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J. Biol. Chem., Vol. 277, Issue 41, 38998-39004, October 11, 2002
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From the Departments of
Received for publication, April 5, 2002, and in revised form, June 24, 2002
The multidrug resistance protein MRP4, a member
of the ATP-binding cassette superfamily, confers resistance to
purine-based antiretroviral agents. However, the antiviral agent
ganciclovir (GCV) has not been shown to be a substrate of MRP4. GCV is
important not only in antiviral therapy, but also in the selective
killing of tumor cells modified to express herpes simplex virus
thymidine kinase (HSV-TK). We therefore tested the effect of MRP4 on
the cytotoxicity of GCV, on the ability of GCV to kill cells
genetically modified to express HSV-TK, and on the bystander effect in
which unmodified target cells are killed by GCV. Cells overexpressing MRP4 had markedly increased resistance to the cytotoxicity of GCV.
Although, expression of recombinant HSV-TK increased the intracellular
concentration of GCV nucleotide, cells were rescued by the
cytoprotective effect of MRP4. In cells that overexpressed MRP4,
intracellular accumulation of GCV metabolites was reduced, efflux of
these metabolites was increased, and resistance to bystander killing
was increased. Therefore, MRP4 can strongly reduce the susceptibility
of HSV-TK-expressing cells to GCV, and its overexpression in adjacent
cells protects them from bystander cell death. These findings indicate
that a nucleotide transporter, such as MRP4, modulates the cellular
response to GCV and thus may influence not only the efficacy of
antiviral therapy, but also prodrug-based gene therapy, which is
critically dependent upon bystander cell killing.
The multidrug resistance proteins
(MRPs)1 are a family of
ATP-binding cassette transporters (ABC transporters; for an overview, see //nutrigene.4t.com/humanabc.html) that mediates drug efflux and multidrug resistance (1). We previously demonstrated that MRP4
(also known as ABCC4) severely reduces the antiviral efficacy of
several nucleoside reverse transcriptase inhibitors, such as zidovudine
(3'-azido-3'-deoxythymidine) (AZT)) and
9-(2-(phosphonomethoxy)ethyl)-adenine (PMEA), in mammalian cells and
that this effect corresponds with increased ATP-dependent
efflux of their nucleotide derivatives (2). These findings were, in
part, replicated by Lee et al. (3). A better understanding
of the role of MRP4 as a nucleotide efflux transporter may lead to
improved nucleoside-based therapies for HIV, herpes viruses, and cancer.
Ganciclovir (9-(1,3-dihydroxy-2-propoxymethyl)guanine (GCV)) is widely
used against cytomegalovirus infection in patients with AIDS
(4-6) but is poorly tolerated in combination with AZT (7). Although
the molecular basis of this interaction is unknown, in vitro
studies indicate that the combination is extremely cytotoxic (8, 9). We
therefore postulated that MRP4 might be involved in the cytotoxicity
induced by the AZT-GCV combination. If so, such an interaction could
have important therapeutic potential because of the widespread use of
GCV as a prodrug in gene therapies for cancer (10, 11). Transduction of
tumor cells with the herpes simplex virus thymidine kinase (HSV-TK)
gene and treatment with GCV is a common anticancer gene therapy
(10-12). HSV-TK specifically converts GCV to its monophosphate
nucleotide form, GCV-monophosphate, which is then further anabolized by
cellular kinases, and accumulation of these metabolites causes cell
death (5, 12). However, the cytotoxicity of this therapy varies
dramatically. The mechanism of this variability is unknown, but it does
not appear to reflect the level of HSV-TK expression (13, 14).
