Mutant P-glycoprotein Research Articles

Long-range communication between transmembrane- and nucleotide-binding domains does not depend on drug binding to mutant P-glycoprotein

In this study, the impact of four P-gp mutations (G185V, G830V, F978A and ΔF335) on drug-binding and efflux-related signal-transmission mechanism was comprehensively evaluated in the presence of ligands within the drug-binding pocket (DBP), experimentally related with changes in their drug efflux profiles. The severe repacking of the transmembrane helices (TMH), induced by mutations and exacerbated by the presence of ligands, indicates that P-gp is sensitive to perturbations in the transmembrane region. Alterations on drug-binding were also observed as a consequence of the TMH repacking, but were not always correlated with alterations on ligands binding mode and/or binding affinity. Finally, and although all P-gp variants holo systems showed considerable changes in the intracellular coupling helices/nucleotide-binding domain (ICH-NBD) interactions, they seem to be primarily induced by the mutation itself rather than by the presence of ligands within the DBP. The data further suggest that the changes in drug efflux experimentally reported are mostly related with changes on drug specificity rather than effects on signal-transmission mechanism. We also hypothesize that an increase in the drug-binding affinity may also be related with the decreased drug efflux, while minor changes in binding affinities are possibly related with the increased drug efflux observed in transfected cells.Communicated by Ramaswamy H. Sarma

Correction to “Loss of Cyclosporin and Azidopine Binding Are Associated with Altered ATPase Activity by a Mutant P-glycoprotein with Deleted Phe335”

Correction to “Loss of Cyclosporin and Azidopine Binding Are Associated with Altered ATPase Activity by a Mutant P-glycoprotein with Deleted Phe³³⁵”

Identification and characterization of the binding sites of P-glycoprotein for multidrug resistance-related drugs and modulators.

A major problem in cancer treatment is the development of resistance to multiple chemotherapeutic agents in tumor cells. A major mechanism of this multidrug resistance (MDR) is overexpression of the MDR1 product P-glycoprotein, known to bind to and transport a wide variety of agents. This review concentrates on the progress made toward understanding the role of this protein in MDR, identifying and characterizing the drug binding sites of P-glycoprotein, and modulating MDR by P-glycoprotein-specific inhibitors. Since our initial discovery that P-glycoprotein binds to vinblastine photoaffinity analogs, many P-glycoprotein-specific photoaffinity analogs have been developed and used to identify the particular domains of P-glycoprotein capable of interacting with these analogs and other P-glycoprotein substrates. Furthermore, significant advances have been made in delineating the drug binding sites of this protein by studying mutant P-glycoprotein. Photoaffinity labeling experiments and the use of site-directed antibodies to several domains of this protein have allowed the localization of the general binding domains of some of the cytotoxic agents and MDR modulators on P-glycoprotein. Moreover, site-directed mutagenesis studies have identified the amino acids critical for the binding of some of these agents to P-glycoprotein. Furthermore, equilibrium binding assays using plasma membranes from MDR cells and radioactive drugs have aided our understanding of the modes of drug interactions with P-glycoprotein. Based on the available data, a topological model of P-glycoprotein and the approximate locations of its drug binding sites, as well as a proposed classification of multiple drug binding sites of this protein, is presented in this review.

Loss of cyclosporin and azidopine binding are associated with altered ATPase activity by a mutant P-glycoprotein with deleted phe(335).

In this study, we further characterize a mutant P-glycoprotein (P-gp) that has a deletion of Phe(335) and is resistant to inhibition by cyclosporins. Photoaffinity labeling with [(3)H]cyclosporine and [(3)H]azidopine revealed markedly decreased binding to the mutant P-gp compared with wild-type P-gp. Expression of the mutant P-gp in multidrug-resistant variant cell line MES-SA/DxP (DxP) cells was associated with a 2-fold higher basal ATPase activity relative to multidrug-resistant cell line MES-SA/Dx5 (Dx5) cells with wild-type P-gp. Cyclosporine inhibited ATPase activity in both cell types, whereas the cyclosporin D analog valspodar (PSC 833), vinblastine, and dactinomycin stimulated ATPase activity in Dx5 but not in mutant DxP cells. Moreover, the cell lines differed in their responses to verapamil, which produced greater stimulation of ATPase in Dx5 than DxP cells. Verapamil significantly reversed the [(3)H]daunorubicin accumulation defect in wild-type Dx5 cells, but it had no significant effect on [(3)H]daunorubicin accumulation in the mutant DxP cells. Verapamil was not transported by cells expressing either mutant or wild-type P-gp. Vanadate trapping of azido-ATP was markedly impaired in mutant P-gp. In conclusion, our data demonstrate that Phe(335) of transmembrane 6 is an important amino acid residue for the formation of cyclosporine and azidopine drug-binding site(s). Phe(335) also plays a role in the coupling of verapamil binding and modulation of daunorubicin intracellular accumulation in wild-type P-gp. In addition, Phe(335) in transmembrane 6 may play a role in coupling drug binding to ATPase activity. The deletion of Phe(335) results in a significant increase in the basal ATPase activity with a concomitant decrease in its ability to trap ATP and transport some P-gp substrates.

