Impaired Membrane Transport in Methotrexate-resistant
CCRF-CEM Cells Involves Early Translation Termination and Increased
Turnover of a Mutant Reduced Folate Carrier*
So C.
Wong
§,
Long
Zhang
,
Teah L.
Witt
,
Susan A.
Proefke
,
Alok
Bhushan¶, and
Larry H.
Matherly
§
From the
Experimental and Clinical Therapeutics
Program, Barbara Ann Karmanos Cancer Institute and the
§ Department of Pharmacology, School of Medicine, Wayne
State University, Detroit, Michigan 48201 and the ¶ Department
of Pharmaceutical Sciences (AB), Idaho State University,
Pocatello, Idaho 83209
 |
ABSTRACT |
The basis for impaired reduced folate carrier
(RFC) activity in methotrexate-resistant CCRF-CEM (CEM/Mtx-1) cells was
examined. Parental and CEM/Mtx-1 cells expressed identical levels of
the 3.1-kilobase RFC transcript. A ~85-kDa RFC protein was detected in parental cells by photoaffinity labeling and on Western blots with
RFC-specific antiserum. In CEM/Mtx-1 cells, RFC protein was undetectable. By reverse transcriptase-polymerase chain reaction and
sequence analysis, G to A point mutations were identified in CEM/Mtx-1
transcripts at positions 130 (P1; changes glycine 44
arginine) and 380 (P2; changes serine 127
asparagine). A 4-base pair (CATG) insertion detected at position 191 (in 19-30% of cDNA clones) resulted in a frameshift and early
translation termination. Wild-type RFC was also detected (0-9% of
clones). Wild-type RFC and double-mutated RFC (RFCP1+P2)
cDNAs were transfected into transport-impaired K562 and Chinese hamster ovary cells. Although RFC transcripts paralleled wild-type protein, for the RFCP1+P2 transfectants, disproportionately
low RFCP1+P2 protein was detected. This reflected an
increased turnover of RFCP1+P2 over wild-type RFC.
RFCP1+P2 did not restore methotrexate transport; however,
uptake was partially restored by constructs with single mutations at
the P1 or P2 loci. Cumulatively, our results
show that loss of transport function in CEM/Mtx-1 cells results from
complete loss of RFC protein due to early translation termination and
increased turnover of a mutant RFC protein.
 |
INTRODUCTION |
Despite the availability of newer antifolates, methotrexate
(Mtx)1 continues to play an
important role as an antineoplastic agent. To reach its intracellular
target, dihydrofolate reductase, the preferred route of Mtx entry
involves the reduced folate carrier (RFC; 1, 2). RFC transport of Mtx
is critical to drug action because of its role in generating sufficient
unbound intracellular antifolate to sustain maximal enzyme inhibition
(1). Furthermore, high levels of Mtx are also necessary for the
synthesis of Mtx polyglutamates (1).
Defective membrane transport of Mtx by RFC has been identified as a
major mechanism of Mtx resistance (1-11). Transport alterations can
manifest as reduced rates of carrier translocation (reduced Vmax), decreased affinities for transport
substrates (increased Kt), or both, and may involve
decreased levels of normal RFC (6) or the expression of structurally
altered RFC proteins (7-11). For instance, in Mtx-resistant K562
(K500E) cells, impaired Mtx transport is accompanied by decreased RFC
transcripts and protein (6). A G to A transition at position 890 of the
murine RFC cDNA resulted in a substitution of serine 297 by
asparagine and a selective decrease in Mtx binding affinity (~4-fold)
without effects on other antifolate analogs (aminopterin,
10-ethyl-10-deazaaminopterin; Ref. 9). Likewise, replacement of serine
46 by asparagine (10) or glutamate 45 by lysine (11) in murine RFC
resulted in greater impairment of uptake for Mtx than
(6S)-5-formyl tetrahydrofolate. In severely transport
defective L1210 cells (MtxrA), loss of transport activity
appeared to reflect a single (G to C) point mutation at nucleotide 429 of the murine RFC cDNA sequence which resulted in the substitution
of proline 130 by alanine (7). However, these cells also contained a
wild-type RFC allele that was not transcribed. A silent wild-type RFC
allele was described for Mtx-resistant MOLT-3 cells
(MOLT-3/Mtx10,000; Ref. 8). Moreover, two mutations in the
RFC coding region were detected which resulted in the creation of new
stop codons and synthesis of truncated nonfunctional RFCs (8).
