From the Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
Received for publication, October 16, 2000, and in revised form, December 1, 2000
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ABSTRACT |
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We investigated the role of phase II
(conjugation) and phase III (efflux) detoxification of the anticancer
drugs melphalan (MLP) and chlorambucil (CHB). Although both
drugs are substrates of Alpha-class glutathione
S-transferases (GST) and the monoglutathionyl conjugates
formed in these enzymatic reactions are transported by MRP1, we found
that GSTA1-1 and MRP1 acted in synergy to confer resistance to CHB but
not to MLP (Morrow, C. S., Smitherman, P. K., Diah, S. K., Schneider, E., and Townsend, A. J. (1998) J. Biol.
Chem. 273, 20114-20120). To explain this selectivity of MRP1/GST-mediated resistance, we report results of side-by-side experiments comparing the kinetics of MLP- versus
CHB-glutathione conjugate: formation, product inhibition of GSTA1-1
catalysis, and transport by MRP1. The monoglutathionyl conjugate of
CHB, CHB-SG, is a very strong competitive inhibitor of GSTA1-1
(Ki 0.14 µM) that is >30-fold more
potent than that of the corresponding conjugate of MLP, MLP-SG
(Ki 4.7 µM). The efficiency of
GSTA1-1-mediated monoglutathionyl conjugate formation is more than
4-fold higher for CHB than MLP. Lastly, both CHB-SG and MLP-SG are
efficiently transported by MRP1 with similar
Vmax although the Km for
CHB-SG (0.37 µM) is significantly lower than for MLP-SG
(1.1 µM). These results indicate that MRP1 is required for GSTA1-1-mediated resistance to CHB in order to relieve potent product inhibition of the enzyme by intracellular CHB-SG formed. The
kinetic properties of MRP1 are well suited to eliminate CHB-SG at
pharmacologically relevant concentrations. For MLP detoxification, where product inhibition of GSTA1-1 is less important, GSTA1-1 does not
confer resistance because of the relatively poorer catalytic efficiency
of MLP-SG formation. Similar analyses can be useful for
predicting the pharmacological and toxicological consequences of MRP
and GST expression on cellular sensitivity to various other electrophilic xenobiotics.
Our laboratory has studied the role in xenobiotic detoxification
of coordinated phase II conjugation reactions and phase III efflux
transport. Among the phase II enzymes, we have been particularly interested in the glutathione S-transferases
(GST),1 a family of enzymes
that catalyze the conjugation of glutathione with a variety of
electrophilic toxins including cancer drugs, carcinogens, and other
xenobiotics (1-4).
Although these conjugation reactions generally render the compound less
chemically reactive and hence less toxic, the overexpression of the
various isozymes of GST is frequently insufficient to confer significant protection from the cyto- and genotoxicities of these electrophiles. Indeed, we have shown that coexpression with GST of the
glutathione conjugate efflux transporters, MRP1 or MRP2, is necessary
to potentiate GST-mediated protection from the toxicities of the cancer
drug chlorambucil (CHB) or the carcinogen 4-nitroquinoline 1-oxide
(5-7). In these and other studies, high intracellular accumulation of
the toxin-glutathione conjugate formed in the absence of MRP1 (or MRP2)
is associated with increased toxicity whereas low intracellular
toxin-conjugate accumulation resulting from MRP-dependent
conjugate efflux is associated with cellular resistance (5, 7, 8).
These results indicate that glutathione conjugates, especially when
they accumulate to high intracellular levels, may themselves be
directly or indirectly toxic. Rarely, the glutathione conjugate may be
more reactive than the parent compound resulting in increased conjugate
toxicity (9-11). However, in most cases, the conjugate is less
reactive than the parent compound. Thus for the majority of glutathione
conjugates, the basis for any apparent conjugate toxicity is unknown
but may involve residual conjugate reactivity, product inhibition of
GST by the conjugate (12, 13), or novel toxicities of the conjugate
when present at high intracellular concentrations.
Previously we showed that expression of both GSTA1-1 and MRP1 are
required to confer resistance to CHB; expression of GSTA1-1 or MRP1
alone afforded no protection from CHB cytotoxicity (6). However,
combined expression of GSTA1-1 and MRP1 failed to confer protection
from the cytotoxicity of the related drug, melphalan (MLP). This was
surprising because both of these structurally similar drugs (Fig.
