ABCMdb

PMID: 24080162

Conseil G, Cole SP
Two polymorphic variants of ABCC1 selectively alter drug resistance and inhibitor sensitivity of the multidrug and organic anion transporter multidrug resistance protein 1.
Drug Metab Dispos. 2013 Dec;41(12):2187-96. doi: 10.1124/dmd.113.054213. Epub 2013 Sep 30., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC1 p.Cys43Ser ABCC1 p.Ser92Phe Mutants C43S and S92F were correctly routed to the HEK cell plasma membrane, but the levels were too low to permit functional characterization. Login to comment 5 ABCC1 p.Arg723Gln ABCC1 p.Gly671Val ABCC1 p.Cys1047Ser ABCC1 p.Arg633Gln ABCC1 p.Ala989Thr In contrast, levels and membrane trafficking of R633Q, G671V, R723Q, A989T, and C1047S were similar to wild-type MRP1. Login to comment 6 ABCC1 p.Cys1047Ser ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr In cell-based assays, all five mutants were equally effective at effluxing calcein, but only two exhibited reduced resistance to etoposide (C1047S) and vincristine (A989T; C1047S). Login to comment 7 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr ABCC1 p.Ala989Thr The GSH-dependent inhibitor LY465803 (LY465803 [N-[3-(9-chloro-3-methyl-4-oxo-4H-isoxazolo- [4,3-c]quinolin-5-yl)-cyclohexylmethyl]-benzamide)] was less effective at blocking calcein efflux by A989T, but in a membrane-based assay, organic anion transport by A989T and C1047S was inhibited by MRP1 modulators as well as wild-type MRP1. Login to comment 8 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr GSH accumulation assays suggest cellular GSH efflux by A989T and C1047S may be impaired. Login to comment 9 ABCC1 p.Gly671Val ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr In conclusion, although six in silico analyses consistently predict deleterious consequences of ABCC1 nsSNPs G671V, changes in drug resistance and inhibitor sensitivity were only observed for A989T and C1047S, which may relate to GSH transport differences. Login to comment 27 ABCC1 p.Cys43Ser ABCC1 p.Gly671Val ABCC1 p.Arg433Ser Previously, we generated and partially characterized recombinant forms of ABCC1 nsSNPs: rs45511401 (2012G.T; G671V), rs60782127 (1299G.T; R433S), and rs41395947 (128G.C; C43S) (Conrad et al., 2001, 2002; Leslie et al., 2003). Login to comment 28 ABCC1 p.Gly671Val We found that the G671V mutation had no deleterious effects on the levels or organic anion (i.e., LTC4, E217bG) transport function of MRP1 when expressed in human embryonic kidney (HEK) cells, despite the fact this mutation is located near NBD1 (Conrad et al., 2001). Login to comment 29 ABCC1 p.Arg433Ser In contrast, the R433S mutant exhibited reduced LTC4 and estrone sulfate transport levels (Conrad et al., 2002). Login to comment 30 ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser Cells expressing MPR1-R433S also showed increased doxorubicin resistance, suggesting R433S can better efflux this drug despite its lower organic anion transport activity. Login to comment 31 ABCC1 p.Cys43Ser Yet another phenotype was observed for the C43S nsSNP in the first TM helix of MSD0. Login to comment 32 ABCC1 p.Cys43Ser Not only did C43S increase organic anion transport activity and reduce resistance to vincristine and arsenite (Leslie et al., 2003), it also mildly disrupted MRP1 membrane trafficking. Login to comment 33 ABCC1 p.Ala989Thr In a subsequent characterization of the organic anion transport activity of 10 additional ABCC1 nsSNPs, only one (rs35529209; 2965G.A; A989T) showed a significant reduction in E217bG transport (L&#e9;tourneau et al., 2005). Login to comment 34 ABCC1 p.Cys43Ser ABCC1 p.Gly671Val ABCC1 p.Arg433Ser ABCC1 p.Ala989Thr In contrast to our experimental data, the predictive algorithms SIFT (Sorting Tolerant From Intolerant) and PolyPhen indicated that G671V would adversely affect MRP1 function but C43S and A989T were less likely to do so, whereas predictions for R433S were mixed (L&#e9;tourneau et al., 2005). Login to comment 40 ABCC1 p.Cys43Ser ABCC1 p.Gly671Val ABCC1 p.Ala989Thr ABCC1 p.Ser92Phe Four (C43S, S92F, G671V, A989T) were