ABCMdb

PMID: 22293538

Jungsuwadee P, Zhao T, Stolarczyk EI, Paumi CM, Butterfield DA, St Clair DK, Vore M
The G671V variant of MRP1/ABCC1 links doxorubicin-induced acute cardiac toxicity to disposition of the glutathione conjugate of 4-hydroxy-2-trans-nonenal.
Pharmacogenet Genomics. 2012 Apr;22(4):273-84., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC1 p.Gly671Val ABCC1 p.Gly671Val ABCC1 p.Gly671Val The G671V variant of MRP1/ABCC1 links doxorubicin-induced acute cardiac toxicity to disposition of the glutathione conjugate of 4-hydroxy-2-trans-nonenal Paiboon Jungsuwadeea,c , Tianyong Zhaoa , Elzbieta I. Stolarczyka , Christian M. Paumia , D. Allan Butterfieldb , Daret K. St Claira and Mary Vorea Objective Doxorubicin-induced acute cardiotoxicity is associated with the Gly671Val (G671V; rs45511401) variant of multidrug resistance-associated protein 1 (MRP1). Login to comment 3 ABCC1 p.Gly671Val ABCC1 p.Gly671Val ABCC1 p.Arg433Ser Methods We established stable HEK293 cell lines overexpressing wild-type MRP1 (HEKMRP1), G671V (HEKG671V), and R433S (HEKR433S), a variant not associated with doxorubicin-induced cardiotoxicity and investigated the sensitivity of HEKG671V cells to doxorubicin and transport capacity of G671V toward GS-HNE. Login to comment 4 ABCC1 p.Gly671Val ABCC1 p.Arg433Ser Results In ATP-dependent transport studies using plasma membrane-derived vesicles, the Vmax (pmol/min/mg) for GS-HNE transport was the lowest for G671V (69 ± 4) and the highest for R433S (972 ± 213) compared with wild-type MRP1 (416 ± 22), whereas the Km values were 2.8 ± 0.4, 6.0 or more, and 1.7 ± 0.2 lmol/l, respectively. Login to comment 9 ABCC1 p.Gly671Val Conclusion These data suggest that decreased MRP1-dependent GS-HNE efflux contributes to increased doxorubicin toxicity in HEKG671V and potentially in individuals carrying the G671V variant. Login to comment 20 ABCC1 p.Gly671Val ABCC1 p.Gly671Val ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser Among several variants, they identified Arg433Ser (R433S), Original article 273 1744-6872 c 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI: 10.1097/FPC.b013e328350e270 located in the second transmembrane spanning domain, and Gly671Val (G671V; rs45511401), near the Walker A motif in the first nucleotide-binding domain. Login to comment 21 ABCC1 p.Arg433Ser The R433S MRP1 variant showed a 50% decreased transport maximum for leukotriene C4 (LTC4) [8]; however, cells expressing this variant were more resistant to doxorubicin than those expressing wild-type (WT) MRP1 [8]. Login to comment 22 ABCC1 p.Gly671Val The G671V variant showed no difference in in-vitro transport assays using LTC4 or estradiol-17b-glucuronide (E217G) as substrates. Login to comment 24 ABCC1 p.Gly671Val The frequency of the G671V variant (exon 16, 2012G > T) was 2.78% for the T allele in Whites and 1.43% in the Indian population, with none reported in the Asian population [9]. Login to comment 26 ABCC1 p.Gly671Val Importantly, in a nested case-control cohort clinical study, patients with the G671V variant showed a significantly increased doxorubicin-induced acute cardiac toxicity that accounted for 6.4% of the incidence of acute cardiac toxicity, with an odds ratio of 3.6 (95% confidence interval: 1.6-8.4) [10]. Login to comment 36 ABCC1 p.Gly671Val In addition, on the basis of the fact that (a) cumulative doses of doxorubicin are a risk for doxorubicin-induced cardiac toxicity, (b) HNE and HNE metabolites (e.g. GS-HNE) have been shown to be associated with oxidative stress in a myocardial ischemic model [21], and (c) MRP1 is highly expressed in the heart [22,23], we postulated that the association of the MRP1 G671V variant with doxorubicin-induced acute cardiac toxicity [10] could be due to a change in its substrate specificity. Login to comment 37 ABCC1 p.Gly671Val ABCC1 p.Gly671Val ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser In this study, we