Ala893ser variant has no effect on p-glycoprotein (P-GP) function.
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CLINICALPI~KICMACOLOGY & THERAPEUTICS VOLUME 73, NUMBEK2 American Society for Clinical Pharmacology and Therapeutics Pl 3 PI-44 STABLE AND TIGHTLY REGULATED EXPRESSION OF HUMAN NA+/TAUROCHOLATE COTRANSPORTING POLY- PEPTIDE (NTCP) USING A TETRACYCLINE-INDUCIBLE SYS- TEM. W. Lee, PhD, R. B. Kim, MD, and Richard B. Kim, MD, Division of Clinical Pharmacology, School of Medicine, Vanderbilt University, Nashville, TN. Transporters are important determinants in the disposition of many exogenous and endogenous molecules. Accordingly, model cell- based systems stably expressing such transporter proteins are likely to significantly aid in the study of drug transporter function. Towards this goat, we constructed a tetracycline-inducible (Tet-on) "gene switch" system, which can induce target transporter expression under the tight control of doxycycline (Dox). Following stable, double transfection of HeLa cells with a vector construct containing the Tet-on response element and the bile acid uptake transporter, human NTCP (Na+/tanrocholate cotransporting polypeptide), a cell line exhibiting highly inducible expression of NTCP in a Dox-dependent manner was identified. Functional studies revealed a sodium- dependent cellular uptake of [3H]-taurocholic acid was approximately 30-fold greater upon addition of Dox (1 Ixg/ml) to the culture media. Accordingly, our results demonstrate that the Tet-on system can be applied to express transporter proteins in an inducible manner and be proved to be useful in the identification of novel drug substrates, and in the high-throughput screening of xenobiotic-transporter interac-" tions. PI-45 ALA893SER VARIANT HAS NO EFFECT ON P-GLYCOPROTEIN (P-GP) FUNCTION. L.M. Hodges, BS, C. I. Lacayo, BS, L. W. Chinn, BS, C. Ho, BS, T. Lillehammer, BS, C. Pauli-Magnus, MD, D. L. Kroetz, PhD, Univ. of CA, San Francisco, San Francisco, CA. The MDRI gene product, P-gp, is an important drug efflux trans- porter that influences the absorption, distribution, and elimination of numerous drugs. Genetic variation in MDR1 has been described but the functional and clinical consequences of this variation remain controversial. The goal of the present study was to examine whether the Ala893Ser variant encoded by the MDR1 G2677T allele has altered function relative to the reference protein. Haplotypes that were studied had the following MDR1 genotypes at positions 1236, 2677 and 3435: CGC (reference), TGT, TGC, TTT and TTC. HEK 293T or HeLa cells were transiently transfected with MDR1 plasmids containing these haplotypes and the accumulation of calcein was measured by flow cytometry in live, P-gp positive cells. There was no difference in calcein accumulation between the Ala893 and Ser893 variants. Comparing the two major haplotypes CGC (reference) and TTT, calcein accumulation in the Ser893 P-gp expressing cells was 107 _+ 49.2 % of that in the Ata893 P-gp cells. There was also no difference in P-gp inhibition by GF 120918 among any of the variants (increase in calcein accumulation: 5.4 _+ 1.4-fold for Ala893 and 6.0 + 2.0-fold for Ser893). Similar results were found using rhoda- mine123 as substrate. These studies do not support a functional difference between the Ala893 and Ser893 P-gp variants. Studies are ongoing to analyze the cellular phenotype of other P-gp variants. Supported by NIGMS 61390. PI-46 THE FUNCTIONAL CHANGE OF P-GLYCOPROTEIN ON LYMPHOCYTES UNDER INDUCTION CONDITIONS AS A FUNCTION OF MDRI POLYMORPHISMS. D.G. Desai, MD, N. Murayama, PhD, M. Lantz, BS, J. M. Woo, BA, W. Chan, BS, L. Z. Benet, PhD, University of California-San Francisco, San Francisco, CA. PURPOSE. To investigate the effects of rifampin induction on the upregulation of P-glycoprotein (P-gp) function in lymphocytes and to determine whether a difference exists among MDR1 polymorphisms. METHODS. Eighteen healthy volunteers were recruited. Subjects were genotyped for exon l I(Gl199A), exon 12(C1236T), exon 13(C[+24]T), exon 14(A[+38]G), exon 21(G2677T,A) and exon 26(C3435T) of the MDR1 gene by polymerase chain reaction. Blood samples were collected before and after a t0-day course of rifampin (600mg/day). Lymphocytes were isolated using density centrifuga- tion and P-gp function was evaluated using a Rhodamine 123 assay. RESULTS. For C3435T, 5 subjects had the CC genotype, 6 subjects had the CT genotype