1) are reportedly substrates of
Alpha-class GST (12, 14-17), and because their monoglutathionyl
conjugates are transported by MRP1 (18). To explain the basis for this selectivity of MRP/GST-mediated resistance, here we report the results
of side-by-side experiments designed to compare the kinetics of CHB-
versus MLP-glutathione conjugate: formation, inhibition of
GSTA1-1, and transport by MRP1. Other investigators have shown that MLP
and CHB are substrates of Alpha-class GST, but to our knowledge no
direct comparisons of the kinetic constants of purified GSTA1-1 toward
the two substrates have been reported. Whereas, the monoglutathionyl
conjugate of CHB, CHB-SG, is known to inhibit Alpha-class GST (12),
again no direct comparison between inhibition by CHB-SG and the
corresponding conjugate of MLP, MLP-SG, has been reported. Finally,
previous studies on MRP1-mediated MLP-SG and CHB-SG transport did not
report kinetic constants for the transport of these two compounds
(18).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structures of chlorambucil
(CHB) and melphalan (MLP).
Our results show that the kinetic parameters for CHB-SG and MLP-SG
formation, efflux, and GSTA1-1 inhibition can explain the selectivity
of GST/MRP1-mediated resistance synergy toward CHB versus
MLP. The data indicate that the most important toxicity of CHB-SG is
indirect and involves the inhibition of GSTA1-1 catalysis, which limits
further drug detoxification.
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EXPERIMENTAL PROCEDURES |
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Cell Lines, Culture, and Cytotoxicity Determinations--
All
cell lines were derived from parental MCF7/WT human breast carcinoma
cells. MCF7/WT cells have low GST activity, no detectable GSTA1-1, and
extremely low level MRP1 expression. The multidrug resistant variant,
MCF7/VP, expresses high level MRP1 but again has low GST activity and
no GSTA1-1 expression. Enforced expression of GSTA1-1 was accomplished
by stable transfection of a GSTA1 expression vector into MCF7/VP cells
to generate MCF7/VP-a derivative expressing high levels of both
GSTA1-1 and MRP1. Development, characterization, and culture of these
cells has been described previously (6, 19).
Cytotoxicity was determined using the sulforhodamine B microtiter plate
method described previously (6, 20). Exposure to varying concentrations
of drugs, CHB or MLP (Sigma), was for 1 h in medium supplemented
with 1% fetal calf serum. Stock solutions of drugs were stored at
80 °C as 100 mM CHB in 95% ethanol and 16.4 mM MLP in acidified ethanol (~0.8 N HCl in 95% ethanol).
Preparation of Glutathione Conjugates of MLP and CHB-- Monoglutathionyl derivatives of CHB and MLP were prepared in 1-5-ml reactions containing 0.13 mM CHB or MLP, 10 mM glutathione (Sigma), 0.1 M sodium phosphate, pH 7.5, and 0.2 M NaCl. Mixtures were incubated for 30 min to 1 h at 37 °C, and the reactions were terminated by the addition of perchloric acid to 6.4% (v/v). Samples were loaded onto Waters (Milford, MA) Oasis HLB 3-cc extraction cartridges and washed with 2 ml each of 0.05% (v/v) trifluoroacetic acid and successively higher concentrations of methanol in 0.05% trifluoroacetic acid. MLP-SG eluted in 40% methanol and CHB-SG in 60% methanol. The purity and identity of each conjugate was verified by analytical HPLC (below) and electrospray mass spectrometry (MLP-SG: m/z [M+H]+ 576.09; CHB-SG: m/z [M+H]+ 575.12). Concentrations of conjugate preparations were estimated by analytical HPLC using integrated absorbance peak areas (254 nm) of conjugates compared with CHB and MLP standards of known concentrations.
Radiolabeled conjugates were prepared as described above with the following modifications. Total reaction volumes were reduced to 100 µl; MLP or CHB was added to 1 mM or 3 mM, respectively; and [glycine-2-3H]glutathione (PerkinElmer Life Sciences) was added to 0.51 mM (specific activity: 0.98 mCi/µmole). Monoglutathionyl conjugates were purified by HPLC using a C18 reverse phase column (Beckman) and a solvent system consisting of a 90-min linear gradient of 5-90% (v/v) methanol in 0.05% trifluoroacetic acid run at 0.5 ml/min. Chromatography was monitored spectrophotometrically at 254 nm with CHB-SG eluting at 57 min and MLP-SG at 41 min. Drug conjugates were dried under nitrogen and dissolved in water just prior to use.