examined whether cells expressing the G671V and R433S variants versus WT MRP1 were more sensitive to doxorubicin, and characterized the transport properties of WT MRP1 relative to the G671V and R433S MRP1 variants, specifically with respect to GS-HNE transport activity. Login to comment 46 ABCC1 p.Gly671Val ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser MRP1 variants of G671Vand R433S were generated using the QuickChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, California, USA) according to the manufacturer`s instructions with the following mutagenic primers (for G671V, forward primer: 50 -TCCATCCCCGAAGTTGCTTTGGTG 274 GCCGTG-30 and reverse primer: 50 -CACGGCCACCAAAG CAACTTCGGGGATGGA-30 ; for R433S, forward primer: 50 -GTGGACGCTCAGAGCTTCATGGACTTGGC-30 and reverse primer: 50 -GCCAAGTCCATGAAGCTCTGAGCGT CCAC-30 ). Login to comment 78 ABCC1 p.Gly671Val Membranes were incubated with the primary antibodies for MRP1 (1:1000) and Na+ /K+ -ATPasea1 (1:20 000), washed three times, each for 5 min with TBS-T, followed by incubation with the secondary MRP1 G671V decreases GS-HNE transport Jungsuwadee et al. 275 antibody (1 : 5000) 1-2 h at room temperature, and finally washed twice with TBS-T buffer for 5 min. Login to comment 113 ABCC1 p.Gly671Val Results HEKG671V cells are more sensitive to doxorubicin cytotoxicity than HEKMRP1 and HEKR433S Patients with the G671V variant showed a significantly increased doxorubicin-induced acute cardiac toxicity [10]. Login to comment 114 ABCC1 p.Gly671Val ABCC1 p.Arg433Ser In an attempt to understand this phenomenon, we developed HEK293 cell lines that stably expressed human MRP1 SNPs G671V, and three different control cells: HEK293 cells transfected with pUSEamp(+) vector (HEKpUSE), WT MRP1 (HEKMRP1), or the R433S variant (HEKR433S), which is not associated with doxorubicin-induced acute cardiac toxicity. Login to comment 117 ABCC1 p.Gly671Val To determine the impact of the G671V SNP on cell viability and to estimate the doxorubicin IC50 value, we cultured cells in the presence of doxorubicin at various concentrations for 48 h (Fig. 2a); IC50 values were calculated from the percent survival curves (Fig. 2b). Login to comment 121 ABCC1 p.Gly671Val To determine whether the G671V variant influenced retention of doxorubicin, we incubated cells with doxorubicin (50 mmol/l, 1 h) and then cultured cells for an additional 30 min in the absence of doxorubicin to determine its MRP1-mediated efflux. Login to comment 141 ABCC1 p.Gly671Val ABCC1 p.Gly671Val ABCC1 p.Gly671Val ABCC1 p.Gly671Val ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser MRP1 G671V decreases GS-HNE transport Jungsuwadee et al. 277 Fig. 1 (b) 3000 MRP1pUSE G671V R433S *** *** *** 2000 Meanfluorescenceintensity 1000 0 pUSE MRP1 G671V R433S (a) (c) 10.0 * * * 7.5 5.0 2.5 MRP1/18S 0.0 pUSE MRP1 G671V R433S Expression in stable cell lines of multidrug resistance-associated protein 1 (MRP1) and its variants. Login to comment 143 ABCC1 p.Arg433Ser Plasmids containing wild-type MRP1 or the MRP1 variants G761V and R433S were transfected into HEK293 cells to generate stable cell lines. Login to comment 149 ABCC1 p.Gly671Val ABCC1 p.Gly671Val ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser Fig. 2 (a) (b) Genotype Doxorubicin IC50 nmol/l (95% CI) pUSE 58.0 (51.4-65.5) MRP1 463.0 (332.7-644.2) G671V 181.4 (137.4-239.5) R433S 645.2 (158.3-2630.0) pUSE125 100 75 50 Percentsurvival 25 0 MRP1 G671V R433S 10 Doxorubicin (μmol/l) -1-2-3 Percent survival of HEKpUSE, HEKMRP1, HEKR433S, and HEKG671V cells in the presence of doxorubicin. Login to comment 154 ABCC1 p.Gly671Val 278 G671V single nucleotide polymorphism maintains LTC4 transport activity but loses glutathione-conjugated 4-hydroxy-2-trans-nonenal transport capacity In view of the cellular GS-HNE retention in HEKG671V cells, we examined further whether the MRP1 polymorphisms would have an impact on their substrate