mutants that our earlier studies showed had a phenotype discordant from that predicted by Polyphen and/or SIFT (L&#e9;tourneau et al., 2005). Login to comment 41 ABCC1 p.Arg723Gln ABCC1 p.Arg633Gln Two others (R633Q, R723Q) were selected because of their location in NBD1. Login to comment 42 ABCC1 p.Cys1047Ser The last, C1047S, was selected because the predicted probability of this mutation having a deleterious effect by SIFT approached statistical significance. Login to comment 58 ABCC1 p.Arg723Gln ABCC1 p.Gly671Val ABCC1 p.Cys1047Ser ABCC1 p.Arg633Gln ABCC1 p.Ala989Thr Lysates (10 mg protein per lane) prepared from HEK293 cell lines expressing wild-type (WT) and mutant (R633Q, G671V, R723Q, A989T, C1047S) MRP1 proteins and the untransfected control cell line (HEK) were immunoblotted, and MRP1 was detected with mAb QCRL-1. Login to comment 96 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr Total cellular glutathione in HEK293 wild-type, A989T and C1047S cells was measured using the enzymatic recycling method of Tietze (1969) and Brehe and Burch (1976). Login to comment 103 ABCC1 p.Ala989Thr Tryptic fragmentation patterns of wild-type and A989T mutant MRP1 were determined as described previously elsewhere (Rothnie et al., 2006; Iram and Cole, 2012). Login to comment 104 ABCC1 p.Ala989Thr Briefly, membrane vesicles (2 mg of protein per lane) enriched for wild-type or A989T mutant MRP1 were incubated in hypotonic buffer (50 mM HEPES, pH 7.4) in the absence or presence of S-methylGSH (10 mM) for 30 minutes on ice. Login to comment 111 ABCC1 p.Gly671Val All the algorithms and matrices are in agreement that the G671V mutation located close to the Walker A motif in NBD1 is the most likely to affect MRP1 activity. Login to comment 114 ABCC1 p.Arg723Gln ABCC1 p.Arg633Gln The analyses of the two other nsSNPs located in NBD1, namely, R633Q and R723Q, yield results very similar to one another (low probability of adverse effect), except that Arg633 is considered to be more critical to MRP1 stability by I-mutant Suite (Table 1), probably because Arg633 is more strictly conserved than Arg723 (Supplemental Table 1). Login to comment 115 ABCC1 p.Cys43Ser ABCC1 p.Ser92Phe The predicted effects of the nsSNPs C43S and S92F in MSD0 are mixed (Table 1). Login to comment 116 ABCC1 p.Cys43Ser ABCC1 p.Ser92Phe ABCC1 p.Ser92Phe They are predicted to be possibly damaging by Polyphen2 but tolerated by SIFT (except for SIFTBLink which places S92F at the threshold), and to cause substantial physicochemical changes and probably low occurrence (Grantham and matrices), yet the I-mutant Suite predicts C43S to be more destabilizing than S92F (Table 1). Login to comment 117 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr The predicted effects of the nsSNPs A989T and C1047S located in TM12 and TM13 of MSD2, respectively, are also mixed. Login to comment 120 ABCC1 p.Gly671Val According to this method, the mutants ranked (from highest to lowest likelihood of an adverse effect) as follows: G671V . Login to comment 121 ABCC1 p.Ser92Phe S92F . Login to comment 122 ABCC1 p.Cys43Ser C43S . Login to comment 123 ABCC1 p.Cys1047Ser C1047S . Login to comment 124 ABCC1 p.Arg633Gln R633Q . Login to comment 125 ABCC1 p.Arg723Gln ABCC1 p.Ala989Thr R723Q = A989T. Login to comment 126 ABCC1 p.Arg723Gln ABCC1 p.Gly671Val ABCC1 p.Cys1047Ser ABCC1 p.Arg633Gln ABCC1 p.Ala989Thr nsSNPs R633Q, G671V, R723Q, A989T, and C1047S Have No Effect on Total on Plasma Membrane MRP1 Levels in HEK293 Cells. Login to comment 127 ABCC1 p.Arg723Gln ABCC1 p.Gly671Val ABCC1 p.Cys1047Ser ABCC1 p.Arg633Gln ABCC1 p.Ala989Thr After we had isolated stably transfected HEK293 cell lines by G418 selection, cell lines expressing R633Q, G671V, R723Q, A989T, and C1047S and wild-type MRP1 were cloned to .90% homogeneity for MRP1 expression. Login to comment 128 ABCC1 p.Arg723Gln ABCC1 p.Gly671Val ABCC1 p.Cys1047Ser ABCC1 p.Arg633Gln ABCC1 p.Ala989Thr Immunoblots of cell lysates showed that the levels of the five mutant proteins were comparable to (R633Q, A989T, C1047S) or somewhat (,50%) higher than (G671V, R723Q) wild-type MRP1, indicating that the mutations do not cause any major misfolding of MRP1 that would result in its degradation (Fig. 1B). Login to comment 129 ABCC1 p.Cys43Ser ABCC1 p.Ser92Phe However, despite our repeated attempts, HEK cell lines homogeneously expressing the two MSD0 mutants, C43S and S92F, could not be isolated. Login to comment 131 ABCC1 p.Cys43Ser ABCC1 p.Ser92Phe Immunoblots of lysates prepared from the S92F and C43S cell lines showed MRP1 protein levels that were much lower than wild-type MRP1 (Supplemental Fig. 1), as might be expected from nonclonal cell lines. Login to comment 132 ABCC1 p.Arg723Gln ABCC1 p.Gly671Val ABCC1 p.Cys1047Ser ABCC1 p.Arg633Gln ABCC1 p.Ala989Thr Confocal fluorescence microscopy experiments showed that the R633Q, G671V, R723Q, A989T, and C1047S mutant proteins in the five clonal HEK cell lines were also routed correctly to the plasma membrane in a manner indistinguishable from wild-type MRP1 (Fig. 2). Login to comment 133 ABCC1 p.Cys43Ser ABCC1 p.Ser92Phe The C43S and S92F mutants in the nonclonal cell populations were also properly routed to the plasma membrane although, as expected because of the heterogeneity of MRP1 expression in these cell lines, significantly fewer cells expressed these proteins (Supplemental Fig. 1). Login to comment 134 ABCC1 p.Cys43Ser ABCC1 p.Ser92Phe Because of the confounding effects of mixed cell populations on the interpretation of subsequent functional assays, the C43S and S92F mutants were not characterized further. Login to comment 136 ABCC1 p.Arg723Gln ABCC1 p.Gly671Val ABCC1 p.Cys1047Ser ABCC1 p.Arg633Gln ABCC1 p.Ala989Thr The HEK cell lines expressing R633Q, G671V, R723Q, A989T, and C1047S were tested for their levels of resistance to five xenobiotics for which human MRP1 is known to confer resistance, including the antineoplastic agents vincristine, etoposide (VP-16), doxorubicin, and the heavy metal oxyanions arsenite and antimony tartrate (Cole et al., 1994). Login to comment 138 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr Levels of vincristine resistance were the most variable, but the differences were only statistically significant (approximately 2.5-fold lower than wild-type MRP1, P , 0.05) in the cell lines expressing A989T and C1047S. Login to comment 139 ABCC1 p.Cys1047Ser Etoposide resistance of the C1047S cell line was also lower than the wild-type MRP1 cell line, but the difference was more modest (1.6-fold; P , 0.05); doxorubicin resistance was also decreased, but the difference was not statistically significant. Login to comment 140 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr Resistance of the A989T and C1047S cell lines to arsenite was comparable to the wild-type MRP1 cell line while resistance to antimony tartrate was reduced (1.7-fold and 3.0-fold, respectively). Login to comment 141 ABCC1 p.Cys43Ser ABCC1 p.Arg723Gln ABCC1 p.Gly671Val ABCC1 p.Cys1047Ser ABCC1 p.Arg633Gln ABCC1 p.Ala989Thr ABCC1 p.Ser92Phe TABLE 1 Predicted effects of MRP1 nsSNPs examined in this study according to various in silico prediction methods nsSNP SIFT/SIFTBLink Probability Scoresa PolyPhen2 Classificationb (Score) I-Mutant Suite "Stability"c (DDG in kcal mol21 ) Grantham Value Difference (D)d Blosum50e PAM250f (Threshold) (,0.05) (.1.000) (,20.5; .0.5) (.50) (,0) (,0) C43S 0.51/0.08 possibly damaging (0.819) decrease (20.74) 112 21 0 S92F 0.11/0.05 possibly damaging (0.303) neutral (20.05) 155 23 23 NBD1-R633Q 0.66/0.57 benign (0.001) decrease (21.16) 43 1 1 NBD1-G671V 0.00/0.02 probably damaging (1.000) decrease (20.57) 109 24 21 NBD1-R723Q 0.49/0.39 benign(0.002) decrease (20.71) 43 1 1 A989T 0.53/0.12 benign (0.000) decrease (20.73) 58 0 1 C1047S 0.07/0.64 benign (0.001) decrease (20.67) 112 21 0 a SIFT (Sorting Intolerant From Tolerant) was used by manually entering a sequence alignment comprising only human homologs of MRP1, and SIFT-BLink probability scores were obtained using 100 aligned computer-selected sequences (threshold for nontolerated substitution set at ,0.05). Login