ABCMdb

PMID: 18287207

Gow JM, Hodges LM, Chinn LW, Kroetz DL
Substrate-dependent effects of human ABCB1 coding polymorphisms.
J Pharmacol Exp Ther. 2008 May;325(2):435-42. Epub 2008 Feb 20., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCB1 p.Asn21Asp ABCB1 p.Val1251Ile The A893S, A893T, and V1251I variants and the N21D/ 1236CϾT/A893S/3435CϾT haplotype altered intracellular accumulation compared with reference P-gp in a substrate-dependent manner. Login to comment 21 ABCB1 p.Ser893Ala The majority of pharmacogenetic studies for P-gp have focused on two polymorphisms, the synonymous 3435CϾT variation and the nonsynonymous 2677GϾT (S893A) variation (Schwab et al., 2003; Marzolini et al., 2004; Pauli-Magnus and Kroetz, 2004; Salama et al., 2006; Schaefer et al., 2006). Login to comment 55 ABCB1 p.Asn21Asp ABCB1 p.Ser1141Thr ABCB1 p.Arg669Cys ABCB1 p.Val1251Ile ABCB1 p.Ser400Thr The following variants were created from the reference ABCB1 plasmid in pCIneo: 61AϾG (N21D), 1199GϾA (S400T), 1596TϾG [a nonfunctional nucleotide-binding domain (NBD) mutant], 2005CϾT (R669C), 2677GϾA (A893T), 3421TϾA (S1141T), and 3751GϾA (V1251I). Login to comment 103 ABCB1 p.Asn21Asp ABCB1 p.Ser1141Thr ABCB1 p.Arg669Cys ABCB1 p.Val1251Ile ABCB1 p.Ser400Thr There are six polymorphic residues that meet these criteria: N21D, S400T, R669C, A893S/T, S1141T, and V1251I. Login to comment 104 ABCB1 p.Asn21Asp Residue 893 is interesting because it is triallelic and occurs naturally in haplotypes, with the two common synonymous SNPs, 1236CϾT and 3435CϾT as well as N21D (Kroetz et al., 2003). Login to comment 111 ABCB1 p.Asn21Asp ABCB1 p.Asn21Asp ABCB1 p.Ser1141Thr ABCB1 p.Arg669Cys ABCB1 p.Val1251Ile ABCB1 p.Ser400Thr Intra-and interexperimental replicates (n ϭ 3) for reference, NBD, and variant P-gp samples have TABLE 2 Allele frequencies, evolutionary conservation, and Grantham values for selected ABCB1 nonsynonymous polymorphisms and haplotypes Variant or Haplotype Allele Frequenciesa Amino Acid Conservationc Grantham Valued Nucleotide Change Amino Acid Change AA (n ϭ 200) CA (n ϭ 200) Reference Variant % 61AϾG N21D 2.5 8 23 1199GϾA S400T 1 2.5 d, ha, mk ms, r 46 2005CϾT R669C 1 0 d, ha, mk, r, s 180 2677GϾT A893S 10 46.4 ha, ms, r d, mk, s 99 2677GϾA A893T 0.5 3.6 ha, ms, r 58 3421TϾA S1141T 11.1 0 d, ha, mk, ms, r, s 58 3751GϾA V1251I 0b 0 d, ha, mk, ms, r s 29 1236CϾT/2677GϾT/3435CϾT A893S 6 34 N.A. N.A. N.A. 61AϾG/1236CϾT/2677GϾT/3435CϾT N21D/A893S 2.5 8 N.A. N.A. N.A. N.A., not applicable. Login to comment 135 ABCB1 p.Ser400Asn ABCB1 p.Asn21Asp ABCB1 p.Asn21Asp ABCB1 p.Ser1141Thr ABCB1 p.Arg669Cys The representative histogram of APC fluorescence for empty vector (gray, dotted), NBD mutant (gray), reference (black), and variant-transfected cells (N21D, yellow; S400N, cyan; R669C, purple; A893S, green; A893T, orange; S1141T, pink; V1251I, blue; 1236CϾT/A893S/3435CϾT, brown; and N21D/1236CϾT/A893S/3435CϾT, red) shows P-gp expression based on the intensity of APC fluorescence, as indicated on the x-axis. Login to comment 141 ABCB1 p.Asn21Asp