ABCMdb

PMID: 10724034

Hafkemeyer P, Licht T, Pastan I, Gottesman MM
Chemoprotection of hematopoietic cells by a mutant P-glycoprotein resistant to a potent chemosensitizer of multidrug-resistant cancers.
Hum Gene Ther. 2000 Mar 1;11(4):555-65., 2000-03-01 [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCB1 p.Phe983Ala We report here that retroviral vectors encoding a mutant P-glycoprotein (MDR1-F983A) protect hematopoietic cells from anticancer drugs even in the presence of trans-(E)-flupentixol, an inhibitor of P-glycoprotein. Login to comment 42 ABCB1 p.Phe983Ala We have introduced a point mutation that leads to a substitution of phenylalanine by alanine in position 983. Login to comment 45 ABCB1 p.Phe983Ala In the present work, we have constructed retroviral vectors containing this mutant MDR1-F983A cDNA. Login to comment 46 ABCB1 p.Phe983Ala With the exception of the inserted point mutation, pHaMA-F983A is identical to a previously described vector, pHaMA (Pastan et al., 1988; Metz et al., 1995), with long-term inal repeats (LTRs) from Harvey sarcoma virus. Login to comment 48 ABCB1 p.Phe983Ala The MDR1-F983A vectors were characterized in KB-3-1 epidermoid cancer cells, and hematopoietic cells from the K562 erythroleukemia line or primary mouse bone marrow. Login to comment 51 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala To construct vector pHaMA-F983A, the NsiI-XhoI fragment of previously described plasmid pTM1MDR1 (Hafkemeyer et al., 1998) containing the F983A-mutant fragment was cloned into the corresponding sites of pHaMA. Login to comment 53 ABCB1 p.Phe983Ala The vectors LZRS- pBMN-MDR1 and LZRS-pBMN-MDR1-F983A, respectively, were obtained after excision of lacZ by EcoRI digest of LZRS- pBMN-lacZ, and blunt ending of the vector backbone with T4 polymerase and Klenow enzyme. Login to comment 54 ABCB1 p.Phe983Ala pHaMA and pHaMA-F983A were then blunt ended after SacII-XhoI digestion. Login to comment 55 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala The thereby obtained MDR1 and MDR1-F983A fragments, respectively, were ligated into the EcoRI-digested and blunt-ended backbone of LZRS-pBMN-lacZ to yield LZRS-pBMN-MDR1 and LZRS- pBMN-MDR1-F983A. Login to comment 61 ABCB1 p.Phe983Ala Cell transfections and transductions KB-3-1 cells were transfected by calcium phosphate coprecipitation with 10 m g of plasmid DNA (pHaMA or pHaMA-F983A) per 1 3 105 cells as described (Germann et al., 1989). Login to comment 64 ABCB1 p.Phe983Ala The same method was used to generate ecotropic retroviral producer cells from GP1 E86 packaging cells (ATCC) with plasmid pHaMA-F983A. Login to comment 67 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala Wild-type and F983A-mutant MDR1 producer lines were obtained by transient transfection with 10 m g of either LZRS- pBMN-MDR1 or LZRS-pBMN-MDR1-F983A, respectively. Login to comment 81 ABCB1 p.Phe983Ala To assess the chemosensitivity of K562 parental cells, and of K562 cells transduced with either the LZRS-pBMN-MDR1 or LZRS- pBMN-MDR1-F983A construct, 200 cells/ml were mixed with daunomycin (Calbiochem , San Diego, CA) and trans-(E)-flupentixol (RBI, Natick, MA) at various concentrations in MethoCult 4330 medium containing 0.8% methylcellulose (Stem Cell Technologies, Vancouver, BC, Canada). Login to comment 85 ABCB1 p.Phe983Ala RESULTS Reversal of the functional activity of wild-type P-gp in KB cancer cells We first analyzed the reversing potential of trans-(E)-flupentixol in multidrug-resist ant cells expressing either the wild-type or the F983A-mutant P-gp. Login to comment 90 ABCB1 p.Phe983Ala We also analyzed KB-3-1 cells stably transfected with MDR1-containing plasmids, pHaMA or pHaMA-F983A, and selected with vincristine; clones G2 (wild-type) and 7A (mutant) expressed similar levels of P-gp (Fig. 1a). Login to comment 97 ABCB1 p.Phe983Ala At 2 m M trans-(E)-flupentixol, 7A cells expressing F983A-mutant P-gp accumulated three- to fivefold less [3H]daunomycin than G2 cells (Fig. 1b). Login to comment 104 ABCB1 p.Phe983Ala (a)DetectionofP-gponthecellsurfaceofparentalKB-3-1cellsandmul- tidrug-resistantKB-8-5cells,andKB-3-1cellsstablytransfectedwithpHaMA(wild-typeMDR1,cloneG2)orpHaMA-F983A(mutantMDR1-F983A,clone7A).Cellswerestained with5mgofmonoclonalantibodyMRK16(specificforhumanP-gp)conjugatedtoFITC,oranequalconcentrationofanisotype-identifiedcontrolantibody.Afterwashing,cells wereanalyzedbyflowcytometry. Login to comment 106 ABCB1 p.Phe983Ala A, another P-gp inhibitor, was comparable in all investigated KB cells, regardless of the presence of the F983A point mutation (data not shown). Login to comment 107 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala Development of retroviruses containing mutant