Impact of probe compound in MRP2 vesicular transport assays.

MRP2 is an efflux transporter that is expressed mainly in the canalicular membrane of hepatocytes, where it expels polar and ionic compounds into the bile. MRP2 is also present in the apical membrane of enterocytes and epithelial cells of proximal tubules of the kidney. Inhibition of MRP2 transport can lead to the accumulation of metabolites and other MRP2 substrates up to toxic levels in these cells. The transport properties of MRP2 are frequently studied with the vesicular transport assay. The assay identifies compounds that interact with MRP2 by measuring the effect of a compound on the transport of a radioactively labeled or fluorescent probe. We have compared the effect of eight selected test compounds (quercetin, disopyramide, paracetamol, indomethacin, diclofenac, estrone-3-sulfate, budesonide, and thioridazine) on the MRP2-mediated transport of three commonly used probes: 5(6)-carboxy-2,7-dichlorofluorescein, leukotriene C4 and estradiol-17-ß-d-glucuronide (E217ßG). Five of the test compounds had different probe-dependent effects on the MRP2-mediated transport, suggesting differences in the transport mechanism of the probes. Our results underline the complexity of substrate recognition by these efflux transporters and the difficulties in directly comparing results obtained with different assays, especially when different probes are used.

Gene expression analysis in SV-40 immortalized human corneal epithelial cells cultured with an air-liquid interface.

PURPOSE: To compare the global gene expression profile of stratified epithelia generated in vitro using simian virus 40 (SV40) immortalized human corneal epithelial cells with the previously reported gene expression of normal human corneal epithelia. METHODS: Immortalized cells expanded in submerged culture were grown in an air-liquid interface of liquid permeable collagen-coated filters to foster stratification and differentiation. Stratified epithelia displaying resistances exceeding 300 Ω·cm2 were dissolved in an RNA purification lysis buffer. Purified RNA was used to globally determine gene expression levels using high-density single-channel oligonucleotide microarrays. Raw hybridization readings were converted into relative gene expression levels using Robust Multi-array Average (RMA) algorithm. Expression levels for selected genes were validated by real-time RT-qPCR. The biologic significance of the gene expression profiles was interpreted with the help of several microarray software analysis tools and ad hoc thematical analysis. RESULTS: The stratified cell culture to native epithelial comparison identified over- and under-expression in 22% and 14% of the probed genes, respectively. The larger expression decreases occurred in genes intimately associated with both the stratified epithelial lineage at large such as keratin 14 and the corneal phenotype, such as keratin 12, connexin 43, aldehyde dehydrogenases (ALDHs), and paired box gene 6 (PAX6) and its whole downstream transcriptome. Overexpression related to genes associated with cell cycling stimulation. CONCLUSIONS: The results indicate that the stratified corneal epithelial cell model generated using SV40 immortalized cells may be useful only in certain research applications. Extrapolations of studies with these cells to actual tissue cells should be done with a great deal of caution.

Effluxing ABC transporters in human corneal epithelium.

ATP-binding cassette (ABC) transporters are able to efflux their substrate drugs from the cells. We compared expression of efflux proteins in normal human corneal epithelial tissue, primary human corneal epithelial cells (HCEpiC), and corneal epithelial cell culture model (HCE model) based on human immortal cell line. Expression of multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1-6 (MRP1-6) and breast cancer resistance protein (BCRP) was studied using quantitative RT-PCR, Western blot, and immunohistochemistry. Only MRP1, MRP5, and BCRP were expressed in the freshly excised human corneal epithelial tissue. Expression of MRP1 and MRP5 was localized predominantly in the basal cells of the central cornea and limbus. Functional efflux activity was shown in the cell models, but they showed over-expression of most efflux transporters compared to that of normal corneal epithelium. In conclusion, MRP1, MRP5, and BCRP are expressed in the corneal epithelium, but MDR1, MRP2, MRP3, MRP4, and MRP6 are not significantly expressed. HCE cell model and commercially available primary cells deviate from this expression profile.

Monocarboxylate transport in human corneal epithelium and cell lines.

Monocarboxylate transporters (MCTs) are transmembrane proteins capable of transferring lactate and other endogenous and exogenous monocarboxylates across the cell membrane. The aim of the present study was to assess the expression and transporter role of human MCT1, MCT3 and MCT4 in the corneal epithelium, corneal epithelial cell lines (primary HCEpiC and immortalized HCE cells) and isolated rabbit corneas. MCT1 and MCT4 were expressed in the human corneal epithelium and the cell lines at mRNA and protein levels. Cellular uptake studies showed saturable and pH-dependent l-lactic acid transport, which was inhibited by various monocarboxylates like diclofenac and flurbiprofen. The permeability of benzoic acid across the rabbit cornea was higher in absorptive direction and this directionality was diminished in the presence of monocarboxylate drug valproic acid. Monocarboxylate transport was functional in the human corneal epithelial cells and rabbit cornea and it may play a role in the ocular drug absorption.

