Cell Surface Transport Research Articles

Base of the Measles Virus Fusion Trimer Head Receives the Signal That Triggers Membrane Fusion

The measles virus (MV) fusion (F) protein trimer executes membrane fusion after receiving a signal elicited by receptor binding to the hemagglutinin (H) tetramer. Where and how this signal is received is understood neither for MV nor for other paramyxoviruses. Because only the prefusion structure of the parainfluenza virus 5 (PIV5) F-trimer is available, to study signal receipt by the MV F-trimer, we generated and energy-refined a homology model. We used two approaches to predict surface residues of the model interacting with other proteins. Both approaches measured interface propensity values for patches of residues. The second approach identified, in addition, individual residues based on the conservation of physical chemical properties among F-proteins. Altogether, about 50 candidate interactive residues were identified. Through iterative cycles of mutagenesis and functional analysis, we characterized six residues that are required specifically for signal transmission; their mutation interferes with fusion, although still allowing efficient F-protein processing and cell surface transport. One residue is located adjacent to the fusion peptide, four line a cavity in the base of the F-trimer head, while the sixth residue is located near this cavity. Hydrophobic interactions in the cavity sustain the fusion process and contacts with H. The cavity is flanked by two different subunits of the F-trimer. Tetrameric H-stalks may be lodged in apposed cavities of two F-trimers. Because these insights are based on a PIV5 homology model, the signal receipt mechanism may be conserved among paramyxoviruses.

NAD+-dependent Sirtuin 1 and 6 Proteins Coordinate a Switch from Glucose to Fatty Acid Oxidation during the Acute Inflammatory Response

The early initiation phase of acute inflammation is anabolic and primarily requires glycolysis with reduced mitochondrial glucose oxidation for energy, whereas the later adaptation phase is catabolic and primarily requires fatty acid oxidation for energy. We reported previously that switching from the early to the late acute inflammatory response following TLR4 stimulation depends on NAD(+) activation of deacetylase sirtuin 1 (SirT1). Here, we tested whether NAD(+) sensing by sirtuins couples metabolic polarity with the acute inflammatory response. We found in TLR4-stimulated THP-1 promonocytes that SirT1 and SirT 6 support a switch from increased glycolysis to increased fatty acid oxidation as early inflammation converts to late inflammation. Glycolysis enhancement required hypoxia-inducing factor-1α to up-regulate glucose transporter Glut1, phospho-fructose kinase, and pyruvate dehydrogenase kinase 1, which interrupted pyruvate dehydrogenase and reduced mitochondrial glucose oxidation. The shift to late acute inflammation and elevated fatty acid oxidation required peroxisome proliferator-activated receptor γ coactivators PGC-1α and β to increase external membrane CD36 and fatty acid mitochondrial transporter carnitine palmitoyl transferase 1. Metabolic coupling between early and late responses also required NAD(+) production from nicotinamide phosphoryltransferase (Nampt) and activation of SirT6 to reduce glycolysis and SirT1 to increase fatty oxidation. We confirmed similar shifts in metabolic polarity during the late immunosuppressed stage of human sepsis blood leukocytes and murine sepsis splenocytes. We conclude that NAD(+)-dependent bioenergy shifts link metabolism with the early and late stages of acute inflammation.

Energy balance and cancer risk at the cellular and whole organism level: modification by metformin

It has long been recognized that dietary restriction (DR) can inhibit carcinogenesis. Recent work has not only clarified the mechanisms involved, which involve the effects of DR on circulating hormones and cytokines, but also demonstrated molecular characteristics of tumors that determine the extent to which they are influenced by variation in host energy intake. Importantly, we and others have also demonstrated that a subset of tumors are growth-stimulated by excess caloric intake, and provided strong circumstantial evidence that hyperinsulinemia is one of the mediating factors. Metformin, a biguanide used in cancer treatment, has been associated with reduced cancer risk in some hypothesis-generating pharmaco-epidemiologic studies. Laboratory studies have provided evidence for some antineoplastic activity. However, this activity is confined to subpopulations including subjects who are obese and/or hyperinsulinemic. Laboratory models suggest that metformin may act in this context because it is well-known to reduce hyperinsulinemia as a consequence of its reduction of gluconeogenesis and hyperglycemia. At the cellular level, metformin acts to inhibit oxidative phosphorylation, and this may provide an additional mechanism that could operate in the subset of cancers or at-risk tissues that express the cell surface transport molecules required for metformin entry to cells. The ATP deficiency induced by metformin may have little effect on some cells, an AMPK-dependent cytostatic effect on others, and still others may suffer an energetic crisis and necrotic death. Current research is providing additional details and mechanistic details underlying these actions, defining the subsets patients for which biguanides may be useful in cancer prevention or treatment, and defining relevant doses for potential new indications. Investigating the possibility of ‘repurposing’ metformin for applications in oncology is ongoing as laboratory and translational research ield data that will help to optimize clinical trial design.

