ABCMdb

PMID: 19002564

Polgar O, Deeken JF, Ediriwickrema LS, Tamaki A, Steinberg SM, Robey RW, Bates SE
The 315-316 deletion determines the BXP-21 antibody epitope but has no effect on the function of wild type ABCG2 or the Q141K variant.
Mol Cell Biochem. 2009 Feb;322(1-2):63-71. Epub 2008 Nov 11., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCG2 p.Gln141Lys The 315-316 deletion determines the BXP-21 antibody epitope but has no effect on the function of wild type ABCG2 or the Q141K variant Orsolya Polgar Æ John F. Deeken Æ Lilangi S. Ediriwickrema Æ Akina Tamaki Æ Seth M. Steinberg Æ Robert W. Robey Æ Susan E. Bates Received: 30 May 2008 / Accepted: 22 October 2008 / Published online: 11 November 2008 Ó Springer Science+Business Media, LLC. 2008 Abstract ABCG2 is a half-transporter initially described in multidrug-resistant cancer cells and lately identified as an important factor in the pharmacokinetics of its substrates. Login to comment 1 ABCG2 p.Gln141Lys Q141K is by far the most intensively studied single nucleotide polymorphism of ABCG2 with potential clinical relevance. Login to comment 2 ABCG2 p.Gln141Lys Here we used stably transfected HEK cells to study the Q141K polymorphism together with the deletion of amino acids 315-316, which were recently reported to coexist in two cancer cell lines (A549 and SK-OV-3). Login to comment 3 ABCG2 p.Gln141Lys Functional studies confirmed our previous report that when normalized to surface expression, Q141K has impaired transport of mitoxantrone. Login to comment 18 ABCG2 p.Gln141Lys The most extensively studied of these SNPs with potential clinical relevance is 421 C [ A resulting in a glutamic acid to lysine substitution (Q141K) in the protein. Login to comment 19 ABCG2 p.Gln141Lys The Q141K SNP was identified with varying frequencies in different ethnic groups and was found to be the most prevalent in the Japanese population (*30%). Login to comment 20 ABCG2 p.Gln141Lys Q141K has been associated with lower levels of protein expression and impaired transport in vitro, though some controversies exist in the publications characterizing this SNP [13-17]. Login to comment 22 ABCG2 p.Gln141Lys Further, Q141K was associated with a higher incidence of diarrhea in non-small cell lung cancer patients treated with gefitinib [21]. Login to comment 23 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Sequencing the ABCG2 gene in the 60 cancer cell lines maintained and used for screening in the National Cancer Institute by various groups revealed that ten of the cell lines carried the Q141K polymorphism. Login to comment 24 ABCG2 p.Gln141Lys The Q141K SNP was identified with varying frequencies in different ethnic groups and was found to be the most prevalent in the Japanese population (~30%). Login to comment 25 ABCG2 p.Gln141Lys Q141K has been associated with lower levels of protein expression and impaired transport in vitro, though some controversies exist in the publications characterizing this SNP [13-17]. Login to comment 26 ABCG2 p.Gln141Lys In the present study, we expanded our previous functional studies on the Q141K SNP [13] using pheophorbide a, a fluorescent compound that was recently reported by our group as an ABCG2-specific substrate [22]. Login to comment 27 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys In addition, we examined the effect of the D315-316 variant with or without the Q141K polymorphism on ABCG2 function. Login to comment 28 ABCG2 p.Gln141Lys Sequencing the ABCG2 gene in the 60 cancer cell lines maintained and used for screening in the National Cancer Institute by various groups revealed that ten of the cell lines carried the Q141K polymorphism. Login to comment 30 ABCG2 p.Gln141Lys Mutagenesis and transfection The D315-316 and the Q141K/D315-316 mutants were generated by site-directed mutagenesis in the pcDNA3.1/ Myc-HisA(-) vector (Invitrogen) as previously described [23]. Login to comment 31 ABCG2 p.Arg482Gly ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys The wild type (R482), Q141K, R482G, and empty vector-transfected (pcDNA) cells were previously reported [13]. Login to comment 32 ABCG2 p.Gln141Lys In addition, we examined the effect of the Δ315-316 variant with or without the Q141K polymorphism on ABCG2 function. Login to comment 35 ABCG2 p.Gln141Lys Mutagenesis and transfection The Δ315-316 and the Q141K/D315-316 