From the Departments of Biology and
§ Molecular Genetics, the Rappaport Institute for Research
in the Medical Sciences and the B. Rappaport Faculty of Medicine, The
Technion-Israel Institute of Technology, Haifa 31096, Israel
Received for publication, September 18, 2002, and in revised form, January 7, 2003
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ABSTRACT |
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The human reduced folate carrier (hRFC) is the
dominant transporter mediating the uptake of reduced folate cofactors
and antifolate anticancer drugs. Defective antifolate uptake due to
inactivating mutations in the hRFC gene is an established mechanism of
drug resistance in various tumor cells. However, while antifolate
transport is frequently impaired, either no or only a single hRFC
allele is inactivated, suggesting that additional mechanism(s) of
resistance are operative. Here we studied the relationship between the
expression and function of transcription factors and antifolate
resistance in transport-defective leukemia cells that poorly express or
completely lack RFC mRNA. Stable transfection with a hRFC
expression construct resulted in restoration of normal RFC mRNA
expression and nearly wild type drug sensitivity in these
antifolate-resistant cells. The loss of RFC gene expression prompted us
to explore transcription factor binding to the hRFC promoter. The hRFC
promoter contains an upstream GC-box and a downstream cAMP-response
element (CRE)/AP-1-like element. Electrophoretic mobility shift
assays and oligonucleotide competition revealed a substantial loss of
nuclear factor binding to CRE and GC-box in these drug-resistant cell
lines. Consistently, antibody-mediated supershift analysis showed a
marked decrease in the binding of CRE-binding protein 1 (CREB-1) and
specificity protein 1 (Sp1) to CRE and GC-box, respectively. Western
blot analysis revealed undetectable expression of CREB-1, decreased ATF-1 levels, parental Sp1 levels, and increased levels of the short
Sp3 isoforms, recently shown to repress hRFC gene expression. Transient
transfections into these antifolate-resistant cells demonstrated a
marked loss of GC-box-dependent, and CRE-driven reporter
gene activities and introduction of CREB-1 or Sp1 expression constructs
resulted in restoration of hRFC mRNA expression. These results
establish a novel mechanism of antifolate resistance that is based on
altered expression and function of transcription factors resulting in
transcriptional silencing of the hRFC promoter.
Mammalian cells are devoid of reduced folate biosynthesis and
therefore meet their folate growth requirement via uptake from exogenous sources (1). Reduced folates are absolutely essential cofactor vitamins involved in a host of one-carbon transfer reactions resulting in the biosynthesis of purines, deoxythymidylate, and methionine (1). Folate antagonists (i.e. antifolates)
including methotrexate (MTX)1
are potent inhibitors of purine and deoxythymidylate biosynthesis and
thereby block DNA synthesis. Consequently, MTX is an integral component
of chemotherapeutic regimens used in the treatment of various human
malignancies (2). Folates and antifolates are hydrophilic divalent
anions that cannot cross membranes via diffusion and are therefore
taken up into mammalian cells by the reduced folate carrier (RFC) (3).
Among the antifolates recognized as transport substrates by the RFC are
the dihydrofolate reductase inhibitor, MTX (2), the novel thymidylate
synthase inhibitors Tomudex (ZD1694, Raltitrexed) (4) and ZD9331 (5),
as well as the glycinamide ribonucleotide transformylase
inhibitor AG2034 (6). Various antifolate resistance phenomena pose a
major obstacle toward curative cancer chemotherapy. For example,
qualitative (i.e. inactivating mutations) (7-11) and
quantitative alterations (i.e. decreased or abolished
expression) in human RFC (hRFC) gene expression and/or function are
documented mechanisms of MTX resistance in acute lymphoblastic leukemia
(ALL) (12, 13) and osteogenic sarcoma patients (14). Although decreased
expression of hRFC mRNA has been suggested as a mechanism of
clinical resistance to MTX, the underlying molecular basis is still
unknown (12-14).
It was recently shown that transcription of the hRFC gene is driven by
at least two promoter elements: an upstream constitutive element
consisting of a GC-box as well as a downstream inducible cAMP-response
element (CRE)/AP-like element (CRE/AP-1) (15). Reporter gene assays
revealed that the binding of Sp1 and the long Sp3 isoforms to the
GC-box as well as that of CRE-binding protein (CREB-1) to the
CRE/AP-1-like element result in activation of hRFC transcription (15).