Functional significance of conserved cysteines in the human organic cation transporter 2.

The significance of conserved cysteines in the human organic cation transporter 2 (hOCT2), namely the six cysteines in the long extracellular loop (loop cysteines) and C474 in transmembrane helix 11, was examined. Uptake of tetraethylammonium (TEA) and 1-methyl-4-phenypyridinium (MPP) into Chinese hamster ovary cells was stimulated >20-fold by hOCT2 expression. Both cell surface expression and transport activity were reduced considerably following mutation of individual loop cysteines (C51, C63, C89, C103, and C143), and the C89 and C103 mutants had reduced Michaelis constants (K(t)) for MPP. The loop cysteines were refractory to interaction with thiol-reactive biotinylation reagents, except after pretreatment of intact cells with dithiothreitol or following cell membrane solubilization. Reduction of disulfide bridge(s) did not affect transport, but labeling the resulting free thiols with maleimide-PEO(2)-biotin did. Mutation of C474 to an alanine or phenylalanine did not affect the K(t) value for MPP. In contrast, the K(t) value associated with TEA transport was reduced sevenfold in the C474A mutant, and the C474F mutant failed to transport TEA. This study shows that some but not all of the six extracellular loop cysteines exist within disulfide bridge(s). Each loop cysteine is important for plasma membrane targeting, and their mutation can influence substrate binding. The effect of C474 mutation on TEA transport suggests that it contributes to a TEA binding surface. Given that TEA and MPP are competitive inhibitors, the differential effects of C474 modification on TEA and MPP binding suggest that the binding surfaces for each are distinct, but overlapping in area.

Ginsenoside Re rapidly reverses insulin resistance in muscles of high-fat diet fed rats

ObjectiveIn a previous study, it was found that a ginseng berry extract with a high content of the ginsenoside Re normalized blood glucose in ob/ob mice. The objective of this study was to evaluate the effect of the ginsenoside Re on insulin resistance of glucose transport in muscles of rats made insulin resistant with a high-fat diet. Material/methodRats were fed either rat chow or a high-fat diet for 5 weeks. The rats were then euthanized, and insulin stimulated glucose transport activity was measured in epitrochlearis and soleus muscle strips in vitro. ResultsTreatment of muscles with Re alone had no effect on glucose transport. The high-fat diet resulted in ~50% decreases in insulin responsiveness of GLUT4 translocation to the cell surface and glucose transport in epitrochlearis and soleus muscles. Treatment of muscles with Re in vitro for 90 min completely reversed the high-fat diet-induced insulin resistance of glucose transport and GLUT4 translocation. This effect of Re is specific for insulin stimulated glucose transport, as Re treatment did not reverse the high-fat diet-induced resistance of skeletal muscle glucose transport to stimulation by contractions or hypoxia. ConclusionsOur results show that the ginsenoside Re induces a remarkably rapid reversal of high-fat diet-induced insulin resistance of muscle glucose transport by reversing the impairment of insulin-stimulated GLUT4 translocation to the cell surface.

A Triple Arg Motif Mediates α2B‐Adrenergic Receptor Interaction with Sec24C/D and Export

Recent studies have demonstrated that cargo exit from the endoplasmic reticulum (ER) may be directed by ER export motifs recognized by components of the coat protein II (COPII) vesicles. However, little is known about ER export motifs and vesicle targeting of the G protein-coupled receptor (GPCR) superfamily. Here, we have demonstrated that a triple Arg (3R) motif in the third intracellular loop functions as a novel ER export signal for α(2B)-adrenergic receptor (α(2B)-AR). The 3R motif mediates α(2B)-AR interaction with Sec24C/D and modulates ER exit, cell surface transport and function of α(2B)-AR. Furthermore, export function of the 3R motif is independent of its position within α(2B)-AR and can be conferred to CD8 glycoprotein. These data provide the first evidence implicating that export of GPCRs is controlled by code-directed interactions with selective components of the COPII transport machinery.

