A phosphoglycoprotein associated with taxol resistance in J774.2 cells.
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, puromycin, 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 Mr 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 Mr 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 Mr 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 [32P]orthophosphate. Phosphoamino acid analysis of the Mr 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 Mr 135,000 phosphoglycoprotein in plasma membranes and resistance to taxol.
Roy
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Horwitz
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D000470:Alkaloids / Q000378:metabolism / Q000494:pharmacology*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D002460:Cell Line; D002846:Chromatography, Affinity; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D005784:Gene Amplification; D006023:Glycoproteins / Q000032:analysis* / Q000302:isolation & purification; D008264:Macrophages / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis*; D051379:Mice; D008970:Molecular Weight; D017239:Paclitaxel; D010750:Phosphoproteins / Q000032:analysis*
D000470:Alkaloids; D000596:Amino Acids; D000972:Antineoplastic Agents, Phytogenic; D006023:Glycoproteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D017239:Paclitaxel