Model system for the analysis of cell surface expression of human ABCA1

<p>Abstract</p> <p>Background</p> <p>The ABCA1 protein plays a pivotal role in reverse cholesterol transport, by mediating the generation of HDL particles and removing cellular cholesterol. Both the proper expression of ABCA1 in the plasma membrane and the internalization along with apoA-I are required for function. Therefore, we developed a model system to investigate the effect of clinically relevant drugs on the cell surface appearance of ABCA1.</p> <p>Results</p> <p>By retroviral transduction system, we established stable mammalian cell lines expressing functional and non-functional ABCA1 variants, tagged with an extracellular hemagglutinin epitope. After characterization of the expression, proper localization and function of different ABCA1 variants, we followed quantitatively their cell surface expression by immunofluorescent staining, using flow cytometry. As expected, we found increased cell surface expression of ABCA1 after treatment with a calpain inhibitor, and observed a strong decrease in plasma membrane ABCA1 expression upon treatment with a trans-Golgi transport inhibitor, Brefeldin A. We tested cholesterol level lowering drugs and other potential inhibitors of ABCA1. Here we demonstrate that ezetimibe affects ABCA1 cell surface expression only in the case of a functional ABCA1.</p> <p>Conclusions</p> <p>Our model system allows a quantitative detection of cell surface expression of ABCA1, screening of substrates or specific inhibitors, and investigating transport regulation.</p

Az ABCG2 multidrog transzporter fehérje szerkezetének és működésének vizsgálata = Structure and function of the multidrug transporter ABCG2

Az ABCG2 multidrog transzporternek fontos szerepe van mind a daganatok kemoterápia-rezisztenciájában, mind a fiziológiás xenobiotikum transzportban. A projektben előállítottuk az ABCG2 fehérje különböző mutáns és polimorf változatait, elvégeztük ezek részletes funkcionális vizsgálatát. Egy sejtfelszínen reagáló, konformáció-érzékeny anti-ABCG2 monoklonális antitest alkalmazásával felderítettük a transzporter funkcionális állapotait, kémiai módosítások és mutációk segítségével elvégeztük az epitópok jellemzését és molekuláris szintű modellezését. Részletesen elemeztük az ABCG2 transzporter és a membrán lipidek kölcsönhatásait, megállapítottuk a membrán koleszterin jelentős szabályozó szerepét. Az ABCG2 transzporter és célzott hatású rákellenes vegyületek kölcsönhatásainak vizsgálata során klinikailag is alkalmazott gyógyszerekre vonatkozóan kaptunk új információkat. Új módszereket fejlesztettünk ki az ABCG2 szabályozásának, lokalizációjának és funkciójának vizsgálatára, elemeztük a transzporter expresszióját humán embrionális őssejtekben. Több, magas impakt faktorú nemzetközi folyóiratban közöltünk a témáról review cikkeket. | The human ABCG2 multidrug transporter plays a key role in the chemotherapy resistance of malignant tumors, as well as in the physiological elimination of xenobiotics. In this project we have prepared and expressed various mutant and polymorphic variants of the transporter, performed their detailed functional characterization. By using a cell-surface reacting, conformation-sensitive monoclonal antibody against ABCG2, we mapped the functional states of the transporter. In these experiments we applied specific chemical modifications and generated site-directed mutations to characterize the extracellular loop epitope region of ABCG2 and constructed a molecular model for this part of the transporter. We have investigated the modulation of ABCG2 by membrane lipids and found a major role for cholesterol in regulating the transport activity of this protein. By examining a number of new targeted anticancer agents we found that ABCG2 interacts with several of these compounds and may be involved in the resistance against clinically applied molecules. We have developed new methods for studying the regulation, localization and function of the ABCG2 protein, examined the expression profile of this transporter in human embryonic stem cells. During this project we have published several review articles in high-impact international journals

ABCG2 Is a Selectable Marker for Enhanced Multilineage Differentiation Potential in Periodontal Ligament Stem Cells.

Periodontal ligament stem cells (PDLSCs) provide an important source for tissue regeneration and may become especially useful in the formation of osteogenic seeds. PDLSCs can be cultured, expanded, and differentiated in vitro; thus, they may be applied in the long-term treatment of the defects in the dental regions. Here we studied numerous potential markers allowing the selection of human PDLSCs with a maximum differentiation potential. We followed the expression of the ATP-binding cassette subfamily G member 2 (ABCG2) membrane transporter protein and isolated ABCG2-expressing cells by using a monoclonal antibody, recognizing the transporter at the cell surface in intact cells. The expression of the ABCG2 protein, corresponding to the so-called side-population phenotype in various tissue-derived stem cells, was found to be a useful marker for the selection of PDLSCs with enhanced osteogenic, chondrogenic, and adipogenic differentiation. These findings may have important applications in achieving efficient dental tissue regeneration by using stem cells from extracted teeth