A Novel ER Export Motif Modulates the ER‐to‐Cell Surface Traffic of α2B‐Adrenergic Receptor

The molecular mechanisms underlying export of the G protein‐coupled receptor (GPCR) superfamily from the endoplasmic reticulum (ER) remain largely unknown. Here we have demonstrated that a triple Arg (3R) motif in the third intracellular loop functions as a novel ER export signal for α2B‐adrenergic receptor (α2B‐AR). The 3R motif mediates α2B‐AR interaction with Sec24C/D which are components of ER‐derived COPII transprot vesilces, and modulates ER exit, cell surface transport, and function of α2B‐AR. Furthermore, export function of the 3R motif is independent of its position within α2B‐AR and can be conferred to CD8 glycoprotein. These data provide the first evidence implicating that export of GPCRs is controlled by code‐directed interactions with selective components of the COPII transport machinery.

Sorting Nexin 27 Interacts with Multidrug Resistance-associated Protein 4 (MRP4) and Mediates Internalization of MRP4

Multidrug resistance-associated protein 4 (MRP4/ABCC4) makes a vital contribution to the bodily distribution of drugs and endogenous compounds because of its cellular efflux abilities. However, little is known about the mechanism regulating its cell surface expression. MRP4 has a PDZ-binding motif, which is a potential sequence that modulates the membrane expression of MRP4 via interaction with PDZ adaptor proteins. To investigate this possible relationship, we performed GST pull-down assays and subsequent analysis with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. This method identified sorting nexin 27 (SNX27) as the interacting PDZ adaptor protein with a PDZ-binding motif of MRP4. Its interaction was confirmed by a coimmunoprecipitation study using HEK293 cells. Knockdown of SNX27 by siRNA in HEK293 cells raised MRP4 expression on the plasma membrane, increased the extrusion of 6-[(14)C]mercaptopurine, an MRP4 substrate, and conferred resistance against 6-[(14)C]mercaptopurine. Cell surface biotinylation studies indicated that the inhibition of MRP4 internalization was responsible for these results. Immunocytochemistry and cell surface biotinylation studies using COS-1 cells showed that SNX27 localized to both the early endosome and the plasma membrane. These data suggest that SNX27 interacts with MRP4 near the plasma membrane and promotes endocytosis of MRP4 and thereby negatively regulates its cell surface expression and transport function.

Regulation of the cell surface traffic of α2‐adrenergic receptors by small GTPases

The molecular mechanisms responsible for the cell surface targeting of nascent G protein‐coupled receptors (GPCRS) remain poorly elucidated. Our previous studies have utilized α2B‐adrenergic receptor (AR) as a model to define the pathways that mediate forward transport of GPCRs by studying the function of small GTPases in the Rab and ARF/Sar1 subfamilies. In this study, we compared the small GTPases‐coordinated cell surface movement of three α2‐ARs and investigated the possible mechanisms. Transient expression of dominant‐negative mutants of Rab, ARF, and Sar1 clearly inhibited the cell surface transport of α2A‐AR and α2C‐AR. Although the cell surface expression of α2A‐AR was much more abundant than that of α2C‐AR, their responsiveness to the small GTPase mutants was similar. In contrast, the cell surface transport of α2B‐AR was inhibited by some, but not all the mutants. More interestingly, three α2‐ARs differentially interacted with some small GTPases and the interaction was identified to be mediated via the third intracellular loops and/or the C‐terminal tails of the receptors. These data have demonstrated that the cell surface traffic of three α2‐ARs is controlled by different mechanisms and their differential interactions with different small GTPases involved in distinct transport steps along the secretory pathway may play an important role.

