György Várady

Multidrug transporter ABCG2 prevents tumor cell death induced by the epidermal growth factor receptor inhibitor Iressa (ZD1839, Gefitinib).

Iressa (ZD1839, Gefitinib), used in clinics to treat non-small cell lung cancer patients, is a tyrosine kinase receptor inhibitor that leads to specific decoupling of epidermal growth factor receptor (EGFR) signaling. Recent data indicate that Iressa is especially effective in tumors with certain EGFR mutations; however, a subset of these tumors does not respond to Iressa. In addition, certain populations have an elevated risk of side effects during Iressa treatment. The human ABCG2 (BCRP/MXR/ABCP) transporter causes cancer drug resistance by actively extruding a variety of cytotoxic drugs, and it functions physiologically to protect our tissues from xenobiotics. Importantly, ABCG2 modifies absorption, distribution, and toxicity of several pharmacologic agents. Previously, we showed that ABCG2 displays a high-affinity interaction with several tyrosine kinase receptor inhibitors, including Iressa. Here, we show that the expression of ABCG2, but not its nonfunctional mutant, protects the EGFR signaling-dependent A431 tumor cells from death on exposure to Iressa. This protection is reversed by the ABCG2-specific inhibitor, Ko143. These data, reinforced with cell biology and biochemical experiments, strongly suggest that ABCG2 can actively pump Iressa. Therefore, variable expression and polymorphisms of ABCG2 may significantly modify the antitumor effect as well as the absorption and tissue distribution of Iressa.

Applying a “Double-Feature” Promoter to Identify Cardiomyocytes Differentiated from Human Embryonic Stem Cells Following Transposon-Based Gene Delivery†‡

Human embryonic stem (HuES) cells represent a new potential tool for cell‐therapy and gene‐therapy applications. However, these approaches require the development of efficient, stable gene delivery, and proper progenitor cell and tissue separation methods. In HuES cell lines, we have generated stable, enhanced green fluorescent protein (EGFP)‐expressing clones using a transposon‐based (Sleeping Beauty) system. This method yielded high percentage of transgene integration and expression. Similarly to a lentiviral expression system, both the undifferentiated state and the differentiation pattern of the HuES cells were preserved. By using the CAG promoter, in contrast to several other constitutive promoter sequences (such as CMV, elongation factor 1α, or phosphoglycerate kinase), an exceptionally high EGFP expression was observed in differentiated cardiomyocytes. This phenomenon was independent of the transgene sequence, methods of gene delivery, copy number, and the integration sites. This “double‐feature” promoter behavior, that is providing a selectable marker for transgene expressing undifferentiated stem cells, and also specifically labeling differentiated cardiomyocytes, was assessed by transcriptional profiling. We found a positive correlation between CAG promoter‐driven EGFP transcription and expression of cardiomyocyte‐specific genes. Our experiments indicate an efficient applicability of transposon‐based gene delivery into HuES cells and provide a novel approach to identify differentiated tissues by exploiting a nontypical behavior of a constitutively active promoter, thereby avoiding invasive drug selection methods. Stem Cells 2009;27:1077–1087

Interaction of the EGFR inhibitors gefitinib, vandetanib, pelitinib and neratinib with the ABCG2 multidrug transporter: Implications for the emergence and reversal of cancer drug resistance

Human ABCG2 is a plasma membrane glycoprotein that provides physiological protection against xenobiotics. ABCG2 also significantly influences biodistribution of drugs through pharmacological tissue barriers and confers multidrug resistance to cancer cells. Moreover, ABCG2 is the molecular determinant of the side population that is characteristically enriched in normal and cancer stem cells. Numerous tumors depend on unregulated EGFR signaling, thus inhibition of this receptor by small molecular weight inhibitors such as gefitinib, and the novel second generation agents vandetanib, pelitinib and neratinib, is a promising therapeutic option. In the present study, we provide detailed biochemical characterization regarding the interaction of these EGFR inhibitors with ABCG2. We show that ABCG2 confers resistance to gefitinib and pelitinib, whereas the intracellular action of vandetanib and neratinib is unaltered by the presence of the transporter. At higher concentrations, however, all these EGFR inhibitors inhibit ABCG2 function, thereby promoting accumulation of ABCG2 substrate drugs. We also report enhanced expression of ABCG2 in gefitinib-resistant non-small cell lung cancer cells, suggesting potential clinical relevance of ABCG2 in acquired drug resistance. Since ABCG2 has important impact on both the pharmacological properties and anti-cancer efficiencies of drugs, our results regarding the novel EGFR inhibitors should provide useful information about their therapeutic applicability against ABCG2-expressing cancer cells depending on EGFR signaling. In addition, the finding that these EGFR inhibitors efficiently block ABCG2 function may help to design novel drug-combination therapeutic strategies.