Using stepwise antifolate selection, the human CCRF-CEM leukemia cell
lines AG2034R2 and ZD9331R1.5 were isolated,
which displayed up to 2300-fold resistance to various antifolates due
to abolished drug transport (11). This was a result of a marked
decrease (or complete loss) of RFC mRNA expression. This major
decrease in RFC mRNA expression was associated with alterations in
the expression of transcription factors, resulting in a prominently
decreased binding to both the constitutive and inducible elements of
the hRFC promoter. This was associated with poor expression of CREB-1
and binding to the inducible CRE as well as substantially diminished
Sp1 binding to the constitutive GC-box element in the hRFC promoter.
The current study constitutes the first demonstration of a novel
mechanism of antifolate resistance that is based on alterations in the
expression of transcription factors and their binding to the hRFC promoter.
Drugs, Biochemicals, and Radiochemicals--
MTX was from Teva
Pharmaceuticals Ltd. Novel antifolate drugs were generous gifts from
the following sources: ZD9331 from Dr. A. L. Jackman, (Institute of
Cancer Research, Sutton, United Kingdom), AG2034 from Dr. T. Boritzki
(Agouron Pharmaceuticals, Inc.), and PT523 from Dr. W. T. McCulloch
(Sparta Pharmaceuticals).
Tissue Culture and Antifolate Drug Selections--
CCRF-CEM, a
human T-cell leukemia line, and its antifolate-resistant sublines were
maintained in RPMI 1640 medium containing 2.3 µM folic
acid (Biological Industries, Beth-Haemek, Israel) supplemented with
10% fetal calf serum (Invitrogen), 2 mM glutamine, 100 units/ml penicillin G (Sigma), and 100 µg/ml streptomycin sulfate
(Sigma). The cell lines AG2034R2 and ZD9331R1.5
were established by stepwise antifolate selection of parental CCRF-CEM
cells in gradually increasing concentrations of AG2034 and ZD9331 as
previously described (11). CCRF-CEM-7A cells with a high RFC
overexpression were cultured in folic acid-free medium containing 10%
dialyzed fetal calf serum and supplemented with 0.25 nM
leucovorin as the sole folate source (16).
Antifolate Growth Inhibition--
Parental cells and their
antifolate-resistant sublines were first grown in antifolate-free
growth medium for five to eight cell doublings. Thereafter, cells were
seeded in 96-well plates (3 × 104/well) in growth
medium (0.15 ml/well) containing various concentrations of different
antifolates. After 3 days of incubation at 37 °C, viable cell
numbers were determined by hemocytometer count using trypan blue
exclusion. The percent inhibition of cell growth was calculated
relative to untreated controls.
[3H]MTX Transport--
To examine the ability of
the antifolate-resistant sublines to transport antifolates, we
determined the initial rates of [3H]MTX uptake relative
to wild type CEM cells. Exponentially growing cells (2 × 107) were washed three times in transport buffer consisting
of HEPES-buffered saline solution (11) and incubated at 37 °C for 3 min in same buffer (1-ml suspensions) containing 2 µM
[3H]MTX. Transport controls contained a 500-fold excess
of unlabeled MTX (1 mM). Transport was terminated by the
addition of 10 ml of ice-cold HEPES-buffered saline solution, after
which cells were centrifuged at 500 × g for 5 min at
4 °C and the cell pellet was washed twice with 10 ml of ice-cold
transport buffer. The final cell pellet was lysed in water and
processed for scintillation counting.