Chemoprotection of hematopoietic cells by a mutant P-glycoprotein resistant to a potent chemosensitizer of multidrug-resistant cancers.

Cancers are frequently chemoresistant because of overexpression of P-glycoprotein. Two different approaches to improve cancer treatment are currently being investigated in clinical trials: inhibition of P-glycoprotein function by reversing agents, and alleviation of leukocytopenia by MDR1 gene transfer to normal bone marrow of patients. 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. Transfer of either mutant or wild-type MDR1 to K562 erythroleukemia cells or primary murine bone marrow resulted in reduced accumulation of daunomycin and vinblastine because of increased drug efflux.trans-(E)-Flupentixol at concentrations up to 10 microM failed to reverse drug efflux mediated by the product of the mutant MDR1 while wild-type P-glycoprotein was inhibited. In the presence of 2 microM trans-(E)-flupentixol chemoresistance to daunomycin was circumvented only in K562 cells transduced with wild-type, but not with mutant, MDR1. Moreover, drug resistance of KB-8-5 epidermoid cancer cells, which express the wild-type MDR1 gene at levels comparable to clinical specimens from multidrug-resistant cancers, was fully overcome in the presence of trans-(E)-flupentixol. Vectors expressing mutant P-glycoprotein may help improve chemotherapy by allowing safe dose intensification under conditions in which multidrug-resistant cancers are rendered drug sensitive by reversing agents.

$3,3',4,4',5$ -Pentachlorobiphenyl Inhibits Drug Efflux Through P-Glycoprotein in KB-3 Cells Expressing Mutant Human P-Glycoprotein.

The effects on the drug efflux of 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126), the most toxic of all coplanar polychlorinated biphenyls (Co-PCBs), were examined in KB-3 cells expressing human wild-type and mutant P-glycoprotein in which the 61st amino acid was substituted for serine or phenylalanine (KB3-Phe61). In the cells expressing P-glycoproteins, accumulations of vinblastine and colchicine decreased form 85% to 92% and from 62% to 91%, respectively, and the drug tolerances for these chemicals were increased. In KB3-Phe61, the decreases in drug accumulation were inhibited by adding PCB-126 in a way similar to that with cyclosporine A: by adding 1 μM PCB-126, the accumulations of vinblastine and colchicine increased up to 3.3- and 2.3-fold, respectively. It is suggested that PCB-126 decreased the drug efflux by inhibiting the P-glycoprotein in KB3-Phe61. Since there were various P-glycoproteins and many congeners of Co-PCBs, this inhibition has to be considered a new cause of the toxic effects of Co-PCBs.

PKC-independent modulation of multidrug resistance in cells with mutant (V185) but not wild-type (G185) p-glycoprotein by bryostatin 1

Bryostatin 1 is a new antitumor agent which modulates the enzyme activity of protein kinase C (PKC, phospholipid-Ca 2+-dependent ATP:protein transferase, EC 2.7.1.37). Several reports have suggested that the pumping activity of the multidrug resistance gene 1 (MDR1)-encoded multidrug transporter P-glycoprotein (PGP) is enhanced by a PKC-mediated phosphorylation. It was shown here that bryostatin 1 was a potent modulator of multidrug resistance in two cell lines over-expressing a mutant MDR1-encoded PGP, namely KB-C1 cells and HeLa cells transfected with an MDR1-V185 construct (HeLa-MDR1-V185) in which glycine at position 185 (G185) was substituted for valine (V185). Bryostatin 1 is not able to reverse the resistance of cells over-expressing the wild-type form (G185) of PGP, namely CCRF-ADR5000 cells and HeLa cells transfected with a MDR1-G185 construct (HeLa-MDR1-G185). Treatment of HeLa-MDR1-V185 cells with bryostatin 1 was accompanied by an increase in the intracellular accumulation of rhodamine 123, whereas no such effect could be observed in HeLa-MDR1-G185 cells. HeLa-MDR1-V185 cells expressed the PKC isoforms α, δ and ζ. Down-modulation of PKC α and δ by 12- O-tetradecanoylphorbol-13-acetate (TPA) did not affect the drug accumulation by bryostatin 1. Bryostatin 1 depleted PKC α completely and PKC δ partially. In HeLa-MDR1-V185 cells, short-term exposure to bryostatin 1, which led to a PKC activation, was as efficient in modulating the pumping activity of PGP as long-term exposure leading to PKC depletion. Bryostatin 1 competed with azidopine for binding to PGP in cells expressing the MDR1-V185 and MDR1-G185 forms of PGP. It is concluded that bryostatin 1: i) interacts with both the mutated MDR1-V185 and the wild-type MDR1-G185; ii) reverses multidrug resistance and inhibits drug efflux only in PGP-V185 mutants; and iii) that this effect is not due to an interference of PKC with PGP. For gene therapy, it is important to reverse the specific resistance of a mutant in the presence of a wild-type transporter and vice versa. Our results show that it is possible to reverse a specific mutant PGP.

