Transient Transfection Of HEK293 Cells Research Articles

Extracellular vesicle depletion and UGCG overexpression mitigate the cell density effect in HEK293 cell culture transfection

The hitherto unexplained reduction of cell-specific productivity in transient gene expression (TGE) at high cell density (HCD) is known as the cell density effect (CDE). It currently represents a major challenge in TGE-based bioprocess intensification. This phenomenon has been largely reported but the molecular principles governing it are still unclear. The CDE is currently understood to be caused by the combination of an unknown inhibitory compounds in the extracellular medium and an uncharacterized cellular change at HCD. This study investigates the role of extracellular vesicles (EVs) as extracellular inhibitors for transfection through the production of HIV-1 Gag virus-like particles (VLPs) via transient transfection in HEK293 cells. EV-depletion from the extracellular medium restored transfection efficiency in conditions suffering from the CDE, also enhancing VLP budding and improving production by 60%. Moreover, an alteration in endosomal formation was observed at HCD, sequestering polyplexes and preventing transfection. Overexpression of UGCG enzyme removed intracellular polyplex sequestration, improving transfection efficiency. Combining EV-depletion and UGCG overexpression improved transfection efficiency by ∼45% at 12x106 cells/mL. These results suggest that the interaction between polyplexes and extracelluar and intracellular vesicles plays a crutial role in the CDE, providing insights for the development of strategies to mitigate its impact.

Erratum: High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension.

An erratum was issued for:High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension. The Authors section was updated. from: Ganesh P. Subedi1 Roy W. Johnson2 Heather A. Moniz2 Kelley W. Moremen2 Adam Barb1 1The Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University 2Complex Carbohydrate Research Center, University of Georgia to Ganesh P. Subedi1 Roy W. Johnson2 Heather A. Moniz2 Kelley W. Moremen2 Adam W. Barb1 1The Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University 2Complex Carbohydrate Research Center, University of Georgia.

Whole exome sequencing and functional characterization increase diagnostic yield in siblings with a 46, XY difference of sexual development (DSD)

Pathogenic biallelic variants in HSD17B3 result in 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) deficiency, variable disruption of testosterone production, and phenotypic diversity among 46, XY individuals with differences of sexual development (DSDs). We performed quad whole exome sequencing (WES) on two male siblings with microphallus, perineal hypospadias, and bifid scrotum and their unaffected parents. Both male siblings were compound heterozygous for a rare pathogenic HSD17B3 variant (c.239 G > A, p.R80Q) previously identified among individuals with 17β-HSD3 deficiency and a HSD17B3 variant (c.641A > G, p.E214 G) of uncertain significance. Following WES, the siblings underwent hCG stimulation testing with measurement of testosterone, androstenedione, and dihydrotestosterone which was non-diagnostic. To confirm pathogenicity of the HSD17B3 variants, we performed transient transfection of HEK-293 cells and measured conversion of radiolabeled androstenedione to testosterone. Both HSD17B3 variants decreased conversion of radiolabeled androstenedione to testosterone. As pathogenic HSD17B3 variants are rare causes of 46, XY DSD and hCG stimulation testing may not be diagnostic for 17β-HSD3 deficiency, WES in 46, XY individuals with DSDs can increase diagnostic yield and identify genomic variants for functional characterization of disruption of testosterone production.

Effects of a Common Eight Base Pairs Duplication at the Exon 7-Intron 7 Junction on Splicing, Expression, and Function of OCT1.