Reliable transgene-independent method for determining Sleeping Beauty transposon copy numbers.

BackgroundThe transposon-based gene delivery technique is emerging as a method of choice for gene therapy. The Sleeping Beauty (SB) system has become one of the most favored methods, because of its efficiency and its random integration profile. Copy-number determination of the delivered transgene is a crucial task, but a universal method for measuring this is lacking. In this paper, we show that a real-time quantitative PCR-based, transgene-independent (qPCR-TI) method is able to determine SB transposon copy numbers regardless of the genetic cargo.ResultsWe designed a specific PCR assay to amplify the left inverted repeat-direct repeat region of SB, and used it together with the single-copy control gene RPPH1 and a reference genomic DNA of known copy number. The qPCR-TI method allowed rapid and accurate determination of SB transposon copy numbers in various cell types, including human embryonic stem cells. We also found that this sensitive, rapid, highly reproducible and non-radioactive method is just as accurate and reliable as the widely used blotting techniques or the transposon display method. Because the assay is specific for the inverted repeat region of the transposon, it could be used in any system where the SB transposon is the genetic vehicle.ConclusionsWe have developed a transgene-independent method to determine copy numbers of transgenes delivered by the SB transposon system. The technique is based on a quantitative real-time PCR detection method, offering a sensitive, non-radioactive, rapid and accurate approach, which has a potential to be used for gene therapy.

Interaction with the 5D3 Monoclonal Antibody Is Regulated by Intramolecular Rearrangements but Not by Covalent Dimer Formation of the Human ABCG2 Multidrug Transporter

Human ABCG2 is a plasma membrane glycoprotein working as a homodimer or homo-oligomer. The protein plays an important role in the protection/detoxification of various tissues and may also be responsible for the multidrug-resistant phenotype of cancer cells. In our previous study we found that the 5D3 monoclonal antibody shows a function-dependent reactivity to an extracellular epitope of the ABCG2 transporter. In the current experiments we have further characterized the 5D3-ABCG2 interaction. The effect of chemical cross-linking and the modulation of extracellular S–S bridges on the transporter function and 5D3 reactivity of ABCG2 were investigated in depth. We found that several protein cross-linkers greatly increased 5D3 labeling in ABCG2 expressing HEK cells; however, there was no correlation between covalent dimer formation, the inhibition of transport activity, and the increase in 5D3 binding. Dithiothreitol treatment, which reduced the extracellular S–S bridge-forming cysteines of ABCG2, had no effect on transport function but caused a significant decrease in 5D3 binding. When analyzing ABCG2 mutants carrying Cys-to-Ala changes in the extracellular loop, we found that the mutant C603A (lacking the intermolecular S–S bond) showed comparable transport activity and 5D3 reactivity to the wild-type ABCG2. However, disruption of the intramolecular S–S bridge (in C592A, C608A, or C592A/C608A mutants) in this loop abolished 5D3 binding, whereas the function of the protein was preserved. Based on these results and ab initio folding simulations, we propose a model for the large extracellular loop of the ABCG2 protein.

Evaluation of ABCG2 expression in human embryonic stem cells: Crossing the same river twice?