and 7 subjects had the TT genotype. There was no significant difference at baseline among the 3 groups in terms of P-gp function (see table). Post induction, P-gp function declined in the CT group (NS). Only the TT group exhibited a significant increase in P-gp function (P<0.005). There was no change in rifampin-induced Pgp function for the MDR1 exon 11,12,14 and 21 polymorphisms studied. CONCLUSIONS. A 10-day course of ri- fampin induction did result in functional upregulation of those with the C3435T TT genotype but not among individuals of the CC and CT genotypes. Mean Intensity of Rhodamine 123 (arbitrary units _+SD) Genotype Pre-rifampin Post-rifampin P value CC (n=5) 1.61±0.28 1.63-+0.18 NS CT (n=6) 1.58-+0.20 1.46+0.16 NS TT (n=7) 1.61 ±0A5 1.82-+0_.20 <0.005 PI-47 A PHARMACOGENETIC STUDY ON UDP-GLUCURONO- SYLTRANSFERASE 2B7 IN HUMAN LIVERS. F. Innocenti, MD, S. Das, Phl), J. W. Chandler, BS, J. Ramirez, MS, E. J. Cooke, MD, M. J. Ratain, MD, University of Chicago, Chicago, IL. UDP-glucuronosyltransferase 2B7 (UGT2B7) is involved in the glucuronidation of many endogenous and exogenous substrates. The genetic basis of interindividual variability in UGT2B7 metabolism is still not fully elucidated. We aimed to use a large panel of human livers to 1) characterize the phenotypic variability, 2) identify phe- notypic extremes fbr gene sequencing, 3) evaluate the correlation between phenotypes and both -161C>T (in the promoter) and 802C>T (missense, exon 2) gene variants. Microsomes (n=74-83) were phenotyped with morphine and epirubicin by HPLC. -161C>T and 802C>T variants were typed by SBE-DHPLC and sequencing, respectively. The level of phenotypic variability was high, with coefficients of variation of 39% and 50% for epirubicin (n=83) and morphine (n=74) glucuronidation, respectively. Linkage disequilib- rium was observed between the two variants in 81 of" the 83 samples. -t61T and 802T allele fi'equencies were 0.55 and 0.40 in Caucasians (n=56) and African Americans (n=15), respectively (p=0.2). No significant correlation was reported between UGT2B7 genotypes and either morphine or epirubicin glucuronidation in the sample popula- tion and after stratification by ethnicity. Although prior studies from our group (ASCPT, 2001) have demonstrated such a relationship in patients, we are unable to provide supportive evidence in vitro. Further clinical studies of these polymorphisms are indicated, as well as SNP discovery based on resequencing of phenotypic extremes.
Transcript of Ala893ser variant has no effect on p-glycoprotein (P-GP) function.
CLINICAL PI~KICMACOLOGY & THERAPEUTICS VOLUME 73, NUMBEK2 American Society for Clinical Pharmacology and Therapeutics P l 3
PI-44 STABLE AND TIGHTLY REGULATED EXPRESSION OF
HUMAN NA+/TAUROCHOLATE COTRANSPORTING POLY- PEPTIDE (NTCP) USING A TETRACYCLINE-INDUCIBLE SYS- TEM. W. Lee, PhD, R. B. Kim, MD, and Richard B. Kim, MD, Division of Clinical Pharmacology, School of Medicine, Vanderbilt University, Nashville, TN.
Transporters are important determinants in the disposition of many exogenous and endogenous molecules. Accordingly, model cell- based systems stably expressing such transporter proteins are likely to significantly aid in the study of drug transporter function. Towards this goat, we constructed a tetracycline-inducible (Tet-on) "gene switch" system, which can induce target transporter expression under the tight control of doxycycline (Dox). Following stable, double transfection of HeLa cells with a vector construct containing the Tet-on response element and the bile acid uptake transporter, human NTCP (Na+/tanrocholate cotransporting polypeptide), a cell line exhibiting highly inducible expression of NTCP in a Dox-dependent manner was identified. Functional studies revealed a sodium- dependent cellular uptake of [3H]-taurocholic acid was approximately 30-fold greater upon addition of Dox (1 Ixg/ml) to the culture media. Accordingly, our results demonstrate that the Tet-on system can be applied to express transporter proteins in an inducible manner and be proved to be useful in the identification of novel drug substrates, and in the high-throughput screening of xenobiotic-transporter interac-" tions.
PI-45 ALA893SER VARIANT HAS NO EFFECT ON
P-GLYCOPROTEIN (P-GP) FUNCTION. L .M. Hodges, BS, C. I. Lacayo, BS, L. W. Chinn, BS, C. Ho, BS, T. Lillehammer, BS, C. Pauli-Magnus, MD, D. L. Kroetz, PhD, Univ. of CA, San Francisco, San Francisco, CA.