Electrophoretic Mobility Shift Assays--
Nuclear extracts were
prepared from exponentially growing cells (2 × 107
cells) as previously described (17). DNA-protein complexes were formed
by incubating nuclear extract proteins (6 µg) with [ Semi-quantitative RT-PCR of hRFC and Various Housekeeping
Genes--
Exponentially growing cells (2 × 107)
were harvested by centrifugation and washed with phosphate-buffered
saline, and total RNA was isolated using the Tri-Reagent kit according
to the instructions of the manufacturer (Sigma). A portion of total RNA
(20 µg in a total volume of 20 µl) was reverse transcribed
using Moloney murine leukemia virus reverse transcriptase (180 units, Promega) in a reaction buffer containing random hexamer primers,
dNTPs, and ribonuclease inhibitor RNasin (Promega). Portions of
cDNA (~50 ng) synthesized from parental CEM cells and their
antifolate-resistant sublines were amplified using Expand polymerase
(Roche Molecular Biochemicals) in a reaction buffer (total volume 25 µl) containing: 10 pmol of each primer, 0.2 mM dNTPs, 1.5 mM MgCl2, and 2% Me2SO according
to the instructions of the manufacturer. The PCR reaction was performed
as follows: initial melting at 95 °C for 5 min, followed by 35 cycles each of 1 min at 95 °C, annealing at 62 °C (56 °C for
Transient Transfections with CRE- and GC-box-luciferase, CREB-1
and Sp1 Expression Constructs, and Reporter
Activity--
Logarithmically growing suspension cells (2 × 107) were harvested by centrifugation and transfected by
electroporation (1000 microfarads, 234 V) with 10 µg of reporter
constructs containing CRE-luciferase, GC-box-luciferase
(pGL3-luciferase) and GC-box-chloramphenicol acetyltransferase (CAT) or
with expression plasmids (10 µg) including pRSV-CREB-1 and pPacSp1
(kindly provided by Drs. M. E. Greenberg and G. Suske, respectively).
Cells were then seeded at 2 × 106/ml in prewarmed
growth medium. For reporter gene experiments, after 24 h of growth
at 37 °C cells were harvested by centrifugation and washed with
phosphate-buffered saline, cell lysates were prepared, and firefly
luciferase activity was assayed using the dual luciferase kit (Promega)
and a luminometer. Firefly luciferase activity was normalized with
Renilla luciferase, following co-transfection with a
Renilla-luciferase plasmid. For transient RFC mRNA
expression, after 24 h of growth cells were harvested and total
RNA was extracted. Results presented were obtained from at least three
independent transfections performed in duplicate cultures and activity determinations.
Stable Transfections with a hRFC Expression
Construct--
Exponentially growing cells (2 × 107)
were harvested by centrifugation and stably transfected by
electroporation (1000 microfarads, 234 V) with 10 µg of an expression
vector (pcDNA3-hRFC1) harboring the hRFC cDNA (10). Following
24 h of growth at 37 °C, cells were exposed to 600 µg/ml
active G-418 (Calbiochem-Novabiochem, San Diego, CA). Stable
transfectants expressing high levels of hRFC mRNA were obtained
after at least 1 month of G-418 selection and were used for further analyses.
Western Blot Analysis--
Nuclear extract proteins (20 µg)
were resolved by electrophoresis on 10% polyacrylamide gels containing
SDS, electroblotted onto a Protran cellulose nitrate membrane
(Schleicher & Schuell), reacted with anti-CREB-1, -ATF-1, -Sp1, and
-Sp3 (Santa Cruz Biotechnology Inc.) according to the instructions of
the manufacturer. To examine RFC expression in parental and
antifolate-resistant cells, microsomes were first isolated (20), and
proteins were extracted in a buffer containing 0.5% Triton X-100 as
previously described (11). Detergent-soluble proteins (12.5-150 µg)
were resolved by electrophoresis, electroblotted onto a nylon membrane
and blocked for 1 h at room temperature in TBS buffer (150 mM NaCl, 0.5% Tween 20, and 10 mM Tris/Cl at pH 8.0) containing 1% skim milk. The blots were then reacted with a
polyclonal antiserum (1:700) prepared in mice against a C-terminal hRFC
peptide (11), rinsed in the same buffer for 10 min in room temperature,
and reacted with horseradish peroxidase-conjugated goat anti-mouse IgG
(1:40,000 dilution, Jackson ImmunoResearch Laboratories, West Grove,
PA) for 1 h at room temperature. Following three washes (10 min
each) in TBS at room temperature, enhanced chemiluminescence (ECL)
detection was performed according to the manufacturer's instructions
(Biological Industries, Beth Haemek, Israel). ECL was recorded on x-ray
films using several exposure times, which were evaluated by scanning densitometry.
RFC Expression, Drug Transport, and Antifolate Resistance--
We
studied the molecular basis of antifolate resistance in the human
leukemia sublines ZD9331R1.5 and AG2034R2.