A recent publication in Stem Cells states that human embryonicstem (ES) cells do not express ABCG2 and ‘‘…absence ofABCG2 isa novelfeatureof humanpluripotentstem cells,whichdistinguishes them from many other stem cells including mouseES cells’’ [1]. This is in sharp contrast to our observations [2]and the report of several other investigators who detectedABCG2 mRNA in various human ES cells [3–6]. The presenceof multidrug resistance ABC (MDR-ABC) transporters may sig-nificantly contribute to stem cell defense mechanisms; thus, thisis an important question that should be addressed properly.Our interest in ABC transporters dates back to the discov-ery of their role in cancer drug resistance over two decadesago. Since then, we have had ample opportunity to experiencehow insufficient methodology and a simplifying approachmay obscure the assessment of the impact of MDR-ABCtransporters on cancer patient survival. Measuring the func-tional expression of ABC transporters proved challengingbecause of the heterogeneity of tumors, the varying levels ofexpression, and the unreliability of the assay systems usedthroughout the trials. As a result, most reports were consid-ered controversial, and the true contribution of MDR-ABCtransporters to therapy failure could only be established onceassay conditions were standardized [7]. The key teaching ofthese extensive studies have immediate relevance to exploringtransporter expression in stem cells. First, MDR-ABC trans-porters are active extrusion pumps that may significantly mod-ify cellular homeostasis or endobiotic and xenobiotic resist-ance even at low levels. Therefore, the assays measuring theirimpact should be sensitive, quantitative, and should preferablytarget the function of the MDR-ABC transporters. Second,samples are often heterogeneous for MDR-ABC expression,as these proteins are rapidly regulated by numerous mecha-nisms, both at the transcriptional and processing levels. How-ever, this initial heterogeneity may be relevant in circumstan-ces of stress, survival, or proliferation. Third, in many cases,the cell type, the mechanism of cell transformation, or differ-entiation does not determine the expression or function ofMDR-ABC transporters. Rather, ABC transporters are modu-lated by numerous environmental conditions [7, 8].In the case of the paper by Zeng et al. [1], the appre-ciation of these features is not possible as there are manyexperimental flaws that are reminiscent of the limitationsthat our field had to overcome to evaluate the MDR ofcancer. First, the reverse transcription polymerase chainreaction (RT-PCR) results are not quantitated, and there isno effort to perform quantitative PCR studies for thedetection of the relevant messages. Second, the Hoechstdye efflux studies lack the essential negative control. Third,instead of using a highly specific ABCG2 inhibitor, theauthors make their case on the basis of the effect of vera-pamil, which is a weak and nonspecific inhibitor ofABCG2. Fourth, the immunostaining studies are not con-vincing, the antibody used requires cell permeabilization,and the membrane localization of ABCG2 is not examined.Fifth, detection of subpopulations is contradictory and isnot evaluated in the context of co-expression of stem cellmarkers. Therefore, this study does not allow conclusionsto be drawn regarding the presence or up- and downregu-lation of ABCG2 in human ES cells.In contrast, we emphasize again that with appropriateexperimental tools, the functional although heterogeneousexpression of membrane ABCG2 is detectable in undifferenti-ated human stem cells. Detailed documentation is not possi-ble here, but the key features of ABCG2 expression in fourdifferent ES cell lines are depicted in Figure 1 and in thesupporting information video. Here we used properly quanti-tated real-time PCR measurements, flow cytometry, and con-focal microscopy with costaining of relevant surface markers.Furthermore, we compare ES cells grown on MEF or Matri-gel, and we also evaluate the expression pattern of a mesen-chymal-like cell line (Figure 1C (F2)). We also document amicroscopic measurement of Hoechst dye uptake in undiffer-entiated stem cells, which is modulated by a specific ABCG2inhibitor. All these measurements suggest that ABCG2 ispresent at relatively high levels in the undifferentiatedhuman ES cells, highlighting its role in the protection of thisvaluable sanctuary against the damage by toxins, drugs, orhypoxia [8, 9].

Dynamic ABCG2 expression in human embryonic stem cells provides the basis for stress response

ABCG2 is a plasma membrane multidrug transporter with an established role in the cancer drug-resistance phenotype. This protein is expressed in a variety of tissues, including several types of stem cell. Although ABCG2 is not essential for life, knock-out mice were found to be hypersensitive to xenobiotics and had reduced levels of the side population of hematopoietic stem cells. Previously we have shown that ABCG2 is present in human embryonic stem cell (hESC) lines, with a heterogeneous expression pattern. In this study we examined this heterogeneity, and investigated whether it is related to stress responses in hESCs. We did not find any difference between expression of pluripotency markers in ABCG2-positive and negative hESCs; however, ABCG2-expressing cells had a higher growth rate after cell separation. We found that some harmful conditions (physical stress, drugs, and UV light exposure) are tolerated much better in the presence of ABCG2 protein. This property can be explained by the transporter function which eliminates potential toxic metabolites accumulated during stress conditions. In contrast, mild oxidative stress in hESCs caused rapid internalization of ABCG2, indicating that some environmental factors may induce removal of this transporter from the plasma membrane. On the basis of these results we suggest that a dynamic balance of ABCG2 expression at the population level has the advantage of enabling prompt response to changes in the cellular environment. Such actively maintained heterogeneity might be of evolutionary benefit in protecting special cell types, including pluripotent stem cells.