The variable toxicity of GCV to HSV-TK gene-modified tumor cells also
markedly affects the efficacy with which bystander (adjacent, non-transduced) tumor cells are killed (15). In vitro
studies show that bystander cell death is associated with the
accumulation of GCV metabolites (16); however, this effect is variable
and idiosyncratic, and the mechanism accounting for variable
accumulation of these metabolites is unclear (17, 25). Some
investigators believe that direct intercellular communication by gap
junctions is a prerequisite for GCV metabolite transfer (18), while
others do not (19, 20). Other studies suggest that diffusion of GCV metabolites and the proximity of bystander cells are important variables (21-23). Although a combination of these mechanisms is likely, it is unknown if nucleotide efflux plays a role. We reasoned that because the efflux transporter MRP4 transports some natural and
modified nucleotides (2, 24) its expression could impact HSV-TK based
gene therapy by hastening the removal of cytotoxic GCV metabolites.
However, it is unknown if MRP4 plays a role in GCV metabolite
accumulation and cellular egress.
We investigated whether MRP4 affects the cellular accumulation and
cytotoxicity of GCV in cells specifically overexpressing MRP4 and in
cells we engineered to overexpress MRP4. The cDNA encoding human
MRP4 was isolated and stably introduced into MCF-7 cells that expressed
negligible immunoreactive MRP4. These cells had enhanced resistance to
GCV cytotoxicity and to bystander cell killing. We also investigated
the effect of MRP4 on the cytotoxicity of GCV in cells engineered to
express HSV-TK. Taken together, our results show that MRP4 plays a key
role in the intracellular accumulation and cytotoxicity of GCV
metabolites. These findings reveal ganciclovir egress by MRP4 as a
novel mechanism contributing to the variable response in gene therapy
based on HSV-TK expression.
Materials--
[8-3H]Ganciclovir,
[adenine-8-3H]bis(pivaloyloxymethyl)-9-[2-(phosphonomethoxy)ethyl]-adenine
(bis-POM-[3H]PMEA), [3H]vinblastine,
[3',5',7-3H]methotrexate (MTX),
[5-3H]gemcitabine, [8-14C]6-mercaptopurine,
and nonlabeled bis-POM-PMEA were purchased from Moravek Biochemicals.
Nonlabeled ganciclovir, vinblastine, and methotrexate ((+)amethopterin)
were obtained from Sigma.
Cell Lines--
The human T-lymphoid cell line CEMss and its
PMEA-resistant variant, CEMr1, were grown as described previously (2).
The human breast tumor cell line, MCF7, and the HSV-TK-expressing cell
line, SW620-HSV-TK, were cultured as previously described (26, 27).
Human osteosarcoma cell line, Saos-2, and 293T human embryonic kidney
were grown in Dulbecco's modified Eagle's medium containing 10%
fetal calf serum and 100 units/ml penicillin, 100 µg/ml streptomycin,
and 2 mM L-glutamine. CEMss and CEMr1 cells were transduced with a recombinant murine retrovirus (pLENTK) encoding
HSV-TK (27).
Immunoblot Analysis--
Immunoblot analyses of MRP4 and MRP1
were performed as described previously (2). N-glycosylation
was assayed by treating the lysates of transfected MCF7 cells with
PNGase F (New England Biolabs) and immunoblotting was as described
(2).
Cytotoxicity Assay--
Cells were plated in 6-cm dishes (2 × 105 cells/dish) and incubated for 24 h at 37 °C.
Medium was removed, drugs were added, and cells were incubated at
37 °C for the indicated intervals. Viability and inhibition of cell
growth were determined by direct counting of trypan blue-dyed cells on
a hemocytometer. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Sigma) was used according to the manufacturer's instructions to determine cellular toxicity. Each assay included duplicate samples for each drug concentration, and all experiments were
performed at least twice.
Flow Cytometry--
After exposure to GCV, cells were harvested,
washed, and resuspended at ~1 × 106 cells/ml in
propidium iodide staining solution (0.05 mg/ml propidium iodide, 0.1%
sodium citrate, 0.1% Triton X-100). Immediately before analysis, cells
were treated for 30 min at room temperature with RNase (Calbiochem, San
Diego, CA) at a final concentration of 14 µg/ml and filtered through
nylon mesh. Fluorescence was measured on a Becton Dickinson FACScan
flow cytometer (BD Biosciences) using laser excitation at 488 nm. The percentages of cells in different phases of the cell cycle were
determined by using the Modfit computer program (Verify Software House,
Topsham, ME).