Organic cation transporter 1 (OCT1, SLC22A1) is localized in the sinusoidal membrane of human hepatocytes and mediates hepatic uptake of weakly basic or cationic drugs and endogenous compounds. Common amino acid substitutions in OCT1 were associated with altered pharmacokinetics and efficacy of drugs like sumatriptan and fenoterol. Recently, the common splice variant rs35854239 has also been suggested to affect OCT1 function. rs35854239 represents an 8 bp duplication of the donor splice site at the exon 7-intron 7 junction. Here we quantified the extent to which this duplication affects OCT1 splicing and, as a consequence, the expression and the function of OCT1. We used pyrosequencing and deep RNA-sequencing to quantify the effect of rs35854239 on splicing after minigene expression of this variant in HepG2 and Huh7 cells and directly in human liver samples. Further, we analyzed the effects of rs35854239 on OCT1 mRNA expression in total, localization and activity of the resulting OCT1 protein, and on the pharmacokinetics of sumatriptan and fenoterol. The 8 bp duplication caused alternative splicing in 38% (deep RNA-sequencing) to 52% (pyrosequencing) of the minigene transcripts when analyzed in HepG2 and Huh7 cells. The alternatively spliced transcript encodes for a truncated protein that after transient transfection in HEK293 cells was not localized in the plasma membrane and was not able to transport the OCT1 model substrate ASP+. In human liver, however, the alternatively spliced OCT1 transcript was detectable only at very low levels (0.3% in heterozygous and 0.6% in homozygous carriers of the 8 bp duplication, deep RNA-sequencing). The 8 bp duplication was associated with a significant reduction of OCT1 expression in the human liver, but explained only 9% of the general variability in OCT1 expression and was not associated with significant changes in the pharmacokinetics of sumatriptan and fenoterol. Therefore, the rs35854239 variant only partially changes splicing, causing moderate changes in OCT1 expression and may be of only limited therapeutic relevance.

Optimizing high-yield production of SARS-CoV-2 soluble spike trimers for serology assays

The SARS-CoV-2 spike trimer is the primary antigen for several serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Until stable cell lines are developed to increase the titer of this secreted protein in mammalian cell culture, the low yield of spike protein produced from transient transfection of HEK293 cells will be a limiting factor for these assays. To improve the yield of spike protein and support the high demand for antigens in serology assays, we investigated several recombinant protein expression variables by altering the incubation temperature, harvest time, chromatography strategy, and final protein manipulation. Through this investigation, we developed a simplified and robust purification strategy that consistently yields 5 mg of protein per liter of expression culture for two commonly used forms of the SARS-CoV-2 spike protein. We show that these proteins form well-behaved stable trimers and are consistently functional in serology assays across multiple protein production lots.

Optimization of Insect Odorant Receptor Trafficking and Functional Expression Via Transient Transfection in HEK293 Cells.

Insect odorant receptors (ORs) show a limited functional expression in various heterologous expression systems including insect and mammalian cells. This may be in part due to the absence of key components driving the release of these proteins from the endoplasmic reticulum and directing them to the plasma membrane. In order to mitigate this problem, we took advantage of small export signals within the human HCN1 and Rhodopsin that have been shown to promote protein release from the endoplasmic reticulum and the trafficking of post-Golgi vesicles, respectively. Moreover, we designed a new vector based on a bidirectional expression cassette to drive the functional expression of the insect odorant receptor coreceptor (Orco) and an odor-binding OR, simultaneously. We show that this new method can be used to reliably express insect ORs in HEK293 cells via transient transfection and that is highly suitable for downstream applications using automated and high-throughput imaging platforms.

Expression of Recombinant LDLR–EGFP Fusion Protein in HEK-293 Cells as a Promising Tool to Assess the Effect of LDLR Gene Mutations

Mutations in the low density lipoprotein receptor gene (LDLR) frequently impair folding and intracellular traffic of the receptor protein, resulting in the development of a monogenic disorder, familial hypercholesterolemia (FH). Identification of novel LDLR mutations requires confirmation of their functional importance in distinguishing pathogenic mutations from neutral changes in the aminoacid sequence. To elaborate a system for evaluation of the effect of mutation on the folding and intracellular transport of the LDLR, as well as its ability to bind low density lipoprotein (LDL), we constructed a plasmid containing LDLR cDNA and the gene of enhanced green fluorescent protein (EGFP). Confocal microscopy has shown that, upon transient transfection of HEK293 cells with the plasmid, the recombinant fusion protein LDLR–EGFP is transported onto the cellular membrane and binds labeled LDL. This construct will be further modified by site-directed mutagenesis to reproduce the LDLR missense mutations most common in the population of northwest Russia so as to study the subcellular localization and function of the modified chimeric protein.

Scalable Production and Purification of Adeno-Associated Viral Vectors (AAV).