Electron Tomography Reveals Rab6 Is Essential to the Trafficking of trans‐Golgi Clathrin and COPI‐Coated Vesicles and the Maintenance of Golgi Cisternal Number

We have shown previously that Rab6, a small, trans-Golgi-localized GTPase, acts upstream of the conserved oligomeric Golgi complex (COG) and ZW10/RINT1 retrograde tether complexes to maintain Golgi homeostasis. In this article, we present evidence from the unbiased and high-resolution approach of electron microscopy and electron tomography that Rab6 is essential to the trans-Golgi trafficking of two morphological classes of coated vesicles; the larger corresponds to clathrin-coated vesicles and the smaller to coat protein I (COPI)-coated vesicles. On the basis of the site of coated vesicle accumulation, cisternal dilation and the normal kinetics of cargo transport from the endoplasmic reticulum (ER) to Golgi followed by delayed Golgi to cell surface transport, we suggest that Golgi function in cargo transport is preferentially inhibited at the trans-Golgi/trans-Golgi network (TGN). The >50% increase in Golgi cisternae number in Rab6-depleted HeLa cells that we observed may well be coupled to the trans-Golgi accumulation of COPI-coated vesicles; depletion of the individual Rab6 effector, myosin IIA, produced an accumulation of uncoated vesicles with if anything a decrease in cisternal number. These results are the first evidence for a Rab6-dependent protein machine affecting Golgi-proximal, coated vesicle accumulation and probably transport at the trans-Golgi and the first example of concomitant cisternal proliferation and increased Golgi stack organization under inhibited transport conditions.

Discovery and structural development of small molecules that enhance transport activity of bile salt export pump mutant associated with progressive familial intrahepatic cholestasis type 2

Progressive familial intrahepatic cholestasis type 2 (PFIC2) is caused by hereditary mutations of bile salt export pump (BSEP), such as E297G BSEP, which is a folding-defective mutant that is unable to traffic beyond the endoplasmic reticulum (ER). 4-Phenylbutyric acid (4-PBA) enhances the cell surface expression and transport capacity of E297G BSEP, but has a relatively high dose (1mM or more) is required to show the effect. Here, we show that bile acids possibly act as pharmacological chaperones, promoting the proper folding and trafficking of E297G BSEP. We also describe the discovery and structural development of non-steroidal compounds with potent pharmacological chaperone activity for E297G BSEP.

Transport of Influenza Virus Neuraminidase (NA) to Host Cell Surface Is Regulated by ARHGAP21 and Cdc42 Proteins

Influenza virus neuraminidase (NA) is transported to the virus assembly site at the plasma membrane and is a major viral envelope component that plays a critical role in the release of progeny virions and in determination of host range restriction. However, little is known about the host factors that are involved in regulating the intracellular and cell surface transport of NA. Here we identified the Cdc42-specific GAP, ARHGAP21 differentially expressed in host cells infected with influenza A virus using cDNA microarray analysis. Furthermore, we have investigated the involvement of Rho family GTPases in NA transport to the cell surface. We found that expression of constitutively active or inactive mutants of RhoA or Rac1 did not significantly affect the amount of NA that reached the cell surface. However, expression of constitutively active Cdc42 or depletion of ARHGAP21 promoted the transport of NA to the plasma membranes. By contrast, cells expressing shRNA targeting Cdc42 or overexpressing ARHGAP21 exhibited a significant decrease in the amount of cell surface-localized NA. Importantly, silencing Cdc42 reduced influenza A virus replication, whereas silencing ARHGAP21 increased the virus replication. Together, our results reveal that ARHGAP21- and Cdc42-based signaling regulates the NA transport and thereby impacts virus replication.

4-Phenylbutyrate modulates ubiquitination of hepatocanalicular MRP2 and reduces serum total bilirubin concentration

Multidrug resistance-associated protein 2 (in humans, MRP2; in rodents, Mrp2) mediates biliary excretion of bilirubin glucuronides. Therefore, upregulation of MRP2/Mrp2 expression may improve hyperbilirubinemia. We investigated the effects of 4-phenylbutyrate (4PBA), a drug used to treat ornithine transcarbamylase deficiency (OTCD), on the cell surface expression and transport function of MRP2/Mrp2 and serum T-Bil concentration. MRP2-expressing MDCKII (MRP2-MDCKII) cells and rats were studied to explore the change induced by 4PBA treatment in the cell surface expression and transport function of MRP2/Mrp2 and its underlying mechanism. Serum and liver specimens from OTCD patients were analyzed to examine the effect of 4PBA on hepatic MRP2 expression and serum T-Bil concentration in humans. In MRP2-MDCKII cells and the rat liver, 4PBA increased the cell surface expression and transport function of MRP2/Mrp2. In patients with OTCD, hepatic MRP2 expression increased and serum T-Bil concentration decreased significantly after 4PBA treatment. In vitro studies designed to explore the mechanism underlying this drug action suggested that cell surface-resident MRP2/Mrp2 is degraded via ubiquitination-mediated targeting to the endosomal/lysosomal degradation pathway and that 4PBA inhibits the degradation of cell surface-resident MRP2/Mrp2 by reducing its susceptibility to ubiquitination. 4PBA activates MRP2/Mrp2 function through increased expression of MRP2/Mrp2 at the hepatocanalicular membrane by modulating its ubiquitination, and thereby decreases serum T-Bil concentration. 4PBA has thus therapeutic potential for improving hyperbilirubinemia.

