntcp and Mrp2 expressions were bilaterally temporarily diminished, whereas LL and NLL structural acinar changes were divergent. In vivo bile and bilirubin-glucuronide excretion mirrored macroscopic changes, whereas serum bilirubin levels remained unaffected. In vivo functional imaging (indocyanine-green clearance test; Tc-mebrofenin hepatobiliary scintigraphy; confocal laser endomicroscopy) indicated transitionally reduced global liver uptake and -excretion. While LL functional involution was permanent, NLL uptake and excretory functions recovered excessively. Following PVL, functioning cells remain even in LL. Despite extensive bilateral morpho-functional changes, NLL functional increment restores temporary declined transport functions, emphasising liver functional assessment.
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http://dx.doi.org/10.1038/s41598-017-18309-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5741735PMC
December 2017

Proteasome Subunit Beta Type 1 P11A Polymorphism Is a New Prognostic Marker in Multiple Myeloma.

Clin Lymphoma Myeloma Leuk 2017 11 30;17(11):734-742. Epub 2017 Jun 30.

3(rd) Department of Internal Medicine, Semmelweis University, Budapest, Hungary.

Background: Proteasome subunit beta type 1 (PSMB1) rs12717 polymorphism, a single nucleotide polymorphism with unknown functional effect, was recently reported to influence response to bortezomib-based therapy in follicular lymphoma.

Patients And Methods: We retrospectively analyzed the prognostic impact of this polymorphism in 211 consecutively diagnosed multiple myeloma cases, and performed in vitro experiments to look into its functional consequences.

Results: On univariate analysis, patients carrying the variant G allele showed significantly shorter progression-free survival (PFS) with a pattern suggestive of a gene-dose effect (PFS 26.4, 22.3, and 16.4 months in C/C, C/G, and G/G patients, respectively, P = .002). On multivariate analysis, carrying the G/G genotype was a significant independent risk factor for relapse (hazard ratio [HR] 2.29, P < .001) with a similar trend in C/G carriers (HR 1.33, P = .097) when compared with the major allele carrier C/C cohort. Our subsequent in vitro analyses demonstrated significantly reduced protease activity in proteasomes of individuals with G/G genotype compared with that of C/C carriers, despite that PSMB1 expression and proteasome assembly remained unaltered. Bortezomib exhibited a lower inhibitory capacity on the caspase- and trypsin-like activity of proteasomes from G/G individuals.

Conclusion: Our results show that carriership of PSMB1 rs12717 minor allele is predictive for suboptimal response with bortezomib treatment, which could be explained by less active proteasomes that are less sensitive to bortezomib, and myeloma cells consequently relying on other escape mechanisms to cope with the abundance of misfolded proteins.
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http://dx.doi.org/10.1016/j.clml.2017.06.034DOI Listing
November 2017

Histone Deacetylase Inhibitor Treatment Increases the Expression of the Plasma Membrane Ca Pump PMCA4b and Inhibits the Migration of Melanoma Cells Independent of ERK.

Front Oncol 2017 24;7:95. Epub 2017 May 24.

2nd Institute of Pathology, Semmelweis University, Budapest, Hungary.

Several new therapeutic options emerged recently to treat metastatic melanoma; however, the high frequency of intrinsic and acquired resistance among patients shows a need for new therapeutic options. Previously, we identified the plasma membrane Ca ATPase 4b (PMCA4b) as a metastasis suppressor in BRAF-mutant melanomas and found that mutant BRAF inhibition increased the expression of the pump, which then inhibited the migratory and metastatic capability of the cells. Earlier it was also demonstrated that histone deacetylase inhibitors (HDACis) upregulated PMCA4b expression in gastric, colon, and breast cancer cells. In this study, we treated one BRAF wild-type and two BRAF-mutant melanoma cell lines with the HDACis, SAHA and valproic acid, either alone, or in combination with the BRAF inhibitor, vemurafenib. We found that HDACi treatment strongly increased the expression of PMCA4b in all cell lines irrespective of their BRAF mutational status, and this effect was independent of ERK activity. Furthermore, HDAC inhibition also enhanced the abundance of the housekeeping isoform PMCA1. Combination of HDACis with vemurafenib, however, did not have any additive effects on either PMCA isoform. We demonstrated that the HDACi-induced increase in PMCA abundance was coupled to an enhanced [Ca] clearance rate and also strongly inhibited both the random and directional movements of A375 cells. The primary role of PMCA4b in these characteristic changes was demonstrated by treatment with the PMCA4-specific inhibitor, caloxin 1c2, which was able to restore the slower Ca clearance rate and higher motility of the cells. While HDAC treatment inhibited cell motility, it decreased only modestly the ratio of proliferative cells and cell viability. Our results show that in melanoma cells the expression of both PMCA4b and PMCA1 is under epigenetic control and the elevation of PMCA4b expression either by HDACi treatment or by the decreased activation of the BRAF-MEK-ERK pathway can inhibit the migratory capacity of the highly motile A375 cells.
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http://dx.doi.org/10.3389/fonc.2017.00095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5442207PMC
May 2017