Multidrug-resistant Human Sarcoma Cells with a Mutant P-Glycoprotein, Altered Phenotype, and Resistance to Cyclosporins

A variant of the multidrug-resistant human sarcoma cell line Dx5 was derived by co-selection with doxorubicin and the cyclosporin D analogue PSC 833, a potent inhibitor of the multidrug transporter P-glycoprotein. The variant DxP cells manifest an altered phenotype compared with Dx5, with decreased cross-resistance to Vinca alkaloids and no resistance to dactinomycin. Resistance to doxorubicin and paclitaxel is retained. The multidrug resistance phenotype of DxP cells is not modulated by 2 microM PSC 833 or cyclosporine. DxP cells manifest a decreased ability to transport [3H]cyclosporine. DNA heteroduplex analysis and sequencing reveal a mutant mdr1 gene (deletion of a phenylalanine at amino acid residue 335) in the DxP cell line. The mutant P-glycoprotein has a decreased affinity for PSC 833 and vinblastine and a decreased ability to transport rhodamine 123. Transfection of the mutant mdr1 gene into drug-sensitive MES-SA sarcoma cells confers resistance to both doxorubicin and PSC 833. Our study demonstrates that survival of cells exposed to doxorubicin and PSC 833 in a multistep selection occurred as a result of a P-glycoprotein mutation in transmembrane region 6. These data suggest that Phe335 is an important binding site on P-glycoprotein for substrates such as dactinomycin and vinblastine and for inhibitors such as cyclosporine and PSC 833.

Membrane Topology of a Cysteine-less Mutant of Human P-glycoprotein

A human P-glycoprotein devoid of cysteine residues was constructed by site-directed mutagenesis for studying its topology. The cDNA for human P-glycoprotein-A52 in which codons for cysteines 137, 431, 717, 956, 1074, 1125, 1227, 1288, and 1304 were changed to Ala, was transfected into NIH 3T3 cells and analyzed with respect to its ability to confer resistance to various drugs. The cysteine-less P-glycoprotein-A52 retained the ability to confer resistance to vinblastine, colchicine, doxorubicin, and actinomycin D with only a small decrease in efficiency relative to wild-type enzyme. Cysteine residues were then reintroduced into predicted extracellular or cytoplasmic loops of the cysteine-less P-glycoprotein-A52, and the topology of the protein was determined using membrane-permeant and impermeant thiol-specific reagents. It was found that 8 of 15 cysteine residues introduced into P-glycoprotein-A52 could be biotinylated, when cells expressing the mutant P-glycoprotein were incubated with membrane-permeant biotin maleimide. Biotinylation of a cysteine residue placed in predicted extracellular loops between transmembrane segment (TM) 5 and TM6, TM7 and TM8, or TM11 and TM12 was blocked by pretreatment of the cells with a membrane-impermeant maleimide, suggesting that these residues have an extracellular location. By contrast, biotinylation of cysteine residues located in the predicted cytoplasmic loops between TM2 and TM3, TM4 and TM5, TM8 and TM9, or TM10 and TM11 were not blocked by pretreatment with membrane impermeant maleimide, suggesting that these residues were in the cytoplasm. These results are consistent with the model of P-glycoprotein, which predicts six transmembrane segments in each of the two homologous halves of the molecule.

Mutations to amino acids located in predicted transmembrane segment 6 (TM6) modulate the activity and substrate specificity of human P-glycoprotein.

