| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Experimental Therapeutics |
Wadsworth Center, New York State Department of Health, Albany, New York 12201 [E. L. V., K. R., M. R., E. S.]; Department of Biomedical Sciences, School of Public Health, University at Albany, Albany, New York 12201 [E. L. V., E. S.]; Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263 [J. J. M.]; Greenbaum Cancer Center of the University of Maryland, Baltimore, Maryland 21201 [L. A. D., D. D. R.]; Department of Medicine, Division of Hematology/Oncology, University of Maryland School of Medicine, Baltimore, Maryland 21201 [L. A. D., D. D. R.]; and Baltimore Veterans Medical Center, Department of Veterans Affairs, Baltimore, Maryland 21201 [D. D. R.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-GH activity and/or expression (8)
, and DHFR
overexpression (9)
or mutation (10, 11, 12)
. In contrast, a putative role for drug efflux in MTX resistance, possibly mediated by an active transport mechanism, is less clear. Although evidence for an ATP-dependent MTX efflux mechanism has accumulated over the years (13, 14, 15, 16, 17) , its molecular identity has not yet been established. Recently, loss of a folate efflux pump was implicated in resistance to the antifolate pyrimethamine, although the effect seems to be indirect through aberrations in the natural folate pools (18) . No evidence for the overexpression of a MTX-specific efflux protein as the cause of MTX resistance has thus far been reported.
A large number of ABC transport proteins have been identified (19 , 20) . These proteins comprise an ever-growing family of efflux pumps, some of which have been implicated in the MDR phenotype. MDR occurs when cells selected for resistance to one compound develop cross-resistance to other structurally and functionally unrelated drugs. The prototype of this class of proteins, Pgp, was first identified by Juliano and Ling (21) in 1976, and was found to produce resistance to drugs such as doxorubicin, MX, Taxol, and vincristine through its action as a drug efflux pump. Overexpression of Pgp was also shown to be associated with MTX resistance in CEM/MTX cells (22) , although cross-resistance to MTX does not appear to be a universal characteristic of cells that overexpress Pgp/MDR1 (23) . Another group of transporters found to be involved in the MDR phenotype are the MRPs (24) , some of which were shown to efflux various compounds conjugated to glutathione, glucuronide, or sulfate (25) and to cause resistance to compounds such as doxorubicin, VP-16, and vincristine (26) . However, cross-resistance to MTX has not been shown. Recently, several reports have been published that demonstrate the potential for some of the MRP family members to actively transport MTX and to cause resistance (27 , 28) , albeit only under certain specific conditions, and, hence, their role in acquired MTX resistance remains unclear.
Here, we investigated the cause of MTX cross-resistance in MCF7 breast cancer cells selected in the presence of 600 nM MX (MCF7/MX), a topoisomerase II inhibitor. These cells exhibit a typical MDR phenotype in that they display an ATP-dependent drug accumulation defect and are highly resistant to not only the drug for which they were selected (MX) but also to other structurally unrelated drugs such as the camptothecin derivatives TPT and CPT-11 (29) . MX resistance in MCF7/MX cells is attributable to the overexpression of the recently identified ABC half-transporter BCRP (30 , 31) , which has also been called MX-resistance protein (MXR; Ref. 32 ) or placental ABC protein (ABC-P; Ref. 33 ). In contrast, MTX resistance in these cells has yet to be characterized. In the present study, we further define this cell line by examining classical mechanisms of MTX resistance as well as the potential role of ATP-dependent drug efflux as the cause of this unusual cross-resistance.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cytotoxicity Assay.
Drugs were stored at -20°C or at 4°C as the following stock
solutions: 6 mM MX in water (Sigma, St. Louis, MO), 10
mM MTX in slightly basic sodium phosphate (Sigma), 100
mM VP-16 in DMSO (Sigma), 10 mM TPT in
water (Smith Kline Beecham Pharmaceuticals, King of Prussia, PA), 18
mM DDATHF in DMSO (Eli Lilly and Company, Indianapolis,
IN), 54 mM trimetrexate in DMSO (provided by Drs. Fry and
Jackson of Warner Lambert Laboratory, Ann Arbor, MI), 115
µM 5-fluorouracil in DMSO (Sigma), 27
mM metoprine in DMSO (Burroughs Wellcome, Research
Triangle, NC), 20 mM AG337 in DMSO (Aguron), and 5.4
mM Tomudex in water (ZD1694; Zeneca Pharmaceuticals,
Wilmington, DE). Trypsinized cells (0.1 ml/well) were seeded in 96-well
microtiter plates at a concentration of 2,500 cells/ml for MCF7/WT;
5,000 cells/ml for MCF7/MX and MCF7/VP; and 10,000 cells/ml for
MCF7/MTX, MCF7/BCRP, and MDA-MB231/BCRP. After 24 h of incubation
at 37°C, cells were treated by adding 0.1 ml of drug solution with
increasing concentration. Corresponding controls received medium
instead of drug solution. Toxicity of the drugs was determined
by sulforhodamine B (SRB) assay as described previously (36
, 37)
.