MDR1-F983A for transduction of hematopoietic cells Ecotropic retroviral producer cells were generated by stable transfection of GP1 E86 packaging cells with plasmid pHaMA-F983A (Fig. 4a). Login to comment 112 ABCB1 p.Phe983Ala Transduction levels for the EBV-retroviral vectors containing mutant or wild-type MDR1 cDNAs were 80% (LZRS-pBMN-MDR1-F983A) and 68% (LZRS-pBMN-MDR1) over background. Login to comment 114 ABCB1 p.Phe983Ala EBV-hybrid vectors with titers of 5-7 3 105 CFU/ml were generated by transient transfection of amphotropic 293T (Phoenix) cells with plasmid LZRS-pBMN-MDR1 (wild-type) or LZRS-pBMN-MDR1-F983A (mutant). Login to comment 118 ABCB1 p.Phe983Ala ABCB1 p.Phe983Ala Modulation of drug transport by trans-(E)-flupentixol in hematopoietic cells transduced with F983A-mutant or wild-type MDR1 vectors Transfer of either the F983A-mutant or the wild-type MDR1 cDNA to K562 cells caused them to accumulate far less [3H]daunomycin and [3H]vinblastine than the non-MDR1 control (Fig. 5b). Login to comment 123 ABCB1 p.Phe983Ala Parental K562 cells formed only a few colonies in the presence of daunomycin (10 ng/ml) but transfer of the wild-type or the F983A-mutant MDR1 cDNA rendered them resistant to this concentration. Login to comment 129 ABCB1 p.Phe983Ala retroviruses containing F983A-mutant MDR1 but reduced the number of resistant colonies from cells expressing wild-type P-gp. Login to comment 130 ABCB1 p.Phe983Ala Moreover, daunomycin at 30 ng/ml, combined with 2 m M trans-(E)-flupentixol, killed all cells transduced with wild-type MDR1 while 10% of the respective cells transduced with the F983A-mutant vector were protected. Login to comment 131 ABCB1 p.Phe983Ala Thus, K562 cells expressing wild-type MDR1 were slightly more chemoresistant than those transduced with the mutant vector, but this resistance was not reversible in cells expressing F983A-MDR1. Login to comment 138 ABCB1 p.Phe983Ala Drug extrusion from K562 cells transduced with the mutant MDR1-F983A vector was not overcome by trans-(E)-flupentixol at concentrations that reverse P-gp function in KB-8-5 cells. Login to comment 139 ABCB1 p.Phe983Ala Correspondingly , daunomycin resistance was reversible only in K562 cells that had been transduced with wild-type but not with the F983A-mutant MDR1 cDNA. Login to comment 141 ABCB1 p.Phe983Ala Although P-gp expression levels and the total amount of drug efflux were lower than in the K562 cell population because no selection was applied, trans-(E)-flupentixol failed to reverse drug efflux from BMCs transduced with the mutant MDR1-F983A vector. Login to comment 153 ABCB1 p.Phe983Ala (a)MapsoftheproviralstructuresofpHaMAandpHaMA-F983Acon- tainingfull-lengthwild-typeormutantMDR1cDNAs,respectively.TheEBV-derivedretroviralhybridvectorsareLZRS-pBMN-MDR1,containingawild-typeMDR1cDNA,and LZRS-pBMN-MDR1-F983A,containingtherespectivemutantMDR1-F983AcDNA. Login to comment 155 ABCB1 p.Phe983Ala (c)ExpressionofP-gpinK562cellstransduced witheitherLZRS-pBMN-MDR1-F983A,LZRS-pBMN-MDR1,orLZRS-pBMN-lacZ(negativecontrol).Aftercoculturewithretroviralproducersfor48hr,transducedK562cells wereselectedwithvincristine(100ng/ml)for18days.Cellswerestainedwith5mgofantibodyMRK16conjugatedtoFITCoranFITC-labeledcontrolantibody.After30min, cellswerewashed,andfluorescenceintensitieswereanalyzedbyflowcytometry.Nonviablecellswereexcludedbypropidiumiodidecounterstaining. Login to comment 162 ABCB1 p.Phe983Ala The advantage of amphotropic, EBV-based hybrid vectors, LZRS-pBMN-MDR1 and LZRS-pBMN-MDR1-F983A, is rapid production of retroviruses after transient transfection (Nolan and Shatzman, 1998). Login to comment 169 ABCB1 p.Phe983Ala (a) Murine BMCs were transduced in cocultures with vectors GP1 E86 MDR1-F983A (d ), GP1 E86 MDR1/A (wild-type) (r ), or packaging cells (h ) that did not confer drug resistance. Login to comment 171 ABCB1 p.Phe983Ala (b) K562 cells, transduced and selected as described in the legend to Fig. 4c, were analyzed under identical conditions (h , negative control; r , LZRS-pBMN-MDR1; d , LZRS-pBMN-MDR1-F983A). Login to comment 183 ABCB1 p.Phe983Ala High concentrations of trans-(E)-flupentixol (20 m M and higher) are not useful because they reduce chemoprotection of hematopoietic cells mediated by the F983A-MDR1 transgene (Fig. 5). Login to comment 185 ABCB1 p.Phe983Ala The mutation F983A in the putative transmembrane domain TM 12 had little effect on drug binding to P-gp but almost abolished cis-(Z)-flupentixol-stim ulated ATPase activity of the transporter (Dey et al., 1999). Login to comment 186 ABCB1 p.Phe978Ala ABCB1 p.Phe978Ser Other mutations in close proximity, e.g., F978A and F978S, affect primarily the drug resistance profile of P-gp (Loo and Clarke, 1993). Login to comment