mutants were generated by site-directed mutagenesis in the pcDNA3.1/Myc-HisA(-) vector (Invitrogen) as previously described [23]. Login to comment 36 ABCG2 p.Arg482Gly ABCG2 p.Gln141Lys The wild type (R482), Q141K, R482G, and empty vector-transfected (pcDNA) cells were previously reported [13]. Login to comment 51 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Once strong inverse correlations were ruled out, then a Kruskal-Wallis test was used to determine if the three clones of particular mutant, or a Wilcoxon rank sum test if 315-316-3 10 0 10 1 10 2 10 3 10 4 FL2-H 0 100 200 300 #Cells 10 0 10 1 10 2 10 3 10 4 FL2-H 0 100 200 300 400 #Cells 10 0 10 1 10 2 10 3 10 4 FL2-H 0 100 200 300 #Cells 10 0 10 1 10 2 10 3 10 4 FL2-H 0 100 200 300 #Cells 10 0 10 1 10 2 10 3 10 4 FL2-H 0 100 200 300 400 #Cells 10 0 10 1 10 2 10 3 10 4 FL2-H 0 100 200 300 400 #Cells 10 0 10 1 10 2 10 3 10 4 FL2-H 0 100 200 300 #Cells 315-316-6 315-316-8 Q141K/ 315-316-5 Q141K/ 315-316-15 Q141K/ 315-316-17 wild type Fig. 1 ABCG2 surface expression in the mutants. Login to comment 52 ABCG2 p.Gln141Lys Flow cytometry with the 5D3 monoclonal antibody recognizing an extracellular epitope of ABCG2 is shown for the wild type and three clones of both mutants (D315-316 and Q141K/D315-316, with clone numbers indicated). Login to comment 62 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Asp620Asn We have previously used the same expression system to study non-synonymous SNPs, such as Q141K, V12M, and D620N, and found that Q141K results in impaired function [13]. Login to comment 63 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Given that Q141K and D315-316 were found to coexist in the A549 and SK-OV-3 carcinoma cell lines, we also generated mutants carrying both the deletion and the Q141K SNP (Q141K/D315-316). Login to comment 64 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Val12Met ABCG2 p.Asp620Asn We have previously used the same expression system to study non-synonymous SNPs, such as Q141K, V12M, and D620N, and found that Q141K results in impaired function [13]. Login to comment 65 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys We selected three clones of both the D315-316 and the Q141K/D315-316 mutants that displayed significant levels of surface expression (Fig. 1) and expanded these clones for further studies. Login to comment 66 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Crude membranes extracted from the D315-316 and the Q141K/D315-316 clones and from the wild type and Q141K transfectants previously generated in our lab were subjected to immunoblotting with the commercially available BXP-21 monoclonal antibody. Login to comment 67 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Surprisingly, despite sufficient surface expression levels seen on flow cytometry, none of the D315-316 and Q141K/D315-316 clones were detectable on immunoblot with the BXP-21 antibody (Fig. 2), while the wild type and the Q141K mutant clones were readily detected. Login to comment 68 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Crude membranes extracted from the Δ315-316 and the Q141K/Δ315-316 clones and from the wild type and Q141K transfectants previously generated in our lab were subjected to immunoblotting with the commercially available BXP-21 monoclonal antibody. Login to comment 69 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Surprisingly, despite sufficient surface expression levels seen on flow cytometry, none of the Δ315-316 and Q141K/Δ315-316 clones were detectable on immunoblot with the BXP-21 antibody (Fig. 2), while the wild type and the Q141K mutant clones were readily detected. Login to comment 70 ABCG2 p.Gln141Lys To prove this, we used the 405 polyclonal antibody created in our laboratory [24] on the immunoblot previously probed with the BXP-21 antibody and found that with this antibody the D315-316 and the Q141K/D315-316 clones were also detectable, though the 405 antibody gives a weaker signal when compared to BXP-21 (Fig. 2). Login to comment 72 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys 72 kDa 72 kDa BXP-21 ab 405 ab wt ∆ 315-316-3,6,8 Q141K/ ∆ 315-316 Q141K-5,8 -5,15,17 Fig. 2 The D315-316 mutant is not detected on immunoblot with the BXP-21 antibody. Login to comment 73 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Membrane proteins extracted from HEK 293 