Abstract B12: The miR-15 family and miR-155 mediate WEE1 and CHEK1 effects on cisplatin-resistance in cancer cells

Abstract A major challenge in the successful treatment of cancer is the ability of cancer cells to acquire a multi-drug resistant (MDR) phenotype, preventing response to chemotherapeutic agents. Cisplatin (cis-diamminedichloroplatinum(II)) is a chemotherapeutic drug commonly used against malignancies including ovarian, and cervical tumors. Cisplatin resistance (CP-r) studied in cultured cancer cells often results from a cellular defense mechanism that results in a highly complex pleiotropic phenotype that confers resistance by reducing apoptosis, up-regulating DNA damage repair mechanisms, altering cell cycle checkpoints, altering protein localization, redirecting cell surface transporters, and disrupting assembly of the cytoskeleton thereby changing protein distribution in the cell. This results in cells that are permanently resistant not only to cisplatin but also show cross-resistance to multiple toxic compounds that piggyback into cells on existing transporters. The highly pleiotropic CP-r phenotype occurs in a single step that has yet to be identified. miRNAs have crucial roles in diverse biological processes such as phenotypic stabilization, and have been shown to also be involved in tumor growth and response to chemotherapy. We hypothesized that miRNAs may play a role in mediating the phenotypic changes associated with a pleiotropic cisplatin resistance mechanism. KB-CP.5 (resistant to 0.5ug/ml CP) cells were screened with whole kinome siRNA (Ambion SilencerSelect, 3 siRNAs per gene) and miRNA mimics (Sanger miRBase 13.0). Cells were then challenged with cisplatin at IC5 and IC30 doses (normally causing minimal toxicity to CP-r cells). Cell viability was determined using the Promega CellTiter-Glo luminescent viability assay, and conditions that sensitized the CP-r cells were examined. The miR-15/16/195/424/497 family miRNA mimics were found to confer >2-fold increased sensitivity to CP.5 cells. miR-16 gave a 5-fold increase in sensitivity and its family member miR-15a caused a 3.4-fold increase, accounting for two of the top three hits in the primary screen. Follow-up dose-response analysis indicated that miR-15a and miR-16 mimics yielded a 20- and 10-fold shift in the cisplatin sensitivity of CP.5 cells, respectively. The two strongest kinome effects were elicited by silencing WEE1 and CHEK1. Using TargetScanHuman (available at www.targetscan.org), an algorithm program for the prediction of miRNA targets, we found that the miR-15 family is predicted to target WEE1 and CHEK1. For the miR-15 family, RT-PCR shows a 2 to 5-fold decrease in expression in the CP-r cells compared to KB-3-1 parental cells. In addition miR-155 has a 3-fold decrease in expression in KB-CP.5 compared to KB-3-1 parental cells. TargetScan analysis of WEE1 indicates that miR-155 is also a potential regulator of the WEE1 kinase. We confirmed the ability of the miR-15 family and miR-155 to sensitize resistant cells to cisplatin by targeting WEE1 and CHEK1 by performing loss-of -function and gain-of-function experiments. Using the MTT cytotoxicity assay and full cisplatin dose response curves, miRNA inhibitors of the miR15 family increased resistance while mimics sensitized resistant cells. miR-155 mimics resulted in sensitivity returning to parental levels and inhibitors of WEE1 and CHEK1 sensitized KB-CP.5 cells to CP. In conclusion, we have identified a miRNA family responsible for regulating the cell-cycle checkpoint kinases WEE1 and CHEK1 associated with cisplatin resistance. Citation Format: Lynn M. Pouliot, Jennifer Bai, Matthew D. Hall, Scott E. Martin, Michael M. Gottesman. The miR-15 family and miR-155 mediate WEE1 and CHEK1 effects on cisplatin-resistance in cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr B12.