Site-directed mutagenesis was used to investigate whether amino acids located in the predicted transmembrane segment, TM6 (residues 330-351), of human P-glycoprotein play essential roles in drug transport. Mutant cDNAs were expressed in mouse NIH 3T3 cells and analyzed with respect to their ability to confer resistance to cytotoxic drugs. Four mutations were found to strongly alter the drug resistance profile conferred by P-glycoprotein. Mutation of Val338 to Ala resulted in a mutant P-glycoprotein which conferred enhanced resistance to colchicine and reduced relative resistance to vinblastine. By contrast, mutant Gly341 to Val conferred little resistance to colchicine or doxorubicin, while its ability to confer resistance to vinblastine or actinomycin D was retained. A reduction in the ability of P-glycoprotein to confer resistance to all four drugs was observed for mutant Ala342 to Leu. Mutation of Ser344 to Ala, Thr, Cys, or Tyr resulted in mutant P-glycoproteins which were unable to confer drug resistance. Photolabeling of P-glycoprotein with azidopine in the presence of varying amounts of vinblastine showed that mutation of Ser344 to Tyr required approximately 15-fold more vinblastine to inhibit photolabeling when compared to wild-type enzyme. All of the Ser344 mutants were found to have reduced drug-stimulated ATPase activity relative to wild-type enzyme. These results, together with our previous demonstration that changes to Phe335 affected dissociation of vinblastine, suggest that TM6 may play an important role in drug--protein interaction and coupling of drug binding to ATPase activity.

Molecular basis of preferential resistance to colchicine in multidrug-resistant human cells conferred by Gly-185----Val-185 substitution in P-glycoprotein.

Expression of P-glycoprotein, encoded by the human MDR1 gene, results in cross-resistance to many lipophilic cytotoxic drugs (multidrug resistance). P-glycoprotein is believed to function as an energy-dependent efflux pump that is responsible for decreased drug accumulation in multidrug-resistant cells. Previous work showed that preferential resistance to colchicine in a colchicine-selected multidrug-resistant cell line was caused by spontaneous mutations in the MDR1 gene that resulted in a Gly-185----Val-185 substitution in P-glycoprotein. We have now compared transfectant cell lines expressing either the wild-type Gly-185 or the mutant Val-185 P-glycoprotein with regard to their levels of resistance to and accumulation and binding of different drugs. In cells expressing the mutant protein, increased resistance to colchicine and decreased resistance to vinblastine correlated with a decreased accumulation of colchicine and increased accumulation of vinblastine. Expression of the mutant P-glycoprotein also resulted in significantly increased resistance to epipodophyllotoxin and decreased resistance to vincristine and actinomycin D; smaller changes in resistance were observed for several other drugs. Unexpectedly, the mutant P-glycoprotein showed increased binding of photoactive analogs of vinblastine and verapamil and the photoactive compound azidopine and decreased binding of a photoactive colchicine analog. These results suggest that the Gly-185----Val-185 substitution affects not the initial drug-binding site of P-glycoprotein but another site, associated with the release of P-glycoprotein-bound drugs to the outside of the cell.

Conformational effects of amino acid substitutions in the P-glycoprotein of themdr 1 gene

The P-glycoprotein of the mdr 1 gene is responsible for the phenomenon of multidrug resistance in human cells. The presumed drug-binding site of the wild-type P-glycoprotein contains a glycine at position 185. A mutant P-glycoprotein which contains valine at this position causes cells to retain resistance to colchicine, but to lose cross-resistance to other drugs such as the chemotherapeutic agents vinblastine and Adriamycin. This has been hypothesized to be due to a conformational change in the protein induced by the amino acid substitution. Using conformational energy analysis, we have determined the allowed three-dimensional structures for the wild-type and mutant proteins in the region of position 185. The results indicate that the wild-type protein adopts a unique left-handed conformation at position 185 which is energically unfavorable for the protein with L-amino acids (including valine) at this position. This conformational change induced by amino acid substitutions for Gly 185 could explain the differences in binding to the P-glycoprotein of various drugs and, hence, the differences in drug resistance exhibited by various cell lines expressing these proteins.

An altered pattern of cross-resistance in multidrug-resistant human cells results from spontaneous mutations in the mdr1 (P-glycoprotein) gene

Multidrug resistance in human cells results from increased expression of the mdr1 (P-glycoprotein) gene, Although the same gene is activated in cells selected with different drugs, multidrug-resistant cell lines can be preferentially resistant to their selecting agent. The mdr1 cDNA sequence from vinblastine-selected KB cells, which are uniformly resistant to different lipophilic drugs, was compared with the corresponding sequence from colchicine-selected KB cells preferentially resistant to colchicine. These sequences differ at three positions, resulting in a single amino acid change in P-glycoprotein. These differences result from mutations that occured during colchicine selection. The appearance of these mutations coincides with the emergence of preferential resistance to colchicine. We have constructed biologically active mdr1 cDNA clones that express either wild-type or mutant P-glycoprotein. Multidrug-resistant transfectants obtained with the mutant sequence were characterized by increased relative resistance to colchicine compared with transfectants obtained with wild-type sequence. mdr1 mutations are therefore responsible for preferential resistance to colchicine in multidrug-resistant KB cells.