These cell lines displayed up to 2300-fold resistance to various antifolates that use RFC as their primary route of uptake (Fig. 1A). This was associated with
a marked decrease (95-97%) in the transport of [3H]MTX
(Fig. 1B). While detectable with 25 µg of microsomal
proteins from parental CEM cells, RFC was not detectable even with 150 µg of proteins from AG2034R2 and ZD9331R1.5
cells (Fig. 2A).
Semiquantitative RT-PCR analysis showed that AG2034R2 cells
expressed ~1/300th of parental RFC mRNA, whereas
ZD9331R1.5 cells had no detectable expression (Fig.
2B). In contrast, AG2034R2 and
ZD9331R1.5 cells expressed normal mRNA levels of
various housekeeping genes including DHFR, FPGS, MRP1, and GAPDH (Fig.
2C), thereby suggesting that these cells did not suffer from
a global transcriptional repression.
Restoration of Antifolate Sensitivity upon Transfection with a hRFC
Expression Vector--
To examine the impact of restoration of RFC
expression on antifolate sensitivity, AG2034R2 and
ZD9331R1.5 cells were stably transfected with an expression
vector harboring the wild type hRFC cDNA (Fig. 2B,
lane 1). This resulted in restoration of normal RFC mRNA
expression in these cell lines (Fig. 2B, lane 1).
Consequently, AG2034R2 and ZD9331R1.5 cells
became up to 170-fold more sensitive to MTX, thereby approaching the sensitivity of wild type cells to this antifolate (Fig.
1C). These results establish that the loss of hRFC gene
expression is a major underlying mechanism of antifolate drug
resistance in these transport defective cell lines.
Nuclear Factor Binding to [32P]-labeled CRE, AP-1,
and GC-box Oligonucleotides--
To explore the basis for the loss of
RFC mRNA expression, nuclear proteins from parental CEM and
antifolate-resistant cells were isolated and examined by an
electrophoretic mobility shift assay. This electrophoretic mobility
shift assay assessed the binding of nuclear factors to
[32P]-labeled CRE, GC-box, and AP-1 double-stranded
oligonucleotides (Table I). A 3-band binding pattern to
[32P]-labeled CRE (Fig.
3A) and
[32P]-labeled GC-box (Fig. 3B) was observed in
parental CEM cells, whereas AG2034R2 and
ZD9331R1.5 cells had a markedly decreased binding to both
oligonucleotides (Fig. 3, A and B) but retained
normal binding to [32P]-labeled AP-1 (Fig.
3C). To examine the specificity of nuclear factor binding to
these consensus binding sites, two independent parameters were used:
(a) competition of binding to CRE and GC-box with unlabeled
oligonucleotides and (b) binding to mutant CRE and GC-box
oligonucleotides (Table I). The binding of nuclear proteins from
parental cells to [32P]-labeled CRE, and GC-box was
competed in a dose-dependent manner, with a 10-100-fold
molar excess of the unlabeled oligonucleotides (data not shown). In
contrast, the residual binding of nuclear proteins from
AG2034R2 and ZD9331R1.5 cells to CRE and GC-box
could not be competed even with a 100-fold molar excess of the
unlabeled oligonucleotides. Furthermore, nuclear proteins from both
parental and antifolate-resistant sublines failed to bind to the mutant
[32P]-labeled CRE and [32P]-labeled GC-box
oligonucleotides and displayed a similar nonspecific basal binding
(data not shown). These results suggest that the residual binding to
CRE and GC-box in these antifolate-resistant sublines was largely
nonspecific.
Restoration of RFC mRNA Expression upon Transient Transfection
with CREB-1 and Sp1 Expression Constructs--
The putative loss of
function of transcription factors that are involved in the binding to
CRE and GC-box prompted us to explore the impact of the
introduction of CREB-1 and Sp1 on RFC mRNA expression (Fig.
2D). Transfection of AG2034R2 and
ZD9331R1.5 cells with expression constructs harboring
CREB-1 (pRSV-CREB1) or Sp1 (pPacSp1) resulted in restoration of normal
RFC mRNA expression (Fig. 2D).
Loss of CRE- and GC-box-driven Reporter Gene Activity--
To
assess CRE- and GC-box-mediated transcription, parental cells and their
antifolate-resistant sublines were transiently transfected with
CRE-luciferase (Fig. 4A),
GC-box luciferase (Fig. 4B), and GC-box-CAT (Fig.