Enzyme Kinetics-- Recombinant human GSTA1-1 was prepared as described previously (6). For the analysis of monoglutathionyl conjugate inhibition of GSTA1-1 activity, an adaptation of the CDNB assay described by Habig et al. (21) was used. Reactions contained 0.1 M potassium phosphate, pH 6.8, nonlimiting concentration (2 mM) glutathione, 0.5-2 mM CDNB (Sigma) as the variable substrate, ± 0.2 µg/ml purified GSTA1-1, and variable concentrations of MLP-SG or CHB-SG. Reactions were initiated at 25 °C by the addition of CDNB, and formation of its conjugate was monitored spectrophotometrically at 340 nm (21). Enzyme-dependent catalysis was determined by subtracting conjugate formed in the absence of GSTA1-1.
GSTA1-1 catalysis of MLP-SG and CHB-SG formation was determined as follows. Reactions contained 0.1 M sodium phosphate, pH 6.5, 140 mM KCl, 2 mM glutathione, ± 0.27 µg/ml recombinant GSTA1-1, and either 50-500 µM CHB or 50-1000 µM MLP as the variable substrate. Reactions were initiated at 25 °C by the addition of CHB or MLP. At 1, 3, 5, 10, and 20 min, aliquots were removed, and reactions were terminated by the addition of perchloric acid to 5% (v/v). Conjugate formation was quantified by analytic HPLC as described above. Enzyme-dependent catalysis was determined by subtracting conjugate formed in the absence of GSTA1-1 from conjugate formed in the presence of GSTA1-1. Kinetic constants were calculated from the initial velocities of enzyme-dependent conjugate formation fitted to the Michaelis-Menten equation using Synergy KaleidaGraph 3.0 software for the Macintosh.
Preparation of Inside-Out Plasma Membrane Vesicles and
Determination of MRP1-dependent Conjugate
Uptake--
Membrane vesicles were prepared by modification of a
method described by Loe and co-workers (22, 23). Frozen cell pellets from ~4 × 108 cells (MRP1
MCF7/WT or MRP1+ MCF7/VP cells) were thawed in 7 ml of the
homogenization mixture (including fresh protease inhibitors) as
described previously (22, 23). Cells were disrupted at 4 °C by
nitrogen cavitation at 1250 psi with constant stirring for 20 min. The
homogenate was centrifuged at 1700 rpm in a Sorvall RT6000 centrifuge
at 4 °C for 15 min. The supernatant was overlaid on a 3-ml sucrose cushion (35% (w/v) in 10 mM Tris, pH 7.5, 1 mM
EDTA). Following centrifugation at 35,000 rpm for 2 h at 4 °C
(Beckman SW41 rotor), the opaque interface was collected, diluted into
5 parts TS (10 mM Tris, pH 7.5, 250 mM
sucrose), and centrifuged at 35,000 rpm for 40 min at 4 °C (Beckman
60 Ti rotor). The pellet was suspended in 1 ml of 50 mM
Tris, pH 7.5, 250 mM sucrose, gently dispersed by 4 passages through a 27-gauge needle, and stored in aliquots at
80 °C. The use of high pressure nitrogen cavitation (24) resulted
in >50% inside-out vesicles as determined by endo- and ecto-enzyme
assays (25, 26).
The kinetics of 3H-labeled conjugate uptake by vesicles was
determined using an adaptation of the membrane rapid filtration method
(27). Briefly, 25-50 µl reaction mixtures contained 50 mM Tris, pH7.5, 10 mM MgCl, 250 mM
sucrose, either 4 mM ATP or 4 mM
,
-methyleneadenosine 5'-triphosphate (nonhydrolyzable ATP control), and varying concentrations of [3H]MLP-SG or
[3H]CHB-SG. Mixtures were warmed to 37 °C, and
reactions were initiated by addition of membrane vesicles (32 µg/50
µl reaction). At 30-s intervals, 10-µl aliquots were removed, and
reactions were terminated in 1 ml of ice-cold TS. Samples were
immediately filtered with vacuum through 25 mm hydrophilic membrane
filters (GVWP, Millipore), and the retained vesicles were washed twice
with 1 ml of ice-cold TS prior to liquid scintillation counting.