specificity or transport capacity. Login to comment 156 ABCC1 p.Gly671Val ABCC1 p.Arg433Ser The G671V variant was comparable with WT MRP1 with respect to LTC4 transport, whereas LTC4 transport was decreased by 75% in the R433S variant compared with WT MRP1 (Fig. 6b), consistent with previous reports [7,8]. Login to comment 157 ABCC1 p.Gly671Val To determine whether the G671V variant might have an altered dependence on glutathione for transport, we examined the effects of glutathione; 0.5 mmol/l of glutathione had no effect on transport, whereas 5 mmol/l of glutathione completely inhibited LTC4 transport by MRP1 and both its variants (Fig. 6b). Login to comment 159 ABCC1 p.Gly671Val GS-HNE was transported by Michaelis-Menten kinetics (Fig. 6c), and showed a markedly reduced transport by the G671V variant such that the Vmax was decreased to about 15% of that by WT MRP1 (Fig. 6c). Login to comment 160 ABCC1 p.Arg433Ser In contrast, the Vmax of the R433S variant was increased over two-fold relative to WT MRP1. Login to comment 161 ABCC1 p.Gly671Val The Km value of the G671V variants did not differ significantly from that of WT MRP1 (Fig. 6c), and agreed well with the Km of 1.6 mmol/l reported previously for MRP1 [20]. Login to comment 162 ABCC1 p.Arg433Ser The estimate of the Km for the R433S variant was higher (Z 6 mmol/l), consistent with its increased Vmax (Fig. 6c). Login to comment 163 ABCC1 p.Gly671Val ABCC1 p.Gly671Val The Vmax/Km of the G671V variant was only about 10% (0.025 mg/l/min) of that of WT MRP1 (0.24 mg/l/min), indicating a markedly decreased transport efficiency of the G671V variant. Login to comment 165 ABCC1 p.Gly671Val ABCC1 p.Gly671Val ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser To investigate whether the cardiac sarcolemma can transport GS-HNE, and the Fig. 3 (a) (b) (c) (d) 1.5 ** ** **** ** ** ** *** ** 1.0 Fractionofcontrol doxorubicinaccumulation Fractionofcontrol doxorubicinaccumulation 0.5 0.0 8 250 200 150 100 GS-HNE(%control) 50 0 pUSE MRP1 G671V R433S 6 4 GS-HNE(nmol/mg) 2 0 Doxorubicin (1.5 μmol/l) - + - + - + - + pUSE MRP1 G671V R433S 1.5 1.0 0.5 0.0 pUSE 1 h M RP1 h G 671V 1 h R433S 1 h pUSE 24 h M RP1 24 h G 671V 24 h R433S 24 h Doxorubicin and glutathione-conjugated 4-hydroxy-2-trans-nonenal (GS-HNE) retention in HEKpUSE, HEKMRP1, HEKR433S, and HEKG671V cells. Login to comment 171 ABCC1 p.Gly671Val MRP1 G671V decreases GS-HNE transport Jungsuwadee et al. 279 importance of Mrp1, we isolated sarcolemma membranes from FVB WT and Mrp1- / - mice that were treated with doxorubicin (20mg/kg, intraperitoneally) and killed 24h later. Login to comment 174 ABCC1 p.Gly671Val Discussion The increased incidence of acute doxorubicin-induced cardiac toxicity in patients who carry the G671V variant of MRP1 suggests that the glycine to valine variant at amino acid 671 of MRP1 affects its transport function for certain substrates. Login to comment 175 ABCC1 p.Gly671Val Consistent with this hypothesis is the result predicted by PolyPhen-2 (http://genetics.bwh.harvard.edu/ pph2/), in which this G671V variant is predicted to be 'probably damaging`, likely due to the proximity of G671 to the Walker A motif. Login to comment 176 ABCC1 p.Gly671Val The present studies provided clear evidence that the ability to transport GS-HNE was markedly decreased by 85% in the G671V MRP1 variant relative to WT MRP1. Login to comment 177 ABCC1 p.Gly671Val In a previous study [7], the G671V variant did not show altered transport of several MRP1 substrates (LTC4, estrone sulfate, and E217G), and in this study, we confirmed that transport of LTC4 was not impacted. Login to comment 182 ABCC1 p.Arg433Ser These data are in contrast with an earlier report [8] showing that HeLa cells expressing R433S are two-fold more resistant than cells expressing WT MRP1. Login to comment 185 ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser We next examined cellular GS-HNE levels