Characterization of monoclonal antibodies recognizing a M(r) 180,000 P-glycoprotein: Differential expression of the M(r) 180,000 and M(r) 170,000 P-glycoproteins in multidrug-resistant human tumor cells

M. B. Meyers, L. Rittmann-Grauer, J. P. O'Brien, Ahmad Safa

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Abstract

P-glycoprotein is a plasma membrane protein believed to mediate resistance to natural product drugs such as vincristine, Adriamycin, and actinomycin D. To facilitate the study of human P-glycoprotein, monoclonal antibodies (designated HYB-612, HYB-241, and HYB-195) were raised against vincristine-resistant human neuroblastoma (SH-SY5Y/VCR) cells. The antibodies recognize a M(r) 180,000 plasma membrane phosphoglycoprotein produced in increased amounts in SH-SY5Y/VCR as well as in vincristine-resistant human neuroepithelioma (MC-IXC/VCR), vinblastine-resistant human leukemia (CEM/VLB100), and actinomycin D- or vincristine-resistant Chinese hamster (DC-3F/AD X and DC-3F/VCRd-5L) cells, as compared to control cells. Radioimmunoprecipitation of proteins in cells metabolically labeled with [35S]methionine, 32P(i), or [3H]glucosamine and Western transfer procedures were used for these studies. Characterization of the HYB-612 or HYB-241 antigen by destructive degradation produced a pattern of results typical of a conformation-dependent protein epitope. HYB-612 recognizes complexes of the M(r) 180,000 antigen with an iodinated photoaffinity analogue of vinblastine or with tritiated azidopine. Furthermore, pretreatment of MC-IXC and MC-IXC/VCR cells with HYB-612 or HYB-241 before measurement of tritium-labeled actinomycin D or vincristine uptake increases the amount of drug accumulation in resistant, but not in sensitive, cells. Of importance is the fact that the M(r) 180,000 protein is expressed in cells which also contain a M(r) 170,000 P-glycoprotein. The relative amounts of the M(r) 180,000 and 170,000 species vary from one drug-resistant cell line to another. Evidence that the M(r) 180,000 protein is a P-glycoprotein and that there is a conserved complex pattern of resistance-related surface proteins in multidrug-resistant cells is presented in this report.

Original languageEnglish (US)
Pages (from-to)3209-3214
Number of pages6
JournalCancer Research
Volume49
Issue number12
StatePublished - 1989
Externally publishedYes

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P-Glycoproteins
P-Glycoprotein
Monoclonal Antibodies
Vincristine
Neoplasms
Dactinomycin
Vinblastine
Membrane Proteins
Cell Membrane
Pharmaceutical Preparations
Antigens
Protein Conformation
Proteins
Tritium
Glucosamine
Cricetulus
Biological Products
Neuroblastoma
Methionine
Doxorubicin

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

@article{7cdf02ea499e4e078808e8a0c116f0eb,
title = "Characterization of monoclonal antibodies recognizing a M(r) 180,000 P-glycoprotein: Differential expression of the M(r) 180,000 and M(r) 170,000 P-glycoproteins in multidrug-resistant human tumor cells",
abstract = "P-glycoprotein is a plasma membrane protein believed to mediate resistance to natural product drugs such as vincristine, Adriamycin, and actinomycin D. To facilitate the study of human P-glycoprotein, monoclonal antibodies (designated HYB-612, HYB-241, and HYB-195) were raised against vincristine-resistant human neuroblastoma (SH-SY5Y/VCR) cells. The antibodies recognize a M(r) 180,000 plasma membrane phosphoglycoprotein produced in increased amounts in SH-SY5Y/VCR as well as in vincristine-resistant human neuroepithelioma (MC-IXC/VCR), vinblastine-resistant human leukemia (CEM/VLB100), and actinomycin D- or vincristine-resistant Chinese hamster (DC-3F/AD X and DC-3F/VCRd-5L) cells, as compared to control cells. Radioimmunoprecipitation of proteins in cells metabolically labeled with [35S]methionine, 32P(i), or [3H]glucosamine and Western transfer procedures were used for these studies. Characterization of the HYB-612 or HYB-241 antigen by destructive degradation produced a pattern of results typical of a conformation-dependent protein epitope. HYB-612 recognizes complexes of the M(r) 180,000 antigen with an iodinated photoaffinity analogue of vinblastine or with tritiated azidopine. Furthermore, pretreatment of MC-IXC and MC-IXC/VCR cells with HYB-612 or HYB-241 before measurement of tritium-labeled actinomycin D or vincristine uptake increases the amount of drug accumulation in resistant, but not in sensitive, cells. Of importance is the fact that the M(r) 180,000 protein is expressed in cells which also contain a M(r) 170,000 P-glycoprotein. The relative amounts of the M(r) 180,000 and 170,000 species vary from one drug-resistant cell line to another. Evidence that the M(r) 180,000 protein is a P-glycoprotein and that there is a conserved complex pattern of resistance-related surface proteins in multidrug-resistant cells is presented in this report.",
author = "Meyers, {M. B.} and L. Rittmann-Grauer and O'Brien, {J. P.} and Ahmad Safa",
year = "1989",
language = "English (US)",
volume = "49",
pages = "3209--3214",
journal = "Journal of Cancer Research",
issn = "0099-7013",
publisher = "American Association for Cancer Research Inc.",
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TY - JOUR