Cell Membrane Preparation.
Cells were seeded at a density of 1,000,000 cells/10-cm dish. After
72 h, or at 70% confluence, cells were washed twice with PBS and
then scraped into PBS. Samples were sonicated (three 10-s bursts at
maximal setting), followed by centrifugation for 10 min at
1000 x g at 4°C. The supernatant was
diluted 1:4 into 10 mM Tris/HCl (pH 7.5) and 250
mM sucrose, and the membrane proteins were
sedimented by centrifugation at 100,000 x g
for 30 min. The pellet was dissolved in 62.5 mM
Tris/HCl (pH 6.8) and 1% CHAPS, sonicated (three 10-s bursts at
maximal setting), and solubilized for 30 min on ice. An equal volume of
2x sample buffer [0.1 M Tris/HCl (pH 6.8) and
4% DTT] was added. The protein concentration was measured by Bradford
assay (38)
. Samples were dispensed into aliquots and
stored at -80°C.
Western Blotting.
Western blots were generated using 100 µg of whole cell or
cytoplasmic cell extracts or 50 µg of cell membrane preparations.
Proteins were fractionated on polyacrylamide running gels (12% for
DHFR, RFC, and -GH and 7.5% for MRPs) with a 4% stacking gel
containing 8 M urea. Multimark (Novex, San Diego, CA)
and/or broad range markers (Bio-Rad, Hercules, CA) were used as
molecular weight standards. Proteins were transferred to a
polyvinylidene difluoride membrane (Millipore, Bedford, MA)
using wet electrophoretic transfer. TBST [20 mM Tris/HCl
(pH 7.5), 0.137 M NaCl, and 0.01% Tween 20] was
used for washing the blot. The nonspecific binding sites were blocked
with Blotto (5% nonfat milk in TBST) overnight. The membrane was then
incubated with primary antibody for 2 h or overnight and with the
appropriate secondary antibody for 1 h. Chemiluminescence using
SuperSignal Substrate (Pierce, Rockford, IL) was used for
detection. To strip the blot, the membrane was incubated in stripping
buffer [62.5 mM Tris/HCl (pH 6.8), 0.2% SDS, and 0.69%
ß-mercaptoethanol) for 30 min at 65°C, followed by brief washing in
TBST. The following antibodies were obtained: anti-DHFR (Research
Diagnostics Inc., Flanders, NJ); anti--GH (Dr. Thomas Ryan,
Wadsworth Center, Albany, NY; Ref. 39
); anti-RFC1 (Dr.
Jeffrey Moscow, University of Kentucky Medical Center, Lexington, KY;
Ref. 40
); anti-MRP1, -2, -3, and -5 (Dr. Rik Scheper,
Department of Pathology, Free Hospital, Amsterdam, the
Netherlands); and anti-MRP4 (Dr. John Schuetz, St. Judes
Children’s Research Hospital, Memphis, TN). SDS-Page and Western blots
for FPGS were performed as described previously (41)
.
-GH Activity Assay.
Cells were grown for 72 h and were then extracted with 0.1
M Tris/HCl (pH 6.0) containing 0.1% Triton X-100, 0.1
M 2-mercaptoethanol, and protease inhibitors. The
supernatant was used for -GH assays with 100 µM
MTXGlu3
(4-NH2-10-CH3PteGlu3)
as substrate and 15 min of incubation at 37°C (42)
.
Activity is expressed as nmol/min/mg protein.
DHFR Activity Assay.
DHFR activity was assayed by the modified method of Mathews and
Huennekens (43)
. The cell extracts were incubated in the
presence of 0.066 mM dihydrofolate
(FH2) and 0.1 mM NADPH in 0.05
M potassium phosphate (pH 7.0)/0.1 M KCl
buffer at 30°C. The decrease of the absorbance was traced at 340 nm.
One unit of DHFR activity is defined as µmol of
FH2 reduced to FH4 per min.
FPGS Activity.
FPGS activity was assayed by the previously described method of McGuire
et al. (44)
. Cells were grown for 72 h and
then extracted into 0.5 M Tris-HCl (pH 8.85)/0.2
M 2-mercaptoethanol. Extracts containing FPGS
were incubated at 37°C in the presence of 0.25
mM MTX substrate and 4 mM
[3H]glutamate (specific activity 24 Ci/mmol;
NEN Life Science Products, Boston, MA). Radiolabeled glutamates were
then separated from unincorporated glutamate on DEAE cellulose columns
and were quantitated using LSC. Activity is expressed as nmol
MTX-Glu(n) formed per hour per mg protein.
MTX Accumulation.
Steady-state MTX accumulation was measured by growing MCF7/WT and
MCF7/MX cells for 96 h, followed by incubation in serum-free
IMEM for an additional 24 h. At this time, 2
µM [3H]MTX (specific activity
1.89 x 105 dpm/nmol; Moravek
Biochemicals, Brea, CA) was added for 3, 6, and 24 h. After the
cells were lysed and neutralized, total MTX accumulation was measured
by LSC.
