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CELLULAR AND MOLECULAR
Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina (P.K.S., A.J.T., C.S.M.); and Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, North Carolina (T.E.K.)
Received July 28, 2003; accepted September 18, 2003.
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Abstract |
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). In particular, we have examined the glutathione (GSH)/glutathione S-transferase (GST)conjugating and the ATP-binding cassette transporter efflux systems. We have shown that, for several drugs, carcinogens, and other toxins, coordinated expression of these systems can effect cellular detoxification that is improved over that of either system alone (Morrow et al., 1998a,b,c, 2000; Paumi et al., 2001).
Although GSTs can catalyze the conjugation with GSH of a variety of electrophilic compounds, rendering these compounds less reactive and generally less toxic, the overexpression of GST alone is often insufficient to afford significant or consistent protection from the cytotoxicities of these electrophilic substrates (Moscow et al., 1989; Leyland-Jones et al., 1991; Townsend et al., 1992; Fields et al., 1994; Hayes and Pulford, 1995). Potential reasons for the failure of GSTs to confer complete protection include reversibility of some conjugation reactions (Witz, 1989), residual, or rarely, increased conjugate toxicities (Van Bladeren, 2000), or product inhibition of GST (Meyer et al., 1992; Meyer, 1993). Previously, our laboratory established that expression of GSTA1-1, which catalyzes the conjugation of GSH with chlorambucil (CHB), confers resistance to CHB in MCF7 cells but only when coexpressed with the ATP-dependent efflux transporter MRP1 (ABCC1) (Morrow et al., 1998b). Subsequent studies suggested that the role for MRP1 in CHB detoxification is to remove the cellular monoglutathionyl conjugate of CHB, CHB-SG, thereby relieving the potent product inhibition of GSTA1-1 by CHB-SG (Paumi et al., 2001).
Work presented herein evaluates the ability of the alternative efflux transporter MRP2 (ABCC2) to also potentiate GSTA1-1-mediated detoxification of CHB-SG. This is important because although MRP1 and MRP2 have considerable substrate overlap, substrate specificities and transport kinetics of common substrates differ (Keppler et al., 1998; Konig et al., 1999; Borst et al., 2000). Moreover, the patterns of cellular and tissue distribution of MRP1 and MRP2 are distinct. MRP1 is ubiquitously expressed and is often overexpressed in drug-resistant tumor cells (Loe et al., 1996; Borst et al., 2000). MRP1 is localized in the plasma membrane and is targeted to the basolateral surface of polarized cells (Mayer et al., 1995; Evers et al., 1996). In contrast, high-level expression of MRP2 is confined to limited tissues such as liver canalicular hepatocytes and renal tubule epithelium where MRP2 is localized on the apical surface of the plasma membrane (Buchler et al., 1996; Schaub et al., 1997). Although MRP2 may be overexpressed in some resistant tumor cells, it is likely to play a more important physiological role in the excretory detoxification of xenobiotics.
In this study, HepG2 cells were used because they target significant levels of MRP2 to the plasma membrane under standard culture conditions yet express negligible MRP1 or cytosolic GST (Jedlitschky et al., 1997; Cantz et al., 2000; Morrow et al., 2000). To evaluate the ability of GSTs to confer alkylating agent resistance when coordinately expressed with MRP2, tetracycline/doxycycline regulatable vectors encoding GSTA1-1, GSTM1a-1a, and GSTP1a-1a were stably introduced into HepG2 cells. To assess the contribution of MRP2 to drug resistance, MRP2-mediated transport function was inhibited by sulfinpyrazone. The validity of this approach was supported by previous studies that showed MRP2 transport function is effectively inhibited by nontoxic doses of sulfinpyrazone (Morrow et al., 2000).
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-3, have been described previously (Morrow et al., 2000).
All other transgenic cell lines were generated by stable transduction of retroviral vectors as described previously (Miller et al., 1993). Briefly, vectors were transiently transfected into PA317 amphotropic packaging cells and the resulting virus-containing supernatants were used to transduce HepG2 and MCF7 cells. Individual clones were selected in either G418 or hygromycin and analyzed separately for transgene expression.
