Doug Stryke

Publications (36) View all

• Article: Genetic and epigenetic regulation of the organic cation transporter 3, SLC22A3.

  L Chen, C Hong, E C Chen, S W Yee, L Xu, E U Almof, C Wen, K Fujii, S J Johns, D Stryke, T E Ferrin, J Simko, X Chen, J F Costello, K M Giacomini
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  ABSTRACT: Human organic cation transporter 3 (OCT3 and SLC22A3) mediates the uptake of many important endogenous amines and basic drugs in a variety of tissues. OCT3 is identified as one of the important risk loci for prostate cancer, and is markedly underexpressed in aggressive prostate cancers. The goal of this study was to identify genetic and epigenetic factors in the promoter region that influence the expression level of OCT3. Haplotypes that contained the common variants, g.-81G>delGA (rs60515630) (minor allele frequency 11.5% in African American) and g.-2G>A (rs555754) (minor allele frequency>30% in all ethnic groups) showed significant increases in luciferase reporter activities and exhibited stronger transcription factor-binding affinity than the haplotypes that contained the major alleles. Consistent with the reporter assays, OCT3 messenger RNA expression levels were significantly higher in Asian (P<0.001) and Caucasian (P<0.05) liver samples from individuals who were homozygous for g.-2A/A in comparison with those homozygous for the g.-2G/G allele. Studies revealed that the methylation level in the basal promoter region of OCT3 was associated with OCT3 expression level and tumorigenesis capability in various prostate cancer cell lines. The methylation level of the OCT3 promoter was higher in 62% of prostate tumor samples compared with matched normal samples. Our studies demonstrate that genetic polymorphisms in the proximal promoter region of OCT3 alter the transcription rate of the gene and may be associated with altered expression levels of OCT3 in human liver. Aberrant methylation contributes to the reduced expression of OCT3 in prostate cancer.The Pharmacogenomics Journal advance online publication, 10 January 2012; doi:10.1038/tpj.2011.60.
  The Pharmacogenomics Journal 01/2012; · 4.54 Impact Factor
• Source

  Available from: salilab.org

  Article: A common 5'-UTR variant in MATE2-K is associated with poor response to metformin.

  J H Choi, S W Yee, A H Ramirez, K M Morrissey, G H Jang, P J Joski, J A Mefford, S E Hesselson, A Schlessinger, G Jenkins, [......], D Stryke, A Sali, T E Ferrin, J S Witte, P-Y Kwok, D M Roden, R A Wilke, C A McCarty, R L Davis, K M Giacomini
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  ABSTRACT: Multidrug and toxin extrusion 2 (MATE2-K (SLC47A2)), a polyspecific organic cation exporter, facilitates the renal elimination of the antidiabetes drug metformin. In this study, we characterized genetic variants of MATE2-K, determined their association with metformin response, and elucidated their impact by means of a comparative protein structure model. Four nonsynonymous variants and four variants in the MATE2-K basal promoter region were identified from ethnically diverse populations. Two nonsynonymous variants-c.485C>T and c.1177G>A-were shown to be associated with significantly lower metformin uptake and reduction in protein expression levels. MATE2-K basal promoter haplotypes containing the most common variant, g.-130G>A (>26% allele frequency), were associated with a significant increase in luciferase activities and reduced binding to the transcriptional repressor myeloid zinc finger 1 (MZF-1). Patients with diabetes who were homozygous for g.-130A had a significantly poorer response to metformin treatment, assessed as relative change in glycated hemoglobin (HbA1c) (-0.027 (-0.076, 0.033)), as compared with carriers of the reference allele, g.-130G (-0.15 (-0.17, -0.13)) (P=0.002). Our study showed that MATE2-K plays a role in the antidiabetes response to metformin.
  Clinical Pharmacology &#38 Therapeutics 09/2011; 90(5):674-84. · 6.04 Impact Factor
• Source

  Available from: Laura B Ramsey

  Article: Functional characterization of liver enhancers that regulate drug-associated transporters.