Sub-G1 Determination--
Analysis of the DNA
content less than G1 was used as an additional measurement
of apoptosis as previously reported (31). Briefly, after drug treatment
both floating and attached cells were harvested in a propidium iodide
solution (50 µg/ml propidium iodide in 0.1% sodium citrate and 0.1%
Triton X-100), treated with 5 µg/ml RNase (Calbiochem) for 30 min at
room temperature, and then analyzed by flow cytometry on a Becton
Dickinson FACscan (BD Biosciences) using laser excitation at 488 nM. The percentage of sub-G1 cells was determined.
DAPI Staining of Apoptotic Cells--
CEMss and CEMr1 cell lines
expressing TK were plated (5 × 104 cells/well) in
8-well plastic chamber slides (Lab-Tek) and treated with the indicated
concentrations of GCV. The cells were then washed with
phosphate-buffered saline, stained in 1 µg/ml DAPI, and examined on a
Zeiss fluorescent microscope at 40× magnification (28).
Assays of Drug Accumulation--
Cells (2 × 105 cells/ml growth medium) were seeded in 6-cm dishes
24 h prior to the assay. They were then washed with Hanks' balanced salt solution and incubated for the desired interval with the
radiolabeled drug dissolved in warmed medium. Aliquots of cells were
removed, washed with chilled phosphate-buffered saline, solubilized in
1 ml of 0.5 N NaOH, and assayed for radioactivity.
Cloning of MRP4 cDNA--
The human MRP4 cDNA
(expressed sequence tag 38,091, described in Ref. 2) was used to
screen a human lung MRP4 Expression in Cell Lines--
HEK293T cells were
transfected with pEQ-PAM2(-E) and pvSVG expression vectors containing
MSCV-IRES-GFP or MRP4-IRES-GFP constructs by calcium phosphate
precipitation (29), and supernatants containing retroviral particles
were used to transfect the MCF7 cell line. GFP+ cells were
identified by FACS. SAOS-2 cells transfected by calcium phosphate
precipitation with either pcDNA3 or pcDNA3-MRP4 were selected
in G418 (1000 µg/ml).
Metabolic Labeling with [3H]GCV--
CEMss HSV-TK
and CEMr1-HSV-TK cells were treated in triplicate with 1 µC of
[3H]GCV (plus 1 µM unlabeled GCV), washed,
and resuspended in fresh medium. Nucleotides were extracted from
pelleted cells in 0.2 ml of 7% methanol at 4 °C for 15 min (22).
Radioactivity was measured in an aliquot of each methanol supernatant
by scintillation counting. The proportion of phosphorylated GCV was
measured in an aliquot of medium by anion exchange chromatography
(22).
Clonal Dilution Assays of Bystander Effect--
The HSV-TK
expressing SW620-TK cells were plated with acceptor cells that did not
express HSV-TK (total, 2 × 105/well) in the following
proportions: (acceptor:HSV-TK cells) 100:0%, 75:25%, 50:50%, and
0:100%. After 2 days, 10 µM GCV was added in 1 ml of
fresh medium. After incubation for 24 h, medium was removed, cells
were trypsinized, and each well was diluted 1:10 to 1:10,000 with fresh
medium. After 7 days, surviving cell colonies were fixed in methanol,
stained, and counted (27).
Effect of MRP4 Overexpression on the Cell Cycle, Sensitivity to
GCV, and Apoptosis--
Immunoblot analysis showed the PMEA-resistant
cell line, CEMr1 (4), expressed 6- to 8-fold more MRP4 and slightly
less MRP1 than did the CEMss parent cell line (Fig
1A). Treatment with GCV caused
an indistinguishable cell cycle arrest in both CEMr1 and CEMss cells,
i.e. the proportion of S-phase cells increased and the
G1-phase decreased (Fig. 1B). However, the
viability of GCV-treated CEMss cells decreased in a
dose-dependent manner, whereas the viability of CEMr1 cells
was only modestly affected by GCV (Fig. 1C); this finding
was confirmed by measuring the population of cells with DNA content
less than G1 indicative of apoptotic cells (31) (Fig.