The MDRI gene product, P-gp, is an important drug efflux trans- porter that influences the absorption, distribution, and elimination of numerous drugs. Genetic variation in MDR1 has been described but the functional and clinical consequences of this variation remain controversial. The goal of the present study was to examine whether the Ala893Ser variant encoded by the MDR1 G2677T allele has altered function relative to the reference protein. Haplotypes that were studied had the following MDR1 genotypes at positions 1236, 2677 and 3435: CGC (reference), TGT, TGC, TTT and TTC. HEK 293T or HeLa cells were transiently transfected with MDR1 plasmids containing these haplotypes and the accumulation of calcein was measured by flow cytometry in live, P-gp positive cells. There was no difference in calcein accumulation between the Ala893 and Ser893 variants. Comparing the two major haplotypes CGC (reference) and TTT, calcein accumulation in the Ser893 P-gp expressing cells was 107 _+ 49.2 % of that in the Ata893 P-gp cells. There was also no difference in P-gp inhibition by GF 120918 among any of the variants (increase in calcein accumulation: 5.4 _+ 1.4-fold for Ala893 and 6.0 + 2.0-fold for Ser893). Similar results were found using rhoda- mine123 as substrate. These studies do not support a functional difference between the Ala893 and Ser893 P-gp variants. Studies are ongoing to analyze the cellular phenotype of other P-gp variants. Supported by NIGMS 61390.
PI-46 THE FUNCTIONAL CHANGE OF P-GLYCOPROTEIN ON
LYMPHOCYTES UNDER INDUCTION CONDITIONS AS A FUNCTION OF MDRI POLYMORPHISMS. D.G. Desai, MD, N. Murayama, PhD, M. Lantz, BS, J. M. Woo, BA, W. Chan, BS, L. Z. Benet, PhD, University of California-San Francisco, San Francisco, CA.
PURPOSE. To investigate the effects of rifampin induction on the upregulation of P-glycoprotein (P-gp) function in lymphocytes and to determine whether a difference exists among MDR1 polymorphisms. METHODS. Eighteen healthy volunteers were recruited. Subjects were genotyped for exon l I(Gl199A), exon 12(C1236T), exon 13(C[+24]T), exon 14(A[+38]G), exon 21(G2677T,A) and exon 26(C3435T) of the MDR1 gene by polymerase chain reaction. Blood samples were collected before and after a t0-day course of rifampin (600mg/day). Lymphocytes were isolated using density centrifuga- tion and P-gp function was evaluated using a Rhodamine 123 assay. RESULTS. For C3435T, 5 subjects had the CC genotype, 6 subjects had the CT genotype and 7 subjects had the TT genotype. There was no significant difference at baseline among the 3 groups in terms of P-gp function (see table). Post induction, P-gp function declined in the CT group (NS). Only the TT group exhibited a significant increase in P-gp function (P<0.005). There was no change in rifampin-induced Pgp function for the MDR1 exon 11,12,14 and 21 polymorphisms studied. CONCLUSIONS. A 10-day course of ri- fampin induction did result in functional upregulation of those with the C3435T TT genotype but not among individuals of the CC and CT genotypes.
Mean Intensity of Rhodamine 123 (arbitrary units _+SD)
Genotype P r e - r i f a m p i n Post-rifampin P value
CC (n=5) 1.61±0.28 1.63-+0.18 NS CT (n=6) 1.58-+0.20 1.46+0.16 NS TT (n=7) 1.61 ±0A5 1.82-+0_.20 <0.005
PI-47 A PHARMACOGENETIC STUDY ON UDP-GLUCURONO-
SYLTRANSFERASE 2B7 IN HUMAN LIVERS. F. Innocenti, MD, S. Das, Phl), J. W. Chandler, BS, J. Ramirez, MS, E. J. Cooke, MD, M. J. Ratain, MD, University of Chicago, Chicago, IL.
UDP-glucuronosyltransferase 2B7 (UGT2B7) is involved in the glucuronidation of many endogenous and exogenous substrates. The genetic basis of interindividual variability in UGT2B7 metabolism is still not fully elucidated. We aimed to use a large panel of human livers to 1) characterize the phenotypic variability, 2) identify phe- notypic extremes fbr gene sequencing, 3) evaluate the correlation between phenotypes and both -161C>T (in the promoter) and 802C>T (missense, exon 2) gene variants. Microsomes (n=74-83) were phenotyped with morphine and epirubicin by HPLC. -161C>T and 802C>T variants were typed by SBE-DHPLC and sequencing, respectively. The level of phenotypic variability was high, with coefficients of variation of 39% and 50% for epirubicin (n=83) and morphine (n=74) glucuronidation, respectively. Linkage disequilib- rium was observed between the two variants in 81 of" the 83 samples. - t61T and 802T allele fi'equencies were 0.55 and 0.40 in Caucasians (n=56) and African Americans (n=15), respectively (p=0.2). No significant correlation was reported between UGT2B7 genotypes and either morphine or epirubicin glucuronidation in the sample popula- tion and after stratification by ethnicity. Although prior studies from our group (ASCPT, 2001) have demonstrated such a relationship in patients, we are unable to provide supportive evidence in vitro. Further clinical studies of these polymorphisms are indicated, as well as SNP discovery based on resequencing of phenotypic extremes.