HepG2 cell lines expressing doxycycline-repressible GSTA1-1 (HepG2/
-10 and HepG2/-25) or GSTM1a-1a (HepG2/µ-2 and HepG2/µ-5) were constructed as follows. Parental HepG2 cells were transduced with pRevTet-Off (BD Biosciences Clontech, Palo Alto, CA). After selection in 0.5 mg/ml G418, one clone, HepG2/RTO-21, which expressed particularly high level doxycycline-repressible transactivator activity was chosen for subsequent transductions with GST expression vectors. EcoRI cDNA fragments encoding the human GSTA1-1 (839 bp; Tu and Qian, 1986; Rhoads et al., 1987) or GSTM1a-1a (1142 bp, DeJong et al., 1988) were blunt-end ligated into the HindIII site of pRevTRE (BD Biosciences Clontech). These vectors were introduced into HepG2/RTO-21 cells, selected in 0.4 mg/ml hygromycin, and two clones expressing each GST vector were used.
MCF7 cells stably transduced with MRP1, MCF7/MRP1-10, have been described previously (Paumi et al., 2003). MCF7 cells expressing MRP2 were generated as follows. A 4689-bp cDNA fragment containing the complete coding sequence of MRP2 from the 5' Kozak consensus to the 3' stop codon was amplified from normal human liver RNA reverse transcription-polymerase chain reaction by using avian myeloblastosis virus reverse transcriptase and Expand High Fidelity DNA polymerase (Roche Diagnostics, Indianapolis, IN) (Ausubel et al., 1997). By using polymerase chain reaction-based site-directed mutagenesis, 9 bp encoding the C-terminal three amino acids were deleted. This modification was based upon a previous report suggesting that elimination of these amino acids, while not effecting the transport properties of MRP2, obviates the requirement of cell polarization to target MRP2 to the plasma membrane (Harris et al., 2001). The veracity of the amplified MRP2 sequence was confirmed by DNA sequence analysis (DNA Synthesis Core Laboratory, Wake Forest University, Winston-Salem, NC) and compared with published database sequences (GenBank accession nos. U63970
[GenBank]
, X96395
[GenBank]
, and U49248
[GenBank]
). The modified MRP2 cDNA was inserted into pLNCX and transduced into MCF7 cells. Individual colonies were selected in 1 mg/ml G418, and one expressing high-level membrane-associated MRP2, MCF7/MRP2-15, was used for transport studies.
Additionally, MCF7 cells expressing GSTA1-1 were generated. A 690-bp cDNA fragment encoding human GSTA1-1 from the Kozak consensus sequence through the stop codon was inserted into the ClaI site of pLHCX (BD Biosciences Clontech). This vector was introduced by transduction into parental MCF7/WT and MCF7/MRP1-10 cells. Cells were selected in 0.4 mg/ml hygromycin. Colonies overexpressing GST activity were identified (Habig et al., 1974), and two colonies from each transduced cell line were chosen for cytotoxicity experiments.
Analysis of Transgenic Cell Lines. GST expression in HepG2 cells was examined by Western and Northern blotting as described previously (Morrow et al., 1998b) and by enzyme activity toward 1-chloro-2,4-dintrobenzene (Habig et al., 1974). GST levels were expressed as milliunits of activity (nanomoles of 2,4-dinitrophenyl glutathione formed per minute per milligram of cellular protein). MRP1 and MRP2 expression was ascertained by Northern blot and Western blot analysis of membrane proteins as described previously (Hipfner et al., 1994) using mouse monoclonal antibodies QCRL1 (Hipfner et al., 1994; MRP1) and M2III-6 (Kool et al., 1997; MRP2) purchased from Alexis Biochemicals (Carlsbad, CA).
Additionally, expression of MRP1 and MRP2 was examined by immunocytochemistry. Cells were fixed with 3.7% formaldehyde in PBS (10 min, room temperature), washed with PBS, and blocked with 1% BSA, 0.1% saponin in PBS (10 min). After washing in PBS, cells were incubated for 30 min at room temperature with 1% BSA, 0.1% saponin in PBS containing the primary mouse monoclonal antibodies: QCRL3 (anti-MRP1; Alexis Biochemicals) at 1 µg/ml or M2III-6 (anti-MRP2) at 10 µg/ml. PBS-washed cells were then incubated 30 min with secondary antibody, 25 µg/ml rhodamine-conjugated goat anti-mouse IgG (Immunotech, Hialeah, FL) in 1% BSA, 0.1% saponin, PBS. Cells were postfixed in 3.7% formaldehyde in PBS. Phase contrast and fluorescent images were captured using a Zeiss Axioplan 2 microscope.