Short-term MTX accumulation in MCF7/WT and MCF7/MX cells was determined in cells grown in 6-well plates to 70% confluency, that were then washed in prewarmed PBS (containing 2 mM each CaCl2 and MgCl2, without EDTA) and incubated for 15 min in either ATP-sustaining control medium (1 g/liter glucose in PBS) or ATP-depletion medium (50 mM deoxyglucose, 15 mM sodium azide, and 1 mM dinitrophenol). Cells were then exposed to 10 µM [3H]MTX (specific activity 2.2 x 105 dpm/nmol) in either control ATP-sustaining conditions or ATP-depleting conditions for 0, 2, 5, 10, 20, and 30 min. Three successive washes in ice-cold PBS were used to stop drug accumulation. Cells were lysed overnight in 0.2 N NaOH and were then neutralized with 0.2 N HCl, and radioactivity in the cells was quantitated by LSC. Values were normalized to total protein content to determine pmol MTX accumulated per mg cell protein.
The kinetics of MTX accumulation was assayed in MCF7/WT and MCF7/MX cells as follows. Cells were grown to confluency in 6-well plates and were washed once in prewarmed PBS, followed by incubation for 2 min in [3H]MTX ranging in concentration from 0.5 to 10 µM. As previously described, uptake was terminated by washing the plates three times in ice-cold PBS, and radioactivity in the cell lysates was counted by LSC. Values were normalized to protein content, and uptake velocity was calculated as pmol/mg/min. These values along with MTX concentration were plotted as a double reciprocal Lineweaver-Burke plot, and approximate Kt and Vmax values were calculated from linear regression curves.
MTX Efflux.
Six-well plates containing cells at 70% confluency were washed in PBS
and incubated for 15 min in either ATP-sustaining control medium (1
g/liter glucose in PBS) or ATP-depletion medium (50 mM
deoxyglucose, 15 mM sodium azide, 1 mM
dinitrophenol). Cells were then loaded with 10 µM
[3H]MTX in either control or depletion medium
for 20 min. Efflux was examined by replacing the drug solution with the
corresponding drug-free medium for 0, 2, 5, 10, 20, and 30 min of
efflux at 37°C. The cells were then washed in ice-cold PBS, lysed,
the remaining radioactivity was determined by LSC, and the protein
concentration was determined as previously described.
The pool of noneffluxable MTX was determined by depleting cells of folate by incubating in folate-free RPMI for 24 h followed by incubation in 2 µM [3H]MTX for an additional 24 h. Cells were then washed in ice-cold PBS and either immediately lysed yielding total MTX values or incubated in prewarmed folate-free RPMI in the absence of drug for a 1-h efflux period, which gave measurements of the noneffluxable MTX pool. After cell lysis, radioactivity was determined by LSC.
MTX Polyglutamylation.
The long-term polyglutamate profile of MTX was measured after 24 h
of exposure to 2 µM [3H]MTX.
Polyglutamates were extracted, separated by high-performance liquid
chromatography and quantitated using LSC as described previously
(8)
.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, a drug not normally associated with MDR phenotypes. To elucidate the
mechanism for this unusual cross-resistance, we tested several
additional antifolate drugs with different modes of entry and different
intracellular targets for their cytotoxic effects on MCF7/MX and
parental MCF7/WT cells (Table 1)
. As controls, we used MTX-selected
MCF7/MTX and etoposide-selected MCF7/VP cells, which are known to
overexpress DHFR and MRP1, respectively (35
, 36)
. As shown
previously, MCF7/MX cells were highly resistant to the
anti-topoisomerase II drug MX and the anti-topoisomerase I drug TPT and
somewhat cross-resistant to another anti-topoisomerase II drug, VP-16.
As expected, MCF7/MTX cells were highly resistant to MTX but,
surprisingly, also showed some cross-resistance to both MX and VP-16,
whereas MCF7/VP cells were resistant to VP-16 and MX. In contrast, only
little, if any, cross-resistance was observed with the other antifolate
drugs tested, with the exception of an approximately 5-fold resistance
to AG337 in MCF7/MX cells. Thus, it seems that the mechanism
responsible for the MTX cross-resistance in MCF7/MX cells is rather
specific for this particular antifolate.
|
. As expected (35)
, 22-fold higher DHFR activity was found
in MCF7/MTX cells. In contrast, no substantial difference could be
measured in the DHFR activity of MCF7/MX and MCF7/VP cells compared
with MCF7/WT cells. These results were confirmed by Western blot using
a polyclonal anti-DHFR antibody (Fig. 1)
. Neither in MCF7/MX cells nor in MCF7/VP cells could a substantial
difference be detected in the expression level of the 22 kDa DHFR
protein compared with MCF7/WT cells, whereas MCF7/MTX cells showed
clear overexpression of DHFR as expected. Thus, it seemed unlikely that
MTX cross-resistance in MCF7/MX cells was attributable to DHFR
overexpression.
|
|
, steady-state levels of total MTX in MCF7/MX cells were
one-half to one-third of those in the parental cells, and short-term
MTX accumulation was also decreased in MCF7/MX cells relative to
MCF7/WT cells (Fig. 2B)
. After 20 min of MTX exposure,
accumulation was approximately 30% lower in the resistant cell line.