Here we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium in orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI) mediated transfection of a 2-plasmid system and is specified for production in milliliter to liter scales. After PEI and plasmid DNA (pDNA) complex formation the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 3-day batch process, cell cultures are further processed using different methods for lysis and recovery. Methods for the purification of viral particles are described, including iodixanol gradient purification, immunoaffinity chromatography, and ultrafiltration, as well as quantitative PCR to quantify vector titer.

Heterodimerization of Organic Anion Transporting Polypeptide 1B3 can affect function and surface expression

OATP1B3 is a drug transporter expressed at the basolateral membrane of human hepatocytes, along with other transporters, including OATP1B1, OCT1 and NTCP. It has a broad spectrum of substrates including endogenous compounds and numerous drugs. Thus, modulation of OATP1B3 function and expression has the potential to affect drug efficacy and disposition. Previous studies in our lab demonstrated that OATP1B3 can form homo‐ and heterodimers, both in cell models and in frozen human liver tissue. However, functional consequences of these interactions have not been investigated so far. In this study, we used transient transfection of HEK293 cells with FLAG‐tagged OATP1B3 in combination with a His‐tagged non‐functional OATP1B3 mutant, or with His‐tagged OATP1B1 or NTCP, surface biotinylation, and cell‐based uptake assays to characterize the functional consequences of the dimerization. Co‐transfection of mutant and wild‐type OATP1B3 resulted in a similar uptake of both estrone‐3‐sulfate and estradiol‐17β‐glucuronide after normalization for surface expression as co‐transfection of empty vector with wild‐type OATP1B3, suggesting that the homodimer of OATP1B3 works as two independent functional subunits. Uptake of the OATP1B3‐specific substrate CCK8 decreased significantly when OATP1B3 was co‐expressed with NTCP, while co‐expression with OATP1B1 had no effect. In contrast, uptake of low concentration estrone‐3‐sulfate by OATP1B1 and taurocholic acid by NTCP was not affected by co‐expression with OATP1B3. Expression of OATP1B3 on the surface of HEK293 cells was reduced when OATP1B3 was co‐expressed with NTCP suggesting that the functional decrease might be a result of disturbed protein production and/or translocation to the plasma membrane. These results indicate that co‐expression of NTCP with OATP1B3 can affect OATP1B3‐mediate drug transport and as a consequence can have an effect on drug therapy.Support or Funding InformationNIH grants GM103549 and GM077336

Manufacturing of recombinant adeno-associated viruses using mammalian expression platforms.

Manufacturing practices for recombinant adeno-associated viruses (AAV) have improved in the last decade through the development of new platforms in conjunction with better production and purification methods. In this review, we discuss the advantages and limitations of the most popular systems and methods employed with mammalian cell platforms. Methods and systems such as transient transfection, packaging and producer cells and adenovirus and herpes simplex virus are described. In terms of best production yields, they are comparable with about 104 -105 vector genomes produced per cell but transient transfection of HEK293 cells is by far the most commonly used. For small-scale productions, AAV can be directly purified from the producing cell lysate by ultracentrifugation on a CsCl or iodixanol-step gradient whereas large-scale purification requires a combination of multiple steps. Micro/macrofiltration (i.e. including tangential flow filtration and/or dead-end filtration) and chromatography based-methods are used for large-scale purification. Purified AAV products must then be quantified and characterized to ensure quality. Recent purification methods and current analytical techniques are reviewed here. Finally, AAV technology is very promising, but manufacturing improvements are still required to meet the needs of affordable, safe and effective AAV vectors essential for licensing of gene therapy clinical protocols.

