ABCMdb

PMID: 21799180

Henderson MJ, Haber M, Porro A, Munoz MA, Iraci N, Xue C, Murray J, Flemming CL, Smith J, Fletcher JI, Gherardi S, Kwek CK, Russell AJ, Valli E, London WB, Buxton AB, Ashton LJ, Sartorelli AC, Cohn SL, Schwab M, Marshall GM, Perini G, Norris MD
ABCC multidrug transporters in childhood neuroblastoma: clinical and biological effects independent of cytotoxic drug Efflux.
J Natl Cancer Inst. 2011 Aug 17;103(16):1236-51. Epub 2011 Jul 28., 2011-08-17 [PubMed]

Sentences

No. Mutations Sentence Comment

66 ABCC1 p.Asp1454Asn For constitutive expression of ABCC1 in SH-SY5Y cells, transfection with pCMV14-3xFLAG-ABCC1, or the ATP-binding site mutants, pCMV14-3xFLAG-ABCC1-D1454N or ABCC1-DE1454LL, was followed by selection of stable clones with 500 µg/ml G418 (Geneticin; Invitrogen Australia Pty Ltd, Mulgrave, Australia), which were subsequently maintained with 150 µg/ml G418. Login to comment 67 ABCC1 p.Asp1454Asn ABCC1 p.Asp1454Asn The ABCC1-D1454N mutation was previously shown to abolish ATP-dependent ABCC1 transporter activity while not affecting correct localization of the transporter (19), and the ABCC1-DE1454LL mutant was predicted to have similar properties (Supplementary Figure 1, available online and http://www.uniprot.org/uniprot/O15439; accessed June, 2010). Login to comment 68 ABCC1 p.Asp1454Asn The ABCC1-D1454N mutation was previously shown to abolish ATP-dependent ABCC1 transporter activity while not affecting correct localization of the transporter (19), and the ABCC1-DE1454LL mutant was predicted to have similar properties (Supplementary Figure 1, available online and http://www.uniprot.org/uniprot/O15439; accessed June, 2010). Login to comment 69 ABCC3 p.Val1322Phe For stable clones expressing ABCC3, BE(2)-C cells were transduced with retroviral pCMV14-3xFLAG-ABCC3 or the ATP-binding site mutant, pCMV14-3xFLAG-ABCC3-V1322F. Login to comment 70 ABCC3 p.Val1322Phe The V1322F mutation was predicted to abolish transporter activity while still being localized in the plasma membrane and is based on studies involving the closely related transporter, ABCC6, in which a single amino acid change in a region N-terminal to the Walker A motif in the second ABC domain abolishes catalytic activity (20) while retaining correct membrane localization in mammalian cell lines (Andras Varadi, personal communication). Login to comment 74 ABCC1 p.Asp1454Asn Generation of ATP-binding site mutants was carried out accordingtotheQuikChangeLightningSite-DirectedMutagenesis Kit (Stratagene, La Jolla, CA) using forward primer 5'-ATC CTT GTG TTG AAT GAG GCC ACG GCA G-3' for ABCC1-D1454N (single mutant), forward primer 5'-ATC CTT GTG TTG CTT CTG GCC ACG GCA G-3' for ABCC1-DE1454/1455LL (double mutant) and forward primer 5'-AGG TGG GGA TCT TCG GCC GCA CTG G-3' for ABCC3-V1322F. Login to comment 75 ABCC1 p.Asp1454Asn ABCC3 p.Val1322Phe Generation of ATP-binding site mutants was carried out accordingtotheQuikChangeLightningSite-DirectedMutagenesis Kit (Stratagene, La Jolla, CA) using forward primer 5'-ATC CTT GTG TTG AAT GAG GCC ACG GCA G-3' for ABCC1-D1454N (single mutant), forward primer 5'-ATC CTT GTG TTG CTT CTG GCC ACG GCA G-3' for ABCC1-DE1454/1455LL (double mutant) and forward primer 5'-AGG TGG GGA TCT TCG GCC GCA CTG G-3' for ABCC3-V1322F. Login to comment 151 ABCC1 p.Asp1454Asn ABCC1 transporter activity is necessary for the observed effects on cell motility, because forced expression