This reduction seemed to be ATP dependent; depletion of ATP in MCF7/MX
cells resulted in increased drug accumulation to levels comparable with
those in MCF7/WT cells, whereas little effect on MTX accumulation was
seen in the parental cell line. To determine whether the differences in
accumulation were caused by alterations in the initial rate of MTX
uptake, cells were exposed to various drug concentrations for 2 min.
Approximate Kt and
Vmax values were inferred from the
double reciprocal plot of initial uptake rate and are as follows (Fig. 2C)
: the
KtWT = 6.8 ± 1.5 µM;
KtMX = 5.4 ± 2.3 µM;
VmaxWT = 17.0 ± 0.9 pmol/mg/min; and
VmaxMX = 16.6 ± 2.8 pmol/mg/min. Thus, it appears that the reduced MTX
accumulation observed in MCF7/MX cells is not likely to be attributable
to differences in drug uptake because parental and resistant cells
exhibited very similar kinetic profiles.
|
. RFC1, a transporter with a molecular
mass of 58 kDa, appeared slightly overexpressed in the drug-resistant
MCF7 sublines tested compared with MCF7/WT and, therefore, did not seem
to be involved in the reduced drug accumulation and MTX
cross-resistance found in the MX-selected cells.
MTX Polyglutamylation.
Decreased polyglutamylation leading to decreased drug retention has
been demonstrated to cause MTX resistance. Therefore, we analyzed the
polyglutamylation pattern of MTX at 24 h and found that the
distribution of the various MTX-polyglutamate species was different in
MCF7/WT and MCF7/MX cells (Fig. 3A)
. Although in MCF7/WT cells the amount of each of the
MTX-Glu2–6 species was approximately equal, in
MCF7/MX cells, there was a gradual decrease in long-chain
MTX-Glu(n) forms, with no detectable levels of
MTX-Glu5 or -Glu6. To
further examine the possible role of MTX polyglutamylation in MTX
resistance, we measured the noneffluxable MTX pools in both MCF7/WT and
MCF7/MX cells. After a 24-h exposure to 2 µM
MTX, the fraction of noneffluxable drug in MCF7/MX compared with
MCF7/WT cells was significantly smaller (Fig. 3B)
. Thus,
both the absence of long-chain polyglutamates as well as the decreased
pool of noneffluxable MTX in the resistant relative to the parental
cells are consistent with altered polyglutamylation playing a role in
the accumulation defect associated with MTX resistance in MCF7/MX
cells.
|
-GH.-GH. FPGS
catalyzes the addition of glutamates, whereas -GH catalyzes their
removal. Thus, reduced FPGS and/or increased -GH activity could be
responsible for the low levels of long-chain MTX-Glu formation.
Therefore, the activities of both FPGS and -GH were measured (Table 2)
. Cell extracts from MCF7/MX cells showed no significant difference
in either FPGS or -GH activity compared with MCF7/WT cells. In
contrast, -GH activity was slightly increased (1.7-fold) and FPGS
activity was slightly decreased in the MTX resistant cell line
MCF7/MTX, whereas -GH activity in the MCF7/VP cells was
substantially lower. These results were confirmed by Western blot using
polyclonal antibodies against the respective human proteins (Fig. 1)
.
MCF7/MX and MCF7/WT cells showed little difference in the expression
levels of FPGS and -GH, whereas there was a slight increase in the
level of -GH in MCF7/MTX cells, in agreement with the activity data.
Interestingly, there appeared to be a clear reduction in -GH protein
in MCF7/VP cells, which was also reflected in the activity. Thus, it
seems unlikely that the reduced formation of MTX polyglutamates in
MCF7/MX cells was attributable to an alteration in these enzymes.
MTX Efflux.
To examine whether the decreased accumulation of MTX observed in
MCF7/MX cells was attributable to enhanced efflux, cells were preloaded
with radiolabeled MTX, washed, and incubated for 0, 2, 5, 10, 20, or 30
min. As shown in Fig. 4
, MCF7/MX cells exhibited increased MTX efflux relative to MCF7/WT
cells. To determine whether this was an energy-dependent phenomenon,
the cells were depleted of ATP before measuring drug efflux. As shown
in Fig. 4
, in the absence of ATP, MCF7/MX cells retained more MTX than
in the presence of ATP, whereas MCF7/WT cells were essentially
unaffected by energy depletion. Under these conditions, there was no
difference in drug efflux between the two cell lines. Thus, these data
suggested the presence of an ATP-dependent efflux mechanism, possibly
an ABC transporter.