341. Nucleic Acid Delivery Using Vesicular Stomatitis Virus-Based Vesicles

Gene delivery methods are essential to understand fundamental cellular mechanisms and to develop new therapies. Recombinant viruses are efficient to transfer nucleic acids but their safety is a concern. In addition, some commercial transfection reagents (including lipids and cationic polymers) and electroporation methods are cytotoxic. Interestingly, the sole expression of G envelope protein of the vesicular stomatitis virus (VSV) in mammalian cells can lead to the formation of VSV-G pseudotyped vesicles (V-VSV-G). In presence of polybrene, these vesicles are able to transfer plasmids in a large panel of animal cells. Unfortunately, the production of V-VSV-G and their use for nucleic acid delivery is poorly documented. Here we propose to improve this promising method of transfection. At first we developed a V-VSV-G production process by transient transfection of HEK-293 cells using polyethylenimine (PEI). Three modes of production were compared: cells cultivated in adherence, in suspension and on micro-carriers. Also we demonstrated that the quantity of vesicles produced depends on the VSV-G sequence used. The harvest of V-VSV-G from cell culture media was also optimized. Then, several parameters potentially involved in the formation and the transfer efficiency of V-VSV-G/DNA complex were studied: polybrene concentration, order of addition of mix transfection components, incubation time of the complexes, medium of transfection, etc. Stability studies also demonstrated that V-VSV-G are robust particles: DNA transfer capacity of V-VSV-G is efficient after 10 freezing and thawing cycles and V-VSV-G can be stored for long term at +4 °C, -20 °C and -80 °C. Finally, V-VSV-G/DNA ratio was optimized for three different cell types. Transfection efficiency of 70 % and 55 % were obtained for HEK-293 and HeLa cells respectively, with 1 µg of V-VSV-G and 0.4 µg of DNA. Transfection of refractory cells such as human myoblasts, reached 25 % with 5 µg of V-VSV-G and 0.8 µg of DNA. V-VSV-G can also deliver large plasmids (18 kb). Furthermore, no cytotoxicity was observed in cells transfected with these complexes. Presently, the potential of V-VSV-G to transfer siRNA is investigated. In conclusion, V-VSV-G is a powerful tool for nucleic acid delivery which could be useful for several applications oriented toward cell and gene therapies.

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension.

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human FcγRIIIa and the rat α2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Human IGF1 pro-forms induce breast cancer cell proliferation via the IGF1 receptor.

IGF1 is a key regulator of tissue growth and development and has been implicated in the initiation and progression of various cancers, including breast cancer. Through IGF1 mRNA splicing different precursor pro-peptides, i.e., the IGF1Ea, IGF1Eb and IGF1Ec pro-forms, are formed whose biological roles in the pathogenesis of breast cancer have not been established yet. The objective of this study was to assess the biological activity of the IGF1 pro-forms in human breast cancer-derived cells. The different IGF1 pro-forms were generated through transient transfection of HEK293 cells with the respective vector constructs. The resulting conditioned media were applied in vitro to MCF7, T47D and ZR751 breast cancer-derived cell cultures. The recombinant human IGF1 pro-forms were also tested for their binding affinity to an anti-IGF1 specific antibody by immunoprecipitation. To determine whether the IGF1 pro-forms induce cell proliferation, mature IGF1 was neutralised in HEK293-derived conditioned media. We found that the IGF1 pro-forms were the only forms that were produced intra-cellularly, whereas both mature IGF1 and the IGF1 pro-forms were detected extra-cellularly. We also found that E peptides can impair the IGF1 pro-form binding affinity for the anti-IGF1 antibody and, thus, hamper an accurate measurement of the IGF1 pro-forms. Additionally, we found that the IGF1 antibody can completely inhibit IGF1-induced breast cancer cell proliferation and IGF1 receptor (IGF1R) phosphorylation, wheras the same antibody was found to only partially inhibit the biological activity of the pro-forms. Moreover, we found that the IGF1 pro-form activities can completely be inhibited by neutralising the IGF1R. Finally, we compared the bioactivity of the IGF1 pro-forms to that of mature IGF1, and found that the IGF1 pro-forms were less capable of phosphorylating the IGF1R in the breast cancer-derived cells tested. Our data indicate that IGF1 pro-forms can induce breast cancer cell proliferation via the IGF1R, independent from the mature IGF1 form. These results underline the importance of an accurate assessment of the presence of IGF1 pro-forms within the breast cancer microenvironment.

Large-Scale Production of Adeno-Associated Viral Vector Serotype-9 Carrying the Human Survival Motor Neuron Gene.

