ABCB6, a member of the adenosine triphosphate–binding cassette (ABC) transporter family, has been proposed to be responsible for the mitochondrial uptake of porphyrins. Here we show that ABCB6 is a glycoprotein present in the membrane of mature erythrocytes and in exosomes released from reticulocytes during the final steps of erythroid maturation. Consistent with its presence in exosomes, endogenous ABCB6 is localized to the endo/lysosomal compartment, and is absent from the mitochondria of cells. Knock-down studies demonstrate that ABCB6 function is not required for de novo heme biosynthesis in differentiating K562 cells, excluding this ABC transporter as a key regulator of porphyrin synthesis. We confirm the mitochondrial localization of ABCB7, ABCB8 and ABCB10, suggesting that only three ABC transporters should be classified as mitochondrial proteins. Taken together, our results challenge the current paradigm linking the expression and function of ABCB6 to mitochondria.

Organic Anion-Transporting Polypeptides are multispecific membrane proteins that regulate the passage of crucial endobiotics and drugs across pharmacological barriers. OATP1B1 and OATP1B3 have been described to play a major role in the hepatic uptake of statins, antivirals and various chemotherapeutics; whereas the pharmacological role of the ubiquitously expressed OATP2B1 is less well characterized. According to current industry standards, in vitro testing for susceptibility to OATP1B1 and 1B3 mediated transport is recommended for drug candidates that are eliminated in part via the liver. Here we show that human OATP1B1, 1B3 and 2B1 transport a series of commercially available viability dyes that are generally believed to be impermeable to intact cells. We demonstrate that the intracellular accumulation of Zombie Violet, Live/Dead Green, Cascade Blue and Alexa Fluor 405 is specifically increased by OATPs. Inhibition of Cascade Blue or Alexa Fluor 405 uptake by known OATP substrates/inhibitors yielded IC50 values in agreement with gold-standard radioligand assays. The fluorescence-based assays described in this study provide a new tool for testing OATP1B/2B1 drug interactions.

Despite significant progress, resistance to chemotherapy is still the main reason why cancer remains a deadly disease. An attractive strategy is to target the collateral sensitivity of otherwise multidrug resistant (MDR) cancer. In this study, our aim was to catalog various compounds that were reported to elicit increased toxicity in P-glycoprotein (Pgp)-overexpressing MDR cells. We show that the activity of most of the serendipitously identified compounds reported to target MDR cells is in fact cell-line specific, and is not influenced significantly by the function of Pgp. In contrast, novel 8-hydroxyquinoline derivatives that we identify in the National Cancer Institute (NCI) drug repository possess a robust Pgpdependent toxic activity across diverse cell lines. Pgp expression associated with the resistance of the doxorubicin-resistant Brca1 À/À ;p53 À/À spontaneous mouse mammary carcinoma cells could be eliminated by a single treatment with NSC57969, suggesting that MDR-selective compounds can effectively revert the MDR phenotype of cells expressing Pgp at clinically relevant levels. The discovery of new MDR-selective compounds shows the potential of this emerging technology and highlights the 8-hydroxyquinoline scaffold as a promising starting point for the development of compounds targeting the Achilles heel of drug-resistant cancer.

ATP-binding cassette, subfamily B (ABCB) 6 is a homodimeric ATP-binding cassette (ABC) transporter present in the plasma membrane and in the intracellular organelles. The intracellular localization of ABCB6 has been a matter of debate, as it has been suggested to reside in the mitochondria and the endo-lysosomal system. Using a variety of imaging modalities, including confocal microscopy and EM, we confirm the endo-lysosomal localization of ABCB6 and show that the protein is internalized from the plasma membrane through endocytosis, to be distributed to multivesicular bodies and lysosomes. In addition to the canonical nucleotide-binding domain (NBD) and transmembrane domain (TMD), ABCB6 contains a unique N-terminal TMD (TMD0), which does not show sequence homology to known proteins. We investigated the functional role of these domains through the molecular dissection of ABCB6. We find that the folding, dimerization, membrane insertion and ATP binding/hydrolysis of the core–ABCB6 complex devoid of TMD0 are preserved. However, in contrast with the full-length transporter, the core–ABCB6 construct is retained at the plasma membrane and does not appear in Rab5-positive endosomes. TMD0 is directly targeted to the lysosomes, without passage to the plasma membrane. Collectively, our results reveal that TMD0 represents an independently folding unit, which is dispensable for catalysis, but has a crucial role in the lysosomal targeting of ABCB6.