|
, the BCRP transfectants were not cross-resistant to MTX; MCF7/BCRP
(clone 8) cells were 0.71-fold resistant relative to the pcDNA3 control
transfectants, whereas MDA-MB231/BCRP (clone 23) cells were 1.45-fold
resistant, differences that were not statistically significant. As
expected, transfected MCF7 and MDA-MB231 cells were 23- and 89-fold,
respectively, resistant to MX. Interestingly, BCRP transfection seemed
to have only a modest effect on TPT sensitivity, an effect that did not
quite reach statistical significance, which raised the question of
whether BCRP can indeed confer cross-resistance to this drug. In
conclusion, these data suggest that BCRP overexpression alone is not
sufficient for MTX cross-resistance in MCF7/MX cells.
|
. However, no evidence for overexpression was found for any of the MRP
proteins in MCF7/MX cells compared with MCF7/WT cells (Fig. 5)
. Thus,
it seems unlikely that these transport proteins are responsible for MTX
resistance in MCF7/MX cells.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Another important feature of MTX metabolism that is involved in
cellular resistance is polyglutamylation (7
, 18)
. The
addition of glutamate residues onto MTX causes the drug to be retained
within the cell at cytotoxic concentrations. Thus, the balance between
the enzyme that catalyzes the addition of glutamates, FPGS, and the
enzyme that functions in the reverse reaction to cleave those glutamate
residues from MTX, -GH, determines the level of MTX
polyglutamylation. Both enzymes have been directly linked to MTX
resistance. Decreased FPGS activity and protein expression were shown
to cause folate analogue resistance in L1210 leukemic cell variants
(7)
. Conversely, H35D cells, a rat hepatoma cell line
selected for DDATHF resistance, were found to exhibit increased
activity of -GH relative to sensitive parental cells
(8)
. Thus, there are multiple mechanisms by which tumor
cells can acquire MTX resistance.
In the present report, we describe MTX cross-resistance in the MX-selected cell line, MCF7/MX, that does not appear to be caused by any of the "classical" mechanisms described above. Overproduction of the target enzyme DHFR does not seem to play a role in MTX resistance in MCF7/MX cells, because both enzyme activity as well as protein expression showed no significant differences between MCF7/MX and MCF7/WT cells. This conclusion is further supported by the lack of cross-resistance to other antifolates that target DHFR, such as metoprine (1.4-fold) and trimetrexate (1.2-fold). Interestingly, MCF7/MTX cells were moderately hypersensitive to trimetrexate, despite the overexpression of DHFR. We have not, however, further investigated the cause for this observation.
The major differences between MCF7/MX and MCF7/WT cells, besides
reduced MTX sensitivity, are the 3-fold reduction in steady-state
accumulation accompanied by reduced formation and accumulation of
long-chain MTX polyglutamates. This suggests that lower intracellular
drug concentration is responsible for MTX resistance. Reduced
accumulation could be attributable to either a quantitative or
qualitative defect in uptake via RFC1, whereas reduced
polyglutamylation could be attributable to alterations in FPGS and/or
-GH. However, neither of these mechanisms seems to play a role.
The RFC is expressed at approximately equal levels in both the MTX-resistant and -sensitive cell lines. In further support of an intact and functional carrier is the lack of cross-resistance of MCF7/MX cells to other RFC1-dependent antifolate drugs such as DDATHF (0.6-fold) and Tomudex (0.2-fold). Moreover, MCF7/MX cells are not more sensitive to trimetrexate, a lipophilic antifolate that does not require RFC1 to gain cellular access. Cells that are MTX-resistant because of RFC1 defects have been shown to be hypersensitive to trimetrexate (50) . The accumulation kinetics data also provide evidence against a role of reduced uptake in MTX resistance because MCF7/WT and MCF7/MX cells have approximately the same transport affinity and uptake velocity for MTX. The slight differences observed between the two cell lines may be attributable to the commencement of efflux activity in the resistant cells.
Both FPGS and -GH proteins were expressed at similar levels relative
to sensitive cells, and, based on activity measurements, there was no
evidence for either decreased FPGS activity or hyperactivity of -GH.
Thus, the reduced formation of MTX polyglutamates in MCF7/MX compared
with MCF7/WT cells does not appear to be attributable to an altered
function of either of those enzymes. This is further supported by the
absence of MCF7/MX cross-resistance to Tomudex (0.2-fold), a TS
inhibitor that is dependent on the glutamylation activity of FPGS for
cytotoxicity.