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RESULTS |
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Previous studies demonstrated that coexpression of MRP1 and
GSTA1-1 confers resistance to CHB (6). Additional experiments showed
that both MRP1 and GSTA1-1 are required for resistance; expression of
GSTA1-1 or MRP1 alone was ineffective for cytoprotection. The
cytotoxicities of CHB versus MLP were compared in the MCF7 derivative cell lines expressing (i) neither MRP1 or GSTA1-1 (MCF7/WT), (ii) MRP1 alone (MCF7/VP), or (iii) MRP1 and GSTA1-1 in combination (MCF7/VP). These data, shown in Fig.
2, revealed that whereas combined
expression of MRP1 and GSTA1-1 conferred resistance to CHB, combined
expression had no effect on cellular sensitivity to MLP. We
hypothesized that this selectivity of MRP1/GSTA1-1 cytoprotection may
result from quantitative differences in (i) GSTA1-1-mediated MLP-SG
versus CHB-SG conjugate formation, (ii) product inhibition
of GSTA1-1 by MLP-SG versus CHB-SG, or (iii) MLP-SG
versus CHB-SG efflux by MRP1.
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Using purified recombinant GSTA1-1 and CDNB as the variable substrate,
experiments shown in Fig. 3, A
and B) demonstrated that both CHB-SG and MLP-SG are
competitive inhibitors of GSTA1-1. CHB-SG is an exceptionally potent
inhibitor with a Ki of 0.14 µM whereas
MLP-SG is considerably less potent with a Ki of 4.7 µM (Fig. 3C). These data indicate that MRP1
may be required to potentiate GSTA1-1-mediated resistance to CHB in
order to relieve product inhibition of the enzyme by CHB-SG.
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For MLP, product inhibition of GSTA1-1 by MLP-SG is relatively less
important. Thus, it is unclear from these data why GSTA1-1 alone or in
combination with MRP1 did not confer resistance to MLP. To address this
issue, the kinetics of GSTA1-1-mediated formation of the
monoglutathionyl conjugates of MLP versus CHB were examined (Fig. 4). Whereas both compounds are
substrates of GSTA1-1, the Vmax is significantly
higher and the Km lower for CHB than for MLP
resulting in a catalytic efficiency
(Vmax/Km) that is more than
4-fold superior for CHB (Table I). These
data predict that GSTA1-1 should be less efficient in the conjugation and detoxification of MLP; a prediction consistent with the
cytotoxicity data (Fig. 2).
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A comparison of CHB-SG and MLP-SG uptake by isolated inside-out
membrane vesicles revealed that both conjugates are efficiently transported by MRP1 in an ATP-dependent manner (Fig.
5). Initial velocities of MRP1-mediated
transport showed that both conjugates are transported with similar
Vmax, but the Km of CHB-SG transport, 0.37 µM, is considerably lower than the
Km of MLP-SG, 1.14 µM (Fig.
6). The relatively low
Km for CHB-SG transport indicates that it is one of
the better substrates so far reported for MRP1 and that MRP1-mediated
efflux of CHB-SG operates efficiently at low, GSTA1-1 inhibitory,
concentrations of CHB-SG.
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DISCUSSION |
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This report characterizes the roles of phase II (conjugation) and
phase III (efflux) in CHB and MLP detoxification in model MCF7 cell
lines. Both drugs share detoxification pathways that involve GSTA1-1
catalysis of monoglutathionyl conjugate formation and
MRP1-dependent conjugate efflux. Despite these shared
processes, differential protection from drug toxicities is observed in
cells expressing both GSTA1-1 and MRP1. In these MCF7/VP cells,
significant resistance is observed only toward CHB, not MLP (Fig. 2).
The preferential detoxification of CHB over MLP can be primarily
attributed to the superior catalytic efficiency of GSTA1
1 toward CHB
(Fig. 4, Table I). The greater efficiency of MRP1-mediated transport of
CHB-SG may make some additional contribution to the differential protection conferred by GSTA1-1 expressing cells (Fig. 6, Table II).