  M J Kim, P Skewes-Cox, H Fukushima, S Hesselson, S W Yee, L B Ramsey, L Nguyen, J L Eshragh, R A Castro, C C Wen, D Stryke, S J Johns, T E Ferrin, P-Y Kwok, M V Relling, K M Giacomini, D L Kroetz, N Ahituv
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  ABSTRACT: Little is known about how genetic variations in enhancers influence drug response. In this study, we investigated whether nucleotide variations in enhancers that regulate drug transporters can alter their expression levels. Using comparative genomics and liver-specific transcription factor binding site (TFBS) analyses, we identified evolutionary conserved regions (ECRs) surrounding nine liver membrane transporters that interact with commonly used pharmaceuticals. The top 50 ECRs were screened for enhancer activity in vivo, of which five--located around ABCB11, SLC10A1, SLCO1B1, SLCO1A2, and SLC47A1--exhibited significant enhancer activity. Common variants identified in a large ethnically diverse cohort (n = 272) were assayed for differential enhancer activity, and three variants were found to have significant effects on reporter activity as compared with the reference allele. In addition, one variant was associated with reduced SLCO1A2 mRNA expression levels in human liver tissues, and another was associated with increased methotrexate (MTX) clearance in patients. This work provides a general model for the rapid characterization of liver enhancers and identifies associations between enhancer variants and drug response.
  Clinical Pharmacology &#38 Therapeutics 03/2011; 89(4):571-8. · 6.04 Impact Factor
• Source

  Available from: Doug Stryke

  Article: Role of organic cation transporter 3 (SLC22A3) and its missense variants in the pharmacologic action of metformin.

  Ligong Chen, Bradley Pawlikowski, Avner Schlessinger, Swati S More, Doug Stryke, Susan J Johns, Michael A Portman, Eugene Chen, Thomas E Ferrin, Andrej Sali, Kathleen M Giacomini
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  ABSTRACT: The goals of this study were to determine the role of organic cation transporter 3 (OCT3) in the pharmacological action of metformin and to identify and functionally characterize genetic variants of OCT3 with respect to the uptake of metformin and monoamines. For pharmacological studies, we evaluated metformin-induced activation of AMP-activated protein kinase, a molecular target of metformin. We used quantitative PCR and immunostaining to localize the transporter and isotopic uptake studies in cells transfected with OCT3 and its nonsynonymous genetic variants for functional analyses. Quantitative PCR and immunostaining showed that OCT3 was expressed high on the plasma membrane of skeletal muscle and liver, target tissues for metformin action. Both the OCT inhibitor, cimetidine, and OCT3-specific short hairpin RNA significantly reduced the activating effect of metformin on AMP-activated protein kinase. To identify genetic variants in OCT3, we used recent data from the 1000 Genomes and the Pharmacogenomics of Membrane Transporters projects. Six novel missense variants were identified. In functional assays, using various monoamines and metformin, three variants, T44M (c.131C>T), T400I (c.1199C>T) and V423F (c.1267G>T) showed altered substrate specificity. Notably, in cells expressing T400I and V423F, the uptakes of metformin and catecholamines were significantly reduced, but the uptakes of metformin, 1-methyl-4-phenylpyridinium and histamine by T44M were significantly increased more than 50%. Structural modeling suggested that these two variants may be located in the pore lining (T400) or proximal (V423) membrane-spanning helixes. Our study suggests that OCT3 plays a role in the therapeutic action of metformin and that genetic variants of OCT3 may modulate metformin and catecholamine action.
  Pharmacogenetics and Genomics 11/2010; 20(11):687-99. · 3.48 Impact Factor
• Article: Genetic variants of human organic anion transporter 4 demonstrate altered transport of endogenous substrates.

  James E Shima, Takafumi Komori, Travis R Taylor, Doug Stryke, Michiko Kawamoto, Susan J Johns, Elaine J Carlson, Thomas E Ferrin, Kathleen M Giacomini
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  ABSTRACT: Apical reabsorption from the urine has been shown to be important for such processes as the maintenance of critical metabolites in the blood and the excretion of nephrotoxic compounds. The solute carrier (SLC) transporter OAT4 (SLC22A11) is expressed on the apical membrane of renal proximal tubule cells and is known to mediate the transport of a variety of xenobiotic and endogenous organic anions. Functional characterization of genetic variants of apical transporters thought to mediate reabsorption, such as OAT4, may provide insight into the genetic factors influencing the complex pathways involved in drug elimination and metabolite reclamation occurring in the kidney. Naturally occurring genetic variants of OAT4 were identified in public databases and by resequencing DNA samples from 272 individuals comprising 4 distinct ethnic groups. Nine total nonsynonymous variants were identified and functionally assessed using uptake of three radiolabeled substrates. A nonsense variant, R48Stop, and three other variants (R121C, V155G, and V155M) were found at frequencies of at least 2% in an ethnic group specific fashion. The L29P, R48Stop, and H469R variants displayed a complete loss of function, and kinetic analysis identified a reduced V(max) in the common nonsynonymous variants. Plasma membrane levels of OAT4 protein were absent or reduced in the nonfunctional variants, providing a mechanistic reason for the observed loss of function. Characterization of the genetic variants of reabsorptive transporters such as OAT4 is an important step in understanding variability in tubular reabsorption with important implications in innate homeostatic processes and drug disposition.
  AJP Renal Physiology 10/2010; 299(4):F767-75. · 4.42 Impact Factor