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) was developed in vitro by growing the cells in increasing drug concentrations. These cells, which are .apprx. 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 .apprx. 90%. Resistant cells grown in the absence of drug for 18 days (J7/TAX-OD10), 1 mo. (J7/TAX-OD30), and 8 mo. (J7/TAX-OD240) regain a major portion (27, 92 and 99%, respectively), of their sensitivity to the drug. However, binding of the drug to the J7/TAX-OD30 and J7/TAX-OD240 cells is increased to only 20 and 70%, respectively, of that measured with sensitive cells. Analysis of plasma membranes by sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by silver staining reveals the presence of a prominent protein with an MW of .apprx. 135,000 in the resistant line that is essentially absent from the parental line and from both of the J7/TAX-OD30 and J7/TAX-OD240 lines. Although this protein can be seen in J7/TAX-OD10, its quantity is diminished. The MW 135,000 protein is also observed in the resistant cells when they are labeled with [3H]eucine, [35S]methionine, [3H]glucosamine or [32P]orthophosphate. Plasma membranes from colchicine- or vinblastine-resistant J774.2 cells also contain prominent phosphoglycoproteins, with MW of .apprx. 145,000 and .apprx. 150,000, respectively. Partial purification of the MW 135,000 glycoprotein by agarose-bound Ricinus communis agglutinin 1-agarose affinity chromatography indicates that it accounts for .apprx. 4-5% of total membrane protein. A MW 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 MW 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 MW 135,000 phosphoglycoprotein in plasma membranes and resistance to taxol.