Distribution of the myrmecoparasitic fungus Rickia wasmannii (Ascomycota: Laboulbeniales) across colonies, individuals, and body parts of Myrmica scabrinodis

The ant species Myrmica scabrinodis plays a markedly important ecological role through much of the humid grasslands of Eurasia. It hosts a species-rich community of pathogens and parasites, including Rickia wasmannii, an enigmatic member of entomoparasitic laboulbenialean fungi. This study provides a descriptive ecology of R. wasmannii by characterizing its prevalence and distribution across several hierarchical levels: colonies, individuals, and anatomic body parts. Infections were restricted to a single ant species, Myrmica scabrinodis, and infected colonies occurred predominantly in wet habitats. Infections tended to be highly prevalent within infected colonies, often reaching 100% sample prevalence among workers. Individual infections exhibited an aggregated distribution typical to host-parasite systems. Workers from the aboveground part of nests (presumably older ones acting as foragers) were more infected than those from the belowground part. Fungal thalli could be found all over the body of the hosts, the head and the abdomen being the most infected parts of the body. The fungi’s distribution among host body parts statistically differed between low versus high-intensity infections: the initial dominance of the head decreased with advancing infection. These findings may provide baseline data for future comparative or monitoring studies

In vitro characterization of human mesenchymal stem cells isolated from different tissues with a potential to promote complex bone regeneration

Bone tissue regeneration is a major, worldwide medical need, and several strategies have been developed to support the regeneration of extensive bone defects, including stem cell based bone grafts. In addition to the application of stem cells with high osteogenic potential, it is important to maintain proper blood flow in a bone graft to avoid inner graft necrosis. Mesenchymal stem cells (MSCs) may form both osteocytes and endothelial cells; therefore we examined the combined in vitro osteogenic and endothelial differentiation capacities of MSCs derived from adipose tissue, Wharton’s jelly, and periodontal ligament. Based on a detailed characterization presented here, MSCs isolated from adipose tissue and periodontal ligament may be most appropriate for generating vascularized bone grafts

Mesenchymal Stem Cells Increase Drug Tolerance of A431 Cells Only in 3D Spheroids, Not in 2D Co-Cultures

Mesenchymal stem cells (MSCs) are an integral part of the tumor microenvironment (TME); however, their role is somewhat controversial: conflicting reports suggest that, depending on the stage of tumor development, MSCs can either support or suppress tumor growth and spread. Additionally, the influence of MSCs on drug resistance is also ambiguous. Previously, we showed that, despite MSCs proliferating significantly more slowly than cancer cells, there are chemotherapeutic drugs which proved to be similarly toxic to both cell types. Here we established 2D co-cultures and 3D co-culture spheroids from different ratios of GFP-expressing, adipose tissue-derived MSCs and A431 epidermoid carcinoma cells tagged with mCherry to investigate the effect of MSCs on cancer cell growth, survival, and drug sensitivity. We examined the cytokine secretion profile of mono- and co-cultures, explored the inner structure of the spheroids, applied MSC-(nutlin-3) and cancer cell-targeting (cisplatin) treatments separately, monitored the response with live-cell imaging and identified a new, double-fluorescent cell type emerging from these cultures. In 2D co-cultures, no effect on proliferation or drug sensitivity was observed, regardless of the changes in cytokine secretion induced by the co-culture. Conversely, 3D spheroids developed a unique internal structure consisting of MSCs, which significantly improved cancer cell survival and resilience to treatment, suggesting that physical proximity and cell–cell connections are required for MSCs to considerably affect nearby cancer cells. Our results shed light on MSC–cancer cell interactions and could help design new, better treatment options for tumors

Comparison of Different Clinical Chemotherapeutical Agents&rsquo; Toxicity and Cell Response on Mesenchymal Stem Cells and Cancer Cells

Mesenchymal stem cells (MSCs) or fibroblasts are one of the most abundant cell types in the tumor microenvironment (TME) exerting various anti- and pro-apoptotic effects during tumorigenesis, invasion, and drug treatment. Despite the recently discovered importance of MSCs in tumor progression and therapy, the response of these cells to chemotherapeutics compared to cancer cells is rarely investigated. A widely accepted view is that these naive MSCs have higher drug tolerance than cancer cells due to a significantly lower proliferation rate. Here, we examine the differences and similarities in the sensitivity of MSCs and cancer cells to nine diverse chemotherapy agents and show that, although MSCs have a slower cell cycle, these cells are still sensitive to various drugs. Surprisingly, MSCs showed similar sensitivity to a panel of compounds, however, suffered fewer DNA double-stranded breaks, did not enter into a senescent state, and was virtually incapable of apoptosis. Our results suggest that MSCs and cancer cells have different cell fates after drug treatment, and this could influence therapy outcome. These findings could help design drug combinations targeting both MSCs and cancer cells in the TME

Synthesis and Anticancer Cytotoxicity of Azaaurones Overcoming Multidrug Resistance

International audienc