to comment 153 ABCC1 p.Arg723Gln ABCC1 p.Gly671Val ABCC1 p.Cys1047Ser ABCC1 p.Arg633Gln ABCC1 p.Ala989Thr To determine whether the five nsSNPs, R633Q, G671V, R723Q, A989T, and C1047S, affected the ability of MRP1 to mediate efflux of calcein, HEK293 cells stably expressing wild-type and mutant MRP1 as well as untransfected HEK cells were incubated with several concentrations of the cell permeable acetoxymethyl ester of calcein (calcein-AM) at 37&#b0;C; 3 hours later, the intracellular hydrolyzed calcein that had not been effluxed by MRP1 was measured. Login to comment 157 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr Thus, unlike the chemosensitivity assay, which showed that the drug-resistance patterns of A989T and C1047S were different from wild-type MRP1 (Table 2), the calcein efflux assay did not detect any differences (Fig. 3). Login to comment 158 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr nsSNPs A989T, But Not C1047S, Affects the Inhibition of MRP1-Mediated Calcein Efflux by LY465803. Login to comment 159 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr To further explore the selective effects of nsSNPs A989T and C1047S on their ability to recognize xenobiotics, we determined whether the two mutations affect the efficacy of MRP1 modulators. Login to comment 167 ABCC1 p.Arg723Gln ABCC1 p.Gly671Val ABCC1 p.Cys1047Ser ABCC1 p.Arg633Gln ABCC1 p.Ala989Thr TABLE 2 Effect of nsSNP mutations on MRP1-mediated resistance to chemotherapeutic agents and metalloids in HEK293 cell lines Cell Line/ nsSNP Relative Resistancea Vincristine Doxorubicin Etoposide Na+ Arsenite K+ Antimony Tartrate Wild-type 12.2 6 0.6 3.9 6 1.6 7.1 6 0.6 1.4; 2.2 5.2; 6.4 R633Q 15.3 6 2.9 3.3 6 1.0 5.1 6 0.9 ND ND G671V 9.3 6 2.8 3.4 6 0.6 7.1 6 0.7 ND ND R723Q 21.3 6 7.2 2.5 6 0.1 6.9 6 0.5 ND ND A989T 4.4 6 1.1b ** 2.5 6 0.6 5.0 6 0.9 1.6; 2.3 3.1; 3.5 C1047S 5.1 6 0.5*** 1.8 6 0.2 4.5 6 0.7* 2.0; 1.8 2.2; 1.7 ND, not determined. Login to comment 172 ABCC1 p.Cys1047Ser Similarly, the calcein efflux activity of the C1047S mutant was completely inhibited. Login to comment 173 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr ABCC1 p.Ala989Thr However, the fluorescence levels in cells expressing A989T were decreased by 50%, indicating that the mutation had diminished the ability of LY465803 to inhibit MRP1. Similarly, in the converse experiment, when cells were preincubated with increasing concentrations of LY465803 before adding a single concentration of calcein-AM (6 mM), the fluorescence levels observed for the cells expressing the A989T mutant were consistently ;50% lower than the fluorescence levels in the control untransfected HEK293 cells as well as the cells expressing wild-type and C1047S mutant MRP1 at all LY465803 concentrations tested (Fig. 4B). Login to comment 174 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr nsSNPs A989T and C1047S Do Not Affect Inhibition of [3 H]E217bG Vesicular Transport by LY465803 and the Leukotriene Modifiers LY171883 and BAYu9773. Login to comment 175 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr ABCC1 p.Ala989Thr To determine whether the nsSNP A989T also affected the sensitivity of MRP1 to the GSH-dependent LY465803 inhibitor in a vesicular transport assay, the ability of LY465803 to inhibit uptake of [3 H]E217bG into membrane vesicles prepared from the HEK cell line expressing A989T was examined; cell lines expressing the C1047S mutant and wild-type MRP1 were included as controls. Login to comment 176 ABCC1 p.Ala989Thr ABCC1 p.Ala989Thr ABCC1 p.Ala989Thr Preliminary experiments confirmed that E217bG vesicular uptake by A989T was partially (50%) reduced in vesicles from the A989T HEK cell line, as it had been shown previously in vesicles prepared from transiently transfected A989T HEK cells (L&#e9;tourneau et al., 2005) (Supplemental Fig. 2). Login to comment 178 ABCC1 p.Cys1047Ser ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr As can be seen in Table 3 from the data summary of multiple experiments, the IC50 values of LY465803 for A989T (0.05 6 