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Expression of recombinant glutathione S-transferase pi, Ya, or Yb1 confers resistance to alkylating agents
1McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Abstract:
Increased levels of glutathione S-transferase (GST; RX:glutathione R-transferase; EC 2.5.1.18) mRNA, protein, and activity in tumor biopsy samples and in drug-resistant cultured cells are associated with resistance to anticancer drugs. We report that each of three full-length cloned GST cDNAs, that for pi (acidic), Ya (basic), and Yb1 (neutral), can confer drug resistance when expressed in cultured mammalian cells. In one approach, stably transfected mouse C3H/10T1/2 cells that express GST pi, Ya, or Yb1 were cloned and analyzed for drug resistance in colony-forming assays. Transiently transfected COS cells that were sorted on a fluorescence-activated cell sorter were used in the second approach to avoid interclonal variation in factors other than the recombinant GST and to show that reversion of transient GST expression correlated with loss of drug resistance. A sorting technique, developed to separate the 20% of the electroporated COS cell population that transiently expressed GST pi, Ya, or Yb1 from the nonexpressing population, was based on a GST-catalyzed intracellular conjugation of glutathione to the fluorescent labeling reagent monochlorobimane. GST Ya conferred the greatest increase in resistance to chlorambucil and melphalan (1.3- to 2.9-fold), Yb1 conferred the greatest increase in resistance to cisplatin (1.5-fold), and pi conferred the greatest increase in resistance to a racemic mixture of 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene and 7 alpha,8 beta-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo [a]pyrene and doxorubicin (1.5- and 1.3-fold) relative to controls. These resistance values to alkylating agents are commensurate with values observed clinically. Cytotoxicity curves representing recombinant GST+ populations were significantly different from their controls with P values ranging from 0.005 to 0.0001. No resistance to vinblastine was detected. Conferred drug resistance was proportional to the magnitude of GST Ya expression, and reversion of transient expression in GST Ya+ COS cell clones to a GST Ya- phenotype was associated with total loss of drug resistance.
Insights
Glutathione S-transferases (GSTs) confer anticancer drug resistance when expressed in mammalian cells. Different GST forms provide varying levels of resistance to specific chemotherapy agents, offering insights into clinical drug resistance.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Elevated glutathione S-transferase (GST) levels correlate with anticancer drug resistance in tumors and cell lines.
- Understanding the specific roles of different GST isozymes in drug resistance is crucial for developing effective cancer therapies.
Purpose of the Study:
- To investigate whether cloned glutathione S-transferase (GST) cDNAs can confer drug resistance when expressed in cultured mammalian cells.
- To determine the relative contribution of specific GST isozymes (pi, Ya, Yb1) to resistance against various anticancer drugs.
Main Methods:
- Stable and transient transfection of mammalian cells (C3H/10T1/2 and COS) with full-length GST cDNAs.
- Drug resistance assays (colony-forming assays) and fluorescence-activated cell sorting (FACS) using a GST-conjugated fluorescent probe (monochlorobimane).
- Analysis of resistance to specific anticancer drugs including alkylating agents (chlorambucil, melphalan), cisplatin, doxorubicin, and vinblastine.
Main Results:
- Expression of GST pi, Ya, and Yb1 in mammalian cells conferred significant resistance to specific anticancer drugs.
- GST Ya provided the greatest resistance to chlorambucil and melphalan (1.3-2.9-fold), Yb1 to cisplatin (1.5-fold), and pi to doxorubicin (1.3-fold) and a benzo[a]pyrene metabolite (1.5-fold).
- Conferred drug resistance was dose-dependent on GST Ya expression, and loss of expression correlated with loss of resistance; no resistance to vinblastine was observed.
Conclusions:
- Individual glutathione S-transferase (GST) isozymes can confer specific patterns of anticancer drug resistance.
- The findings provide a molecular basis for GST-mediated drug resistance observed in clinical settings.
- Targeting specific GST isozymes may offer novel strategies to overcome chemotherapy resistance.