Multidrug Resistance of DNA-Mediated Transformants is Linked to Transfer of the Human <i>mdr</i>1 Gene

Shen, Donglai; Fojo, Antonio Tito; Roninson, Igor B.; Chin, Janice E.; Soffir, R; Pastan, Ira; Gottesman, Michael M.

doi:10.1128/mcb.6.11.4039-4045.1986

Cited by 62 publications

(10 citation statements)

References 24 publications

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“…Previous studies demonstrated that NIH 3T3 cells were rendered drug resistant after transformation with high molecular weight DNA from multidrug-resistant KB cells (Shen et al, 1986c) or by transfection with a full-length cDNA derived from the human MDR 1 gene (Ueda et al, 1987). To demonstrate that human P-glycoprotein was synthesized in these cells and that the protein produced was of the appropriate size, antibody P7 was used to immunoprecipitate labeled cell extracts.…”

Section: Resultsmentioning

confidence: 99%

“…From the specific activity of total cell protein, the amount of P-glycoprotein (nanograms per milligram of extract) was made. ' NIFI 3T3 cells selected for multidrug resistance in colchicine have an amplified mouse mdr gene and are designated NIH-mrfr (Shen et al, 1986c). ''The MDR\ RNA expression for NIH 3T3 and NT-12 is estimated from Ueda et al (1987) and for T2C1 and NI-mrfr cells are from Shen et al (1986c).…”

mentioning

confidence: 99%

“…NIFI 3T3 cells selected for multidrug resistance in colchicine have an amplified mouse mdr gene and are designated NIH-mrfr (Shen et al, 1986c). ''The MDR\ RNA expression for NIH 3T3 and NT-12 is estimated from Ueda et al (1987) and for T2C1 and NI-mrfr cells are from Shen et al (1986c). The mdr RNA level in NIH-Wr may be underestimated, since a human MDR\ hybridization probe was used.…”

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confidence: 99%

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Stability and covalent modification of P-glycoprotein in multidrug-resistant KB cells

Richert¹,

Aldwin²,

Nitecki³

et al. 1988

Biochemistry

113

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An antipeptide antibody (P7) to P-glycoprotein has been produced by immunizing rabbits with a synthetic peptide. Antibody P7 is directed against the amino-terminal region of P170 (residues 28-35). The antibody immunoprecipitates a 170-kDa P-glycoprotein from extracts of drug-resistant KB-V1 cells that is not present in the drug-sensitive cell line KB-3-1. Antibody P7 was used to quantitate the amount of P-glycoprotein present in drug-resistant KB lines at various levels of resistance and to demonstrate the presence of P-glycoprotein in NIH 3T3 cells transfected with a cloned MDR1 cDNA or human genomic DNA encoding MDR1. Pulse-chase labeling experiments demonstrated that P-glycoprotein is synthesized as a 140-kDa precursor which is slowly converted over 2-4 h to a 170-kDa glycoprotein. Tunicamycin treatment blocks the conversion of the precursor to the mature form, and removal of N-linked oligosaccharides with Endo F reduces the relative molecular weight of P-glycoprotein to 140K. The mobility of mature P-glycoprotein is unaffected by treatment with neuraminidase and Endo H. These data indicate that P-glycoprotein is N-glycosylated and contains little or no neuraminic acid. P-Glycoprotein is also phosphorylated, and the extent of phosphate incorporated is proportional to the amount of protein present in drug-resistant cells.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Stability and covalent modification of P-glycoprotein in multidrug-resistant KB cells

Richert¹,

Aldwin²,

Nitecki³

et al. 1988

Biochemistry

113

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“…Cell Transfections and Drug Selection. Drug-sensitive NIH3T3 cells were transfected with variants of the wildtype pHaMDR1/A (G185) retroviral vector (22,26) by the calcium phosphate coprecipitation method (27). Ten micrograms of plasmid DNA was used to transfect 3.5 × 10 5 cells/25 cm 2 tissue culture flask that were fed with fresh DMEM 3 h before transfection.…”

