Scintillation proximity assay for measuring uptake by the human drug transporters hOCT1, hOAT3, and hOATP1B1

Lohmann, Christina; Gelius, Birgitta; Danielsson, Jeanette; Skoging-Nyberg, Ulrica; Hollnack, Evelyn; Dudley, Amanda; Wahlberg, Johanna; Hoogstraate, Janet; Gustavsson, Lena

doi:10.1016/j.ab.2007.04.038

Cited by 16 publications

(11 citation statements)

References 18 publications

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“…However, to our knowledge, the effect of the transfection procedure on the background expression of drug-transporting proteins has not been investigated. Therefore, we studied whether stable transfection, using the methods previously described by Lohmann et al (2007) with each of four different transporters (BCRP, OAT3, OCT1, OATP1B1) (Fig. 2a), and transient transfection using polyethyleneimine as transfection agent of HEK293 cells with OCT1 (data not shown) affected the endogenous gene expression of transporters.…”

Section: Gene and Protein Expression Of Transporters In Cellsmentioning

confidence: 99%

Endogenous Gene and Protein Expression of Drug-Transporting Proteins in Cell Lines Routinely Used in Drug Discovery Programs

Ahlin

Hilgendorf²,

Karlsson³

et al. 2009

Drug Metab Dispos

116

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ABSTRACT:The aim of this study was to investigate the gene and protein expression profiles of important drug-transporting proteins in human cell lines commonly used for studies of drug transport mechanisms. Human cell lines used to transiently or stably express single transporters [HeLa, human embryonic kidney (HEK) 293] and leukemia cell lines used to study drug resistance by ATP-binding cassette transporters (HL-60, K562) were investigated and compared with organotypic cell lines (HepG2, Saos-2, Caco-2, and Caco-2 TC7). For gene expression studies, real-time polymerase chain reaction was used, whereas monospecific polyclonal antibodies were generated and used to investigate protein expression by immunohistochemistry. Thirty-six transporters were studied for gene expression, and nine were studied for protein expression. The antibodies were validated using expression patterns in human tissues. Finally, the function of one ubiquitously expressed transporter, MCT1/SLC16A1, was investigated using [ 14 C]lactic acid as a substrate. In general, the adherent cell lines (HeLa, HEK293) displayed low transporter expression, and the expression patterns were barely affected by transfection. The leukemia cell lines (K562, HL-60) and Saos-2 also had low endogenous transporter expression, whereas the organotypic cell lines (HepG2 and Caco-2) showed higher expression of some transporters. Comparison of gene and protein expression profiles gave poor correlations, but better agreement was obtained for antibodies with a good validation score, indicating that antibody quality was a significant variable. It is noteworthy that the monocarboxylic acid-transporting protein MCT1 was significantly expressed in all and was functional in most of the cell lines, indicating that MCT1 may be a confounding factor when the transport of small anionic drugs is investigated.

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Section: Gene and Protein Expression Of Transporters In Cellsmentioning

confidence: 99%

Endogenous Gene and Protein Expression of Drug-Transporting Proteins in Cell Lines Routinely Used in Drug Discovery Programs

Ahlin

Hilgendorf²,

Karlsson³

et al. 2009

Drug Metab Dispos

116

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“…Anastrozole transport assays were modified based on previous studies . Specifically, 48 hours after cDNA plasmid transfection, cells were washed once with warm Hank's Balanced Salt Solution (HBSS) buffer (HBSS buffered with 25 mM 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid at pH 7.4) and were then preincubated with fresh HBSS buffer at 37°C for 30 minutes.…”

Section: Methodsmentioning

confidence: 99%

Anastrozole Aromatase Inhibitor Plasma Drug Concentration Genome‐Wide Association Study: Functional Epistatic Interaction Between SLC38A7 and ALPPL2

Dudenkov

Liu

Cairns

et al. 2019

Clin Pharma and Therapeutics

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Anastrozole is a widely prescribed aromatase inhibitor for the therapy of estrogen receptor positive ( ER +) breast cancer. We performed a genome‐wide association study ( GWAS ) for plasma anastrozole concentrations in 687 postmenopausal women with ER + breast cancer. The top single‐nucleotide polymorphism ( SNP ) signal mapped across SLC 38A7 (rs11648166, P = 2.3E‐08), which we showed to encode an anastrozole influx transporter. The second most significant signal (rs28845026, P = 5.4E‐08) mapped near ALPPL 2 and displayed epistasis with the SLC 38A7 signal. Both of these SNP s were cis expression quantitative trait loci ( eQTL )s for these genes, and patients homozygous for variant genotypes for both SNP s had the highest drug concentrations, the highest SLC 38A7 expression, and the lowest ALPPL 2 expression. In summary, our GWAS identified a novel gene encoding an anastrozole transporter, SLC 38A7 , as well as epistatic interaction between SNP s in that gene and SNP s near ALPPL 2 that influenced both the expression of the transporter and anastrozole plasma concentrations.

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“…The resulting light emission signals were measured on-line in a microplate scintillation counter. In the transfected cells, time-dependent uptake of substrates of hOCT1, OATP1B1 or OAT3 were measured readily [78]. Because of the difficulty of synthesizing radiolabeld compounds, this assay is not likely to be used for substrate screening.…”

Section: In Vitro Methods For Identifying the Sub-strates And Inhibitmentioning

confidence: 99%

Permeability – In Vitro Assays for Assessing Drug Transporter Activity

Jin¹,

Di²

2008

CDM

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The accumulating evidence has revealed that drug transporters have essential roles in the delivery and excretory processes of drugs and their metabolites. Inhibition or induction of drug transporters can affect pharmacokinetic properties and therapeutic efficacy of a drug. Thus, the characterization of drug-transporter interactions becomes important for the selection of compounds to avoid transporter associated absorption, distribution, metabolism, excretion and toxicity (ADME/Tox) issues. Additionally, the potential use of absorptive transporters for drug delivery has been recognized for drug design. In vitro and in vivo approaches have been developed for studying the transporter activities. In vitro assays can rapidly provide the information for identifying interaction of a compound and a particular transporter and have proved to be amenable to high throughput approaches. Therefore, the studies are conducted in early drug discovery. In this article, in vitro methods are reviewed, including cell free and cell-based assays. Their applications, limitations and impact on drug discovery are discussed.

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Scintillation proximity assay for measuring uptake by the human drug transporters hOCT1, hOAT3, and hOATP1B1

Cited by 16 publications

References 18 publications

Endogenous Gene and Protein Expression of Drug-Transporting Proteins in Cell Lines Routinely Used in Drug Discovery Programs

Endogenous Gene and Protein Expression of Drug-Transporting Proteins in Cell Lines Routinely Used in Drug Discovery Programs

Anastrozole Aromatase Inhibitor Plasma Drug Concentration Genome‐Wide Association Study: Functional Epistatic Interaction Between SLC38A7 and ALPPL2

Permeability – In Vitro Assays for Assessing Drug Transporter Activity

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