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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, 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.
Insights
Taxol resistance in J774.2 cells is linked to a prominent 135,000 molecular weight phosphoglycoprotein in plasma membranes. This protein
Area of Science:
- Cell Biology
- Molecular Pharmacology
- Cancer Research
Background:
- Taxol (paclitaxel) is a microtubule-stabilizing agent that inhibits cell replication.
- Taxol resistance can develop in cancer cells, limiting therapeutic efficacy.
- Understanding the molecular mechanisms of taxol resistance is crucial for developing effective cancer treatments.
Purpose of the Study:
- To investigate the molecular basis of taxol resistance in a murine macrophage cell line (J774.2).
- To identify potential protein markers associated with taxol resistance.
- To explore the relationship between drug binding, cellular sensitivity, and membrane protein composition.
Main Methods:
- Development of a taxol-resistant subline (J7/TAX-50) from J774.2 cells.
- Assessment of drug sensitivity and radiolabeled drug binding in parental and resistant cells.
- Analysis of plasma membrane proteins using SDS-PAGE and silver staining.
- Affinity chromatography and metabolic labeling to characterize specific membrane proteins.
- Phosphoamino acid analysis to determine phosphorylation sites.
Main Results:
- The taxol-resistant J7/TAX-50 subline exhibited approximately 800-fold resistance to taxol and reduced drug binding.
- A prominent 135,000 molecular weight (Mr) phosphoglycoprotein was identified in the plasma membranes of resistant cells but was absent in sensitive cells.
- Loss of drug sensitivity correlated with the presence of this Mr 135,000 protein.
- Reversion of drug sensitivity upon prolonged drug withdrawal was accompanied by a decrease in the Mr 135,000 protein.
- Other resistant cell lines showed distinct membrane phosphoglycoproteins.
Conclusions:
- A specific Mr 135,000 phosphoglycoprotein in plasma membranes is strongly correlated with taxol resistance in J774.2 cells.
- This protein may play a significant role in the mechanism of taxol resistance.
- Further research into this protein could lead to novel strategies for overcoming taxol resistance in cancer therapy.