1D). CEMr1 and CEMss cells were equally sensitive to
vinblastine and, as expected, retained their respective resistance and
sensitivity to PMEA (Fig. 1E) (4). These results indicate
that cells overexpressing MRP4 are sensitive to cell cycle arrest
induced by GCV but are substantially more resistant to its cytotoxic
effects.
Efflux of GCV Is Enhanced in HSV-TK-transduced Cells That
Overexpress MRP4--
Sensitivity to GCV can be enhanced by modifying
targeted cells to express HSV-TK, but identically modified cells vary
markedly in their sensitivity to GCV (13). To evaluate whether MRP4
modulates the GCV sensitivity, we modified CEMss and CEMr1 cells
to stably express HSV-TK, isolated cells that expressed similar levels
of HSV-TK (Fig 2B), and
treated them with GCV. Although expression of HSV-TK increased the
sensitivity of both sets of cells to GCV, their relative sensitivity
was preserved (Fig 2A). The IC50 of GCV was 60 µM in the CEMr1-TK cells, a value five times that in the
CEMss-TK cells (12 µM). Thus, MRP4 overexpression
strongly influences sensitivity to GCV in cells that are and are not
modified to express HSV-TK.
We compared efflux of GCV and its metabolites from CEMss-HSV-TK
and CEMr1-HSV-TK cells by pretreating the cells with GCV, suspending
them in drug-free medium for 4 h, and quantifying GCV and
phosphorylated GCV in the medium by anion exchange chromatography (27).
Sixty-one percent of GCV metabolites in the CEMr1-HSV-TK cells and 44%
in the CEMss-HSV-TK cells were released (Fig. 2C). Thirty
percent more of the GCV metabolites exported by CEMr1-HSV-TK cells than
by the CEMss-HSV-TK cells were phosphorylated. These findings indicate
that cells that overexpress MRP4 readily remove GCV metabolites.
Accumulation of GCV Is Reduced in Cells That Overexpress
MRP4--
The initial uptake of GCV (0-5 min after exposure) was very
rapid and was essentially identical in CEMss and CEMr1 cells
(not shown). However, marked differences in GCV accumulation were
observed after 60 min or more. CEMr1 cells accumulated substantially
less drug than CEMss cells at every GCV concentration tested (Fig. 2D). Treatment with indomethacin, an inhibitor of MRP4
transport (see below), dramatically increased GCV accumulation in the
CEMr1 cells (and to a lesser extent in the CEMss cells; Fig.
2D) and decreased their efflux of radiolabeled GCV
nucleotides into the media to the level observed in CEMss cells (not shown).
Isolation and Expression of Human MRP4--
A cDNA encoding
human MRP4 was isolated from two independent human lung cDNA
libraries (see "Materials and Methods"). Its coding sequence was
identical to the reported sequence (GenBankTM/EBI
accession number AF071202) with the exception of seven single-base
substitutions. Of these, only four caused amino acid changes. These
differences may reflect polymorphisms or may be related to derivation
of the reported MRP4 cDNA from a drug-selected cell line; drug
selection has been noted to cause such alterations in other mammalian
ABC transporters (e.g. MDR1 and BCRP). These differences
clearly do not affect the transport of PMEA (see Fig. 3B).
Uptake of bis-POM-PMEA, Vinblastine, and Methotrexate by Cells
Modified to Express MRP4--
The human breast carcinoma cell line,
MCF-7, was shown by Western blotting to express very low levels of
MRP4. We transduced MCF-7 cells with either the MSCV-MRP4-IRES-GFP or
the control MSCV-IRES-GFP and isolated GFP+ cell
populations. We then quantified MRP4 in crude membrane fractions from
each cell population by Western blotting (Fig. 3A). As
expected, MRP4 was highly expressed in cells transduced with
MSCV-MRP4-IRES-GFP, whereas it was expressed at very low levels in
cells transduced with MSCV-IRES-GFP. However, treatment of plasma
membrane fractions with PNGase F to de-glycosylate MRP4 yielded a band
of ~150 kDa (a value close to that predicted from the amino acid
sequence) in both cell lines, which suggests that posttranslational
modifications may affect detection of MRP4 with this antibody.