In this report, the molecular mechanisms responsible for the
transport-impaired phenotype (~3% of wild-type) of Mtx-resistant (~243-fold) CCRF-CEM (CEM/Mtx-1;12) cells were examined. We show that
although the levels of RFC transcripts are essentially unchanged from
wild-type cells, there is a complete loss of RFC protein due to early
translation termination and increased turnover of a double mutant RFC
protein. The residual transport activity previously described in this
transport-impaired line (12) presumably reflects extremely low levels
of wild-type RFC and/or, possibly, non-RFC modes of Mtx uptake
(13-15).
 |
EXPERIMENTAL PROCEDURES |
Materials--
[
-32P]dCTP (3000 Ci/mmol) and
[
-35S-thiol]dATP (1400 Ci/mmol) were obtained from NEN
Life Science Products Inc. [3',5',7-3H]Mtx (20 Ci/mmol)
and [4,5-3H]leucine (120 Ci/mmol) were purchased from
Moravek Biochemicals (Brea, CA). Unlabeled Mtx was provided by the Drug
Development Branch, NCI, National Institutes of Health, Bethesda, MD.
Both labeled and unlabeled Mtx were purified by high performance liquid chromatography prior to use (16). GW1843U89 (17) was obtained from
Glaxo-Wellcome Pharmaceuticals (Research Triangle Park, NC). Sequenase
version 2.0 and reagents for dideoxynucleotide sequencing were from
U. S. Biochemical Corp. (Cleveland, OH). Restriction and modifying
enzymes were obtained from Promega (Madison, WI). Synthetic
oligonucleotides were obtained from Genosys Biotechnologies, Inc. (The
Woodlands, TX).
Cell Culture--
Wild-type CCRF/CEM and transport-impaired
CEM/Mtx (18) lymphoblastic leukemia lines were gifts of Dr. Andre
Rosowsky (Boston, MA). Cells were cloned in soft agar (6, 19) and
clonal lines (designated CEM-4 and CEM/Mtx-1 for the parental and
Mtx-resistant cells, respectively) were used for all experiments.
Transport-deficient K500E cells were selected from wild-type K562 cells
by cloning in soft agar with 500 nM Mtx (6). K500E cells
were transfected with wild-type RFC (KS43) to generate the K43-1 and
K43-6 sublines, as described previously (6). The cell lines were
maintained in RPMI 1640 medium as described previously (6, 12).
Transport-defective Mtx-resistant Chinese hamster ovary (CHO) cells,
MtxRIIOuaR2-4 (20), were a gift of Dr. Wayne Flintoff
(London, Ontario, Canada). Cells were grown in
-minimal essential
medium with 10% iron-supplemented bovine calf serum, penicillin (100 units/ml), and streptomycin (100 µg/ml). The pC43/10 CHO line was
derived from MtxRIIOuaR2-4 cells by transfection with the
full-length human RFC cDNA (KS43; Ref. 21). CHO cells were grown as
monolayers for transfection and general maintenance; for transport
experiments, cells were grown in suspension in spinner flasks.
Southern and Northern Analysis--
Genomic DNAs were isolated
from cultured cells using the PuregeneTM DNA isolation kit
from Gentra Biosystem, Inc. (Minneapolis, MN). Aliquots (10 µg) were
digested with restriction enzymes (either BamHI or
HindIII), fractionated on a 0.6% agarose gel, and blotted onto a nylon membrane (Genescreen Plus, DuPont), following standard protocols (22).
Total RNA was isolated from log phase cells using the TRIzol Reagent
(Life Technologies, Inc.). RNA samples were analyzed on a
formaldehyde-agarose gel, exactly as described previously (21).
Equal loading was established by probing with 32P-labeled
-actin cDNA or by staining with ethidium bromide. All membranes
were hybridized with 32P-labeled full-length RFC cDNA
and processed as described previously (21).
