Abstract
To examine the role of multidrug resistance protein 1 (MRP1) and glutathione S-transferases (GSTs) in cellular resistance to antineoplastic drugs, derivatives of MCF7 breast carcinoma cells were developed that express MRP1 in combination with one of three human cytosolic isozymes of GST. Expression of MRP1 alone confers resistance to several drugs representing the multidrug resistance phenotype, drugs including doxorubicin, vincristine, etoposide, and mitoxantrone. However, co-expression with MRP1 of any of the human GST isozymes A1-1, M1-1, or P1-1 failed to augment MRP1-associated resistance to these drugs. In contrast, combined expression of MRP1 and GST A1-1 conferred approximately 4-fold resistance to the anticancer drug chlorambucil. Expression of MRP1 alone failed to confer resistance to chlorambucil, showing that the observed protection from chlorambucil cytotoxicity was absolutely dependent upon GST A1-1 protein. Moreover, using inhibitors of GST (dicumarol) or MRP1 (sulfinpyrazone), it was shown that in MCF7 cells resistance to chlorambucil requires both intact MRP1-dependent efflux pump activity and, for full protection, GST A1-1 catalytic activity. These results are the first demonstration that GST A1-1 and MRP1 can act in synergy to protect cells from the cytotoxicity of a nitrogen mustard, chlorambucil.
Highlights
To examine the role of multidrug resistance protein 1 (MRP1) and glutathione S-transferases (GSTs) in cellular resistance to antineoplastic drugs, derivatives of MCF7 breast carcinoma cells were developed that express MRP1 in combination with one of three human cytosolic isozymes of GST
Characterization of Cell Lines—MCF7 breast carcinoma cells were chosen to study the effects of expression of MRP1 alone or in combination with isozymes of GST
Our results establish that MRP1 and GST A1-1 act in synergy to confer resistance to chlorambucil in MCF7 cells
Summary
Drugs and Chemicals—Mitoxantrone, 1,3-bis(2-chloroethyl)-1-nitrosourea, thiotepa, and hepsulfan were provided by the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program of the NCI, National Institutes of Health (Bethesda, MD). Melphalan, 1,3-bis(2-chloroethyl)-1-nitrosourea, thiotepa, and hepsulfan, cells were exposed to drugs or vehicle for 1 h in either serum-free DMEM or DMEM supplemented with 1% fetal calf serum. Enzyme kinetic and in vitro inhibition analyses were accomplished using affinity-purified, recombinant human GST A1-1 For these assays, CDNB served as the varied substrate, and glutathione concentration was fixed at 2 mM. 24 h later, cells were incubated in DMEM plus 1% fetal calf serum Ϯ 2 mM sulfinpyrazone or in HBSS Ϯ 0.1 mM dicumarol at 37 °C in 5% CO2. Cells were washed and incubated in HBSS equilibrated to 25 °C containing inhibitor (2 mM sulfinpyrazone or 0.1 mM dicumarol) or vehicle plus 10 M CDNB. Relative in vivo GST activities were determined as the rate of total DNP-SG formation (DNP-SG in medium plus cell lysates) for 2 and 5 min at 25 °C. The levels of DNP-SG formation increased linearly with time throughout the 5 min incubation periods
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