cells stably transfected with the wild type (482R) and the mutants (Q141K, D315-316, and Q141K/D315-316) were separated on SDS/PAGE (18 lg/lane), transferred onto a PVDF membrane, and incubated overnight with the mouse monoclonal anti-ABCG2 antibody BXP-21 (upper panel) or the 405 rabbit polyclonal anti-ABCG2 antibody (lower panel). Login to comment 74 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Two clones are shown for the Q141K mutant and three clones for the D315-316 and Q141K/D315-316 mutants, with clone numbers indicated. Login to comment 76 ABCG2 p.Arg482Gly Figure 3 shows a representative example of surface expression, mitoxantrone and pheophorbide a efflux for each mutant compared to the wild type and the R482G mutant which is a previously characterized, so-called gain-of-function mutant with a wider substrate specificity than wild type ABCG2 [25]. Login to comment 78 ABCG2 p.Gln141Lys For the Q141K variant, where a reduced surface expression has been confirmed [13,16,17], our previous studies also demonstrated impaired transport when mitoxantrone efflux was normalized to surface expression [13]. Login to comment 79 ABCG2 p.Gln141Lys Efflux is represented by the shift between the histograms with (dashed line) and without (solid line) FTC c wildtype 10 0 10 1 10 2 10 3 10 4 FL2-H 0 100 200 300 #Cells 10 0 10 1 10 2 10 3 10 4 FL2-H 0 100 200 300 400 #Cells 10 0 10 1 10 2 10 3 10 4 FL2-H 0 100 200 300 400 #Cells 10 0 10 1 10 2 10 3 10 4 FL2-H 0 100 200 300 #Cells 10 0 10 1 10 2 10 3 10 4 FL2-H 0 100 200 300 #Cells 5D3 mitoxantrone pheophorbide a 10 0 10 1 10 2 10 3 10 4 FL4-H 0 100 200 300 400 #Cells 10 0 10 1 10 2 10 3 10 4 FL4-H 0 100 200 300 400 500 #Cells 10 0 10 1 10 2 10 3 10 4 FL4-H 0 100 200 300 400 500 #Cells 10 0 10 1 10 2 10 3 10 4 FL4-H 0 100 200 300 400 500 #Cells 10 0 10 1 10 2 10 3 10 4 FL4-H 0 100 200 300 400 #Cells 10 0 10 1 10 2 10 3 10 4 FL4-H 0 100 200 300 400 #Cells 10 0 10 1 10 2 10 3 10 4 FL4-H 0 100 200 300 400 500 #Cells 10 0 10 1 10 2 10 3 10 4 FL4-H 0 100 200 300 400 #Cells 10 0 10 1 10 2 10 3 10 4 FL4-H 0 100 200 300 400 #Cells 10 0 10 1 10 2 10 3 10 4 FL4-H 0 100 200 300 400 #Cells Q141K/315-316-5315-316-6Q141K-8R482G-2 We next utilized a flow cytometry-based assay measuring the efflux of mitoxantrone and pheophorbide a to determine the activity of the mutants. Login to comment 81 ABCG2 p.Arg482Gly Figure 3 shows a representative example of surface expression, mitoxantrone and pheophorbide a efflux for each mutant compared to the wild type and the R482G mutant which is a previously characterized, so-called gain-of-function mutant with a wider substrate specificity than wild type ABCG2 [25]. Login to comment 83 ABCG2 p.Gln141Lys For the Q141K variant, where a reduced surface expression has been confirmed [13, 16, 17], our previous studies also demonstrated impaired transport when mitoxantrone efflux was normalized to surface expression [13]. Login to comment 84 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Using a Wilcoxon rank sum test, differences with unadjusted P-values <0.05 were found when the wild type was compared to the Q141K and Q141K/Δ315-316 mutants for both mitoxantrone and pheophorbide a. Login to comment 85 ABCG2 p.Arg482Gly In agreement with previous reports, the transport efficacy of the R482G mutant was found to be significantly higher than that of the wild type for mitoxantrone [13]. Login to comment 86 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys The average ratio calculated for pheophorbide a for the Δ315-316 mutant was significantly higher than that for the Q141K and the Q141K/Δ315-316 mutants. Login to comment 87 ABCG2 p.Gln141Lys In summary, the mutant carrying the deletion was similar in transport efficacy to the wild type for both substrates, while any mutant carrying the Q141K was impaired. Login to comment 89 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Using a Wilcoxon rank sum test, differences with unadjusted P-values \0.05 were found when the wild type was compared to the Q141K and Q141K/D315-316 mutants for both mitoxantrone and pheophorbide a. Login to comment 90 ABCG2 p.Arg482Gly ABCG2 p.Gln141Lys In agreement with previous reports, the transport efficacy of the R482G mutant was found to be significantly higher than that of the wild type for mitoxantrone [13]. Login to comment 91 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys The average ratio calculated for pheophorbide a for the D315-316 mutant was significantly higher than that for the Q141K and the Q141K/D315-316 mutants. Login to comment 92 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys In summary, the mutant carrying the deletion was similar in transport efficacy to the wild type for both substrates, while any mutant carrying the Q141K was impaired. Login to comment 93 ABCG2 p.Gln141Lys Additionally, the experiments reconfirmed that the Q141K SNP has significantly impaired function compared to the wild type protein when normalized by surface expression. Login to comment 94 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys Of the numerous SNPs identified so far in the ABCG2 gene, 421 C [ A, resulting in a mutant protein with a glutamine to lysine substitution at residue 141 (Q141K), is the most extensively studied and is suggested to have impaired transport function. Login to comment 95 ABCG2 p.Gln141Lys Interestingly, some cell lines of the NCI-60 panel were reported to have the Q141K SNP together with a splicing variant resulting in the deletion of amino acids 315 and 316. Login to comment 96 ABCG2 p.Gln141Lys Here, we investigated the functional consequences of the 315-316 deletion with/or without the Q141K mutation in stably transfected HEK 293 cells using a flow cytometry-based assay to analyze mitoxantrone and pheophorbide a efflux. Login to comment 97 ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys After normalizing the measured efflux values to ABCG2 expression by the 5D3 monoclonal antibody, we have found that the D315-316 mutant has equivalent efflux 0.331 0.289 0.261 0.256 0.520 0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 482R Q141K 316 R482G p=0.028 p<0.0001 p=0.0099 Mitoxantroneefflux/5D3antibody n=14 n=15n=23n=23n=22 0.445 0.410 0.263 0.313 0.504 0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 482R Q141K 316 R482G p=0.00075 p=0.0000052 p=0.013 p=0.039 Pheophorbideaefflux/5D3antibody n=6 n=10n=14n=15 n=12 A B ∆315-316 Q141K/∆315- ∆315-316 Q141K/∆315- Fig. 4 Efflux normalized to cell surface expression. Login to comment 98 ABCG2 p.Arg482Gly ABCG2 p.Gln141Lys The mean FTC-inhibitable mitoxantrone (panel A) and pheophorbide a (panel B) efflux was plotted for the wild type (482R) and each mutant (Q141K, D315-316, aQ141K/D315-316, and R482G). Login to comment 102 ABCG2 p.Gln141Lys ABCG2 p.Gln141Lys The numbers above the bars represent the mean values plotted capacity to the wild type, while the double mutant (Q141K/ D315-316) was similar to the Q141K SNP. Login to comment 103 ABCG2 p.Gln141Lys Additionally, the experiments reconfirmed that the Q141K SNP has significantly impaired function compared to the wild type protein when normalized by surface expression. Login to comment 107 ABCG2 p.Arg482Gly Finally, it should be noted that the gain-of-function feature of the R482G mutant was again demonstrated, although the latter was only significant when mitoxantrone was used. Login to comment 110 ABCG2 p.Arg482Gly This could mean that similar to the R482G mutation [34] this splicing variant will be found only in cell culture. Login to comment 115 ABCG2 p.Gln141Lys In conclusion, the results presented here confirm our previous finding that the clinically relevant, frequently occurring Q141K SNP impairs transport of the chemotherapeutic agent mitoxantrone by ABCG2 [13]. Login to comment 117 ABCG2 p.Gln141Lys We believe the Q141K mutation results in decreased surface expression due to impaired trafficking of ABCG2 [13], which, combined with impaired transporter function, effects a clinically detectable reduction in transporter function [19]. Login to comment 120 ABCG2 p.Arg482Gly This could mean that similar to the R482G mutation [34] this splicing variant will be found only in cell culture. Login to comment 125 ABCG2 p.Gln141Lys In conclusion, the results presented here confirm our previous finding that the clinically relevant, frequently occurring Q141K SNP impairs transport of the chemotherapeutic agent mitoxantrone by ABCG2 [13]. Login to comment 127 ABCG2 p.Gln141Lys We believe the Q141K mutation results in decreased surface expression due to impaired trafficking of ABCG2 [13], which, combined with impaired transporter function, effects a clinically detectable reduction in transporter function [19]. Login to comment