Analysis of RFC Coding Region and Genomic Sequence--
RFC
cDNAs from parental and CEM/Mtx-1 cells were synthesized from total
RNA with random hexamers using a RT-PCR kit from Perkin-Elmer. Four
sets of PCR primers were used to generate overlapping partial cDNAs
spanning the entire RFC coding region. The PCR primers for RFC cDNA
amplification are shown in Table I. PCR conditions were 94 °C for
30 s, 63 °C for 45 s, and 72 °C for 1 min (35 cycles), and ending with 72 °C for 7 min (1 cycle). PCR products were
subcloned into the pCR 2.1 plasmid using the T-A cloning kit
(Invitrogen) and the nucleotide sequences were determined by
dideoxynucleotide sequencing (23). RT-PCR reactions were repeated 2-3
times for regions containing the P1 and P2
mutations and for each primer set, multiple cDNA clones were sequenced.
Genomic fragments containing point mutations identified in the
CEM/Mtx-1 cDNAs were PCR amplified and the PCR products subcloned and sequenced as described above. Primers for genomic amplification were based on the human RFC cDNA (21) and gene (24, 25) nucleotide
sequences. For amplifying the fragment containing the P1
mutation, a nested PCR approach was used. In the primary PCR reaction,
two RFC intron-specific primers, RFC-IP1
(5'-ctgcagaccatcttccaaggtgccctga; upstream of the splice acceptor site
at
49) and RFC-IP2 (5'-gcagaccatcttccaaggtgccctga; downstream of the
splice donor site at 189), were used. For the secondary nested PCR
reaction, the primers used were the exon-specific primer P8 (Table I)
and another intron-specific primer RFC-IP3 (5'-acctactggtgctgctgcccctgc; downstream of the splice donor site at
189). The fragment containing the P2 mutation was amplified with intron-specific RFC-IP4 (5'-gcggcagcattgctaacacctggtg; upstream of
the splice acceptor site at 190) and exon-specific P7 (Table I)
primers. PCR conditions for amplifying genomic DNAs were 94 °C for
10 s, 63 °C for 60 s, and 72 °C for 60 s (35 cycles), and 1 cycle of 72 °C for 7 min.
Preparation of Mutant RFC Constructs and Transfection of
Transport-defective K562 and CHO Cells--
Constructs containing the
P1 (RFCP1) or P2
(RFCP2) mutations were prepared by restriction digesting
CEM/Mtx-1 RFC mutant cDNAs with Eco72/ApaI
(for P1) or NcoI/ApaI (for
P2). The fragments were subcloned into
Eco72/ApaI- or
NcoI/ApaI-digested wild-type (KS43) RFC in
pBluescript SK(
) (21). The mutated RFC cDNAs were excised with
BamHI and XhoI, and the ~2-kb fragments
subcloned into a BamHI/XhoI-digested pCDNA3
expression vector. The construct containing both
P1 and P2 mutations (RFCP1+P2) was
prepared by ligating the Eco72/ApaI
RFCP1 fragment into
Eco72/ApaI-digested RFCP2 in
pCDNA3. Mutant constructs were transfected into transport defective
MtxRIIOuaR2-4 and K500E cells as described previously (6,
21). G418-resistant clones were expanded and screened for RFC
transcripts (Northern), immunoreactive RFC protein (Western), and
[3H]Mtx uptake.
Preparation of Recombinant RFC Antiserum--
The complete
coding sequence of the KS43 RFC cDNA (21) was subcloned into a pGEX
glutathione S-transferase (GST) fusion vector (Pharmacia
Biotech, Piscataway, NJ). Following transformation of Escherichia
coli (BL21) cells and induction by
isopropyl-
-D-thiogalactoside (0.5 mM) for
4 h at room temperature, GST-RFC fusion proteins were purified
from bacterial lysates by affinity chromatography using
glutathione-Sepharose 4B (Pharmacia Biotech), as recommended by the
manufacturer. Authenticity and purity of the purified RFC fusion
protein were confirmed by Coomassie Blue staining and Western analysis
with anti-GST (Pharmacia Biotech) and RFC peptide-specific (RFC/ps;
Ref. 26) antibodies. Anti-GST-RFC antiserum was raised in rabbits using
purified GST-RFC fusion protein as antigen (Pocono Rabbit Farms and
Laboratories, Canadensis, PA). Both immune and preimmune sera were
purified on protein A-agarose columns prior to use (27).