Recombinant AAV (rAAV) vectors are a suitable vector for gene therapy studies because of desired characteristics such as low immunogenicity, transfection of non-dividing and dividing cells, and long-term expression of the transgene. In this study, the large-scale production of single stranded (ss) and self-complementary (sc) AAV9 carrying the human survival motor neuron (SMN) gene (AAV9-SMN) suitable for in vivo gene therapy studies of SMA was described. SMN cDNA has been cloned into pAAV-CB6-PI and pAAVsc-CB6-PI with and without its specific UTRs, respectively. Both plasmids bear CMV enhancer/beta-actin (CB) promoter, CMV IE enhancer, and polyadenylation signal sequences. 2.5μg of constructed pAAV-CB6-PI-SMN and pAAVsc-CB6-PI-SMN cause to, respectively, 4.853- and 2.321-fold increases in SMN protein levels in transfected cells compared to untransfected cells. Ss and scAAV9-SMN vectors were also produced from these plasmids by transient transfection of HEK293 cells using CaCl2 solution. The silver staining and electron microscopy analysis demonstrated good quality of both isolated vectors, ssAAV9-SMN and scAAV9-SMN, with the titers of 2.00E+13 and 1.00E+13 GC/ml. The results of this study show that, the plasmid containing UTR elements causes to twice more SMN gene expression in transfected cells. The quality control results show that both produced ss and scAAV9-SMN are suitable for in vivo studies.

Abstract B16: Development of a high-throughput imaging screen for the functional assessment of cell-linking moieties using effector and target cells in a cell kill assay

Abstract Cell Linking Moieties (CLMs) are bi-specific antibody fragments that are capable of recognizing epitopes on tumor cells and specific effector cells via unique cell surface receptors. They are comprised of 2 scFv elements recognizing unique epitopes, bound by a linking element. CLMs offer a distinct advantage in that they do not rely on the expression of MHC class I molecules, generation of specific antigenic peptide, or ex vivo manipulation of cells to exert their tumor cell killing effect. To identify the proper orientation of scFv elements and to define the most potent and selective CLM, we have established an image-based screening platform capable of interrogating a large number of CLMs based on their ability to activate effectors and specifically kill tumor cells. We demonstrate the ability to screen a number of CLMs targeting wild type EGFR and HER2 receptors on fluorescently-tagged tumor cell lines for their ability to recruit CD3+ T-cells from unstimulated PBMC pools and mediate selective cell killing. CLMs were created by transient transfection of HEK293 cells with PCR-generated DNA fragments in 96 well plates. Supernatant was harvested 3 days post transfection. Dilutions of crude supernatant were applied to A549, MDA-MB-468, and SW620 (negative) target cells expressing EGFR or HER2 receptors in the presence of unstimulated PBMCs at 10:1 PBMC:Target ratio in 384 well plates. PBMC to target ratios were pre-optimized by running a titration from 2:1 through 1:30 Target:PBMCs in a kill assay. Resulting target cell loss (cell death) was compared to control wells (mock transfected supernatant). In the screening campaign, a primary screen first selects CLM hits based on killing ability, and then a secondary screen is employed to provide dose-responses, IC50s, and additional information, such as non-specific killing of negative lines. Using this image-based screen, consistent Z-scores above 0.5 were obtained, which corresponds to 12 standard deviations between the positive and negative controls, indicating an excellent dynamic range and low variability in the assay. In a proof of concept study using this platform, two EGFR-CD3 CLMs were selected from a larger list and characterized further in the secondary screen. In the top candidate, secondary screening resulted in IC50 average values of 1632 and 12,971 - fold dilution of CLM-containing HEK293 supernatant in A549 cells, and MDA-MB-468 cells, respectively. At high doses of top-candidate CLMs, no target cells remained in PBMC co-cultures, representing a near 100% killing effect. No killing was observed in the negative control line, SW620. Consistent ranking of CLM candidates was observed among 4 donors of PBMCs evaluated. In conclusion, this image-based screening platform allows for well- and cell-level killing information to be obtained with specific loss of target cells in complex mixtures to be accurately calculated without the presence of radioactive compounds like 51Cr. Furthermore, this method allows for kinetic analysis where the same well can be scanned and analyzed at multiple timepoints in the assay to understand the dynamics of killing activity. Lastly, the development of a specific and selective high throughput functional screening assay using non-terminal high content fluorescent imaging to rapidly select CLM-T cell hits results in a shortening of the developmental time line. Citation Format: Jeffrey Rosenbloom, Jason Isaacson, Srinivas Rengarajan, Paul Szymanski, Jennifer Buenviaje, Kristi Elliott, Francois Lebel, John A. Barrett, Richard Einstein. Development of a high-throughput imaging screen for the functional assessment of cell-linking moieties using effector and target cells in a cell kill assay. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B16.