ABCB1 p.Arg669Cys The effects of cyclosporin A on calcein-AM accumulation are displayed in a representative histogram for P-gp reference (black, shaded), N21D (yellow), R669C (purple), and A893S (green). Login to comment 151 ABCB1 p.Asn21Asp ABCB1 p.Arg669Cys It is interesting that the N21D, R669C, and A893S variants are more sensitive to cyclosporin A (136 Ϯ 28, 139 Ϯ 43, and 130 Ϯ 22%, respectively; p Ͻ 0.05) compared to reference (Fig. 2c). Login to comment 156 ABCB1 p.Ser400Asn ABCB1 p.Asn21Asp ABCB1 p.Arg669Cys The N21D, S400N, R669C, and A893T variants and the 1236CϾT/A893S/3435CϾT haplotype were within 5% of the reference (Fig. 3c). Login to comment 157 ABCB1 p.Asn21Asp In contrast, the A893S and V1251I P-gp variants and the N21D/1236CϾT/A893S/ 3435CϾT haplotype showed intracellular paclitaxel levels that were 114 Ϯ 13, 118 Ϯ 12, and 124 Ϯ 13% of reference, respectively (p Ͻ 0.01), indicating decreased P-gp function. Login to comment 159 ABCB1 p.Ser1141Thr Three variants (A893S, A893T, and S1141T) were 30 Ϯ 5, 30 Ϯ 8, and 27 Ϯ 17%, respectively, less sensitive to cyclosporin A inhibition than reference, whereas V1251I was 65 Ϯ 26% less sensitive than reference (p Ͻ 0.01). Login to comment 160 ABCB1 p.Asn21Asp The haplotype containing N21D/1236CϾT/A893S/3435CϾT was 53 Ϯ 8% less sensitive to cyclosporin A inhibition than reference (p Ͻ 0.01). Login to comment 163 ABCB1 p.Ser400Asn ABCB1 p.Asn21Asp ABCB1 p.Ser1141Thr ABCB1 p.Val1251Ile Six of 13 previously described nonsynonymous variants were chosen for study based on an allele frequency Ͼ2%: N21D, S400N, A893S, A893T, S1141T, and V1251I. Login to comment 164 ABCB1 p.Arg669Cys The R669C variant has an allele frequency of only 1% in African Americans but was chosen because the variant amino acid causes a drastic chemical change (Grantham value ϭ 180). Login to comment 170 ABCB1 p.Asn21Asp ABCB1 p.Val1251Ile The data collected in the absence of cyclosporin A for empty vector (gray, dotted), P-gp reference (black), NBD mutant (gray), A893S (green), V1251I (blue), and haplotype N21D/1236CϾT/A893S/3534CϾT (red) are shown in a representative fluorescence histogram (a). Login to comment 171 ABCB1 p.Asn21Asp ABCB1 p.Ser1141Thr ABCB1 p.Val1251Ile The effects of cyclosporin A on BODIPY-FL-paclitaxel accumulation are displayed in a representative histogram in b for P-gp reference (black, shaded), A893S (green), A893T (orange), S1141T (pink), V1251I (blue), and haplotype N21D/1236CϾT/A893S/3534CϾT (red). Login to comment 177 ABCB1 p.Asn21Asp These haplotypes contained A893S, 1236CϾT, and 3435CϾT either with or without N21D. Login to comment 189 ABCB1 p.Val1251Ile The A893T and V1251I variants had lower intracellular levels of calcein, indicating increased efflux of calcein-AM by P-gp (Table 3). Login to comment 193 ABCB1 p.Ser400Asn ABCB1 p.Asn21Asp Previous work consistent with the current findings has shown that N21D, S400N, and A893S do not change calcein-AM transport (Kimchi-Sarfaty et al., 2002; Kroetz et al., 2003). Login to comment 198 ABCB1 p.Ser400Asn This