Cytotoxicity Determinations. Cells were plated in 96-well plates at a density of 1200 cells/well (HepG2 and derivatives) or 300 cells/well (MCF7 and derivatives). Twenty-four hours later, cells were treated with alkylating agents chlorambucil (Sigma-Aldrich, St. Louis, MO), melphalan (Sigma-Aldrich), or thiotepa (Drug Synthesis and Chemistry Branch, National Cancer Institute, Bethesda, MD), or with vehicle control in serum-free medium for 1 h. Medium was replaced with Dulbecco's modified Eagle's medium plus 10% fetal calf serum. Cells were fixed and stained using sulforhodamine B (Skehan et al., 1990). For some experiments, MRP2 activity was inhibited by the inclusion of 2 mM sulfinpyrazone (Sigma-Aldrich) in serum-free medium added 15 min before alkylating drug exposure and continued for the 1-h period of drug exposure as described previously (Morrow et al., 2000). In other experiments, GST activity was suppressed in transgenic HepG2 cells by prior growth in medium supplemented with 1 µg/ml doxycycline for 7 to 10 days. Doxycycline-suppressed cells were grown without doxycycline for 24 h before plating and cytotoxicity experiments. This treatment completely suppressed GST expression (residual GST activity <5 mU/min · mg cellular protein). Because the IC50 values toward the three alkylating agents were identical in the doxycycline-suppressed transduced cell lines and in parental HepG2 cells, doxycycline-suppressed and parental HepG2 cells were used interchangeably for minus GST controls.
MRP1- and MRP2-Dependent Transport of Monoglutathionyl-Chlorambucil (CHB-SG) in Isolated Membrane Vesicles. Inside-out membrane vesicles were prepared from HepG2, MCF7/MRP1-10, and MCF7/MRP2-15 cells as described previously (Paumi et al., 2001). Uptake of [3H]CHB-SG was determined and kinetic parameters calculated as described previously (Paumi et al., 2001). ATP-dependent transport was calculated by subtracting uptake observed in minus ATP controls containing 4 mM of the nonhydrolyzable analog 
,
-methylene adenosine 5'-triphosphate, from uptake observed in the presence of 4 mM ATP as described.
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; Morrow et al., 2000), HepG2 cells do not express significant levels of MRP1 (Fig. 3A) but do express high levels of MRP2 (Fig. 3B). Moreover, coexpression with GSTA1-1, GSTM1a-1a, or GSTP1a-1a had no effect on MRP2 expression (Fig. 3B). Culture of HepG2 cells in 1 µg/ml doxycycline to suppress transgenic GST activity had no effect on MRP2 expression (not shown).
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Western blot analysis showed that high level expression of membrane-associated MRP1 or MRP2 was achieved in the transduced MCF7 derivatives MCF7/MRP1-10 (Fig. 3A) or MCF7/MRP2–15 (Fig. 3C), respectively. The summary in Table 1 describes all of the cell lines used in the following studies.
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Verification That MRP1, Expressed as a Transgene, Potentiates GSTA1-1-Mediated Resistance to CHB. Previous studies using etoposide-selected MCF7 cells overexpressing MRP1 (MCF7/VP cells) showed that coexpression of MRP1 and GSTA1-1, but not MRP1 or GST alone, conferred significant levels of resistance to CHB cytotoxicity (Morrow et al., 1998b). To eliminate any potential confounding, unrecognized alterations in gene expression that may have occurred during cytotoxic drug selection, we examined the role of MRP1 expressed as a transgene in GSTA1-1-associated resistance to CHB. As shown in Table 2, expression of GSTA1-1 alone (MCF7/7 and MCF7/8) resulted in very little resistance to CHB (1.5–2.3-fold relative resistance). In contrast, coexpression of MRP1 and GSTA1-1 (MRP1-10/2 and MRP1-10/13) conferred considerable resistance to CHB cytotoxicity (5.8–7.1-fold relative resistance). These results confirm the importance of MRP1 in potentiation and augmentation of GSTA1-1-mediated resistance to CHB.