0.01 mM) and C1047S (0.03 6 0.03 mM) were not significantly different from the IC50 for wild-type MRP1 (0.07 6 0.01 mM) although the difference approached statistical significance for C1047S (P = 0.07). Login to comment 180 ABCC1 p.Ala989Thr BAYu9773, a nonselective antagonist of the CysLT receptors 1 and 2 and LY171883, a selective antagonist of CysLT receptor 1, inhibited E217bG transport by A989T with IC50 values of 0.32 6 0.19 mM and 19.58 6 6.15 mM, respectively. Login to comment 181 ABCC1 p.Cys1047Ser ABCC1 p.Cys1047Ser These values were not significantly different from the IC50 values obtained for BAYu9773-mediated and LY171883-mediated inhibition of E217bG transport by either wild-type or C1047S MRP1 (Table 3) (0.69 6 0.18 mM and 0.62 6 0.16 mM [for BAYu9773] and 16.10 6 1.28 mM and 23.90 6 9.85 mM [for LY171883] for wild-type and C1047S, respectively; P . Login to comment 183 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr Effect of nsSNPs A989T and C1047S on [3 H]GSH Transport and Cellular GSH Levels. Login to comment 184 ABCC1 p.Ala989Thr Because the A989T nsSNP was associated with a decreased inhibitory potency of the GSH-dependent LY465803 inhibitor in a cell-based (where its effects are dependent on intracellular GSH concentrations) but not in a membrane-based transport assay (where exogenous GSH is provided and is not limiting), it was of interest to determine whether vesicular transport of GSH and/or cellular levels of GSH were affected by this nsSNP. Login to comment 185 ABCC1 p.Cys1047Ser The C1047S mutant was included for comparative purposes. Login to comment 186 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr As shown in Fig. 6A, levels of apigenin-stimulated GSH uptake by A989T and C1047S were 16% and 26% lower than for wild-type MRP1, but in neither case were these differences statistically significant (P = 0.2 and 0.16, respectively). Login to comment 187 ABCC1 p.Ala989Thr Thus, the difference in results obtained with LY465803 and the nsSNP A989T in the cell-based versus membrane-based Fig. 3. Login to comment 189 ABCC1 p.Arg723Gln ABCC1 p.Gly671Val ABCC1 p.Cys1047Ser ABCC1 p.Arg633Gln ABCC1 p.Ala989Thr HEK293 cells stably expressing wild-type (WT-MRP1) and mutant (R633Q, G671V, R723Q, A989T, C1047S) MRP1 were incubated in the presence of increasing concentrations of calcein-AM (0-6mM), and the intracellular calcein remaining in the cells after 3 hours was measured by fluorometry as described in Materials and Methods. Login to comment 191 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr Effect of the MRP1 inhibitor LY465803 on calcein accumulation in HEK293 cells expressing wild-type and A989T and C1047S mutant MRP1 proteins. Login to comment 198 ABCC1 p.Cys1047Ser ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr ABCC1 p.Ala989Thr In contrast to the wild-type MRP1 cell line, the GSH levels present in the A989T and C1047S cell lines were comparable (106% and 94%, respectively) to those of the control untransfected cell line, suggesting that both nsSNPs adversely affect the ability of MRP1 to efflux GSH from intact cells (P = 0.08 and 0.02, respectively, for A989T and C1047S cells compared with wild-type MRP1 cells). Login to comment 199 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr nsSNPs A989T and C1047S Have No Major Impact on the Protease Susceptibility of MRP1. Login to comment 200 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr Because the functional changes detected for A989T and C1047S were the most significant among all the nsSNPs tested, we determined whether any of these changes were associated with any differences in protein conformation as reflected by a change in protease susceptibility. Login to comment 203 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr As shown in Supplemental Fig. 3A, when blots were probed with mAbs MRPr1 and 42.4 that detect epitopes in the NH2-half of MRP1, no differences in the tryptic fragment patterns obtained were observed between A989T (and C1047S, data not shown) and wild-type MRP1. Similarly, no differences