4C), and reporter gene activity was determined. The
activities of firefly luciferase and CAT were normalized to that of a
promoterless Renilla construct. Antifolate-resistant cells
had a substantial decrease in luciferase activity driven by the
CRE-containing construct, relative to parental cell transfectants (Fig.
4A). Reporter gene activity driven by two independent
constructs containing GC-box was markedly reduced in these
antifolate-resistant cells (Fig. 4, B and C).
Thus, these drug-resistant cell lines had a major loss in GC-box- and
CRE-dependent transcription, resulting in a marked or
complete loss of hRFC mRNA expression.
Antibody-mediated Supershift Analysis--
It was recently shown
that CREB-1 and Sp1 bind to CRE and GC-box in the hRFC promoter,
respectively (15). To identify alterations in the binding of specific
transcription factors to CRE (Fig. 5A) and GC-box (Fig.
5B), antibody-mediated supershift analysis was used.
Preincubation of nuclear proteins either with antibodies to CREB-1 and
ATF-1 or a combination of the two followed by
[32P]-labeled CRE-protein-antibody complex formation
revealed supershifts in parental CEM cells (Fig. 5A,
complexes A-C in lanes 2-4, respectively; note
the elimination of bands 1 and/or 2). Likewise,
[32P]-labeled GC-box supershifts in parental CEM cells
were obtained with anti-Sp1 (Fig. 5B, complex A
in lane 2; note the elimination of band 2) and anti-Sp3
antibodies (Fig. 5B; note the elimination of bands
1 and 3 in lane 3). Furthermore, the
combination of anti-Sp1 and -Sp3 antibodies eliminated all three
[32P]-labeled GC-box bands and formed a high molecular
weight complex (Fig. 5B, complex B in lane
4). In contrast, nuclear proteins from AG2034R2
(lanes 5-8) and ZD9331R1.5 cells (lanes
9-12) neither showed [32P]-labeled CRE supershifts
with anti-CREB-1 or ATF-1 antibodies (Fig. 5A) or
[32P]-labeled GC-box supershifts with antibodies to Sp1
(Fig. 5B). However, anti-Sp3 antibodies eliminated
band 3 of [32P]-labeled GC-box in both
AG2034R2 and ZD9331R1.5 cells (Fig.
5B, lanes 7 and 11, respectively).
Alterations in the Expression and Function of Transcription
Factors--
As alterations in the function of transcription factors
could also result from changes in their expression, we determined CREB-1, ATF-1 (Fig. 6A), Sp1
(Fig. 6B), and Sp3 levels (Fig. 6C) by Western
blot analysis. Parental CEM cells expressed substantial levels of
CREB-1 and ATF-1 (Fig. 6A, lane 1), whereas
AG2034R2 and ZD9331R1.5 cells did not contain
detectable levels of CREB-1 (Fig. 6A, lanes 2 and
3, respectively). In addition, these cells had decreased ATF-1 levels (Fig. 6A, lanes 2 and 3,
respectively). Reprobing the Western blot with anti-Sp1 antibodies
revealed similar Sp1 levels in parental cells and their
antifolate-resistant sublines (Fig. 6B, lanes
1-3). Parental CEM cells expressed both the long and short
isoforms of Sp3 (Fig. 6C, lane 1), whereas
AG2034R2 and ZD9331R1.5 cells showed increased
levels of the short Sp3 isoforms (Fig. 6C, lanes
2 and 3, respectively). As the short Sp3 isoforms were recently shown to act as inhibitors of hRFC transcription (15), we
examined the possibility that a repressor activity was present in these
antifolate-resistant cells that could interfere with GC-box binding. A
nuclear protein mixture (vol:vol) derived from parental and
ZD9331R1.5 cells showed a normal binding to
[32P]-labeled CRE (Fig. 3D, compare lane
3 with 1) but a poor binding (<15% of wild type) to
the [32P]-labeled GC-box (Fig. 3E, compare
lane 3 with 1). These results suggest that an
inhibitory component was present in the nuclear extract from
ZD9331R1.5 cells that appeared to disrupt GC-box
binding.