Abstract 5112: In vivo molecular imaging of HIF-1α and septin 9 interaction by bimolecular fluorescence complementation

Abstract Hypoxia is a common feature of many solid tumors that promotes tumor progression and leads to radiation and chemotherapy resistance. Hypoxia-inducible factor 1 (HIF-1), as one of the major mediators of the hypoxic response, has been shown to activate hypoxia-responsive genes, which are involved in multiple aspects of tumorigenesis and cancer progression. Previously, we have described an interaction between septin 9 isoform 1 (SEPT9_i1) protein and HIF-1α, the oxygen regulated subunit of HIF-1. SEPT9_i1 is a member of the conserved family of septins, which are GTP-binding proteins that form cytoskeleton-like filaments essential for many functions in eukaryotic organisms including neoplasia. The interaction of SEPT9_i1 with HIF-1α increases protein stability and HIF-1 transcriptional activity in vitro and promotes proliferation, tumor growth and angiogenesis in vivo. Recent data indicated that SEPT9_i1 was also involved in the trafficking of cytoplasmic HIF-1α into the nucleus by direct interaction to the nuclear transporter importin-α. Herein, we utilized split YFP bimolecular fluorescence complementation (BiFC) methodology in order to monitor HIF-1α/SEPT9_i1 interactions in vivo. First, we generated split YFP protein chimeras fused to HIF-1α and to SEPT9_i1 on both their amino and carboxyl termini. Different pairs of HIF-1α and SEPT9_i1 chimeras were tested to functional complementation of the split YFP fragments using transient transfection in HEK293 cells. The complemented proteins capable of fluorescence were identified as SEPT9_i1-YN (the amino terminus of YFP fused to the carboxyl terminal of SEPT9_i1) and YC-HIF-1α (the carboxyl terminus of YFP fused to the N-terminal of HIF-1α). This pair was selected for further studies in PC-3 human prostate cancer cells. YFP complementation fluorescence derived from these chimeras was increased in the presence of hypoxia mimicking agents, such as CoCl2 and the iron chelators deferoxamine (DFO) and dibenzoylmethane (DBM). The chimera, YCΔHLH lacking the HLH domain that is essential for the interaction with SEPT9_i1 gave a significant reduction in YFP complementation fluorescence. Furthermore, expression of SEPT9_i1 252-379aa fragment, the domain essential for the interaction with HIF-1α, abolished almost completely the YFP complementation fluorescence. These results reconfirm our previous studies on HIF-1α/SEPT9_i1 interactions and make this system attractive for identifying new compounds capable of disrupting this complex in order to inhibit tumor growth and angiogenesis. Citation Format: Maya Golan, Nicola J. Mabjeesh. In vivo molecular imaging of HIF-1α and septin 9 interaction by bimolecular fluorescence complementation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5112. doi:10.1158/1538-7445.AM2015-5112