after treatment of cells with 1.5 mmol/l of doxorubicin for 24 h and Fig. 4 20(a) (b) (c) 15 12.5 10.0 7.5 5.0Glutathione (nmol/mgprotein) GSSG(nmol/mgprotein) 2.5 0.0 15 10 5 Glutathione/GSSGratio 0 pUSE pUSE 0.8 *** *** *** ** *** ***0.6 0.4 0.2 0.0 M RP1 G 671V R433S M RP1 G 671V R433S pUSE M RP1 G 671V R433S 10 Glutathione(nmol/mg) Glutathione/GSSGratio 5 0 0 16 14 12 10 8 0 10 20 30 40 50 Time (h) 25 50 Time (h) 1.5 1.0 0.5 0.0 0 10 20 30 40 50 Time (h) GSSG(nmol/mg) Time course of intracellular glutathione and GSSG in HEKpUSE, HEKMRP1, HEKR433S, and HEKG671V cells. Login to comment 197 ABCC1 p.Gly671Val On the basis of Vmax values, the GS-HNE transport capacity of the G671V variant was decreased 85% relative to WT MRP1 (Fig. 6c), and exhibited a 10-fold decrease in Vmax/Km. Login to comment 204 ABCC1 p.Gly671Val These are the first data demonstrating that the G671V variant has a decreased capacity to efflux GS-HNE, despite retention of the ability of this MRP1 variant to transport other classic MRP1 substrates, that is, LTC4, estrone sulfate, and E217G [7]. Login to comment 205 ABCC1 p.Arg433Ser It is also interesting to note that the R433S variant showed significantly increased transport of GS-HNE, despite decreased transport of LTC4 (Fig. 6b) and estrone sulfate, and unaltered transport of E217G [8]. Login to comment 212 ABCC1 p.Gly671Val MRP1 G671V decreases GS-HNE transport Jungsuwadee et al. 281 MRP3, Grant et al. [30] substituted amino acids 425-516 of MRP1 in the region spanning transmembrane helices 8 and with those of amino acids 411-502 of MRP3, and found complete loss of LTC4 transport, but a modest enhancement in E217bG transport, with minimal effects on transport of methotrexate, a substrate common to both MRP1 and MRP3 [30]. Login to comment 218 ABCC1 p.Gly671Val In conclusion, cells expressing the G671V MRP1 variant were more sensitive to doxorubicin than cells expressing WT MRP1, most likely due to an increase in the accumulation of intracellular GS-HNE, together with a decrease in the glutathione/GSSG ratio, indicating oxidative stress that can lead to cytotoxicity. Login to comment 219 ABCC1 p.Gly671Val ABCC1 p.Gly671Val ABCC1 p.Gly671Val ABCC1 p.Gly671Val ABCC1 p.Gly671Val ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser ABCC1 p.Arg433Ser Although increased retention of doxorubicin could also contribute to the increased oxidative stress in cells expressing the G671V variant, because amino acids in the third membrane spanning domain, especially between amino acids 959 and 1187, are considered most critical for doxorubicin transport [35], it seems less Fig. 6 Genotype Km ± SE (μmol/l) Vmax± SE (pmol/min/mg) Vmax/Km (mg/l/min) pUSE (95% CI) 6.3 ±12.9 (0.0-33.5) 28.4± 36.4 (0.0-104.8) 4.5 MRP1 (95% CI) 1.7 ±0.2 (1.2-2.1) 416.1±21.7 (371.1-461.2) 244.7 G671V (95% CI) 2.8± 0.4 (2.1-3.6) 69.1 ±4.1 (60.5-77.7) 24.7 R433S (95% CI) ≥6 972.1± 212.7 (531.0-1413) Approximately 160 pUSE MRP1 600 500 Protein(pmol/min/mg) 400 300 200 100 0 0 1 2 3 GS-HNE (μmol/l) 4 5 6 7 G671V R433S 125 *** *** 100 75 50 25 0 PercentLTC4efflux relativetoWT M RP1G 671VR433S M RP1+glutathione 5 G 671V+glutathione 5 R433S+glutathione 5 M RP1+glutathione 0.5 G 671V+glutathione 0.5 R433S+glutathione 0.5 2.5 (a) (b) (c) MRP1 pUSE/293 MRP1 G671V R433S Na/K-ATPase MRP1/NaK-ATPase banddensity (arbitraryunit) 2.0 1.5 1.0 0.5 0.0 MRP1 G671V R433S Multidrug resistance-associated protein 1 (MRP1) expression and transport activities of HEKMRP1, HEKR433S, and HEKG671V. Login to comment 228 ABCC1 p.Gly671Val 282 likely that the G671V variant alters MRP1 recognition of doxorubicin. Login to comment 229 ABCC1 p.Gly671Val The decreased GS-HNE transport capacity of the G671V variant further indicates that MRP1 polymorphisms can play a significant role in MRP1 activity, and that these findings may be clinically important in patients receiving chemotherapy, particularly doxorubicin. Login to comment