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The role of MRP1, or other glutathione conjugate efflux transporters, is an important issue in GST-mediated detoxification. GSTs catalyze the conjugation with glutathione of reactive electrophilic centers of several toxic xenobiotics, including MLP and CHB (2). These reactions generally decrease the reactivity and presumably the toxicity of the electrophile. However, as we have also noted for the cyto- and genotoxic compound 4-nitroquinoline 1-oxide, expression of GST alone is insufficient to afford measurable protection from CHB cytotoxicity (5-7). In these studies, GST-mediated resistance required the coexpression of a glutathione conjugate efflux transporter such as MRP1 (CHB and 4-nitroquinoline 1-oxide) or MRP2 (4-nitroquinoline 1-oxide). These results suggest that the glutathione conjugates, which accumulate within the cell in the absence of MRP, have some residual or novel toxicities. Indeed, in the absence of MRP1, GST-expressing MCF7 cell derivatives treated with only micromolar concentrations of CDNB or 4-nitroquinoline 1-oxide can rapidly accumulate millimolar concentrations of the respective glutathione conjugates, S-(2, 4- dinitrophenyl)-glutathione or 4-(glutathione-S-yl)-quinoline 1-oxide (5, 7, 8). Under similar conditions, MRP1-expressing cells are able to maintain very low intracellular levels of these conjugates.
There are at least two plausible mechanisms for the apparent toxicity of CHB-SG in cells lacking MRP1. The first is the potential direct toxicity of this monoglutathionyl conjugate of CHB. GSTA1-1 catalyzes the formation of CHB-SG but leaves the second reactive chloroethyl group intact. Whereas CHB-SG can no longer form DNA strand cross-linkages, it retains the ability to form monovalent adducts with cellular nucleophiles including proteins and nucleic acids. Therefore, it would not be surprising to find that the monoglutathionyl derivative of CHB retained significant cytotoxicity at high intracellular concentrations. Several findings argue that this mechanism contributes relatively little to the CHB toxicity overcome by combined expression of GSTA1-1 and MRP1. First, it is known that finite levels of CHB-SG are formed nonenzymatically, in the absence of GSTA1-1. MRP1 will support efflux of this conjugate (Figs. 5 and 6), yet MRP1 alone does not confer resistance (MCF7/VP, Fig. 2). Indeed, expression of MRP1 alone consistently results in either no change or a modest sensitization to CHB toxicity (6, 19).
The data suggest that a second potential mechanism of CHB-SG toxicity is more important. This mechanism is indirect and involves product inhibition of GSTA1-1. As originally described by Meyer et al. (12) and shown here (Fig. 3, Table I), CHB-SG is a potent inhibitor of GSTA1-1 (Ki 0.14 µM). In contrast, MLP-SG is a much less effective inhibitor of GSTA1-1 (Ki 4.7 µM). Hence, removal of intracellular CHB-SG is particularly critical to maintain GSTA1-1 activity and continued detoxifying conjugation of CHB. MRP1 efficiently supports CHB-SG efflux (Figs. 5 and 6 and Table II). The Km of transport is remarkably low (0.37 µM), which indicates that MRP1-mediated transport is kinetically suited to remove CHB-SG at low, GSTA1-1-inhibitory concentrations of the conjugate.
In summary, MRP1 is essential to potentiate GSTA1-1 detoxification of
CHB in order to relieve product inhibition of this conjugating enzyme.
In contrast to its effect on CHB toxicity, GSTA1-1 is unable to confer
measurable protection from MLP toxicity because of its relatively
poorer efficiency toward this substrate. These studies indicate that
the determination of quantitative differences in the kinetics of phase
II conjugation and phase III efflux processes can be used to accurately
predict the sensitivities of cells and tissues to the toxicities of
some drugs and other xenobiotics.
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ACKNOWLEDGEMENTS |
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We thank Emmanuelle Rocchi and Erasmus Schneider for helpful suggestions on vesicle preparation.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA70338 and CA64579.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
Wake Forest University School of Medicine, Medical Center Blvd.,
Winston-Salem, NC 27157. Tel.: 336-716-9478; Fax: 336-716-7671; E-mail:
cmorrow@wfubmc.edu.
Published, JBC Papers in Press, December 13, 2000, DOI 10.1074/jbc.M009400200
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ABBREVIATIONS |
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The abbreviations used are: GST, GSTA1, and GSTA1-1, glutathione S-transferase, human GST isoform A1 monomer, and A1-1 dimer; CHB and CHB-SG, chlorambucil and monoglutathionyl conjugate of chlorambucil; CDNB, 1-chloro-2,4-dinitrobenzene; MLP and MLP-SG, melphalan and monoglutathionyl melphalan; MRP, MRP1, and MRP2, multidrug resistance protein or multidrug resistance-associated protein, MRP isoform 1, MRP isoform 2; HPLC, high performance liquid chromatography; WT, wild-type.
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