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MRP2 Supports CHB-SG Transport and GSTA1-1-Mediated Resistance to CHB in HepG2 Cells. MRP2 is the dominant MRP-family transporter expressed on the plasma membrane of HepG2 cells (Jedlitschky et al., 1997; Morrow et al., 2000). To determine whether MRP2 could support CHB-SG efflux, uptake of radiolabeled CHB-SG was examined in inside-out membrane vesicles prepared from HepG2 cells. Vesicles derived from HepG2 cells showed substantial ATP-dependent uptake of CHB-SG, whereas MRP-minus control vesicles derived from MCF7/WT cells did not (Fig. 4). To evaluate whether this capacity of MRP2-mediated CHB-SG export present in HepG2 cells was sufficient to support GSTA1-1-dependent resistance to CHB, CHB cytotoxicity was examined in two independent HepG2 clones overexpressing GSTA1-1, HepG2/-10 (70–110 mU of GST/mg), and HepG2/-25 (110–153 mU of GST/mg). Compared with parental HepG2 cells (<5 mU GST/mg), expression of GSTA1-1 in HepG2/-10 and HepG2/-25 cells conferred 3.5- and 4.5-fold resistance, respectively, to CHB cytotoxicity (Fig. 5 and Table 3). That this resistance is dependent upon GSTA1-1 was confirmed by additional experiments in which GSTA1-1 expression was completely suppressed by culture in 1 µg/ml doxycycline. Suppression of GST expression completely reversed CHB resistance, resulting in cytotoxicity profiles indistinguishable from those of parental HepG2 cells (not shown). Moreover, GST-mediated resistance was GSTA1-1 isozyme-specific in that overexpression of GSTP1a-1a (HepG2/-3) or GSTM1a-1a (HepG2/µ-2 and HepG2/µ-5) did not confer resistance to CHB (Table 3).
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The contribution of MRP2 to GSTA1-1-associated CHB resistance was evaluated by the use of sulfinpyrazone to inhibit MRP2. Previous studies showed that 2 mM sulfinpyrazone can inhibit MRP2 transport of glutathione conjugates and does so without significant toxicity (Morrow et al., 2000). As shown in Fig. 6, 2 mM sulfinpyrazone also inhibits MPR2-dependent transport of CHB-SG in isolated membrane vesicles. The inclusion of sulfinpyrazone just before and during the period of exposure to CHB had no effect on the sensitivity of parental HepG2 cells (Fig. 7A). However, sulfinpyrazone strongly sensitized GSTA1-1-expressing HepG2/-10 and HepG2/-25 cells, yielding cytotoxicity profiles that were nearly identical to parental HepG2 control cells (Fig. 7, B and C). These results indicate that CHB resistance in this system is dependent upon the simultaneous expression of both GST and MRP2 activities.
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The ability of GST and MRP2 to influence sensitivities to other alkylating agents was examined. As shown in Table 3, MRP2 expressed in HepG2 cells was unable to support cytoprotection from melphalan or thiotepa toxicities in cells expressing any of the three GST isozymes tested. Thus, resistance conferred by combined GST and MRP2 expression is also specific for the alkylating drug used.
Kinetics of MRP1- versus MRP2-Dependent Transport of CHB-SG. The relative efficacy of MRP1 and MRP2 transport of CHB-SG was compared in MCF7 cells transduced with MRP1 (MCF7/MRP1-10) and MRP2 (MCF7/MRP2-15). Immunocytochemistry revealed that the majority of MRP1 was correctly targeted to the plasma membrane (Fig. 8A, bottom). Neither MRP1 nor MRP2 was detected in parental MCF7/WT cells (Fig. 8, A and B, top). MCF7/MRP2-15 cells were generated using an expression vector containing a C-terminal three-amino acid truncation in an effort to promote plasma membrane targeting in these nonpolarized cells (Harris et al., 2001). Although much of the MRP2 was indeed found associated with the plasma membrane, considerable protein remained in cytoplasmic compartments despite this modification (Fig. 8B, bottom). Nevertheless, inside-out vesicles derived from MCF7/MRP2-15 cells supported robust transport of CHB-SG as did vesicles from MCF7/MRP1-10, but not MCF7/WT, cells (Fig. 9). At the levels of protein expressed, MRP1 and MRP2 transported CHB-SG with similar Vmax (82 and 99 nmol/min · mg, respectively) but with a significantly lower Km value for MRP1 (0.39 µM, MRP1; 10 µM, MRP2). This Km value for MRP1 is very close to the value determined previously (0.37 µM) for MRP1 expressed in the drug-selected MCF7/VP cell line (Paumi et al., 2001).
