A phosphoglycoprotein associated with taxol resistance in J774.2 cells

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Abstract

Taxol is an inhibitor of cell replication that promotes the assembly of microtubules in vitro and in cells. In the murine macrophage-like cell line J774.2, a taxol-resistant subline (J7/TAX-50) has been developed in vitro by growing the cells in increasing drug concentrations. These cells, which are approximately 800-fold resistant to taxol, display some cross-resistance to colchicine, vinblastine, pyromycin, doxorubicin, and actinomycin D but remain sensitive to bleomycin. Binding of radiolabeled drug to the resistant cells is reduced by approximately 90%. Resistant cells grown in the absence of drug for 10 days (J7/TAX-0D10), 1 month (J7/TAX-0D30), and 8 months (J7/TAX-0D240) regain a major portion (27, 92, and 99%, respectively) of their sensitivity to the drug. However, binding of the drug to the J7/TAX-0D30 and J7/TAX-0D240 cells is increased to only 20 and 70%, respectively, of that measured with sensitive cells. Analysis of plasma membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining reveals the presence of a prominent protein with an approximate molecular weight of 135,000 in the resistant line that is essentially absent from the parental line and from both of the J7/TAX-0D30 and J7/TAX-0D240 lines. Although this protein can be seen in J7/TAX-0D10, its quantity is diminished. The M(r) 135,000 protein is also observed in the resistant cells when they are labeled with [3H]leucine, [35S]methionine, [3H]glucosamine, or [32P]orthophosphate. Plasma membranes from colchicine- or vinblastine-resistant J774.2 cells also contain prominent phosphoglycoproteins, with approximate molecular weights of 145,000 and 150,000, respectively. Partial purification of the M(r) 135,000 glycoprotein by agarose-bound ricinus communis agglutinin I-agarose affinity chromatography indicates that it accounts for approximately 4 to 5% of total membrane protein. A M(r) 100,000 phosphoglycoprotein, present in the membranes of J774.2 cells, is essentially absent in J7/TAX-50 cells after labeling with [3H]glucosamine or [33P]orthophosphate. Phosphoamino acid analysis of the M(r) 135,000 and 100,000 phosphoglycoproteins reveals that the phosphorylation sites are serine and threonine residues. There appears to be a good correlation between the presence of the M(r) 135,000 phosphoglycoprotein in plasma membranes and resistance to taxol.

Original languageEnglish (US)
Pages (from-to)3856-3863
Number of pages8
JournalCancer Research
Volume45
Issue number8
StatePublished - 1985

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Paclitaxel
Cell Membrane
Pharmaceutical Preparations
Vinblastine
Glucosamine
Colchicine
Molecular Weight
Phosphates
Phosphoamino Acids
Agarose Chromatography
Silver Staining
Proteins
Bleomycin
Dactinomycin
Threonine
Affinity Chromatography
Leucine
Microtubules
Sodium Dodecyl Sulfate
Methionine

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

A phosphoglycoprotein associated with taxol resistance in J774.2 cells. / Roy, S. N.; Band Horwitz, Susan.

In: Cancer Research, Vol. 45, No. 8, 1985, p. 3856-3863.

Research output: Contribution to journalArticle

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title = "A phosphoglycoprotein associated with taxol resistance in J774.2 cells",
abstract = "Taxol is an inhibitor of cell replication that promotes the assembly of microtubules in vitro and in cells. In the murine macrophage-like cell line J774.2, a taxol-resistant subline (J7/TAX-50) has been developed in vitro by growing the cells in increasing drug concentrations. These cells, which are approximately 800-fold resistant to taxol, display some cross-resistance to colchicine, vinblastine, pyromycin, doxorubicin, and actinomycin D but remain sensitive to bleomycin. Binding of radiolabeled drug to the resistant cells is reduced by approximately 90{\%}. Resistant cells grown in the absence of drug for 10 days (J7/TAX-0D10), 1 month (J7/TAX-0D30), and 8 months (J7/TAX-0D240) regain a major portion (27, 92, and 99{\%}, respectively) of their sensitivity to the drug. However, binding of the drug to the J7/TAX-0D30 and J7/TAX-0D240 cells is increased to only 20 and 70{\%}, respectively, of that measured with sensitive cells. Analysis of plasma membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining reveals the presence of a prominent protein with an approximate molecular weight of 135,000 in the resistant line that is essentially absent from the parental line and from both of the J7/TAX-0D30 and J7/TAX-0D240 lines. Although this protein can be seen in J7/TAX-0D10, its quantity is diminished. The M(r) 135,000 protein is also observed in the resistant cells when they are labeled with [3H]leucine, [35S]methionine, [3H]glucosamine, or [32P]orthophosphate. Plasma membranes from colchicine- or vinblastine-resistant J774.2 cells also contain prominent phosphoglycoproteins, with approximate molecular weights of 145,000 and 150,000, respectively. Partial purification of the M(r) 135,000 glycoprotein by agarose-bound ricinus communis agglutinin I-agarose affinity chromatography indicates that it accounts for approximately 4 to 5{\%} of total membrane protein. A M(r) 100,000 phosphoglycoprotein, present in the membranes of J774.2 cells, is essentially absent in J7/TAX-50 cells after labeling with [3H]glucosamine or [33P]orthophosphate. Phosphoamino acid analysis of the M(r) 135,000 and 100,000 phosphoglycoproteins reveals that the phosphorylation sites are serine and threonine residues. There appears to be a good correlation between the presence of the M(r) 135,000 phosphoglycoprotein in plasma membranes and resistance to taxol.",
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T1 - A phosphoglycoprotein associated with taxol resistance in J774.2 cells

AU - Roy, S. N.