Immunohistochemical and subcellular fractionation studies revealed that
most of the MRP4 expression in MCF-7 cells was localized to the plasma
membrane (not shown).
Uptake of PMEA, an acyclic nucleoside phosphonate that contains a
nonhydrolyzable bond and resembles a nucleoside 5'-monophosphate (4),
was decreased in cells transfected with MRP4. However, because PMEA
uptake is slow, we also incubated cells with bis-POM-PMEA, which is
taken up and is converted to PMEA intracellularly before efflux (4).
Fig. 3B shows that uptake of bis-POM PMEA by MCF-7-MRP4 cells was less than one-third of the uptake by control cells at every
concentration tested. In contrast, vinblastine uptake was identical in
test and control cells (Fig. 3C). Interestingly, whereas
earlier studies suggested that MRP4 plays a role in MTX uptake and
efflux (3), we found that the steady-state intracellular concentration
of methotrexate was the same in test and control cells (Fig.
3D).
We next compared MRP4 function with the quantity of MRP4 protein
expressed. Saos-2 human osteosarcoma cells, which express very little
MRP4, were transfected with pcDNA3-MRP4 and selected in G418.
Immunoblot analysis revealed a wide range of MRP4 expression (Fig.
3E). Although there appeared to be an inverse relationship between expression of MRP4 and expression of MRP1 in CEMss cells (Fig.
1A), MRP1 and MRP4 expression were completely unrelated in
Saos-2 transfectants (Fig. 3E). Intracellular accumulation of PMEA was inversely proportional to the quantity of MRP4 expressed: accumulation of PMEA was decreased (~41%) at low levels of MRP4 expression and substantially (more than 95%) reduced at very high levels of MRP4 expression (Fig. 3F). Accumulation of PMEA in
the Saos-2 MRP4 transfectants was increased over 600% by treatment with 100 µM indomethacin (Fig. 3G). In
contrast, 100 µM probenecid treatment had no effect (Fig.
3G). Indomethacin-treated Saos-2 control cells also showed a
modest increase (56%) in PMEA accumulation, possibly reflecting
inhibition of an endogenous transporter.
Accumulation, Efflux, and Cytotoxicity of GCV in MCF7 Cells
Modified to Express MRP4--
We observed no difference between
control and MRP4-transfected MCF-7 cells in the initial rates of uptake
(<5 min) of GCV, gemcitabine (a cytidine analog). This finding is
consistent with the patterns of mRNA expression we observed for the
major nucleoside transporters (hCNT1a, ENT2, and hENT1) (not shown).
However, there were striking differences between test and control cells
in GCV accumulation: the steady-state concentration of GCV in the
MRP4-transfected cells was only ~50% that in the vector cells (Fig.
4A). This disparity was
observed over a wide range of concentrations of GCV (Fig. 4B).
We investigated whether efflux was altered in the cells expressing
MRP4. First, we treated these cells with ~1.5 times the amount
radiolabeled GCV used to treat control cells to obtain a comparable
intracellular GCV concentration. We then washed the cells, placed them
in drug-free medium, and measured intracellular radioactive GCV (Fig.
4C). The decline of intracellular GCV was more rapid in the
MRP4-transfected cells compared with vector cells
(t1/2 s of ~16 and 32 min, respectively). This
increased efflux was accompanied by a striking decrease in GCV-induced
cytotoxicity (Fig. 4D) but not in MTX-induced cytotoxicity
(Fig. 4E). Similar results were obtained in parallel
experiments with Saos-2 cells modified to express MRP4 (not shown).
