ABCMdb

PMID: 21935580

Eddabra L, Wenner T, El Btaouri H, Baranek T, Madoulet C, Cornillet-Lefebvre P, Morjani H
Arginine 482 to glycine mutation in ABCG2/BCRP increases etoposide transport and resistance to the drug in HEK-293 cells.
Oncol Rep. 2012 Jan;27(1):232-7. doi: 10.3892/or.2011.1468. Epub 2011 Sep 20., [PubMed]

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No. Mutations Sentence Comment

5 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly HEK293 cells were transfected with an empty vector (HEK/V), the vector bearing the wild-type BCRP (HEK/R482), the mutant arginine-482-glycine (HEK/R482G) or the mutant arginine-482-threonine (HEK/R482T). Login to comment 7 ABCG2 p.Arg482Gly Data show that HEK/R482G cells displayed the highest levels of resistance to etoposide. Login to comment 8 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Cellular [3 H]-etoposide uptake was lower in HEK/R482, HEK/R482G and HEK/R482T cells compared to HEK/V cells. Login to comment 9 ABCG2 p.Arg482Gly In addition, cellular [3 H]-etoposide uptake in HEK/R482G was the lowest. Login to comment 10 ABCG2 p.Arg482Gly Drug efflux measurements showed that fumitremorgin C was able to increase the residual cellular [3 H]-etoposide uptake in BCRP-transfected cells and especially in HEK/R482G ones. Login to comment 11 ABCG2 p.Arg482Gly Our data show that the R482G mutation in BCRP is able to increase efflux of etoposide and that mutation analysis at codon 482 may be of clinical importance in cancers treated with etoposide. Login to comment 19 ABCG2 p.Arg482Thr In these cells the BCRP gene has been shown to be mutated in the exon 5 leading to the substitution of the arginine 482 for a threonine (R482T). Login to comment 20 ABCG2 p.Arg482Gly The same amino acid is also mutated in another cell line but substituted for a glycine (R482G) (9,10). Login to comment 23 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly We studied the resistance to etoposide and its cellular transport in HEK293 cells transfected by the human wild-type BCRP or its R482G and R482T mutants. Login to comment 24 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly We show that cells expressing the R482G mutant display higher resistance level to etoposide than the wild-type BCRP or the mutant R482T genes. Login to comment 29 ABCG2 p.Arg482Gly Fumitremorgin C (FTC) Arginine 482 to glycine mutation in ABCG2/BCRP increases etoposide transport and resistance to the drug in HEK-293 cells LAHCEN EDDABRA1 , THOMAS WENNER2 , HASSAN EL BTAOURI1 , THOMAS BARANEK3 , CLAUDIE MADOULET1 , PASCALE CORNILLET-LEFEBVRE4 and HAMID MORJANI1 1 MEDyC Unit&#e9; CNRS UMR6237, UFR de Pharmacie, IFR53, 51096 Reims, France; 2 CRP-Sant&#e9;, Luxembourg; 3 IIER EA 4303, UFR M&#e9;decine, IFR53; 4 EA3801, Laboratoire d'H&#e9;matologie, CHU de Reims, 51096 Reims, France Received July 13, 2011; Accepted August 16, 2011 DOI: 10.3892/or.2011.1468 Correspondence to: Dr Hamid Morjani, MEDyC Unit&#e9; CNRS UMR6237, UFR de Pharmacie, IFR53, 51096 Reims, France E-mail: hamid.morjani@univ-reims.fr Key words: ABCG2/BCRP, mutation, etoposide transport, resistance was kindly provided by R.W. Robey and Professor S. Bates (National Cancer Institute, NIH, Bethesda, MD, USA). Login to comment 31 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly The human embryonic kidney HEK293 cells transfected with empty vector (HEK/V) or BCRP (HEK/R482 and HEK/R482G and HEK/R482T) were kindly provided by R.W. RobeyandS.Bates(12).Cells weregrownas monolayerin MEM (Invitrogen, Paris, France) supplemented with 10% fetal calf serum and 100 &#b5;g/ml penicillin, and 100 &#b5;g/ml streptomycin. Login to comment 79 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly The relative expression level of BCRP mRNA in HEK/R482, HEK/R482G and HEK/R482T cell lines was 347-, 247- and 315-fold and is higher than in HEK/V cells, respectively. Login to comment 90 ABCG2 p.Arg482Gly Interestingly, the resistance factor was significantly increased to 30-fold in the glycine mutant (HEK/R482G). Login to comment 91 ABCG2 p.Arg482Thr In contrast, resistance to etoposide in HEK/ R482T cell line remains in the same range of HEK/R482 cell line. Login to comment 93 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Relative mRNA expression levels of BCRP and MRP1 as estimated by real-time RT-PCR in HEK293 and 2008 transfected cells.a HEK/V HEK/R482 HEK/R482G HEK/R482T 2008 2008/MRP1 Ct TBP 21.20 20.59 20.43 20.07 25.13 25.31 Ct BCRP 28.41 19.36 19.69 18.98 - - Ct MRP1 - - - - 27.20 22.23 ࢞Ct 7.21 -1.23 -0.74 -1.09 2.07 -3.08 ࢞࢞Ct - -8.44 -7.95 -8.30 - -5.15 2-࢞࢞Ct - 347.29 247.28 315.17 - 35.51 a The fold change of BCRP or MRP1 expression in the BCRPand MRP1-transfected cell lines was determined as described in Materials and methods. Login to comment 98 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly HEK/V HEK/R482 HEK/R482G HEK/R482T 2008 2008/MRP1 BCRP 1.56 20 25 21 - - MRP1 - - - - 1.02 4.44 Figure 1. Login to comment 102 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly (A) Cytotoxic effect of etoposide in HEK293 (c6;), HEK/R482 (fc;), HEK/R482T (cf;) and HEK/R482G (b2;) cell lines. Login to comment 109 ABCG2 p.Arg482Gly By contrast, FTC was able to reverse resistance to etoposide moderately in HEK/R482G cell line as compared to novobiocin. Login to comment 110 ABCG2 p.Arg482Thr Novobiocin was not able to modulate resistance to etoposide in HEK/R482T cell line. Login to comment 115 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly The [3 H]-etoposide uptake represented 63 (p<0.02), 40 (p<0.01) and 54% (p<0.02), respectively, in HEK/R482, HEK/ R482G and HEK/R482T, of the value measured in HEK/V cells (Fig. 3A and B). Login to comment 116 ABCG2 p.Arg482Gly Moreover, the [3 H]-etoposide uptake is significantly reduced in HEK/R482G cells as compared to HEK/ R482 cells (p<0.05). Login to comment 117 ABCG2 p.Arg482Thr The [3 H]-etoposide uptake is also reduced in HEK/R482T cells as compared to HEK/R482 cells, although Table III. Login to comment 118 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly IC50 of mitoxantrone and etoposide in HEK293 and 2008 cells in the presence or not of novobiocin or FTC.a Cell line -------------------------------------------------------------------------- HEK/V HEK/R482 HEK/R482G HEK/R482T 2008 2008/MRP1 ------ ----------- ------------ ----------- ---- ---------- Drug Inhibitor IC50 IC50 RF IC50 RF IC50 RF IC50 IC50 RF Mitoxantrone - 0.15 1.17 11.33 3.10 20.67 3.00 20.00 Novobiocin 0.08 0.08 1.00 0.50 6.25 0.90 11.25 FTC 0.12 0.11 0.92 0.21 1.75 0.32 2.67 Etoposide - 1.00 5.00 5.00 30.00 30.00 8.00 8.00 0.70 6.00 8.57 Novobiocin 1.00 2.00 2.00 4.50 4.50 8.20 8.20 FTC 1.00 1.30 1.30 8.20 8.20 3.20 3.20 a IC50 values are the means of three independent experiments, each performed in triplicate. Login to comment 138 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly [3 H]-etoposide uptake reached 92 (p<0.05), 77 (p<0.02) and 81% (p<0.05) in HEK/R482, HEK/R482G and HEK/R482T cells, respectively, when compared to the values observed in the absence of novobiocin. Login to comment 146 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly However, in transfected cells it represented 44 (p<0.05), 35 (p<0.01) and 41% (p<0.02) of the initial concentration in HEK/ R482, HEK/R482G and HEK/R482T cells, respectively (Fig. 4). Login to comment 147 ABCG2 p.Arg482Gly The intracellular [3 H]-etoposide concentration is significantly lower in HEK/R482G cells as compared to HEK/R482 cells (p<0.05). Login to comment 148 ABCG2 p.Arg482Thr However, this does not reach stastical significance when HEK/R482T cells were compared to HEK/R482 cells. Login to comment 150 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly However, in HEK/R482, HEK/R482G and HEK/R482T cells treated with FTC, the cellular residual [3 H]-etoposide was increased to 73 (p<0.02), 77 (p<0.01) and 68% (p<0.01) of the initial concentration, and was significantly higher than that observed in the absence of FTC (Fig. 4). Login to comment 152 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly In drug-selected cell lines, two different mutations leading to a transition from arginine 482 to threonine (R482T) and glycine (R482G) respectively, have been observed (9,10). Login to comment 153 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly R482T mutation confers high-level resistance to anthracyclines (21), and cells with R482G or R482T mutation are able to efflux more efficiently rhodamine 123 (9). Login to comment 154 ABCG2 p.Arg482Gly Moreover, R482G mutation seems to confer relatively less resistance to camptothecins. Login to comment 158 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly In order to study the role of this protein in resistance to etoposide in human, we used the embryonic HEK293 cells transfected with the wild-type BCRP (HEK/ R482) or its two mutants R482G (HEK/R482G) and R482T (HEK/R482T) (5). Login to comment 160 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Determination of rhodamine 123 uptake confirmed that HEK/R482G and HEK/R482T cells transported efficiently this probe when compared with HEK/R482 cells (data not shown). Login to comment 161 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly In our study, we have shown that wild-type BCRP, R482T and especially R482G mutant can confer significant resistance to etoposide, the HEK/R482G cells showing an IC50 value six-fold higher than in HEK/R482 cells (Table III). Login to comment 163 ABCG2 p.Arg482Thr Nevertheless, HEK/R482 and HEK/R482T cells were only 5and 8-fold resistant, respectively, to etoposide (Table III). Login to comment 164 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly This suggest that R482G mutation confer to the BCRP protein a better affinity for etoposide than the mutation R482T, or a better efficiency in drug efflux, as suggested by our results (Fig. 3). Login to comment 166 ABCG2 p.Arg482Gly In the presence of FTC, cellular uptake of etoposide was significantly increased and particularly in HEK/R482 and HEK/R482G cells. Login to comment 170 ABCG2 p.Arg482Gly Our data show clearly that R482G mutation is able to increase resistance to etoposide. Login to comment