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).
MRP2/GST detoxification is GST isozyme-specific. GSTA1-1, but not GSTP1a-1a or GSTM1a-1a, can confer resistance to CHB in MRP2-expressing HepG2 cells (Table 3). In contrast, no GST isozyme/MRP2 pair tested conferred resistance to thiotepa or melphalan, alkylating agents that can also form conjugates with glutathione (Dulik et al., 1986; Dulik and Fenselau, 1987; Dirven et al., 1995; Srivastava et al., 1999). Therefore, MRP2/GST resistance is drug-selective. Differences in MRP2-mediated transport of thiotepa- or melphalan-glutathione conjugates may have contributed to the drug selectivity but were not examined in the present study. Alternatively, or additionally, the selectivity of drug resistance may be attributable to differences in the catalytic efficiency of GST-dependent conjugate formation or in the potency of conjugate product inhibition of GST, differences previously observed for GSTA1-1 and its substrates melphalan and CHB (Paumi et al., 2001).
In transgenic MCF7 cells, the apparent efficiency of CHB-SG transport (Vmax/Km) is 
25-fold greater for MRP1 than MRP2, a difference due to the higher Km value of MRP2 for CHB-SG (Fig. 9). Nevertheless, MRP2 effectively potentiates GSTA1-1 resistance in HepG2 cells. This suggests that the level of MRP2 in cells that naturally express the protein is more than sufficient to relieve CHB-SG product inhibition despite the relatively higher Km of transport.
Harris et al. (2001) reported that the C-terminal three amino acids of MRP2 are required for proper targeting of MRP2 to the apical plasma membrane of polarized cells. Although trafficking of MRP2 to the plasma membrane is generally poor in nonpolarized cells, these investigators found that deletion of the C-terminal three amino acids resulted in efficient expression of MRP2 on the plasma membrane of murine L1210 leukemia cells. We attempted to take advantage of this observation to target MRP2 to the plasma membrane of nonpolarized MCF7 cells. Although significant MRP2 associated with the plasma membrane, considerable protein remained in the cytosolic compartments (Fig. 8). The failure to more efficiently target MRP2 to the plasma membrane may represent inherent differences in membrane protein trafficking between MCF7 and L1210 cells. Additionally, more recent findings by Mateus Fernandez et al. (2002) indicate that the N-terminal transmembrane domain of MRP2, a region not modified in the MRP2 protein expressed in MCF7/MRP2-15 cells, is crucial for correct and stable targeting of MRP2. It may be that modification in this N-terminal region would be required to efficiently target MRP2 to the plasma membrane of nonpolarized cells.
In summary, the GSH conjugate efflux activity of MRP2 can support resistance to CHB mediated by GSTA1-1. Although the resistance observed was drug- and GST-isozyme specific, it is likely that a variety of drugs and other xenobiotics are similarly detoxified by various GST isozyme/MRP2 pairs (Morrow et al., 2000). Overexpression of MRP2 has been observed in some cancer cells (Konig et al., 1999) and may therefore play a role in the emergence of resistance to chemotherapeutic drugs, including some alkylating agents. Moreover, high level MRP2 expression is observed on the apical, excretory surfaces of tissues such as the tubular epithelium of kidney and the canalicular hepatocytes of liver (Buchler et al., 1996; Schaub et al., 1997), the latter representing a particularly rich source of several cytosolic isozymes of GST (Hayes and Pulford, 1995). Using the model cell lines developed in our laboratory, we have shown how the interplay between MRP2 and the GSH/GST-conjugating systems can effect detoxification of reactive, electrophilic toxins, including some alkylating agents and other xenobiotics.
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ABBREVIATIONS: GSH, glutathione; GST, glutathione S-transferase; CHB, chlorambucil; MRP, multidrug resistance protein; MRP1 and MRP2, multidrug resistance-associated protein, MRP isoform 1 (ABCC1), MRP isoform 2 (ABCC2); CHB-SG, monoglutathionyl-chlorambucil; bp, base pair(s); PBS, phosphate-buffered saline; BSA, bovine serum albumin.
Address correspondence to: Dr. Charles S. Morrow, Department of Biochemistry, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. E-mail: cmorrow{at}wfubmc.edu
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