Section: Methodsmentioning

confidence: 99%

Structural Flexibility of the Linker Region of Human P-Glycoprotein Permits ATP Hydrolysis and Drug Transport

et al. 1998

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P-Glycoprotein (Pgp), an energy-dependent drug efflux pump responsible for multidrug resistance of many cancer cells, is comprised of two homologous halves connected by a peptide segment approximately 75 amino acids (aa) in length. The effects of length and composition of this connecting region on Pgp cell surface expression and the ability of the two halves to interact were explored using both stable transfections of Pgp mutants in mammalian cell lines and a vaccinia virus transient expression system. A 17 aa insertion of predicted flexible structure between amino acids 681 and 682 resulted in a functional Pgp molecule that was capable of conferring drug resistance. In contrast, an 18 aa peptide insertion with a predicted alpha-helical structure was unstable when expressed transiently. A 34 aa deletion from the central core of the linker region (Delta653-686) resulted in a protein expressed at the cell surface in amounts comparable to that of wild-type Pgp but unable to confer drug resistance. No apparent differences in drug or [alpha-32P]-8-azido-ATP photoaffinity labeling were observed. However, both ATP hydrolysis and drug transport activities of the deletion mutant were completely abrogated, indicating that the linker deletion disconnected substrate binding from ATP hydrolysis and transport. This mutant also failed to exhibit an ATP hydrolysis-dependent enhancement of binding of a conformation-sensitive monoclonal antibody, UIC2. Upon replacement with a 17 aa linker peptide having a predicted flexible secondary structure, but bearing no homology to the deleted 34 aa segment, normal Pgp transport and basal and drug-stimulated ATPase activities were restored along with increased UIC2 binding in the presence of substrate, suggesting a dramatic conformational change between the nonfunctional and functional molecules. Taken together, these data suggest a flexible secondary structure of the connector region is sufficient for the coordinate functioning of the two halves of Pgp, likely specifically required for the proper interaction of the two ATP binding sites.

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“…In MDR cell lines, MDRl expression is increased as a result of gene amplification or increased transcription (Fairchild etal., 1987;Fojoet al, 1985;Riordan et al, 1985;Roninson et al, 1986;Scotto et al, 1986), and overexpression of the MDRl gene, but not the MDRl gene, confers MDR (Schinkel et al, 1991). Several studies have shown that expression of either the genomic or the cDNA sequence of MDRl confers the MDR phenotype to the recipient cells (Ueda etal., 1987;Croop etal., 1987;Debenham et al, 1982;Gros et al, 1986;Shen et al, 1986;Sugimoto & Tsuruo, 1987), although the degree of resistance is generally less when compared to drug-selected MDR cells.…”

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confidence: 99%

Modulation of P-Glycoprotein by Protein Kinase C.alpha. in a Baculovirus Expression System

1994

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The modulation of P-glycoprotein by protein kinase C alpha (PKC alpha) was examined in a baculovirus expression system. PGP was phosphorylated in membrane vesicle preparations in vitro only when coexpressed with PKC alpha, and phosphorylation was Ca(2+)-dependent and inhibited by the PKC inhibitor Ro 31-8220. PGP and PKC alpha were tightly associated in membrane vesicles and were coimmunoprecipitated with antibodies against either PGP or PKC alpha. Photoaffinity labeling of membrane vesicles with [3H]azidopine indicated that drug binding to PGP was slightly increased in the presence of PKC alpha. In contrast, PGP ATPase activity was increased by PKC alpha as well as by verapamil, but only PKC-stimulated activity in the presence of verapamil was inhibited by Ro 31-8220. Mutation of serine-671 to asparagine in the linker region of PGP abolished PKC alpha-stimulated ATPase activity, and also inhibited to a lesser degree verapamil-stimulated ATPase activity. These results indicate that PKC alpha in a positive regulator of PGP ATPase activity and suggest that this mechanism may account for the increased multidrug resistance observed in MDR1-expressing cells when PKC alpha activity is elevated.

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Multidrug Resistance of DNA-Mediated Transformants is Linked to Transfer of the Human mdr1 Gene

Cited by 62 publications

References 24 publications

Stability and covalent modification of P-glycoprotein in multidrug-resistant KB cells

Stability and covalent modification of P-glycoprotein in multidrug-resistant KB cells

Structural Flexibility of the Linker Region of Human P-Glycoprotein Permits ATP Hydrolysis and Drug Transport

Modulation of P-Glycoprotein by Protein Kinase C.alpha. in a Baculovirus Expression System

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