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N2 - Taxol is an inhibitor of cell replication that promotes the assembly of microtubules in vitro and in cells. In the murine macrophage-like cell line J774.2, a taxol-resistant subline (J7/TAX-50) has been developed in vitro by growing the cells in increasing drug concentrations. These cells, which are approximately 800-fold resistant to taxol, display some cross-resistance to colchicine, vinblastine, pyromycin, doxorubicin, and actinomycin D but remain sensitive to bleomycin. Binding of radiolabeled drug to the resistant cells is reduced by approximately 90%. Resistant cells grown in the absence of drug for 10 days (J7/TAX-0D10), 1 month (J7/TAX-0D30), and 8 months (J7/TAX-0D240) regain a major portion (27, 92, and 99%, respectively) of their sensitivity to the drug. However, binding of the drug to the J7/TAX-0D30 and J7/TAX-0D240 cells is increased to only 20 and 70%, respectively, of that measured with sensitive cells. Analysis of plasma membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining reveals the presence of a prominent protein with an approximate molecular weight of 135,000 in the resistant line that is essentially absent from the parental line and from both of the J7/TAX-0D30 and J7/TAX-0D240 lines. Although this protein can be seen in J7/TAX-0D10, its quantity is diminished. The M(r) 135,000 protein is also observed in the resistant cells when they are labeled with [3H]leucine, [35S]methionine, [3H]glucosamine, or [32P]orthophosphate. Plasma membranes from colchicine- or vinblastine-resistant J774.2 cells also contain prominent phosphoglycoproteins, with approximate molecular weights of 145,000 and 150,000, respectively. Partial purification of the M(r) 135,000 glycoprotein by agarose-bound ricinus communis agglutinin I-agarose affinity chromatography indicates that it accounts for approximately 4 to 5% of total membrane protein. A M(r) 100,000 phosphoglycoprotein, present in the membranes of J774.2 cells, is essentially absent in J7/TAX-50 cells after labeling with [3H]glucosamine or [33P]orthophosphate. Phosphoamino acid analysis of the M(r) 135,000 and 100,000 phosphoglycoproteins reveals that the phosphorylation sites are serine and threonine residues. There appears to be a good correlation between the presence of the M(r) 135,000 phosphoglycoprotein in plasma membranes and resistance to taxol.

AB - Taxol is an inhibitor of cell replication that promotes the assembly of microtubules in vitro and in cells. In the murine macrophage-like cell line J774.2, a taxol-resistant subline (J7/TAX-50) has been developed in vitro by growing the cells in increasing drug concentrations. These cells, which are approximately 800-fold resistant to taxol, display some cross-resistance to colchicine, vinblastine, pyromycin, doxorubicin, and actinomycin D but remain sensitive to bleomycin. Binding of radiolabeled drug to the resistant cells is reduced by approximately 90%. Resistant cells grown in the absence of drug for 10 days (J7/TAX-0D10), 1 month (J7/TAX-0D30), and 8 months (J7/TAX-0D240) regain a major portion (27, 92, and 99%, respectively) of their sensitivity to the drug. However, binding of the drug to the J7/TAX-0D30 and J7/TAX-0D240 cells is increased to only 20 and 70%, respectively, of that measured with sensitive cells. Analysis of plasma membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining reveals the presence of a prominent protein with an approximate molecular weight of 135,000 in the resistant line that is essentially absent from the parental line and from both of the J7/TAX-0D30 and J7/TAX-0D240 lines. Although this protein can be seen in J7/TAX-0D10, its quantity is diminished. The M(r) 135,000 protein is also observed in the resistant cells when they are labeled with [3H]leucine, [35S]methionine, [3H]glucosamine, or [32P]orthophosphate. Plasma membranes from colchicine- or vinblastine-resistant J774.2 cells also contain prominent phosphoglycoproteins, with approximate molecular weights of 145,000 and 150,000, respectively. Partial purification of the M(r) 135,000 glycoprotein by agarose-bound ricinus communis agglutinin I-agarose affinity chromatography indicates that it accounts for approximately 4 to 5% of total membrane protein. A M(r) 100,000 phosphoglycoprotein, present in the membranes of J774.2 cells, is essentially absent in J7/TAX-50 cells after labeling with [3H]glucosamine or [33P]orthophosphate. Phosphoamino acid analysis of the M(r) 135,000 and 100,000 phosphoglycoproteins reveals that the phosphorylation sites are serine and threonine residues. There appears to be a good correlation between the presence of the M(r) 135,000 phosphoglycoprotein in plasma membranes and resistance to taxol.

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