Effect of clinical mutations on functionality of the human riboflavin transporter-2 (hRFT-2)

The Brown–Vialetto–Van Laere syndrome (BVVLS) is a rare neurological disease characterized by ponto-bulbar palsy, bilateral sensorineural deafness, and respiratory insufficiency. Recent genetic studies have identified mutations in the C20orf54 gene, which encodes the human riboflavin (RF) transporter -2 (hRFT-2) and suggested their link to the manifestation of BVVLS. However, there is nothing currently known about the effect of these mutations on functionality of hRFT-2, a protein that is expressed in a variety of tissues with high expression in the intestine. We addressed this issue using the human-derived intestinal epithelial Caco-2 cells. Our results showed significant (P<0.01) impairment in RF uptake by Caco-2 cells transiently expressing W17R, P28T, E36K, E71K, and R132W (but not L350M) hRFT-2 mutants. This impairment in RF transport was not due to a decrease in transcription and/or translation of hRFT-2, since mRNA and protein levels of the carrier were similar in cells expressing the mutants and wild-type hRFT-2. Confocal images of live Caco-2 cells transiently transfected with hRFT-2 mutants (fused with green fluorescent protein) showed the P28T, E36K, E71K, and R132W mutants were retained within the endoplasmic reticulum, while the W17R and L350M mutants were expressed at the cell membrane; cell surface expression of the W17R mutant was further confirmed by direct determination of cell surface transporter density. These results show for the first time that some of the BVVLS associated mutations in hRFT-2 affect the transporter functionality and that this effect is mediated via alterations in membrane targeting and/or activity of the transporter.

Multiple post-translational modifications regulate E-cadherin transport during apoptosis

E-cadherin is synthesized as a precursor and then undergoes cleavage by proprotein convertases. This processing is essential for E-cadherin maturation and cell adhesion. Loss of cell adhesion causes detachment-induced apoptosis, which is called anoikis. Anoikis can be inhibited despite loss of cell-matrix interactions by preserving E-cadherin-mediated cell-cell adhesion. Conversely, acute loss of E-cadherin sensitizes cells to apoptosis by unknown post-translational mechanisms. After treatment of breast cancer cells with drugs, we found that two independent modifications of E-cadherin inhibit its cell surface transport. First, O-linked β-N-acetylglucosamine (O-GlcNAc) modification of the cytoplasmic domain retains E-cadherin in the endoplasmic reticulum. Second, incomplete processing by proprotein convertases arrests E-cadherin transport late in the secretory pathway. We demonstrated these E-cadherin modifications (detected by specific lectins and antibodies) do not affect binding to α-catenin, β-catenin or γ-catenin. However, binding of E-cadherin to Type I gamma phosphatidylinositol phosphate kinase (PIPKIγ), a protein required for recruitment of E-cadherin to adhesion sites, was blocked by O-GlcNAc glycosylation (O-GlcNAcylation). Consequently, E-cadherin trafficking to the plasma membrane was inhibited. However, deletion mutants that cannot be O-GlcNAcylated continued to bind PIPKIγ, trafficked to the cell surface and delayed apoptosis, confirming the biological significance of the modifications and PIPKIγ binding. Thus, O-GlyNAcylation of E-cadherin accelerates apoptosis. Furthermore, cell-stress-induced inactivation of proprotein convertases, inhibited E-cadherin maturation, further exacerbating apoptosis. The modifications of E-cadherin by O-GlcNAcylation and lack of pro-region processing represent novel mechanisms for rapid regulation of cell surface transport of E-cadherin in response to intoxication.

Impact of growth conditions on transport behavior of E. coli

The aim of this investigation is to determine the effect that growth solution has on the cell surface properties and transport behavior of eleven Escherichia coli isolates through saturated porous media. The two growth solutions used were a standard laboratory growth medium (LB) and a dairy manure extract solution. In general, cells grown in manure extract were more hydrophobic, had a more negative zeta potential, had lower amounts of surface macromolecules, and had lower attachment efficiencies than isolates grown in LB. An inverse relationship between the natural log of zeta potential and the attachment efficiency of the isolates for the cells grown in LB media was the only statistically significant correlation observed between transport behavior and cell characteristics of the isolates. This study shows the need to consider growth conditions when studying bacteria to better mimic the environmental stresses that bacteria undergo in the natural environment. This approach could lead to a better understanding of the behavior of manure-derived bacteria in aquatic and terrestrial environments.