Ezrin interacts with S100A4 via both its N- and C-terminal domains.

PLoS One 2017 11;12(5):e0177489. Epub 2017 May 11.

Department of Biochemistry, Eötvös Loránd University, Budapest, Hungary.

Ezrin belongs to the ERM (ezrin, radixin, moesin) protein family that has a role in cell morphology changes, adhesion and migration as an organizer of the cortical cytoskeleton by linking actin filaments to the apical membrane of epithelial cells. It is highly expressed in a variety of human cancers and promotes metastasis. Members of the Ca2+-binding EF-hand containing S100 proteins have similar pathological properties; they are overexpressed in cancer cells and involved in metastatic processes. In this study, using tryptophan fluorescence and stopped-flow kinetics, we show that S100A4 binds to the N-terminal ERM domain (N-ERMAD) of ezrin with a micromolar affinity. The binding involves the F2 lobe of the N-ERMAD and follows an induced fit kinetic mechanism. Interestingly, S100A4 binds also to the unstructured C-terminal actin binding domain (C-ERMAD) with similar affinity. Using NMR spectroscopy, we characterized the complex of S100A4 with the C-ERMAD and demonstrate that no ternary complex is simultaneously formed with the two ezrin domains. Furthermore, we show that S100A4 co-localizes with ezrin in HEK-293T cells. However, S100A4 very weakly binds to full-length ezrin in vitro indicating that the interaction of S100A4 with ezrin requires other regulatory events such as protein phosphorylation and/or membrane binding, shifting the conformational equilibrium of ezrin towards the open state. As both proteins play an important role in promoting metastasis, the characterization of their interaction could shed more light on the molecular events contributing to this pathological process.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0177489PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5426754PMC
September 2017

Comparative study of CYP2B1/2 induction and the transport of bilirubin and taurocholate in rat hepatocyte-mono- and hepatocyte-Kupffer cell co-cultures.

J Pharmacol Toxicol Methods 2016 Nov - Dec;82:1-8. Epub 2016 May 25.

Department of Functional Pharmacology, Institute of Organic Chemistry, Research Centre for Natural Sciences, HAS, 1117 Budapest, Magyar tudósok körútja 2, Hungary. Electronic address:

Introduction: Hepatocyte-Kupffer cell (KC) co-cultures represent a promising approach for in vitro modeling of complex interactions between parenchymal and non-parenchymal cells in the liver, responsible for drug-induced liver injury (DILI). In this study we aimed to compare hepatocyte monocultures with hepatocyte-KC co-cultures regarding some basic liver functions associated with the chemical defense system. These pathways involve transporters and enzymes the function of which is highly sensitive towards hepatotoxic events.

Methods: CYP2B1/2 induction and the biliary and sinusoidal elimination of bilirubin (B) and taurocholate (TC) were studied in rat hepatocyte sandwich cultures compared with rat hepatocyte-KC sandwich co-cultures of 1:0, 6:1, 2:1 and 1:1 cell combinations representing the physiologic and pathologic conditions of the liver.

