Resistance to mitoxantrone in multidrug-resistant MCF7 breast cancer cells: evaluation of mitoxantrone transport and the role of multidrug resistance protein family proteins.

We examined the role of multidrug resistance protein (MRP) 1 (ABCC1) in the emergence of mitoxantrone (MX) cross-resistance in a MCF7 breast cancer cell line selected for resistance to etoposide. The resistant cell line, MCF7/VP, expresses high levels of MRP1, whereas the parental cell line, MCF7/WT, does not. MCF7/VP cells are 6-10-fold cross-resistant to MX when compared with MCF7/WT cells. Drug transport studies in intact MCF7/VP cells revealed that MX resistance is associated with reduced MX accumulation due to enhanced MX efflux. MX efflux is ATP dependent and inhibited by sulfinpyrazone and cyclosporin A. Inhibition of MX efflux with these agents sensitizes cells to MX cytotoxicity and partially reverses MX resistance in MCF7/VP cells. Whereas resistance is partially attributable to increased MX efflux in MRP1-expressing MCF7/VP cells, we found no evidence for glutathione or other conjugates of MX in these cells. Moreover, glutathione depletion with buthionine sulfoximine had no effect on MX transport or sensitivity in MCF7/VP cells. MRP1 substrates are generally amphiphilic anions such as glutathione conjugates or require the presence of physiological levels of glutathione for MRP1-mediated transport. Therefore we conclude that MRP1 overexpression is unlikely to be responsible for increased MX efflux and resistance in MCF7/VP cells. In considering the potential involvement of other MRP family isoforms, a 3-fold increase in the expression of MRP5 was observed in MCF7/VP cells. However, stable expression of a transduced MRP5 expression vector in MCF7/WT cells failed to confer MX resistance. Because other transporters known to be associated with MX resistance, including P-glycoprotein and BCRP/MXR (ABCG2), are not expressed in MCF7/VP cells, we conclude that increased MX efflux and resistance in MCF7/VP cells is attributable to a novel transport mechanism or that MX represents a novel class of cationic, glutathione-independent MRP1 substrates.

Molecular cloning of the rat TA1/LAT-1/CD98 light chain gene promoter.

The rat LAT-1 (L-amino acid transporter-1) gene is a CD98 light chain highly expressed in cancer and development. As an initial study of the molecular basis underlying regulation of its expression, we cloned 2 kb of the LAT-1 5' flanking region. Inverse RACE and primer extension methods were used to define the transcription initiation site at 80 bp upstream from the translational start site. Functional studies carried out in normal hepatic cells using constructs containing progressive 5' deletion from region -1958 to -185 showed 3-5-fold beta-galactosidase activities over control. The presence of an activator site(s) between -52 and -185 was indicated by low activities conferred by the construct spanning this region.

Coordinated action of glutathione S-transferases (GSTs) and multidrug resistance protein 1 (MRP1) in antineoplastic drug detoxification. Mechanism of GST A1-1- and MRP1-associated resistance to chlorambucil in MCF7 breast carcinoma cells.

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.

Detoxification of 1-chloro-2,4-dinitrobenzene in MCF7 breast cancer cells expressing glutathione S-transferase P1-1 and/or multidrug resistance protein 1.

We examined the roles of glutathione S-transferase (GST) P1-1 and the glutathione S-conjugate (GS-X) transporter, multidrug resistance protein 1 (MRP1), singly or in combination, in the detoxification of 1-chloro-2,4-dinitrobenzene (CDNB). Derivatives of MCF7 breast carcinoma cells expressing GST P1-1 and MRP1 alone or in combination were developed. Detoxification was measured in cells as formation of the glutathione conjugate of CDNB, S-(2,4-dinitrophenyl)-glutathione (DNP-SG), efflux of DNP-SG, and ultimately protection from CDNB cytotoxicity. MRP1 expression in the absence of GST P1-1 confers a three- to fourfold resistance to CDNB, which is associated with a >10-fold increase in the maximum rate of DNP-SG efflux. DNP-SG efflux in MRP1-expressing MCF7 cells was ATP-dependent and exhibited an apparent Km for DNP-SG of 95 microM. MRP1 expression alone, however, had no effect on DNP-SG formation. Combined expression of GST P1-1 and MRP1 increased the rates of DNP-SG formation when cells were exposed to 10 microM CDNB. Moreover, combined expression of GSTP1-1 with MRP1 moderately augmented MRP1-mediated resistance to CDNB but only during short term (10 min) exposures to CDNB where IC50 values were in the 8-10 microM range. In contrast, expression of GST P1-1 in the absence of MRP1 slightly sensitized cells to the toxicity of CDNB (10 min exposures), despite increasing rates of DNP-SG formation. The sensitization to CDNB in cells expressing GST P1-1 alone was associated with increased intracellular accumulation of DNP-SG, indicating that DNP-SG may contribute to CDNB toxicity. The potential toxicity of DNP-SG is also suggested by the finding that inhibition of DNP-SG formation by prior glutathione depletion confers resistance to CDNB cytotoxicity in MRP1-poor MCF7 cells. Altogether, our results demonstrate that glutathione conjugation and MRP1-mediated conjugate efflux can operate together to confer resistance to CDNB. The data indicate that MRP1-mediated conjugate efflux is required for cytoprotection from CDNB because its conjugate (DNP-SG), when present at high intracellular levels, may also be toxic to cells.

High-molecular-weight polypeptide substrates for phospholysine phosphatases.

The detection and characterization of the phosphatases responsible for the dephosphorylation of N-phosphoryl groups, such as phospholysine, in proteins has been frustrated in large measure by a lack of suitable substrates for the assay of these enzymes. Herein we describe the preparation of phospholysine-containing amino acid homo- and heteropolymers by chemical means, and their use in a simple, nonradioactive assay for phospholysine phosphatase activity.