were detected when the blots were probed with mAbs MRPm5, 897.2, and MRPm6, which detect epitopes located in the COOH-half of MRP1 (Supplemental Fig. 3B). Login to comment 210 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr Effect of MRP1 modulators on ATP-dependent vesicular uptake of [3 H]E217bG by wild-type and A989T and C1047S mutant MRP1. Login to comment 211 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr Membrane vesicles prepared from HEK293 cells expressing wild-type [WT (d)], A989T (s), and C1047S (m) mutant MRP1 were preincubated with a range of concentrations of (A) BAYu9773, (B) LY171883, and (C) LY465308 (+ GSH), and then ATP-dependent [3 H]E217bG uptake was measured after 5 minutes in the continued presence of the modulators as described in Materials and Methods. Login to comment 214 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr TABLE 3 Effect of MRP1 modulators on ATP-dependent vesicular uptake of E217bG by A989T and C1047S mutant MRP1 The values shown represents the mean IC50 values 6 S.D. obtained from three independent experiments performed using two different preparations of membrane vesicles. Login to comment 215 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr IC50 (mM) Wild-Type A989T C1047S LY465803 (+ GSH) 0.07 6 0.01 0.05 6 0.01 0.03 6 0.03 BAYu9773 0.62 6 0.16 0.32 6 0.19 0.69 6 0.18 LY171883 16.10 6 1.28 19.58 6 6.15 23.90 6 9.85 and compared the experimental data obtained with stable HEK cell lines with the consequences of the amino acid substitutions predicted by various in silico methods. Login to comment 217 ABCC1 p.Cys43Ser ABCC1 p.Ser92Phe The first category includes the two nsSNPs in TM1 (C43S) and TM2 (S92F) that could not be functionally characterized because stable homogeneously expressing clonal HEK cell lines for these mutants could not be isolated. Login to comment 220 ABCC1 p.Cys43Ser ABCC1 p.Ser92Phe However, most in silico prediction programs based on chemical properties, amino acid conservation, and structural data classify the C43S and S92F nsSNPs as very likely to be deleterious to MRP1 function. Login to comment 221 ABCC1 p.Cys43Ser Previously, we showed that C43S expressed in HeLa cells was associated with decreased arsenite and vincristine resistance, and moderately impaired MRP1 trafficking to the plasma membrane (Leslie et al., 2003). Login to comment 225 ABCC1 p.Ser92Phe ABCC1 p.Ser92Phe The inability of the S92F mutant to be established as a stable homogenously expressing HEK cell line is also somewhat unexpected because S92F was readily expressed in transiently transfected HEK cells (L&#e9;tourneau et al., 2005). Login to comment 226 ABCC1 p.Ser92Phe In this latter system, S92F transported organic anions LTC4, E217bG, and methotrexate at levels only modestly below those of wild-type MRP1. Login to comment 227 ABCC1 p.Arg723Gln ABCC1 p.Gly671Val ABCC1 p.Arg633Gln The second category of mutants examined includes the three nsSNPs located in NBD1 (R633Q, G671V, R723Q), which exhibited little, if any, change in transport levels or sensitivity to MRP1 substrates or inhibitors. Login to comment 228 ABCC1 p.Arg633Gln For R633Q, this is not surprising because Arg633 is not located in or close to any functionally important motifs in NBD1. Login to comment 230 ABCC1 p.Arg633Gln Thus, our results here showing little or no effect of R633Q on MRP1 expression or function are consistent with in silico predictions and with our earlier study on transiently transfected cells (L&#e9;tourneau et al., 2005). Login to comment 231 ABCC1 p.Gly671Val This contrasts with G671V, which all prediction algorithms, physicochemical parameters, and structure analyses concur should be deleterious (Table 2). Login to comment 232 ABCC1 p.Gly671Val Indeed, the G671V mutation ranked as the most damaging of all seven nsSNPs examined here. Login to comment 235 ABCC1 p.Gly671Val T (G671V) nsSNP has been associated with an altered response to certain drugs, including adverse reactions, in several studies. Login to comment 240 ABCC1 p.Gly671Val These findings are consistent