Expression Levels of the ABCG2 Multidrug Transporter in Human Erythrocytes Correspond to Pharmacologically Relevant Genetic Variations

We have developed a rapid, simple and reliable, antibody-based flow cytometry assay for the quantitative determination of membrane proteins in human erythrocytes. Our method reveals significant differences between the expression levels of the wild-type ABCG2 protein and the heterozygous Q141K polymorphic variant. Moreover, we find that nonsense mutations on one allele result in a 50% reduction in the erythrocyte expression of this protein. Since ABCG2 polymorphisms are known to modify essential pharmacokinetic parameters, uric acid metabolism and cancer drug resistance, a direct determination of the erythrocyte membrane ABCG2 protein expression may provide valuable information for assessing these conditions or for devising drug treatments. Our findings suggest that erythrocyte membrane protein levels may reflect genotype-dependent tissue expression patterns. Extension of this methodology to other disease-related or pharmacologically important membrane proteins may yield new protein biomarkers for personalized diagnostics.

Cell surface membrane proteins as personalized biomarkers: where we stand and where we are headed.

Personalized medicine requires the development of a wide array of biomarker diagnostic assays, reflecting individual variations and thus allowing tailored therapeutic interventions. Membrane proteins comprise approximately 30% of total human proteins; they play a key role in various physiological functions and pathological conditions, although, currently, only a limited number of membrane proteins are applied as biomarkers. In many normal tissues, cell surface membrane proteins are not easily accessible for diagnostic sampling, and tumor-derived membrane preparations - while serving as potential tumor biomarkers - may not reflect physiological protein expression. In addition to post-translational modifications, which may include glycosylation, phosphorylation and lipid modifications, the trafficking of membrane proteins is also regulated. Moreover, a tight cellular quality control monitors membrane protein maturation, and continuous removal and reinsertion, involving special signaling systems, occurs in many cases. However, cell surface membrane proteins already serve as valuable prognostic and predicative biomarkers, for example, in hematological and immunological diseases, by the determination of the cluster of differentiation markers. In this review, we demonstrate the relevance of cell surface membrane biomarkers in various diseases and call attention to the potential application of red blood cell (erythrocyte) membrane proteins in this regard. Surprisingly, red blood cells express hundreds of membrane proteins, which seem to reflect a general genetic and regulatory background, and may serve as relatively stable and easily accessible personalized membrane biomarkers. Quantitative membrane protein detection in red blood cells by flow cytometry may bring a breakthrough in this regard.

Screening the Expression of ABCB6 in Erythrocytes Reveals an Unexpectedly High Frequency of Lan Mutations in Healthy Individuals

Lan is a high-incidence blood group antigen expressed in more than 99.9% of the population. Identification of the human ABC transporter ABCB6 as the molecular basis of Lan has opened the way for studies assessing the relation of ABCB6 function and expression to health and disease. To date, 34 ABCB6 sequence variants have been described in association with reduced ABCB6 expression based on the genotyping of stored blood showing weak or no reactivity with anti-Lan antibodies. In the present study we examined the red blood cell (RBC) surface expression of ABCB6 by quantitative flow cytometry in a cohort of 47 healthy individuals. Sequencing of the entire coding region of the ABCB6 gene in low RBC ABCB6 expressors identified a new allele (IVS9+1G>A, affecting a putative splice site at the boundary of exon 9) and two nonsynonymous SNPs listed in the SNP database (R192Q (rs150221689) and G588 S (rs145526996)). The R192Q mutation showed co-segregation with reduced RBC ABCB6 expression in a family, and we found the G588 S mutation in a compound heterozygous individual with undetectable ABCB6 expression, suggesting that both mutations result in weak or no expression of ABCB6 on RBCs. Analysis of the intracellular expression pattern in HeLa cells by confocal microscopy indicated that these mutations do not compromise overall expression or the endolysosomal localization of ABCB6. Genotyping of two large cohorts, containing 235 and 1039 unrelated volunteers, confirmed the high allele frequency of Lan-mutations. Our results suggest that genetic variants linked to lower or absent cell surface expression of ABCB6/Langereis may be more common than previously thought.