Expression of MRP4 Affects Bystander Cell Killing--
Because
MRP4 transports nucleotide derivatives (1-3, 24, 26, 32) including GCV
anabolites from cells, we reasoned that the killing of bystander cells
(those that do not express TK) by GCV could be significantly influenced
by MRP4 expression. To explore this possibility, we used an
SW620-HSV-TK cell line that activates GCV and readily effluxes GCV
nucleotides (27). We performed a clonal dilution assay (27) in which
either MCF-7 or MCF-7-MRP4 cells were co-cultured with increasing
percentages of the SW620 HSV-TK cells (see "Materials and Methods")
(Fig. 5). As the proportion of
HSV-TK-expressing cells increases the delivery of GCV metabolite
increases (30) leading to greater cell death of bystander cells. The
MCF-7-MRP4 cells are more resistant to bystander killing. However, with
very high proportions of HSV-TK cells we see that MRP4 overexpression
offers less protection. These studies indicate that MRP4 plays a strong
role in bystander cell survival, but suggest its protection may be
overcome by increasing the proportion of HSV-TK expressing cells.
We have shown that expression of MRP4 affects the sensitivity of
cells to the therapeutically important antiviral guanine analog GCV and
that it modulates the direct and bystander cytotoxicity in which cells
modified to express HSV-TK are treated with GCV. Cells that
overexpressed MRP4 accumulated smaller quantities of GCV metabolites
than did controls. Although the sensitivity to GCV-induced cell cycle
arrest was not reduced in these cells, they were protected from GCV
cytotoxicity. This finding is consistent with a relationship between
GCV cytotoxicity and the intracellular concentration of the drug (7,
14). However, the effect did not extend to all cytotoxins: sensitivity
to methotrexate and vinblastine was not affected.
Because we and others have demonstrated that GCV metabolites are
readily transported from cells expressing HSV-TK (17, 27, 30), we
evaluated the effect of MRP4 expression on GCV cytotoxicity of cells
gene-modified to express HSV-TK. HSV-TK cells that overexpressed MRP4
had significantly enhanced resistance to GCV (Fig. 2, A and B). This finding has strong implications for gene therapy
with HSV-TK because varying MRP4 expression among cells gene-modified with HSV-TK would lead to decreased cytotoxicity.
The success of gene therapy requires not only that the HSV-TK-modified
cells export GCV nucleotides but also that the adjacent bystander cells
accumulate and retain sufficient nucleotides to induce apoptosis. An
increase in the extracellular concentration of GCV can induce the
killing of otherwise intractable bystander cells (13), suggesting the
existence of a mechanism that excludes GCV nucleotides or decreases
their intracellular concentration in these cells. Bystander killing by
GCV can also be enhanced by prolonged exposure to the drug (13). Our
results strongly suggest that expression of MRP4 by bystander cells
protects them from GCV cytotoxicity when they are co-cultured with
initiator cells expressing HSV-TK (Fig. 5). However, this protection
decreases as the proportion of HSV-TK-expressing cells increases,
implying that a threshold level of GCV nucleotide is exceeded and
overrides the protective effect of MRP4. Nevertheless, this result
suggests that cells overexpressing MRP4 would be less susceptible to
the bystander effect because of their reduced accumulation of GCV nucleotides.
Our results demonstrate that cellular retention and accumulation of the
guanine analog ganciclovir is strongly influenced by MRP4. Expression
of this nucleotide transporter may reduce the antiviral efficacy of GCV
therapy, which is often used to treat or prevent cytomegalovirus
disease in transplant recipients and patients with AIDS. It can also
attenuate both direct GCV cytotoxicity and bystander cell killing in
cells modified to express HSV-TK to induce selective cell killing. We
have also shown that cells that express MRP4 are much less susceptible
to GCV despite modification with an HSV-TK expression vector. These
studies provide the mechanistic basis for future studies evaluating the
role of MRP4 expression in antiviral response to GCV and in HSV-TK and GCV gene therapy with solid malignancies (e.g. brain tumors)
Finally, a challenge for the future will be to determine the degree to which MRP4 modulates the levels of toxic metabolites near bystander cells.