Preparation of Plasma Membranes and Western Analysis--
Plasma
membranes were prepared by differential centrifugation (19, 28). Where
noted, particulate membrane fractions were additionally purified on
discontinuous sucrose gradients (19, 28). Plasma membrane purity and
endoplasmic reticulum contamination of crude particulate and sucrose
density gradient-purified membranes were established by 5'-nucleotidase
(29) and NADPH-cytochrome c reductase (30) assays, respectively.
Membrane proteins were electrophoresed on 7.5% gels in the presence of
SDS (31) and electroblotted onto polyvinylidene difluoride membranes
(DuPont) for detection with protein A-purified GST-RFC antibody and
enhanced chemiluminescence (Pierce, Rockford, IL). A few experiments
employed RFC peptide-specific (RFC/ps) antibody (26). Light emission
was recorded on x-ray film with various exposure times, and the signal
was analyzed with a computing densitometer and ImageQuant software
(Molecular Dynamics, Sunnyvale, CA). For some experiments, the
heterogeneously glycosylated RFCs were enzymatically deglycosylated
with N-glycosidase F (Boehringer Mannheim), as described
previously (6, 28).
Photoaffinity Labeling of Cell Surface RFC Proteins--
Cell
surface RFC proteins in wild-type CCRF-CEM and CEM/Mtx-1 cells (1 × 108 cells/labeling condition) were photoaffinity labeled
using
N
-(4-amino-4-deoxy-10-methylpteroyl)-N
-(4-azido-5-[125I]iodosalicylyl)-L-lysine
(APA-[125I]ASA-Lys), as described previously (6,
21). Specificity of labeling was established by performing identical
incubations in the presence of 100 µM aminopterin. Equal
aliquots of labeled proteins were electrophoresed on a 4-10% gradient
gel in the presence of SDS (31). The gel was dried and exposed to x-ray film.
Transport of [3H]Mtx in Transfected
Cells--
Initial [3H]Mtx uptake rates were determined
over 180 s using 1-2 × 107 cells/ml (6, 12, 19,
21) and a Mtx concentration of 0.5 µM. The levels of
intracellular radioactivity were expressed as picomoles/mg of protein,
calculated from direct measurements of radioactivity and protein
contents of the cell homogenates. Protein assays were by the method of
Lowry et al. (32). Kinetic constants (Kt
and Vmax) were calculated from Lineweaver-Burk plots.
 |
RESULTS |
Impaired Mtx Transport in CEM/Mtx-1 Cells Is Independent of
Changes in RFC Transcripts or Gene Structure
Northern analysis of
total RNAs from parental CCRF-CEM (CEM-4) and Mtx-resistant CEM/Mtx-1
cells showed that essentially identical levels of a major 3.1-kb RFC
mRNA transcript were expressed (Fig.
1) despite a ~33-fold difference in
relative Mtx transport (12). Although a 1-kb RNA species hybridized
with the RFC cDNA in parental cells and a unique 9.5-kb band was
detected in CEM/Mtx-1 cells (Fig. 1), the significance of these forms
is not clear. These bands were still present even when poly(A)
mRNAs were used for Northern analysis (data not shown). Restriction
analysis (BamHI or HindIII) of genomic DNAs from
CEM-4 and CEM/Mtx-1 cells did not reveal any major alterations in RFC
gene organization or copy number between the lines (data not
shown).

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Fig. 1.
RFC transcript levels in parental CCRF/CEM
(CEM-4) and CEM/Mtx-1 cells. Total RNAs (20 µg) were
fractionated on a 0.9% formaldehyde-agarose gel and transferred to a
nylon membrane. The blot was hybridized with 32P-labeled
full-length RFC cDNA (upper panel). After
autoradiography, the blot was stripped and reprobed with
32P-labeled -actin to demonstrate equal loading
(lower panel). Molecular size standards are indicated.