Results: KCs decreased phenobarbital inducibility of CYP2B1/2 in a cell ratio dependent manner and activation of KCs by lipopolisacharide (LPS) amplified this effect. Similarly, KCs decreased the transport of B and its glucuronides (BG) in both sinusoidal and canalicular directions resulting in its intracellular accumulation. In contrast, the uptake and the efflux of TC were greater in the co-cultures than in the hepatocyte monocultures. Immuno-labelling of sodium-dependent taurocholate transporter (Ntcp) revealed increased expression of the transporter in the presence of KCs.

Discussion: Here we presented that KCs have a direct impact on some hepatocyte functions suggesting that the co-culture model may be more suitable for drug related hepatotoxicity studies than hepatocyte monocultures.
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http://dx.doi.org/10.1016/j.vascn.2016.05.015DOI Listing
May 2017

A transgenic rat hepatocyte - Kupffer cell co-culture model for evaluation of direct and macrophage-related effect of poly(amidoamine) dendrimers.

Toxicol In Vitro 2017 Feb 4;38:159-169. Epub 2016 Oct 4.

Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.

Increasing number of papers demonstrate that Kupffer cells (KCs) play a role in the development of drug induced liver injury (DILI). Furthermore, elevated intracellular Ca level of hepatocytes is considered as a common marker of DILI. Here we applied an in vitro model based on hepatocyte mono- and hepatocyte/KC co-cultures (H/KC) isolated from transgenic rats stably expressing the GCaMP2 fluorescent Ca sensor protein to investigate the effects of polycationic (G5), polyanionic (G4.5) and polyethylene-glycol coated neutral (G5 Peg) dendrimers known to accumulate in the liver, primarily in KCs. Following dendrimer exposure, hepatocyte homeostasis was measured by MTT cytotoxicity assay and by Ca imaging, while hepatocyte functions were studied by CYP2B1/2 inducibility, and bilirubin and taurocholate transport. G5 was significantly more cytotoxic than G4.5 for hepatocytes and induced Ca oscillation and sustained Ca signals at 1μM and10 μM, respectively both in hepatocytes and KCs. Dendrimer-induced Ca signals in hepatocytes were attenuated by macrophages. Activation of KCs by lipopolysaccharide and G5 decreased the inducibility of CYP2B1/2, which was restored by depleting the KCs with gadolinium-chloride and pentoxyphylline, suggesting a role of macrophages in the hindrance of CYP2B1/2 induction by G5 and lipopolysaccharide. In the H/KC, but not in the hepatocyte mono-culture, G5 reduced the canalicular efflux of bilirubin and stimulated the uptake and canalicular efflux of taurocholate. In conclusion, H/KC provides a good model for the prediction of hepatotoxic potential of drugs, especially of nanomaterials known to be trapped by macrophages, activation of which presumably contributes to DILI.
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http://dx.doi.org/10.1016/j.tiv.2016.09.016DOI Listing
February 2017

Application of fluorescent dye substrates for functional characterization of ABC multidrug transporters at a single cell level.

Cytometry A 2016 09 7;89(9):826-34. Epub 2016 Sep 7.

Research Centre for Natural Sciences, Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary.

ABC multidrug transporters are key players in cancer multidrug resistance and in determining the ADME-Tox properties of drugs and xenobiotics. The most sensitive and specific detection of these transporters is based on functional assays. Assessment of the transporter-dependent reduction of cellular uptake of the fluorescent dyes, such as Hoechst 33342 (Ho) and more recently DyeCycle Violet (DCV), have been widely advocated for the characterization of both ABCB1 and ABCG2 multidrug transporters. Detailed comparison of these supravital DNA-binding dyes revealed that DCV is less toxic to ABCG2- and ABCB1-expressing cells than Ho. ATPase measurements imply that DCV and Ho are similarly handled by ABCB1, whereas ABCG2 seems to transport DVC more effectively. In addition, we have developed an image-based high content microscopy screening method for simultaneous in situ measurement of the cellular activity and expression of the ABCG2 multidrug transporter. We demonstrated the applicability of this method for identifying ABCG2-positive cells in heterogeneous cell population by a single dye uptake measurement. These results may promote multidrug transporter studies at a single cell level and allow the quantitative detection of clinically important drug-resistant sub-populations. © 2016 International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.22931DOI Listing
September 2016