Taken together, these data indicate that both MTX import and metabolism are intact and functional in MCF7/MX cells. Nevertheless, our data indicate that the critical factor in the MTX resistance of MCF7/MX cells is reduced drug accumulation accompanied by decreased polyglutamylation. This observation is reminiscent of one previously reported by Moscow et al. (52) , who showed that melphalan-selected MelRMCF7 cells displayed MTX cross-resistance attributable to a lack of polyglutamylation associated with a decreased drug accumulation. We show here that the reduced MTX accumulation found in MCF7/MX cells could be attributable to an enhanced efflux that is sensitive to ATP levels, analogous to what has been shown for various natural product drugs. Although MTX is not a classical MDR-drug, our data support the hypothesis that the presence of an ABC transporter that may be specific for MTX is the cause of the cross-resistance in the MCF7/MX cell line. An enhanced efflux mechanism is also consistent with reduced polyglutamylation; if the drug is rapidly removed from the cell, it does not become available as a substrate for FPGS. Here, we demonstrate a rapid drug efflux in which 50% of the drug has been exported within the first 2 min of efflux. Although the rate of efflux is faster than seen for many natural product drugs, it is not without precedent inasmuch as previous studies have shown that VP-16 efflux via MRP1 in MCF7/VP cells occurred at a similarly rapid rate (53 , 54) .
Although it is not a classical mechanism of MTX resistance, there are other reports of ATP-dependent MTX efflux, and ABC transport proteins have been implicated. Data with L1210 cells demonstrated that energy depletion resulted in increased short-term drug uptake and reduced efflux, similar to results reported here (55) . Furthermore, using inside-out vesicles from L1210 cells Schlemmer and Sirotnak (14 , 15) and Saxena and Henderson (17) have demonstrated the existence of an as-yet-unidentified ATP-dependent transporter for MTX. Several known ABC transporters have been shown to transport MTX under certain conditions. Pgp, an efflux pump with broad substrate specificity that is often responsible for the MDR phenotype, has been associated with MTX efflux and resistance in 3T6-C26 and SW-1573/2R10 cells (56 , 57) . Hooijberg et al. (28) described resistance to short-term, but not long-term, exposure to MTX in ovarian carcinoma 2008 cells transfected with MRP1 and -2. Also, Masuda et al. (47) reported that the MRP2 protein is involved in excretion of MTX into bile, and Bakos et al. (48) recently presented evidence from in vitro experiments that MTX was actively transported by both MRP1 and MRP2. Another member of this protein family that has been linked to antifolate resistance is MRP3. LLC-PK1 cells, transfected with MRP3, were found to transport MTX as well as other organic anions (27) and to be resistant to short-term MTX exposure. Interestingly, in each of these reports, transport of only the native form of MTX was shown, whereas its polyglutamylated derivatives did not appear to be substrates for the various MRPs studied. Thus, there are several ABC protein candidates that could cause MTX resistance. The obvious candidate in our cells is BCRP, which was recently shown to be highly overexpressed in MCF7/MX cells and to be responsible for MX resistance (31 , 58) . However, whereas MCF7 and MDA-MB231 cells that were transfected with BCRP are 23-fold and 89-fold resistant, respectively, to MX, these cells did not display the same MTX cross-resistance pattern that was observed in MCF7/MX cells. This suggests that BCRP is not responsible for MTX cross-resistance, although the possibility that the MCF7/MX cells contain a variant form of BCRP cannot be excluded. This is currently under active investigation in our lab. A survey of MDR1 (34) and MRP1–5 expression gave, however, no evidence that any of these proteins are overexpressed in MCF7/MX cells. Furthermore, MTX resistance in these cells was also seen when cells were continuously exposed to the drug for 7 days, conditions under which neither MRP1 nor MRP3 were able to confer resistance (27 , 28) . It is presently unclear why those MRP proteins were able to confer resistance only to short-term MTX exposure, whereas MCF7/MX cells are also resistant to long-term treatment. One possibility is that the efflux pump responsible for the export of MTX in MCF7/MX cells is a more efficient transporter than the aforementioned MRP proteins, such that the majority of the drug has been extruded before polyglutamylation occurs. Furthermore, additional, not yet identified, factors may also be involved. Thus, there is no evidence that any of the known MRP or Pgp proteins cause MTX resistance in the MCF7/MX cells.
In conclusion, MX-selected MCF7/MX cells display an unusual cross-resistance pattern specific to the antifolate MTX. These cells exhibit an accumulation defect caused by an enhanced ATP-dependent efflux. The highly overexpressed BCRP protein does not seem to be involved, and, thus far, we have been unable to associate any of the other transport proteins that have been implicated in MTX transport with resistance. Studies are under way to identify the putative transporter involved, which is expected to provide valuable insight into the cause of MTX resistance in tumor cells.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This work has been supported in part by NIH
Grants CA72455 (to E. S.), CA25933 (to J. G.), and CA43500 (to
J. J. M.); Roswell Park Cancer Institute Core Grant CA16056
(to J. J. M.); and a Veterans Affairs merit review grant (to
D. D. R.). 
2 To whom requests for reprints should be
addressed, at Wadsworth Center, Empire State Plaza, Albany, NY
12201-0509. Phone: (518) 474-2088; Fax: (518) 474-1850; E-mail: schneid{at}wadsworth.org
3 The abbreviations used are: MTX, methotrexate;
ABC, ATP binding cassette; BCRP, breast cancer resistance protein; Pgp,
P-glycoprotein; RFC, reduced folate carrier; TS, thymidylate synthase;
DHFR, dihydrofolate reductase; FPGS, folylpolyglutamate synthetase;
-GH, -glutamyl hydrolase; LSC, liquid scintillation counting;
MDR, multidrug resistance; MRP, multidrug resistance-associated
protein; MX, mitoxantrone; NE, noneffluxable; TPT, topotecan; DDATHF,
5,10-dideazatetrahydrofolate; WT, wild type; VP-16, etoposide.