We thank Dr. Richard Ashmun and Sam Lucas for
FACScan analysis, Dr. Dan Hua Pan for excellent technical assistance,
and Vicki Gray for help in preparing this manuscript. We also thank
Drs. Phil Potter and Brian Sorrentino for their critical comments and Sharon Naron for excellent editorial advice.
*
This work was supported by National Institutes of Health
Research Grants GM-60904, CA-63203, CA-23099, CA53535, P30 CA-21765, and CA83843 and by the American Lebanese Syrian Associated Charities (ALSAC).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY081219.
§
Both authors contributed equally to this work.
¶¶
To whom correspondence should be addressed: Dept. of
Pharmaceutical Sciences, St. Jude Children's Research Hosp., 332 N. Lauderdale Ave., Memphis, TN 38105-2794. Tel.: 901-495-2174; Fax:
901-525-6869; E-mail: John.schuetz@stjude.org.
Published, JBC Papers in Press, June 24, 2002, DOI 10.1074/jbc.M203262200
The abbreviations used are:
MRP, multidrug
resistance protein;
ABC, ATP-binding cassette;
AZT, (3'-azido-3'-deoxythymidine);
PMEA, 9-(2-(phosphonomethoxy)ethyl)-adenine;
GCV, ganciclovir;
HSV, herpes
simplex virus;
TK, thymidine kinase;
MTX, methotrexate;
POM, pigeon
ovomucoid;
DAPI, 4',6-diamidino-2-phenylindole;
GFP, green fluorescent
protein;
MSCV, murine stem cell virus;
IRES, internal ribosomal entry
site;
FACS, fluorescence-activated cell sorting.
Expression of MRP4 Confers Resistance to Ganciclovir and
Compromises Bystander Cell Killing*
§,§,,,
,,,,**,¶¶
Pharmaceutical
Sciences and ¶ Hematology-Oncology, St. Jude Children's Research
Hospital, Memphis, Tennessee 38105, the Department of
Pharmacology and the ** Barbara Ann Karmanos Cancer
Institute, Wayne State University School of Medicine, Detroit, Michigan
48201, the Department of Biochemistry,
University of Arkansas Center for Medical Sciences, Little Rock,
Arkansas 72205, and the §§ Department of
Biochemistry, Eastern Virginia Medical School,
Norfolk, Virginia 23529
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ZAPII cDNA library (Stratagene) by plaque
hybridization. A 4.5-kb cDNA that lacked the initiator methionine
was identified, and a 5' portion of this cDNA was used to screen a
human lung 5'-STRETCH PLUS cDNA library (Clontech). Ten positive clones that contained only
the 5' portion of MRP4 were isolated. The 5' portion of MRP4, together
with the overlapping 4.5-kb clone of the 3' portion, yielded a 5.8-kb
MRP4 cDNA (GenBankTM accession number: AY081219). The
5'- and 3'-untranslated regions of this cDNA fragment were removed,
and the complete 4.2-kb cDNA encoding the 1325 amino acids of MRP4
was used to construct the expression vector MSCV-MRP4-IRES-GFP
(contains the murine stem cell virus (MSCV) long terminal repeat as
well as a ribosomal entry site (IRES) to permit expression of green
fluorescent protein (GFP) and was kindly provided by Dr. Robert Hawley,
Holland Laboratory, American Red Cross, Rockville, MD). The complete
MRP4 cDNA was also subcloned into the EcoRI site of
pcDNA3 (Invitrogen) to generate pcDNA3-MRP4.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Overexpression of MRP4 decreases the
cytotoxicity but not the cell cycle arrest induced by GCV.