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Analysis of RFC Protein Expression in CEM/Mtx-1 Cells by Western
Blotting and Photoaffinity Labeling--
Expression of RFC protein in
plasma membranes from parental and CEM/Mtx-1 cells was analyzed by
Western blotting using antibody to recombinant RFC fusion protein
(GST-RFC) and chemiluminescence detection, and by photoaffinity
labeling with APA-[125I]ASA-Lys (6, 21). For both
methods, a broadly migrating RFC band centered at ~85 kDa was
identified in parental cells (Fig. 2,
left panel, and Fig. 3,
respectively). Identical results were obtained on Western blots with
peptide-specific (RFC/ps) antiserum (not shown). Slight differences
were seen in the relative migrations for RFC, reflecting the different
gel systems used for separation (7.5% for the Western
versus 4-10% for the photoprobe experiments). By both
approaches, the major bands identified as RFC were converted to a
single ~65-kDa deglycosylated form by treatment with
N-glycosidase F (shown for the immunoblotted RFC in parental
CCRF-CEM cells; Fig. 2, right panel). This is the size
predicted from the RFC cDNA sequence (21, 33, 34). By contrast, in
CEM/Mtx-1 cells none of the ~85-kDa RFC protein was
detected either by Western blotting with anti-GST-RFC (Fig. 2) or
peptide-specific antiserum (not shown), or by photoaffinity labeling
with APA-[125I]ASA-Lys (Fig. 3). However, an unidentified
42-kDa protein was specifically labeled with the photoprobe (Fig. 3).
Although there were no changes in the background staining on Western
blots following treatment of CEM/Mtx-1 proteins with
N-glycosidase F, the 42-kDa photolabeled band was converted
to ~37 kDa by this treatment (not shown).

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Fig. 2.
Immunoreactive RFC protein in parental
CCRF/CEM (CEM-4) and CEM/Mtx-1 cells. Left panel, CEM-4
and CEM/Mtx-1 proteins (50 µg), solubilized from sucrose density
gradient-purified plasma membranes, were fractionated on a 7.5%
polyacrylamide gel with SDS and electroblotted onto polyvinylidene
difluoride membranes. Immunoreactive RFC protein was detected with
anti-GST-RFC antibody and an enhanced chemiluminescence kit. Migrations
of the molecular weight markers are shown in kDa. Right
panel, membrane proteins (100 µg) from CEM-4 cells were digested
in the presence (+) and absence ( ) of N-glycosidase F for
18 h at 37 °C prior to SDS-gel electrophoresis and
electrotransfer. Detection was with anti-GST-RFC antibody, as described
above. DgRFC designates the 65-kDa deglycosylated RFC. There
was no change in the banding pattern for the CEM/Mtx-1 protein upon
N-glycosidase F treatment (not shown), demonstrating that
the bands in the CEM/Mtx-1 lane (left panel) are
nonspecific.
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Fig. 3.
Photoaffinity labeling of RFC. Equal
numbers (1 × 108) of parental CCRF-CEM (CEM-4) and
CEM/Mtx-1 cells were labeled with APA-[125I]ASA-Lys in
the presence (+) or absence ( ) of 100 µM aminopterin
(AMT) at 0 °C. Labeled proteins were extracted in 1%
Triton X-100 and equal aliquots were analyzed on 4-10% gradient gel.
The gel was dried and exposed to x-ray film. Molecular masses of
protein standards are indicated.
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Identification of Mutations in the RFC Coding Sequence in CEM/Mtx-1
Cells--
The RFC coding sequences from parental CCRF-CEM and
CEM/Mtx-1 cells were examined by RT-PCR and dideoxynucleotide
sequencing of the PCR products. Four primer sets were used to amplify
the entire RFC coding sequence (P1/P3, P4/P7, P8/P7, and P9/P10; Table I). Three alterations were identified in
a 572-bp segment (positions
23 to 549, where 1 is the translational
start site) amplified from CEM/Mtx-1 cells by primer set P7/P8 and
encoding the RFC amino terminus. These include two G to A point
mutations at positions 130 (designated P1; nucleotide
position 1 is the translation start) and 380 (P2) in all of
the 16 CEM/Mtx-1 clones sequenced, and a 4-bp (CATG) insertion at
position 191 in 3 of the clones. By contrast, none of the 9 cDNA
clones amplified with P7/P8 from parental CCRF-CEM cells contained any
alterations from wild-type RFC sequence (21, 33, 34).