Received 9/13/99. Accepted 4/28/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-glutamyl hydrolase activity. Cancer Res., 53: 2227-2230, 1993.-glutamyl hydrolase. Mol. Pharmacol., 53: 1040-1046, 1998.-glutamyl hydrolase in vitro. Cancer Res., 51: 3874-3881, 1991.This article has been cited by other articles:
|
|
 |
|
  E Kis, T Nagy, M Jani, E Molnar, J Janossy, O Ujhellyi, K Nemet, K Heredi-Szabo, and P Krajcsi Leflunomide and its metabolite A771726 are high affinity substrates of BCRP: implications for drug resistance Ann Rheum Dis, July 1, 2009; 68(7): 1201 - 1207. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Lagas, C. M. M. van der Kruijssen, K. van de Wetering, J. H. Beijnen, and A. H. Schinkel Transport of Diclofenac by Breast Cancer Resistance Protein (ABCG2) and Stimulation of Multidrug Resistance Protein 2 (ABCC2)-Mediated Drug Transport by Diclofenac and Benzbromarone Drug Metab. Dispos., January 1, 2009; 37(1): 129 - 136. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Glavinas, E. Kis, A. Pal, R. Kovacs, M. Jani, E. Vagi, E. Molnar, S. Bansaghi, Z. Kele, T. Janaky, et al. ABCG2 (Breast Cancer Resistance Protein/Mitoxantrone Resistance-Associated Protein) ATPase Assay: A Useful Tool to Detect Drug-Transporter Interactions Drug Metab. Dispos., September 1, 2007; 35(9): 1533 - 1542. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Takenaka, J. A. Morgan, G. L. Scheffer, M. Adachi, C. F. Stewart, D. Sun, M. Leggas, K. F.K. Ejendal, C. A. Hrycyna, and J. D. Schuetz Substrate Overlap between Mrp4 and Abcg2/Bcrp Affects Purine Analogue Drug Cytotoxicity and Tissue Distribution Cancer Res., July 15, 2007; 67(14): 6965 - 6972. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. K. W. To, Z. Zhan, and S. E. Bates Aberrant Promoter Methylation of the ABCG2 Gene in Renal Carcinoma Mol. Cell. Biol., November 15, 2006; 26(22): 8572 - 8585. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Wang, L. Zhou, A. Gupta, R. R. Vethanayagam, Y. Zhang, J. D. Unadkat, and Q. Mao Regulation of BCRP/ABCG2 expression by progesterone and 17beta-estradiol in human placental BeWo cells Am J Physiol Endocrinol Metab, May 1, 2006; 290(5): E798 - E807. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Shafran, I. Ifergan, E. Bram, G. Jansen, I. Kathmann, G. J. Peters, R. W. Robey, S. E. Bates, and Y. G. Assaraf ABCG2 Harboring the Gly482 Mutation Confers High-Level Resistance to Various Hydrophilic Antifolates Cancer Res., September 15, 2005; 65(18): 8414 - 8422. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.L. R. Ee, X. He, D. D. Ross, and W. T. Beck Modulation of breast cancer resistance protein (BCRP/ABCG2) gene expression using RNA interference Mol. Cancer Ther., December 1, 2004; 3(12): 1577 - 1584. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Breedveld, N. Zelcer, D. Pluim, O. Sonmezer, M. M. Tibben, J. H. Beijnen, A. H. Schinkel, O. van Tellingen, P. Borst, and J. H. M. Schellens Mechanism of the Pharmacokinetic Interaction between Methotrexate and Benzimidazoles: Potential Role for Breast Cancer Resistance Protein in Clinical Drug-Drug Interactions Cancer Res., August 15, 2004; 64(16): 5804 - 5811. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Ifergan, A. Shafran, G. Jansen, J. H. Hooijberg, G. L. Scheffer, and Y. G. Assaraf Folate Deprivation Results in the Loss of Breast Cancer Resistance Protein (BCRP/ABCG2) Expression: A ROLE FOR BCRP IN CELLULAR FOLATE HOMEOSTASIS J. Biol. Chem., June 11, 2004; 279(24): 25527 - 25534. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Nakayama, A. Kanzaki, K. Terada, M. Mutoh, K. Ogawa, T. Sugiyama, S. Takenoshita, K. Itoh, N. Yaegashi, K. Miyazaki, et al. Prognostic Value of the Cu-Transporting ATPase in Ovarian Carcinoma Patients Receiving Cisplatin-Based Chemotherapy Clin. Cancer Res., April 15, 2004; 10(8): 2804 - 2811. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J van der Heijden, M C de Jong, B A C Dijkmans, W F Lems, R Oerlemans, I Kathmann, C G Schalkwijk, G L Scheffer, R J Scheper, and G Jansen Development of sulfasalazine resistance in human T cells induces expression of the multidrug resistance transporter ABCG2 (BCRP) and augmented production of TNF{alpha} Ann Rheum Dis, February 1, 2004; 63(2): 138 - 143. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. L. Volk and E. Schneider Wild-Type Breast Cancer Resistance Protein (BCRP/ABCG2) is a Methotrexate Polyglutamate Transporter Cancer Res., September 1, 2003; 63(17): 5538 - 5543. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Stark, L. Rothem, G. Jansen, G. L. Scheffer, I. D. Goldman, and Y. G. Assaraf Antifolate Resistance Associated with Loss of MRP1 Expression and Function in Chinese Hamster Ovary Cells with Markedly Impaired Export of Folate and Cholate Mol. Pharmacol., August 1, 2003; 64(2): 220 - 227. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nakanishi, J. E. Karp, M. Tan, L. A. Doyle, T. Peters, W. Yang, D. Wei, and D. D. Ross Quantitative Analysis of Breast Cancer Resistance Protein and Cellular Resistance to Flavopiridol in Acute Leukemia Patients Clin. Cancer Res., August 1, 2003; 9(9): 3320 - 3328. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z.-S. Chen, R. W. Robey, M. G. Belinsky, I. Shchaveleva, X.-Q. Ren, Y. Sugimoto, D. D. Ross, S. E. Bates, and G. D. Kruh Transport of Methotrexate, Methotrexate Polyglutamates, and 17{beta}-Estradiol 17-({beta}-D-glucuronide) by ABCG2: Effects of Acquired Mutations at R482 on Methotrexate Transport Cancer Res., July 15, 2003; 63(14): 4048 - 4054. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Allen, S. C. van Dort, M. Buitelaar, O. van Tellingen, and A. H. Schinkel Mouse Breast Cancer Resistance Protein (Bcrp1/Abcg2) Mediates Etoposide Resistance and Transport, but Etoposide Oral Availability Is Limited Primarily by P-glycoprotein Cancer Res., March 15, 2003; 63(6): 1339 - 1344. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. L. Volk, K. M. Farley, Y. Wu, F. Li, R. W. Robey, and E. Schneider Overexpression of Wild-Type Breast Cancer Resistance Protein Mediates Methotrexate Resistance Cancer Res., September 1, 2002; 62(17): 5035 - 5040. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Allen and A. H. Schinkel Multidrug Resistance and Pharmacological Protection Mediated by the Breast Cancer Resistance Protein (BCRP/ABCG2) Mol. Cancer Ther., April 1, 2002; 1(6): 427 - 434. [Full Text] [PDF]
|
|
|
|
|
|
J. Worm, A. F. Kirkin, K. N. Dzhandzhugazyan, and P. Guldberg Methylation-dependent Silencing of the Reduced Folate Carrier Gene in Inherently Methotrexate-resistant Human Breast Cancer Cells J. Biol. Chem., October 19, 2001; 276(43): 39990 - 40000. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Komatani, H. Kotani, Y. Hara, R. Nakagawa, M. Matsumoto, H. Arakawa, and S. Nishimura Identification of Breast Cancer Resistant Protein/Mitoxantrone Resistance/Placenta-Specific, ATP-binding Cassette Transporter as a Transporter of NB-506 and J-107088, Topoisomerase I Inhibitors with an Indolocarbazole Structure Cancer Res., April 1, 2001; 61(7): 2827 - 2832. [Abstract] [Full Text]
|
|
|
|
|
|
J. E. De Larco, B. R. K. Wuertz, J. C. Manivel, and L. T. Furcht Progression and Enhancement of Metastatic Potential after Exposure of Tumor Cells to Chemotherapeutic Agents Cancer Res., April 1, 2001; 61(7): 2857 - 2861. [Abstract] [Full Text]
|
|
|
|
|
|
C. Erlichman, S. A. Boerner, C. G. Hallgren, R. Spieker, X.-Y. Wang, C. D. James, G. L. Scheffer, M. Maliepaard, D. D. Ross, K. C. Bible, et al. The HER Tyrosine Kinase Inhibitor CI1033 Enhances Cytotoxicity of 7-Ethyl-10-hydroxycamptothecin and Topotecan by Inhibiting Breast Cancer Resistance Protein-mediated Drug Efflux Cancer Res., January 1, 2001; 61(2): 739 - 748. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
) and MCF7/MX (•) cells. Total
MTX accumulation was measured at 0, 3, 6, and 24 h as described in
"Materials and Methods." B, time course for the
short-term accumulation of [3H] MTX by MCF7/WT (
)
and MCF7/MX (•, 
) cells in the presence (
