ABCMdb

PMID: 20623213

Crouthamel MH, Wu D, Yang Z, Ho RJ
A novel MDR1 GT1292-3TG (Cys431Leu) genetic variation and its effect on P-glycoprotein biologic functions.
AAPS J. 2010 Dec;12(4):548-55. Epub 2010 Jul 10., [PubMed]

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0 ABCB1 p.Cys431Leu Research Article A Novel MDR1 GT1292-3TG (Cys431Leu) Genetic Variation and Its Effect on P-glycoprotein Biologic Functions Matthew H. Crouthamel,1 Daniel Wu,2 Ziping Yang,1,3 and Rodney J. Y. Ho1,4 Received 11 February 2010; accepted 18 June 2010; published online 10 July 2010 Abstract. Login to comment 4 ABCB1 p.Cys431Leu We have identified a novel MDR1GT1292-3TG (Cys431Leu) genetic variation through systematic profiling of subjects with leukemia. Login to comment 8 ABCB1 p.Cys431Leu The effects of the Cys431Leu variation, due to MDR1GT1292-3TG nucleotide transition, on P-gp-dependent intracellular substrate accumulation appeared to be substrate dependent where doxorubicin, vinblastine, and paclitaxel exhibit an increased accumulation (p<0.05), while verapamil and Hoechst33342 exhibit a decreased intracellular concentration compared with wild type (p<0.05). Login to comment 30 ABCB1 p.Gly191Arg MDR1G571A (Gly191Arg) is located in the third transmembrane (TM) domain of P-gp and decreased resistance to vinblastine, vincristine, and paclitaxel with no significant difference with doxorubicin. Login to comment 99 ABCB1 p.Cys431Leu Allelic Frequency of MDR1 1292-3 Variants in Leukemia Subjects Sequence/variation Amino acid variation Estimated allelic frequency in leukemia patients Copies per ng RNA Wild type Cys 431 0.9865 459±293 GT 1292-3 TG Cys 431 Leu 0.0135 1126±137* The RNA from 74 leukemia patients was collected, and the MDR1 RNA transcripts were reverse transcribed into complementary DNA. Login to comment 131 ABCB1 p.Cys431Leu White bars rhodamine123, black bars rhodamine123+GF120918 ATPase Activity ATPase activity was measured to determine if the change from cysteine to leucine at amino acid 431 in the nucleotide binding domain (NBD) would alter the ability of P-gp to hydrolyze ATP to ADP. Login to comment 135 ABCB1 p.Cys431Leu Based on our MDR1 sequence variation analysis, we discovered the novel MDR1GT1292-3TG nucleotide variation that translates to an amino acid substitution from cysteine to leucine at position 431. Login to comment 137 ABCB1 p.Cys431Leu In this study, we used MDR1GT1292-3TG recombinant HEK cells that express a P-gp variant, with a Cys to Leu substitution at amino acid 431, to evaluate functional changes. Login to comment 146 ABCB1 p.Cys431Ala In addition, the Cys431Ala mutation did not impact ATPase activity. Login to comment 147 ABCB1 p.Cys431Ala When the cysteine was re-inserted at amino acid 431 as the only cysteine within the Cys-less (Cys431Ala) P-gp molecule, covalent modification of the thiol side chain with N-ethylmaleimide inhibited activity of P-gp, suggesting that both NBDs must be active for function (22). Login to comment 156 ABCB1 p.Cys431Ala ABCB1 p.Cys431Leu Although a direct comparison of Cys431Ala and Cys431Leu with WT P-gp in HEK recombinant cells remained, such a study is beyond the scope of this report. Login to comment 159 ABCB1 p.Cys431Leu Our cytotoxicity studies showed that Cys431Leu substitution resulted in a significant increase in sensitivity to chemotherapeutic drugs. Login to comment 193 ABCB1 p.Cys431Leu The substrate-specific differences in intracellular accumulation do not appear to link with ATPase activity. Our studies of the ATPase activity showed no difference between the wild-type P-gp and Cys431Leu variant. Login to comment 211 ABCB1 p.Cys431Leu In our MDR1GT1292-3TG variant, there are no amino acid changes within the TM segments; the MDR1GT1292-3TG variation results in a Cys431Leu substitution in the ATP binding domain. Login to comment 213 ABCB1 p.Cys431Leu We hypothesize that the Cys431Leu change may affect the communication between the NBD and the TM domains, resulting in altered conformation of the TM segments during ATP binding and/or ATP hydrolysis. Login to comment 218 ABCB1 p.Cys431Leu It must also be noted that this Cys431Leu variation is the result of two mutations occurring simultaneously at nucleotides 1292 and 1293. Login to comment 220 ABCB1 p.Cys431Phe ABCB1 p.Cys431Trp If these mutations were to occur independently of one another, they would result in a Cys431Phe transition (G1292T) or Cys431Trp transition (T1293G). Login to comment 222 ABCB1 p.Cys431Leu ABCB1 p.Cys431Phe ABCB1 p.Cys431Trp Regardless, if our hypothesis is correct in that the change in P-gp-mediated resistance and uptake is due to the increase in size and hydrophobicity of the resulting amino acid 431 compared with Cys, we believe that the Cys431Phe and Cys431Trp may have similar results to the Cys431Leu transition, and remains to be investigated. Login to comment 223 ABCB1 p.Cys431Leu CONCLUSION We investigated a novel human MDR1GT1292-3TG (Cys431Leu) variation. Login to comment 226 ABCB1 p.Cys431Leu Drug uptake studies showed that the effect of the Cys431Leu variation results in a substrate-specific effect where doxorubicin, vinblastine, and paclitaxel had an increased uptake, while verapamil and Hoechst33342 had a decreased uptake compared with wild type. Login to comment