of catalytically inactive ABCC1 (ABCC1-D1454N clone B4 or ABCC1-DE1454LL clone B7) had no impact on cell motility (mean % of wound open ± 95% CI: ABCC1 B4, 49.6 ± 4.5 vs empty vector, 59.9 ± 9.6, P = .08; ABCC1 B7, 55.6 ± 9.0 vs empty vector, 59.9 ± 9.6, P = .54, one-way ANOVA and two-sided t test, three independent Figure 2.Impact of ABCC1 and ABCC4 suppression in BE(2)-C human neuroblastoma cells. Login to comment 152 ABCC1 p.Asp1454Asn ABCC1 transporter activity is necessary for the observed effects on cell motility, because forced expression of catalytically inactive ABCC1 (ABCC1-D1454N clone B4 or ABCC1-DE1454LL clone B7) had no impact on cell motility (mean % of wound open ± 95% CI: ABCC1 B4, 49.6 ± 4.5 vs empty vector, 59.9 ± 9.6, P = .08; ABCC1 B7, 55.6 ± 9.0 vs empty vector, 59.9 ± 9.6, P = .54, one-way ANOVA and two-sided t test, three independent Figure 2.Impact of ABCC1 and ABCC4 suppression in BE(2)-C human neuroblastoma cells. Login to comment 163 ABCC1 p.Asp1454Asn This effect was completely abrogated in cells overexpressing the mutant ABCC1 carrying a single amino acid change in the ABC domain (D1454N) or the catalytically inactive double mutant of ABCC1 (DE1454LL) (mean number of colonies ± 95% CI: ABCC1 B4, 74.8 ± 1.6 vs empty vector, 73.9 ± 4.3, P = .96; ABCC1 B7, 73.5 ± 5.9 vs empty vector, 73.9 ± 4.3, P = .88; one-way ANOVA and two-sided t test, four independent experiments; Figure 3, D). Login to comment 164 ABCC1 p.Asp1454Asn This effect was completely abrogated in cells overexpressing the mutant ABCC1 carrying a single amino acid change in the ABC domain (D1454N) or the catalytically inactive double mutant of ABCC1 (DE1454LL) (mean number of colonies ± 95% CI: ABCC1 B4, 74.8 ± 1.6 vs empty vector, 73.9 ± 4.3, P = .96; ABCC1 B7, 73.5 ± 5.9 vs empty vector, 73.9 ± 4.3, P = .88; one-way ANOVA and two-sided t test, four independent experiments; Figure 3, D). Login to comment 182 ABCC1 p.Asp1454Asn A) Western blot analysis of ABCC1 protein expression following stable transduction of SH-SY5Y cells with either empty vector, wild-type (wt) ABCC1 (clones D5, H7), ABCC1 D1454N single mutant (clone B4), or ABCC1 DD1454LL double mutant (clone B7) constructs. Login to comment 183 ABCC1 p.Asp1454Asn A) Western blot analysis of ABCC1 protein expression following stable transduction of SH-SY5Y cells with either empty vector, wild-type (wt) ABCC1 (clones D5, H7), ABCC1 D1454N single mutant (clone B4), or ABCC1 DD1454LL double mutant (clone B7) constructs. Login to comment 281 ABCC3 p.Val1322Phe ABCC3 transport activity is necessary for the observed effects on cell motility and clonogenicity, because the wound closure and colony-forming ability of cells expressing catalytically inactive ABCC3 (ABCC3-V1322F) were indistinguishable from cells transfected with empty vector (mean % of wound open ± 95% CI: ABCC3 C4, 5.4 ± 4.3 vs ABCC3 D1, 3.0 ± 2.4 vs empty vector, 1.5 ± 2.0, P = .34, one-way ANOVA, three independent experiments; Figure 6, C and mean numberof colonies ± 95% CI: ABCC3 C4, 91.1 ± 6.5 vs ABCC3 D1, 92.0 ± 7.4 vs empty vector, 90.9 ± 9.4, P = .98, one-way ANOVA, three independent experiments; Figure 6, D). Login to comment 283 ABCC3 p.Val1322Phe A) Western blot analysis of ABCC3 protein expression following stable transduction of BE(2)-C cells with either empty vector, wild-type (wt) ABCC3 (clones A12, B12) or ABCC3 V1322F mutant (clones C4, D1) constructs. Login to comment