T1 - Characterization of monoclonal antibodies recognizing a M(r) 180,000 P-glycoprotein

T2 - Differential expression of the M(r) 180,000 and M(r) 170,000 P-glycoproteins in multidrug-resistant human tumor cells

AU - Meyers, M. B.

AU - Rittmann-Grauer, L.

AU - O'Brien, J. P.

AU - Safa, Ahmad

PY - 1989

Y1 - 1989

N2 - P-glycoprotein is a plasma membrane protein believed to mediate resistance to natural product drugs such as vincristine, Adriamycin, and actinomycin D. To facilitate the study of human P-glycoprotein, monoclonal antibodies (designated HYB-612, HYB-241, and HYB-195) were raised against vincristine-resistant human neuroblastoma (SH-SY5Y/VCR) cells. The antibodies recognize a M(r) 180,000 plasma membrane phosphoglycoprotein produced in increased amounts in SH-SY5Y/VCR as well as in vincristine-resistant human neuroepithelioma (MC-IXC/VCR), vinblastine-resistant human leukemia (CEM/VLB100), and actinomycin D- or vincristine-resistant Chinese hamster (DC-3F/AD X and DC-3F/VCRd-5L) cells, as compared to control cells. Radioimmunoprecipitation of proteins in cells metabolically labeled with [35S]methionine, 32P(i), or [3H]glucosamine and Western transfer procedures were used for these studies. Characterization of the HYB-612 or HYB-241 antigen by destructive degradation produced a pattern of results typical of a conformation-dependent protein epitope. HYB-612 recognizes complexes of the M(r) 180,000 antigen with an iodinated photoaffinity analogue of vinblastine or with tritiated azidopine. Furthermore, pretreatment of MC-IXC and MC-IXC/VCR cells with HYB-612 or HYB-241 before measurement of tritium-labeled actinomycin D or vincristine uptake increases the amount of drug accumulation in resistant, but not in sensitive, cells. Of importance is the fact that the M(r) 180,000 protein is expressed in cells which also contain a M(r) 170,000 P-glycoprotein. The relative amounts of the M(r) 180,000 and 170,000 species vary from one drug-resistant cell line to another. Evidence that the M(r) 180,000 protein is a P-glycoprotein and that there is a conserved complex pattern of resistance-related surface proteins in multidrug-resistant cells is presented in this report.

AB - P-glycoprotein is a plasma membrane protein believed to mediate resistance to natural product drugs such as vincristine, Adriamycin, and actinomycin D. To facilitate the study of human P-glycoprotein, monoclonal antibodies (designated HYB-612, HYB-241, and HYB-195) were raised against vincristine-resistant human neuroblastoma (SH-SY5Y/VCR) cells. The antibodies recognize a M(r) 180,000 plasma membrane phosphoglycoprotein produced in increased amounts in SH-SY5Y/VCR as well as in vincristine-resistant human neuroepithelioma (MC-IXC/VCR), vinblastine-resistant human leukemia (CEM/VLB100), and actinomycin D- or vincristine-resistant Chinese hamster (DC-3F/AD X and DC-3F/VCRd-5L) cells, as compared to control cells. Radioimmunoprecipitation of proteins in cells metabolically labeled with [35S]methionine, 32P(i), or [3H]glucosamine and Western transfer procedures were used for these studies. Characterization of the HYB-612 or HYB-241 antigen by destructive degradation produced a pattern of results typical of a conformation-dependent protein epitope. HYB-612 recognizes complexes of the M(r) 180,000 antigen with an iodinated photoaffinity analogue of vinblastine or with tritiated azidopine. Furthermore, pretreatment of MC-IXC and MC-IXC/VCR cells with HYB-612 or HYB-241 before measurement of tritium-labeled actinomycin D or vincristine uptake increases the amount of drug accumulation in resistant, but not in sensitive, cells. Of importance is the fact that the M(r) 180,000 protein is expressed in cells which also contain a M(r) 170,000 P-glycoprotein. The relative amounts of the M(r) 180,000 and 170,000 species vary from one drug-resistant cell line to another. Evidence that the M(r) 180,000 protein is a P-glycoprotein and that there is a conserved complex pattern of resistance-related surface proteins in multidrug-resistant cells is presented in this report.

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