with our earlier observation that lymphocytes from normal individuals bearing this G671V nsSNP expressed ABCC1 mRNA at relatively low levels (Conrad et al., 2001), and they suggest a change in mRNA structure is more important than the amino acid change caused by this nSNP. Login to comment 244 ABCC1 p.Gly671Val ABCC1 p.Gly671Val The reasons for the discordance between most of the experimental observations with the G671V mutant protein versus the uniformly adverse effects predicted for the Val substitution of Gly671 by the in silico analytical methods and the genetic association studies are unclear but may well be related to the cellular context in which the Fig. 6. Login to comment 245 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr Apigenin-stimulated vesicular transport of [3 H]GSH by A989T and C1047S mutant MRP1 and intracellular glutathione content. Login to comment 246 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr (A) ATP-dependent uptake of [3 H]GSH (100 mM/120 nCi) in the presence of apigenin (30 mM) was measured in membrane vesicles prepared from HEK293 cells expressing wild-type, A989T and C1047S MRP1 for 20 minutes at 37&#b0;C. Login to comment 250 ABCC1 p.Gly671Val It is also possible that analyses in a cell culture setting may not be adequate to discern all aspects of G671V function. Login to comment 251 ABCC1 p.Arg723Gln With respect to the R723Q nsSNP, a conserved positive charge is lost (Supplemental Table 1), and the side chain has been shortened. Login to comment 253 ABCC1 p.Arg723Gln Consequently, in this case, the prediction that R723Q would be a benign substitution by five out of six in silico methods used is consistent with the experimental data reported here in a stable HEK cell line as well as in our earlier study in transiently transfected HEK cells (L&#e9;tourneau et al., 2005). Login to comment 254 ABCC1 p.Arg723Gln ABCC1 p.Arg723Gln However, in contrast with our observations, Yin et al. (2009) reported that HEK cells stably expressing R723Q exhibited varying degrees of reduced resistance to natural product drugs and that the plasma membrane trafficking of the R723Q protein was mildly impaired. Login to comment 256 ABCC1 p.Arg723Gln It is of interest that in a small phase III study of Caucasian patients with advanced multiple myeloma, the presence of the R723Q nsSNP in 5 of 279 patients being treated with bortezomib and pegylated liposomal doxorubicin was associated with a different overall response rate and durability (Buda et al., 2010). Login to comment 258 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr The third and final category consists of A989T and C1047S which, despite the fact that most predictive methods indicated they were unlikely to affect MRP1 activity, exhibited altered phenotypes that distinguished them not only from wild-type MRP1 but to some degree from each other. Login to comment 259 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr Thus, HEK cells expressing A989T or C1047S differed in their drug-resistance profiles and their sensitivity to LY465803- mediated inhibition of calcein efflux, as well as their cellular GSH levels as detected in cell-based assays. Login to comment 260 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr The C1047S affected both vincristine and etoposide resistance while the A989T mutation affected only vincristine resistance. Login to comment 262 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr Inhibition of calcein efflux by the GSH-dependent LY465803 was also reduced in A989T cells but not C1047S cells. Login to comment 263 ABCC1 p.Cys1047Ser ABCC1 p.Ala989Thr In contrast, membrane-based vesicular transport assays indicated that A989T and C1047S did not affect the ability of LY465803 to inhibit MRP1-mediated E217bG uptake. Login to comment 269 ABCC1 p.Gly671Val Typically, either a cell-based or membrane-based functional assay is used to measure MRP1 function, but experience with the 2012C.T (G671V) allele suggests that additional nucleic acid-based methods should also be included. Login to comment