ABCMdb

PMID: 19880777

Chapuy B, Panse M, Radunski U, Koch R, Wenzel D, Inagaki N, Haase D, Truemper L, Wulf GG
ABC transporter A3 facilitates lysosomal sequestration of imatinib and modulates susceptibility of chronic myeloid leukemia cell lines to this drug.
Haematologica. 2009 Nov;94(11):1528-36., [PubMed]

Sentences

No. Mutations Sentence Comment

32 ABCA3 p.Asn568Asp Enforced expression and small interfering RNA of ABCA3 pEGFP-N1-ABCA3 wild-type or pEGFP-N1-ABCA3 N568D mutant plasmids16 as well as the pre-designed short interfering RNA (siRNA) against ABCA3 and scrambled siRNA (Qiagen) were delivered by electroporation as previously described.15 Using siRNA knock-down, the nadir of ABCA3 mRNA and ABCA3 protein expression was documented 72 h after electroporation, as tested by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting (Online Supplementary Appendix Figures S2). Login to comment 91 ABCA3 p.Asn568Asp ABCA3 p.Asn568Asp normal BM non-SP SP Hoechst red Hoechstblu ABCA3abundance [foldofnon-SP] CML normal CD34+ CD34- CD34+ CD34- CML normal 0 100 250 500 1000 0 100 250 500 1000 0 100 250 500 10000 1 5 10 0 0.5 2.5 5 0 0.5 2.5 5 scrambled siRNAABCA3 siRNA scrambled siRNA ABCA3 siRNA Imatinib [µM] p-Crkl protein Imatinib [µM] Imatinib [µM] Imatinib[nM] Colonies[fractionofcontroll] Colonies[fractionofcontroll] Viability[fractionofcontroll] K562 K562 K562 LAMA84 LAMA84 BV173 LAMA84 Imatinib [µM] 0 1 5 10 ABCA3 siRNA scrambled siRNA ABCA3 siRNA scrambled siRNA ABCA3 siRNA scrambled siRNA ABCA3 siRNA scrambled siRNA ABCA3 siRNA scrambled siRNA ABCA3-N568D ABCA3-wt ABCA3-N568D ABCA3-wt 0 100 250 500 1000 0 100 250 500 1000 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 0 250 500 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 n=6 n=19 n=11 p=0.0255 0 1023 01023 ABCA3positive[%ofall] transcriptabundance 80 60 40 20 0 108 107 106 105 104 103 102 101 100 10-1 10-2 10-3 40 30 20 10 0 BC+AP CP BM CD34+ CD34- CML BM A A B C D B D C significantly increased the susceptibility of all three cell lines to the suppressive effects of imatinib. Login to comment 92 ABCA3 p.Asn568Asp ABCA3 p.Asn568Asp normal BM non-SP SP Hoechst red Hoechst blu ABCA3 abundance [fold of non-SP] CML normal CD34+ CD34- CD34+ CD34- CML normal 0 100 250 500 1000 0 100 250 500 1000 0 100 250 500 1000 0 1 5 10 0 0.5 2.5 5 0 0.5 2.5 5 scrambled siRNA ABCA3 siRNA scrambled siRNA ABCA3 siRNA Imatinib [&#b5;M] p-Crkl protein Imatinib [&#b5;M] Imatinib [&#b5;M] Imatinib [nM] Colonies [fraction of controll] Colonies [fraction of controll] Viability [fraction of controll] K562 K562 K562 LAMA84 LAMA84 BV173 LAMA84 Imatinib [&#b5;M] 0 1 5 10 ABCA3 siRNA scrambled siRNA ABCA3 siRNA scrambled siRNA ABCA3 siRNA scrambled siRNA ABCA3 siRNA scrambled siRNA ABCA3 siRNA scrambled siRNA ABCA3-N568D ABCA3-wt ABCA3-N568D ABCA3-wt 0 100 250 500 1000 0 100 250 500 1000 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 0 250 500 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 n=6 n=19 n=11 p=0.0255 0 1023 0 1023 ABCA3 positive [% of all] transcript abundance 80 60 40 20 0 108 107 106 105 104 103 102 101 100 10-1 10-2 10-3 40 30 20 10 0 BC+AP CP BM CD34 + CD34 - CML BM A A B C D B D C significantly increased the susceptibility of all three cell lines to the suppressive effects of imatinib. Login to comment 106 ABCA3 p.Asn568Asp B C D A -1 2 3 4 5 6 7 8 9 101112 131415 16171819 20 21 2223 35 30 25 20 15 10 5 0 35 30 25 20 15 10 5 0 25 20 15 10 5 0 25 20 15 10 5 0 1 2 3 4 5 6 7 8 9 101112 131415 16171819 20 21 2223 1 2 3 4 5 6 7 8 9 101112 131415 16171819 20 21 2223 β-Hexosaminidase [14 C]-Imatinib [%ofall][%ofall][%ofall] Activity[%ofall] Fraction LAMP1 EEA1 ABCA3-GFP NaKATPase HEK293 + ABCA3 HEK293 LAMA84 K562 Activity[%ofall]Activity[%ofall]Activity[%ofall] [14 C] Imatinib β-Hexosaminidase [14 C] Imatinib β-Hexosaminidase [14 C] Imatinib β-Hexosaminidase [14 C] Imatinib β-Hexosaminidase [%ofall] 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 [14 C]-Imatinib [14 C]-Imatinib Free Membrane bound Cytosol/Organelles Nucleus LL+ LL+ 1 2 3 4 5 6 7 8 9 101112 131415 16171819 20 21 2223 LAMA84 K562 HEK293-ABCA3 HEK293 HEK293-ABCA3 HEK293 HEK293-ABCA3 HEK293 with ectopic expression of wild-type ABCA3 and cells expressing the non-functional ABCA3-N568D variant carrying a mutation at the Walker A motif of the transporter (Figure 2C). Login to comment 107 ABCA3 p.Asn568Asp B C D A -1 2 3 4 5 6 7 8 9 101112 131415 16171819 20 21 2223 35 30 25 20 15 10 5 0 35 30 25 20 15 10 5 0 25 20 15 10 5 0 25 20 15 10 5 0 1 2 3 4 5 6 7 8 9 101112 131415 16171819 20 21 2223 1 2 3 4 5 6 7 8 9 101112 131415 16171819 20 21 2223 b2;-Hexosaminidase [14 C]-Imatinib [% of all] [% of all] [% of all] Activity [% of all] Fraction LAMP1 EEA1 ABCA3-GFP NaKATPase HEK293 + ABCA3 HEK293 LAMA84 K562 Activity [% of all] Activity [% of all] Activity [% of all] [14 C] Imatinib b2;-Hexosaminidase [14 C] Imatinib b2;-Hexosaminidase [14 C] Imatinib b2;-Hexosaminidase [14 C] Imatinib b2;-Hexosaminidase [% of all] 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 [14 C]-Imatinib [14 C]-Imatinib Free Membrane bound Cytosol/Organelles Nucleus LL+ LL+ 1 2 3 4 5 6 7 8 9 101112 131415 16171819 20 21 2223 LAMA84 K562 HEK293-ABCA3 HEK293 HEK293-ABCA3 HEK293 HEK293-ABCA3 HEK293 with ectopic expression of wild-type ABCA3 and cells expressing the non-functional ABCA3-N568D variant carrying a mutation at the Walker A motif of the transporter (Figure 2C). 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