The aim of the current study was to identify the molecular basis
underlying the loss of RFC mRNA expression in antifolate-resistant human leukemia cell lines displaying impaired MTX transport. We find
substantial alterations in the expression of CREB-1, ATF-1, and the
short isoforms of Sp3. These alterations were associated with a
prominent decrease in the binding of transcription factors to the
inducible (CRE) and constitutive (GC-box) elements in the hRFC
promoter, presumably leading to a marked decrease in RFC mRNA
expression. Consistently, introduction of CREB-1 and Sp1 into these
antifolate-resistant cells resulted in restoration of RFC mRNA
expression. Importantly, stable transfection of a hRFC cDNA driven
by a potent cytomegalovirus promoter, resulted in restoration of
normal RFC mRNA expression and nearly wild type sensitivity to MTX.
These results provide the first demonstration of a novel mechanism of
antifolate drug resistance that is based upon altered expression and
function of transcription factors resulting in transcriptional
silencing of the hRFC promoter.
It was recently found that the hRFC promoter is driven by an inducible
CRE/AP-1-like element and a constitutive GC-box element (15). This
study showed that CREB-1 and c-Jun in human fibrosarcoma HT1080 cells,
as well as CREB-1 and ATF-1 in human hepatocellular carcinoma HepG2
cells, were the cell-specific transcription factors involved in CRE
binding (the inducible element) in the hRFC promoter. We consistently
find here that CREB-1 and ATF-1 were expressed at substantial levels in
wild type human leukemia CEM cells and displayed a functional binding
to CRE. The marked decrease or loss of RFC mRNA expression in
AG2034R2 and ZD9331R1.5 cells, respectively,
was associated with a decreased expression (or lack) of CREB-1 and
ATF-1. These results strongly suggest that CREB-1 and ATF-1 are
essential components for the transcriptional activation of the
inducible element in the hRFC promoter in leukemia cells. However,
antifolate-resistant cancer cell lines and malignant tumors of
different cell lineages may potentially express alternative, cell-specific transacting factors of the basic region-leucine zipper
(bZip) family, which may also contribute to hRFC transcription.
Transient transfections of either Sp1 or the long Sp3 isoform resulted
in a potent activation of the constitutive GC-box element in the hRFC
promoter (15). In contrast, introduction of the short Sp3 isoform
resulted in a potent repression of the Sp1-dependent activation of the hRFC promoter. Consistently, ZD9331R1.5
and AG2034R2 cells showed elevated levels of the short Sp3
isoforms; the latter is likely to act as a repressor of the
Sp1-mediated activation of the constitutive GC-box element in the hRFC
promoter. This suggestion could gain support from the following results
and considerations. First, the 1:1 mixing of nuclear proteins from
parental and ZD9331R1.5 cells markedly blocked GC-box
binding but did not interfere with CRE binding. Second, it is likely
that transfection of ZD9331R1.5 and AG2034R2
cells with an Sp1 expression construct should result in Sp1 levels that
exceed those of the short Sp3 isoforms, resulting in restoration of RFC
mRNA expression. Thus, the short Sp3 isoforms that are expressed at
elevated levels in these antifolate-resistant cells appear to
competitively block Sp1 binding to the GC-box, thereby repressing
transcriptional activation of the hRFC gene.
We have recently shown that a predominant mechanism of resistance to
MTX and various novel hydrophilic antifolates is the loss of
RFC-mediated drug transport in multiple human leukemia sublines (8, 10,
11). These transport-defective sublines harbored various inactivating
mutations that primarily clustered in the first transmembrane domain of
the hRFC (8, 10, 11). In contrast, AG2034R2 cells were
devoid of RFC mutations (11), had an extremely poor expression of RFC
mRNA, and had no detectable levels of RFC protein. These cells
consequently displayed an abolished antifolate transport. Thus, the
loss of RFC expression in AG2034R2 cells constitutes the
first mechanism of antifolate-resistance that appears to rely solely on
the loss of transcriptional activation of the hRFC promoter. In
contrast, ZD9331R1.5 cells harbored a heterozygous
premature translation termination mutation (Glu257Stop) but retained a
wild type hRFC allele (11). Although this wild type allele could have
been substantially transcribed, ZD9331R1.5 cells were
devoid of RFC mRNA expression. These results suggest that although
mutations are frequently observed in the human (7-11) and the murine
(21) RFC genes, the mutational inactivation of a single allele is
insufficient and may be accompanied by transcriptional silencing of
both alleles via alterations in the expression and function of certain
transcription factors. A strong support to this suggestion derives from
the fact that various antifolate-resistant human (7-11) and mouse (21)
leukemia sublines harbored both RFC-inactivating mutations and
prominently decreased RFC mRNA levels.