Quantitative 3D Tracing of Gene-delivery Viral Vectors in Human Cells and Animal Tissues

Trafficking through a variety of cellular structures and organelles is essential for the interaction between gene-delivery vectors (i.e., adeno-associated virus (AAV) and liposomes) and host cells/tissues. Here, we present a method of computer-assisted quantitative 3D biodistribution microscopy that samples the whole population of fluorescently-labeled vectors and document their trafficking routes. Using AAV as a working model, we first experimentally defined numerical parameters for the singularity of Cy5-labeled particles by combining confocal microscopy and atomic force microscopy (AFM). We then developed a robust approach that integrates single-particle fluorescence imaging with 3D deconvolution and isosurface rendering to quantitate viral distribution and trafficking in human cells as well as animal tissues at the single-particle level. Using this quantitative method, we uncovered an as yet uncharacterized rate-limiting step during viral cell entry, while delineating nuclear accumulation of virions during the first 8 hours postinfection. Further, our studies revealed for the first time that following intramuscular injection, AAV spread progressively across muscle tissues through endomysium between myofibers instead of traversing through target cells. Such 3D resolution and quantitative dissection of vector-host interactions at the subcellular level should significantly improve our ability to resolve trafficking mechanisms of gene-delivery particles and facilitate the development of enhanced viral vectors.

Use of Baculovirus BacMam Vectors for Expression of ABC Drug Transporters in Mammalian Cells

ATP-binding cassette (ABC) drug transporters ABCB1 [P-glycoprotein (Pgp)] and ABCG2 are expressed in many tissues including those of the intestines, the liver, the kidney and the brain and are known to influence the pharmacokinetics and toxicity of therapeutic drugs. In vitro studies involving their functional characteristics provide important information that allows improvements in drug delivery or drug design. In this study, we report use of the BacMam (baculovirus-based expression in mammalian cells) expression system to express and characterize the function of Pgp and ABCG2 in mammalian cell lines. BacMam-Pgp and BacMam-ABCG2 baculovirus-transduced cell lines showed similar cell surface expression (as detected by monoclonal antibodies with an external epitope) and transport function of these transporters compared to drug-resistant cell lines that overexpress the two transporters. Transient expression of Pgp was maintained in HeLa cells for up to 72 h after transduction (48 h after removal of the BacMam virus). These BacMam-baculovirus-transduced mammalian cells expressing Pgp or ABCG2 were used for assessing the functional activity of these transporters. Crude membranes isolated from these cells were further used to study the activity of these transporters by biochemical techniques such as photo-cross-linking with transport substrate and adenosine triphosphatase assays. In addition, we show that the BacMam expression system can be exploited to coexpress both Pgp and ABCG2 in mammalian cells to determine their contribution to the transport of a common anticancer drug substrate. Collectively, these data demonstrate that the BacMam-baculovirus-based expression system can be used to simultaneously study the transport function and biochemical properties of ABC transporters.

Molecular and cellular pharmacology of the novel noncamptothecin topoisomerase I inhibitor Genz-644282.

Camptothecin derivatives are powerful anticancer drugs because of their ability to trap topoisomerase I (Top1)-DNA cleavage complexes. However, they exhibit clinical limitations due to the instability of their α-hydroxylactone six-membered E-ring structure. In addition, they exhibit bone marrow and intestinal toxicity, especially in adults, and are drug efflux substrates. Here, we report a novel Top1 inhibitor, Genz-644282. We show that Genz-644282 and its metabolites induce Top1 cleavage at similar, as well as unique genomic positions, compared with camptothecin. The compound also induces protein-linked DNA breaks and Top1-DNA cleavage complexes that persist longer after compound removal than camptothecin. Concentration-dependent and persistent γH2AX formation was readily observed in cells treated with Genz-644282, and was present in greater than 50% of the cell population following 24 hours compound exposure. The compound shows partial cross-resistance in cell lines resistant to camptothecin. These cell lines include the human prostate DU145RC0.1 and the leukemic CEM/C2 cells. Limited cross-resistance to Genz-644282 was also found in the Top1 knockdown colon cancer (HCT116) and breast cancer (MCF7) cell lines and in human adenocarcinoma cells (KB31/KBV1) that overexpress (P-glycoprotein, ABCB1), a member of the ATP-binding cassette family of cell surface transport proteins known to confer MDR. Together, our results provide the first molecular and cellular characterization of Genz-644282 and its clinically relevant metabolites.