Analogous results were obtained by amplification of a fragment
containing the P2 locus (positions 141-549) with the P4/P7 primer set (21/23 with a P2 mutation, including 7 with
insertion at position 191). However, 2 of 23 clones derived from
CEM/Mtx-1 also contained wild-type sequence at this position. All of
the 15 clones amplified from parental CCRF-CEM cells with P4/P7 primers contained wild-type sequence at the P2 locus; however, for
3 of these wild-type clones, the 4-bp CATG insertion was detected.
PCR amplification of CEM/Mtx-1 genomic DNA with both intron- and
exon-specific primers, and sequencing of the PCR products confirmed
both P1 and P2 mutations at the genomic level.
Again, neither of the mutations was detected in parental cells.
Notably, the 4-bp (CATG) insertion could not be found in any of the
genomic DNAs amplified with the P8 and RFC-IP3 primers, suggesting that this probably arose from alternative splicing of intron sequence at the
splice donor junction at position 189 (24, 25). Not surprisingly, a
number of CEM/Mtx-1 clones (1 of 8 for P1 and 3 of 4 for
P2, amplified with separate primer sets) exhibited wild-type genomic sequence.
The P1 mutation would result in a change of glycine
44 to arginine and, by computer prediction
(Garnier-Robson-Osguthorpe; 35), introduce an altered secondary
structure in the region immediately upstream from this locus. The
P2 mutation results in a substitution of serine 127 by
asparagine in a conserved putative transmembrane domain (residues 124 to 144), yet secondary structure is seemingly unaffected. The CATG
insertion at position 191 of the RFC coding sequence generates a
frameshift and early translation termination at position 1176, resulting in a truncated RFC protein (~48 kDa) with only 11% of
recognizable primary sequence. However, this would appear to account
for no more than 30% of the loss of the full size RFC protein.
Characterization of Mutant RFCP1+P2 Protein--
The
lack of detectable RFC protein in CEM/Mtx-1 cells may reflect its
inefficient synthesis, membrane targeting, or decreased stability of
the double-mutated RFC. To evaluate these possibilities, wild-type RFC
and a mutant construct containing G to A mutations at both positions
130 and 380 (designated RFCP1+P2) were transfected into
transport-defective K562 (K500E) and CHO (MtxRIIOuaR2-4)
cells, for comparison to wild-type RFC (6, 21). As described previously
for K500E and MtxRIIOuaR2-4 transfectants (designated K43-1
and K43-6, and pC43/10, respectively) expressing wild-type
RFC, RFC protein in plasma membranes is reflective of the high levels
of RFC transcripts (Refs. 6 and 21; data are shown in Fig.
4 for the human K43-6 (left
panel) and hamster pC43/10 (right panel)
transfectants).

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Fig. 4.
Expression and transport of wild-type RFC and
RFCP1+P2 cDNA constructs in K500E (left
panels) and CHO (right panels)
transfectants. Panels A and D, equal amounts of total
RNAs (15 µg) from K500E (K43-6, K43P1+P2/4, and
K43P1+P2/22) and CHO (pC43/10 and RIIP1+P2/13)
transfectants, and untransfected cells (K500E and
MtxRIIOuaR2-4) were analyzed on Northern blots probed with
a 32P-labeled human RFC cDNA (KS43; Ref. 20). Equal
loading was established by staining with ethidium bromide (not shown).
Size markers (18 S and 28 S, or molecular standards) are noted.