The present findings have potentially important implications for the
development of clinical resistance to MTX and novel antifolates, including ZD1694 (Raltitrexed, Tomudex) (4) and multitargeted antifolate (Pemetrexed) (22), which use RFC as their primary route of
entry. Several studies with specimens derived from ALL (12, 13) and
osteosarcoma (14) have shown that defective drug transport via the RFC
may be a mechanism of resistance to MTX. Consistently, decreased RFC
mRNA expression has also been documented in specimens from ALL (12,
13) and osteosarcoma patients (14). Surprisingly however, while
inactivating mutations in the RFC gene were a frequent mechanism of
antifolate resistance in human (7-11) and animal tumor cell lines
(21), the frequency of RFC mutations in ALL specimens was extremely
low.2 These results suggest
that transcriptional silencing of the hRFC gene either by altered
expression and function of transcription factors or alternatively by
hRFC promoter methylation (23) may prove as factors that contribute to
antifolate resistance. Importantly, the collateral sensitivity of
various transport-defective phenotypes to lipophilic antifolates
including trimetrexate (11) may be exploited to eradicate
antifolate-resistant tumors (24).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP or [
-32P]dATP end-labeled
CRE, AP-1, or GC-box double-stranded oligonucleotides (Table
I) as detailed elsewhere (18).
Oligonucleotide competition was performed with a 10-100-fold molar
excess of unlabeled oligonucleotides (Table I). For supershift
analysis, an aliquot of nuclear proteins (6 µg) was incubated for
1 h at 4 °C with anti-CREB-1, -ATF-1, -Sp1, and -Sp3 (Santa
Cruz Biotechnology) antibodies (2-4 µg) in binding buffer according
to the instructions of the manufacturer. Then, the radiolabeled
oligonucleotide was added and DNA-protein immunocomplexes were allowed
to form for 30 min at 4 °C. Complexes consisting of DNA, nuclear
protein(s), and a specific antibody were resolved by electrophoresis on
6% non-denaturing polyacrylamide gels in 0.5× Tris borate-EDTA, pH
8.4, at 4 °C. The gels were then dried, and DNA-protein complexes
were visualized by phosphorimaging. Protein concentration was
determined by the colorimetric method of Bradford (19).
Oligonucleotides used for electrophoretic mobility shift assay
-actin) for 45 s, elongation at 72 °C for 1 min, followed by
a 10-min extension at 72 °C. Then, the PCR products were resolved on
2% agarose gels. The primers used for the semiquantitative RT-PCR of
hRFC, DHFR, FPGS, MRP1, GAPDH, and
-actin are depicted in Table
II.
Oligonucleotides used for RT-PCR of various housekeeping genes
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Antifolate resistance and
[3H]MTX transport in parental CEM cells and
their antifolate-resistant sublines. Levels of antifolate
resistance in AG2034R2 and ZD9331R1.5 cells
prior to (A) or after stable transfection with a hRFC
cDNA (C). Initial rates of [3H]MTX
transport (B) were determined as described under
"Materials and Methods." The influx of [3H]MTX in
parental CEM cells was: 4.0 ± 0.6 pmol
[3H]MTX/min × 107 cells. Results shown
are the means of four independent experiments ± S.D.
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Fig. 2.
Expression of RFC and various housekeeping
genes in wild type and antifolate-resistant cells. Western blot
analysis with microsomal proteins from RFC-overexpressing CEM-7A cells
(50 µg, lane 1), wild type CEM cells (100, 50, 25, and
12.5 µg, lanes 2-5, respectively), as well as
AG2034R2 and ZD9331R1.5 cells (150 µg,
lanes 6 and 7, respectively) was performed with
anti-hRFC antibodies as detailed under "Materials and Methods."