A, a representative immunoblot analysis of MRP4 and MRP1 in
CEMss and CEMr1 cells (100 µg of total cell lysate). B,
the proportions of CEMss and CEMr1 cells in G1 or S phase
were determined after 48 h of exposure to various concentrations
of GCV. C, viability (by trypan blue dye exclusion) of CEMss
and CEMr1cells after 48 h of exposure to GCV. D, the
proportion of cells with sub-G1 DNA content (an indicator
of apoptosis) was quantified by FACS after staining with propidium
iodide. E, CEMss and CEMr1 cells were counted after exposure
for 48 h to 80 µM PMEA or 5 nM
vinblastine. The error bars represent the standard deviation
from the mean value of two to three independent experiments with
triplicate determinations.
View larger version (18K):
[in a new window]
Fig. 2.
Cells expressing MRP4 show increased efflux
of GCV metabolites and remain GCV-resistant after transduction with
HSV-TK. A, CEMss and CEMr1 were transduced with a
plasmid encoding recombinant HSV-TK. Cells expressing HSV-TK were
treated with GCV and assayed for viability. B, Western blot
analysis of HSV-TK in CEMss and CEMr1 cells transduced with HSV-TK.
C, efflux of GCV metabolites from CEMss-HSV-TK and
CEMr1-HSVTK cells. D, cells were incubated with various
concentrations of radiolabeled GCV, and intracellular accumulation of
GCV was measured. Some cells were preincubated with indomethacin. The
error bars represent one standard deviation from the mean,
and their absence indicates the error was smaller than the
symbol.
View larger version (28K):
[in a new window]
Fig. 3.
Accumulation of MRP4 substrate is reduced in
proportion to the MRP4 protein expressed. A, immunoblot
analysis of MRP4 and MRP1 in MCF-7 cells transduced with either empty
vector (MSCV-IRES-GFP) or MSCV-MRP4-IRES-GFP. B, uptake of
bis-POM-[3H]PMEA was measured in cells transduced with
either MRP4 or empty vector. C, accumulation of
[3H]vinblastine was measured in cells transduced with
either MRP4 or empty vector. D, steady-state
[3H]MTX concentration was measured as previously
described (33). E, the osteosarcoma cell line Saos-2, which
expresses minimal MRP4, was transfected with either pcDNA3 or
pcDNA3-MRP4, and cells were selected in G418. Asterisks
indicate the clones that were expanded for functional analysis.
F, uptake of bis-POM-[3H]PMEA was inversely
proportional to the expression of MRP4. G, indomethacin
inhibited MRP4 function, but probenecid did not. Uptake of
bis-POM-[3H]PMEA was measured in cells preincubated with
no drug, with indomethacin, or with probenecid.
View larger version (17K):
[in a new window]
Fig. 4.
Ectopic expression of MRP4 in MCF-7 cells
reduces GCV uptake and cytotoxicity. A, MCF-7 and
MCF-7-MRP4 cells were incubated for various intervals with radiolabeled
GCV, and intracellular radioactivity was measured. B, MCF-7
and MCF-7-MRP4 cells were incubated with various concentrations of
radiolabeled GCV, and intracellular radioactivity was measured.
C, MCF-7 and MCF-7-MRP4 cells were pretreated with
radiolabeled GCV, washed in ice-cold buffer, and resuspended in warmed
drug-free medium. Intracellular radioactivity was measured at the
indicated intervals. D, MCF-7 and MCF-7-MRP4 cells were
exposed for 72 h to GCV at the indicated concentrations, and an
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
assay was performed to determine viability. E, MCF-7 and
MCF-7-MRP4 cells were exposed for 4 h to various concentrations of
MTX. No difference in MTX sensitivity was seen at either 4 h or
after 72 h of exposure (not shown).
View larger version (18K):
[in a new window]
Fig. 5.
Clonal dilution bystander assay reveals MRP4
overexpressing MCF-7 cells are more resistant to cell killing by HSV-TK
expressing cells. SW620-TK cells mixed in various proportions with
MCF7 or MCF7-MRP4 cells were treated with 10 µM GCV for
24 h (7, 16). Surviving cell colonies were quantified (27). The
values are obtained from triplicate determinations in a single
experiment that was repeated twice with similar results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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