Chemokine-derived peptides as carriers for gene delivery to CXCR4 expressing cells.

BACKGROUND: Cell and tissue-specific DNA delivery can be achieved by derivatizing vehicles with a targeting ligand for certain receptor. CXCR4 is a receptor of chemokine stromal cell-derived factor (SDF)-1 and viral protein viral macrophage inflammatory protein (vMIP)-II. It is expressed on some types of stem and cancer cells. The present study aimed to design and characterize the group of CXCR4 targeted peptides for receptor-mediated gene delivery. We focused on bifunctional peptide carriers: two derived from N-terminal sequences of SDF-1 and one from vMIP-II. METHODS: Three synthetic chemokine-derived peptides, designated long CDP (KPVSLSYRSPSRFFESH-K9-biotin), short CDP (KPVSLSYR-K9-biotin) and viral CDP (D-LGASWHRPDK-K9-biotin), were evaluated for gene delivery to CXCR4 positive and negative cells. Oligolysine K9-biotin was used as a control. The Lys 8 moiety binds DNA electrostatically, whereas C-terminal lysine was modified with biotin to study intracellular uptake of the peptides. Complex formation with DNA was monitored by ethidium bromide fluorescence quenching. RESULTS: All peptides condensed plasmid DNA. Gene delivery by CDP/DNA complexes is glycerol-dependent and the level of luciferase expression with signal modified carriers was comparable with the efficacy of polyethylenimine (PEI) in CXCR4 expressing cell lines (A172, HeLa) and was ten- to 50-fold higher compared to unmodified peptide. By contrast, CDP transfection efficacy on CXCR4-negative cells (chinese hamster ovary) was much lower than in PEI. Intracellular uptake analysis of biotin-labeled peptides indicated that CDPs entered cells more efficiently than oligolysine. CONCLUSIONS: The small, bifunctional peptides reported in the present study may be useful in gene delivery to (and gene therapy of) the different tumors and other cells expressing CXCR4.

SUMO-1 promotes association of SNURF (RNF4) with PML nuclear bodies.

Small nuclear RING finger protein SNURF (RNF4) is involved in transcriptional and cell growth regulation. We show here that a significant portion of endogenous SNURF localizes to nuclear bodies (NBs) that overlap with or are adjacent to domains containing endogenous promyelocytic leukemia (PML) protein and small ubiquitin-like modifier-1 (SUMO-1). In biochemical assays, SNURF efficiently binds SUMO-1 in a noncovalent fashion. SNURF is also covalently modified by SUMO-1 at nonconsensus attachment sites. Ectopic expression of SUMO-1 markedly enhances the interaction between PML3 (PML IV) and SNURF, but covalent attachment of SUMO-1 to neither protein is required. Moreover, overexpression of PML3, but not PML-L (PML III), abolishes the coactivation function of SNURF in transactivation assays, which parallels the ability of PML3 to recruit SNURF to nuclear bodies. In sum, we have identified SNURF as a novel component in PML bodies and suggest that SUMO-1-facilitated sequestration into these nuclear domains regulates the transcriptional activity of SNURF.

Small nuclear RING finger protein stimulates the rat luteinizing hormone-beta promoter by interacting with Sp1 and steroidogenic factor-1 and protects from androgen suppression.

GnRH controls expression of the LH subunit genes, alpha and LHbeta, with the LHbeta subunit regulated most dramatically. Two enhancer regions, distal and proximal, on the rat LHbeta gene promoter cooperate for full basal expression and GnRH stimulation. It has been hypothesized that the transcription factors binding to these regions, Sp1, Egr-1, and steroidogenic factor 1 (SF-1), may interact directly or indirectly via a coactivator. One such coactivator may be small nuclear RING finger protein (SNURF), which is expressed in pituitary tissue and the LbetaT2 gonadotrope cell line. In transfection experiments in LbetaT2 cells, SNURF stimulated basal expression of LHbeta and increased overall GnRH stimulation. SNURF specifically stimulated LHbeta, with no effect on the alpha-subunit promoter. SNURF interacts with Sp1 and SF-1, but not Egr-1, in pull-down experiments. Point mutations or deletions of SNURF functional domains demonstrated that Sp1 and SF-1 interactions with SNURF are required for SNURF stimulatory effects on the LHbeta promoter. Endogenous SNURF is associated with the LHbeta promoter on native chromatin, suggesting that it plays a physiological role in LHbeta gene expression. SNURF also binds the androgen receptor, and SNURF overexpression overcomes androgen suppression of GnRH-stimulated LHbeta but not alphasubunit promoter activity. SNURF mutations that disrupt Sp1 or SF-1 binding eliminate rescue by SNURF. We conclude that SNURF may mediate interactions between the distal and proximal GnRH response regions of the LHbeta promoter to stimulate transcription and can also protect the promoter from androgen suppression.