Messenger RNA levels of RFC (B), DHFR, GAPDH, FPGS, and MRP1
(C) were determined by semiquantitative RT-PCR as detailed
under "Materials and Methods." RFC mRNA expression determined
by various dilutions of total cDNA prior to or after transient
transfection of parental and antifolate-resistant cells with the
expression vectors pRSV-CREB-1 and pPacSp1 (D) was
normalized to -actin (B) or GAPDH (D). RFC
mRNA expression in wild type and antifolate-resistant cells stably
transfected with an expression vector harboring the hRFC cDNA is
also shown (B, lane 1). The H2O group
represents a negative PCR control in which no cDNA was present
(B, lane 2).
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Fig. 3.
Electrophoretic mobility shift assay of
[32P]-labeled CRE, GC-box, and AP-1
oligonucleotides. Nuclear proteins (6 µg) from parental CEM,
AG2034R2, and ZD9331R1.5 cells were first
incubated with [32P]-labeled CRE (A), GC-box
(B), and AP-1 (C) oligonucleotides, resolved by
electrophoresis on polyacrylamide gels, and viewed by phosphorimaging.
A portion of nuclear proteins (6 µg total) from parental CEM cells
was mixed with an equal amount of nuclear proteins from
ZD9331R1.5 cells, and the binding to
[32P]-labeled CRE (D) and
[32P]-labeled GC-box (E) oligonucleotides was
examined. The nuclear protein-CRE/GC-box/AP-1 complexes are denoted on
the left by 1, 2, and
3.
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Fig. 4.
CRE and GC-box reporter gene
activities following transient transfection into parental and
antifolate-resistant cells. CRE-luciferase (A),
GC-box-luciferase (B), GC-box-CAT (C), and
control Renilla constructs were transfected by
electroporation into parental CEM and antifolate-resistant cells. Cells
were then lysed and reporter gene activities were normalized to
Renilla luciferase. Results presented are averages ± S.D. derived from three experiments. The 100% control activity was
obtained from parental CEM cells.
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Fig. 5.
Antibody-mediated supershift analysis with
[32P]-labeled CRE and
[32P]-labeled GC-box oligonucleotides.
Nuclear proteins (6 µg) from parental CEM and antifolate-resistant
cell lines were first incubated with antibodies (2-4 µg) to CREB-1,
ATF-1, Sp1, and Sp3 for 1 h at 4 °C. Then, the binding to
[32P]-labeled CRE (A) or
[32P]-labeled GC-box (B) oligonucleotides was
examined by phosphorimaging. The antibody-nuclear
protein(s)-oligonucleotide complexes are denoted on the left
by A, B, and C.
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Fig. 6.
Western blot analysis of transcription factor
expression in parental and antifolate-resistant cell lines.
Nuclear proteins (20 µg) from parental CEM and antifolate-resistant
sublines were resolved by polyacrylamide gels containing SDS,
transferred to a cellulose nitrate Protran membrane, and reacted with
antibodies to human CREB-1/ATF-1 (A) as detailed under
"Materials and Methods." The blots were then reprobed with
antibodies to Sp1 (B) and Sp3 (C).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Yaffa Both for technical assistance, Dr. Michael E. Greenberg for the pRSV-CREB-1 expression vector, and Dr. Guntram Suske for the pPacSp1 construct.
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FOOTNOTES |
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* This work was supported by research grants from The Israel Cancer Association and the Star Foundation (to Y. G. A.).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 Biology, The Technion-Israel Institute of Technology, Haifa 32000, Israel. Tel.: 972-4-829-3744; Fax: 972-4-822-5153; E-mail: assaraf@tx.technion.ac.il.
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M209578200
2 S. Drori, Y. Kaufman, P. D. Cole, B. A. Kamen, J. Sirota, G. Rechavi, A. Toren, M. Ben-Arush, R. Elhasid, D. Sahar, G. J. L. Kaspers, L. H. Matherly, G. Jansen, and Y. G. Assaraf, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: MTX, methotrexate; RFC, reduced folate carrier; hRFC, human RFC; CRE, cyclic AMP-response element; CREB-1, CRE-binding protein 1; Sp1, specificity protein 1; DHFR, dihydrofolate reductase; FPGS, folylpolyglutamate synthetase; MRP1, multidrug resistance protein 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ALL, acute lymphoblastic leukemia; RT, reverse transcription; CAT, chloramphenicol acetyltransferase.
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