suggests that the S400N variation alters P-gp function in a substrate-specific manner. Login to comment 199 ABCB1 p.Val1251Ile Likewise, we found that the functional effects of A893S, A893T, V1251I, and N21N/1236CϾT/A893S/3435CϾT were substrate-dependent. Login to comment 202 ABCB1 p.Ser400Asn The ABCB1 S400N variant has been shown to confer higher drug resistance to paclitaxel, but the stable cell line overexpressing this P-gp was selected for G418 resistance (Crouthamel et al., 2006). Login to comment 206 ABCB1 p.Ser400Asn ABCB1 p.Ser400Asn ABCB1 p.Ser400Asn ABCB1 p.Asn21Asp ABCB1 p.Asn21Asp ABCB1 p.Phe103Leu In one study, the N21D, F103L, S400N, A893S, and A998T SNPs and three double mutants (N21N/S400N, N21D/A893S, and S400N/ A893S) were investigated using a vaccinia virus expression system with BODIPY-FL-paclitaxel, and no differences in function were noted among the variant and reference P-gps (Kimchi-Sarfaty et al., 2002). Login to comment 207 ABCB1 p.Ser400Asn ABCB1 p.Asn21Asp ABCB1 p.Asn21Asp Our results are similar for N21D and S400N, but we found that A893S and N21D/A893S have decreased transport of BODIPY-FL-paclitaxel (Table 3). Login to comment 209 ABCB1 p.Val1251Ile It should be noted that V1251I P-gp displays decreased function with BODIPY-FL-paclitaxel but increased function with calcein-AM. Login to comment 211 ABCB1 p.Val1251Ile There is no other data on the function of the V1251I variant, and testing additional substrates may elucidate the importance of this amino acid change. Login to comment 213 ABCB1 p.Arg669Cys The R669C variant showed highly increased function for all substrates, whereas our data showed no difference for calcein-AM and BODIPY-FL-paclitaxel. Login to comment 214 ABCB1 p.Asn21Asp ABCB1 p.Asn21Asp ABCB1 p.Asn21Asp ABCB1 p.Asn21Asp ABCB1 p.Ser1141Thr ABCB1 p.Ser1141Thr ABCB1 p.Ser1141Thr ABCB1 p.Arg669Cys ABCB1 p.Arg669Cys ABCB1 p.Val1251Ile ABCB1 p.Val1251Ile ABCB1 p.Ser400Thr ABCB1 p.Ser400Thr There was increased function for A893S and S1141T depending on the substrate, TABLE 3 Substrateand inhibitor-dependent effects of P-gp variants on transport function Variant or Haplotype Calcein-AM BODIPY-FL-Paclitaxel -CsAa ϩCsAb -CsA ϩCsA N21D 1 S400T R669C 1 A893S 1 2 2 A893T 1 2 S1141T 2 V1251I 1 2 2 1236CϾT/A893S/3435CϾT N21D/1236CϾT/A893S/3435CϾT 2 2 a Arrows indicate statistically significant changes in P-gp function relative to reference in the absence of cyclosporin A (-CsA). Login to comment 224 ABCB1 p.Asn21Asp ABCB1 p.Arg669Cys The N21D, R669C, and A893S P-gp variants show increased inhibition of calcein-AM transport by cyclosporin A. Login to comment 225 ABCB1 p.Asn21Asp ABCB1 p.Ser1141Thr In contrast, the A893S, A893T, and S1141T variants and the N21D/1236CϾT/A893S/3435CϾT haplotype are less sensitive to cyclosporin A inhibition of BODIPY-FL-paclitaxel transport. Login to comment 229 ABCB1 p.Asn21Asp ABCB1 p.Val1251Ile Intracellular accumulation of calcein-AM and/or BODIPY-FL-paclitaxel was altered by A893S, A893T, V1251I, and N21D/1236CϾT/A893S/3435CϾT. Login to comment