Molecular cloning and functional characterization of a rainbow trout liver Oatp

Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.g. microcystins, MCs), which constrain fish health or even cause fish death. However the toxicokinetics of the most abundant toxin, microcystin-LR (MC-LR), are not yet fully understood. To investigate the uptake mechanism, the novel Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. The cDNA isolated from a clone library consisted of 2772bp containing a 2115bp open reading frame coding for a 705 aa protein with an approximate molecular mass of 80kDa. This fish specific transporter belongs to the OATP1 family and has most likely evolved from a common ancestor of OATP1C1. Real time PCR analysis showed that rtOatp1d1 is predominantly expressed in the liver, followed by the brain while expression in other organs was not detectable. Transient transfection in HEK293 cells was used for further characterization. Like its human homologues OATP1A1, OATP1B1 and OATP1B3, rtOatp1d1 displayed multi-specific transport including endogenous and xenobiotic substrates. Kinetic analyses revealed a Km value of 13.9μM and 13.4μM for estrone-3-sulfate and methotrexate, respectively and a rather low affinity for taurocholate with a Km value of 103μM. Furthermore, it was confirmed that rtOatp1d1 is a MC-LR transporter and therefore most likely plays a key role in the susceptibility of rainbow trout to MC intoxications.

Interaction of environmental contaminants with zebrafish organic anion transporting polypeptide, Oatp1d1 (Slco1d1)

Polyspecific transporters from the organic anion transporting polypeptide (OATP/Oatp) superfamily mediate the uptake of a wide range of compounds. In zebrafish, Oatp1d1 transports conjugated steroid hormones and cortisol. It is predominantly expressed in the liver, brain and testes. In this study we have characterized the transport of xenobiotics by the zebrafish Oatp1d1 transporter. We developed a novel assay for assessing Oatp1d1 interactors using the fluorescent probe Lucifer yellow and transient transfection in HEK293 cells. Our data showed that numerous environmental contaminants interact with zebrafish Oatp1d1. Oatp1d1 mediated the transport of diclofenac with very high affinity, followed by high affinity towards perfluorooctanesulfonic acid (PFOS), nonylphenol, gemfibrozil and 17α-ethinylestradiol; moderate affinity towards carbaryl, diazinon and caffeine; and low affinity towards metolachlor. Importantly, many environmental chemicals acted as strong inhibitors of Oatp1d1. A strong inhibition of Oatp1d1 transport activity was found by perfluorooctanoic acid (PFOA), chlorpyrifos-methyl, estrone (E1) and 17β-estradiol (E2), followed by moderate to low inhibition by diethyl phthalate, bisphenol A, 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4 tetrahydronapthalene and clofibrate. In this study we identified Oatp1d1 as a first Solute Carrier (SLC) transporter involved in the transport of a wide range of xenobiotics in fish. Considering that Oatps in zebrafish have not been characterized before, our work on zebrafish Oatp1d1 offers important new insights on the understanding of uptake processes of environmental contaminants, and contributes to the better characterization of zebrafish as a model species.

GNAQ and BRAF mutations show differential activation of the mTOR pathway in human transformed cells

Somatic mutations in GNAQ gene were described as being the main oncogenic activation in uveal melanomas, whereas mutations in BRAF gene have been described as a key genetic alteration that contributes to skin melanoma development. We have previously reported differential activation of the MAPK and AKT/mTOR signalling pathways in uveal and skin melanomas harbouring, respectively, GNAQ and BRAF mutations. The aim of this work was to compare the functional effect of GNAQ and BRAF mutations in mTOR and MAPK pathway activation, cell proliferation and apoptosis. In this work, we performed transient transfection of HEK293 cells with BRAFWT, BRAFV 600E, GNAQWT, GNAQQ209P and GNAQQ209L vectors. We treated melanoma cell lines displaying different BRAF and GNAQ mutational status with the mTOR inhibitor RAD001 and with the MEK1/2 inhibitor U0126 and evaluated the effects in the growth of the cell lines and in mTOR and MAPK pathway effectors expression. At variance with the significant increase in the level of pmTOR Ser2448 and pS6 Ser235/236 proteins observed in cells transfected with BRAF vectors, no significant alteration in mTOR pathway effectors was observed in cells transfected with the three GNAQ expressing vectors. Also, GNAQ overexpression enhances Stat3 activation, which might mediate GNAQ oncogenic effects. None of the vectors led to significant differences in proliferation or apoptosis in the transfected cell lines. Cell lines harbouring a BRAF mutation were more sensitive to RAD001 treatment. U0126 leads to the reduction of MAPK and mTOR pathways activation in all cell lines tested. Our results indicate that GNAQ and BRAF activation drive distinct intracellular signalling pathways that may be useful for therapeutic decisions in human melanomas.