Organic anion‐transporting polypeptide 1A2 (OATP1A2), expressed in the human blood–brain barrier, promotes drug uptake from the blood and hence can be exploited for central nervous system‐targeted drug delivery. The thyroid transporter OATP1C1, expressed in the choroid plexus and in astrocytes, is also a potential pharmacological target. Based on their established pharmacological relevance, screening the drug interaction profile of OATP1A2 and OATP1C1 is highly desirable. However, drug interaction screens require suitable model systems and functional assays. In the current study, uptake of a set of cell‐impermeable fluorescent dyes was screened in HEK‐293 and A431 cell lines overexpressing OATP1A2 and OATP1C1. Based on the uptake of fluorescent dye substrates, a functional assay was developed, which was used to characterize OATP inhibitors/substrates. We identify Live/Dead Green (LDG), Live‐or‐Dye 488, and sulforhodamines 101, G, and B as novel fluorescent substrates of OATP1A2 and OATP1C1. We show that LDG uptake is proportional to OATP1A2/1C1 expression, allowing the isolation of cells expressing high transporter levels. Additionally, dye uptake can be used to characterize the drug interaction pattern of OATP1A2 and OATP1C1. We demonstrate that third‐generation P‐glycoprotein inhibitors elacridar, tariquidar, and zosuquidar inhibit OATP1A2 function. Increased toxicity of elacridar in OATP1A2‐expressing cells suggests that OATP1A2 may modulate the distribution of this compound. The fluorescence‐based assays developed in the current study are a good alternative of radioligand‐based tests and pave the way toward high‐throughput screens for OATP1A2/1C1 drug interaction studies.

Rapid Evaporative Ionization Mass Spectrometry (REIMS) was used for the rapid mass spectrometric profiling of cancer cell lines. Spectral reproducibility was assessed for three different cell lines and extent of inter-class differences and intraclass variance were found to allow the identification of these cell lines based on the REIMS data. Subsequently, the NCI60 cell line panel was subjected to REIMS analysis and the resulting dataset was investigated for its distinction of individual cell lines and different tissue types of origin. Information content of REIMS spectral profiles of cell lines were found to be similar to those obtained of mammalian tissues although pronounced differences in relative lipid intensity were observed. Ultimately, REIMS was shown to detect changes in lipid content of cell lines due to Mycoplasma infection. The data show that REIMS is an attractive means to study cell lines involving minimal sample preparation and analysis times in the range of seconds.

Resistance to chemotherapeutic agents is a major obstacle in cancer treatment. A recently proposed strategy is to target the collateral sensitivity of multidrug resistant (MDR) cancer. Paradoxically, the toxicity of certain metal chelating agents is increased, rather than decreased, by the function of P-glycoprotein (Pgp), which is known to confer resistance by effluxing chemotherapeutic compounds from cancer cells. We have recently characterized and compared the solution’s chemical properties including ligand protonation and the metal binding properties of a set of structurally related 8-hydroxyquinoline derived Mannich bases. Here we characterize the impact of the solution stability and redox activity of their iron(III) and copper(II) complexes on MDR-selective toxicity. Our results show that the MDR-selective anticancer activity of the studied 8-hydroxyquinoline derived Mannich bases is associated with the iron deprivation of MDR cells and the preferential formation of redox-active copper(II) complexes, which undergo intracellular redox-cycling to induce oxidative stress.

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.