Panels B and E, particulate membrane fractions from the
K500E and CHO transfectants were analyzed on Western blots with
untransfected cells. The protein amounts (in µg) analyzed for each of
the sublines are noted. Detection was with anti-GST-RFC antibody and
enhanced chemiluminescence. Migrations of wild-type RFC (~85 kDa) and
RFCP1+P2 (70 kDa) are noted. In the wild-type RFC
transfectants, a 70-kDa band comigrating with RFCP1+P2 is
seen at some exposures, likely reflecting less glycosylated variants of
wild-type RFC. Panels C and F, initial uptake rates for
[3H]Mtx (0.5 µM) were assayed over 180 s as described under "Experimental Procedures." Mean data are shown
for duplicate incubations from a single representative
experiment.
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RFCP1+P2 transcripts were, likewise, detected on Northern
blots for the majority of both K500E (8 of 12) and
MtxRIIOuaR2-4 (11 of 18) transfectants (not shown),
invariably as multiple hybridizing bands (Fig. 4, A and
D, shows representative data). The smallest band
approximated the size (~2 kb) of the RFCP1+P2 cDNA so
that all transcript forms were of sufficient size to encode the
RFCP1+P2 protein. For only two K500E
(K43P1+P2/4 and K43P1+P2/22) clones and one
MtxRIIOuaR2-4 (RIIP1+P2/13) clone was
RFCP1+P2 protein detected on Western blots (shown in Fig.
4, panels B and E). For these, the levels of
mutant protein were exceedingly low (estimated by densitometry as
4-8% of the wild-type value relative to levels of total RFC or
RFCP1+P2 transcripts). Furthermore, RFCP1+P2
protein was distinctly smaller (~70 kDa) than the wild-type carrier (Fig. 4). Both wild-type RFC and RFCP1+P2 completely
reverted to 65-kDa deglycosylated forms upon treatment with
N-glycosidase F (Fig. 5),
establishing that these differences in carrier size reflected their
extents of N-glycosylation.

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Fig. 5.
Deglycosylation of wild-type RFC and
RFCP1+P2. Sucrose density gradient-purified membranes
were prepared from K43-6 and K43P1+P2/22 cells. Membrane
proteins (20 µg) were digested in the presence (+) and absence ( )
of N-glycosidase F for 18 h at 37 °C, then
fractionated on SDS gels and electrotransferred to a polyvinylidene
difluoride membrane. Detection was with anti-GST-RFC antibody and
enhanced chemiluminescence. The arrows denote wild-type RFC
(A, ~85 kDa), RFCP1+P2 (B, 70 kDa),
and the deglycosylated wild-type RFC and RFCP1+P2
(C, 65 kDa).
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Turnover of Wild-type RFC and RFCP1+P2
Proteins--
The decreased levels of mutant RFCP1+P2
compared with wild-type RFC in transfected cells (and by extension,
CEM/Mtx-1) may, in part, reflect differential rates of carrier
degradation. To explore this possibility, K43-6 and
K43P1+P2/22 transfectants were treated with 0.2 mg/ml
cycloheximide (results in >98% inhibition of protein synthesis, as
reflected in trichloroacetic acid-precipitable
[3H]leucine). Rates of exponential decline of wild-type
RFC and RFCP1+P2 were assayed over 24 h on Western
blots (Fig. 6). By this analysis, the
level of wild-type RFC decreased by approximately 50% over 24 h.
In contrast, RFCP1+P2 protein exhibited a rapid turnover
(Fig. 6). The half-life of RFCP1+P2 was calculated as
2.0 ± 0.56 h (mean ± S.E.; n = 3) and
no RFCP1+P2 protein could be detected after 8 h
following addition of cycloheximide.

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Fig. 6.
Turnover of wild-type RFC and
RFCP1+P2 protein. K43-6 and K43P1+P2/22
cells were treated with cycloheximide (0.2 mg/ml) and cells processed
to particulate plasma membrane fractions at the indicated times for
Western blot analysis of wild-type RFC and RFCP1+P2 protein
turnover. Detection was with anti-GST-RFC antibody and enhanced
chemiluminescence, followed by computer densitometry. For the K43-6 and
K43P1+P2/22 sublines, 5 and 20 µg, respectively, of
membrane protein were analyzed.
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Functional Properties of the RFCP1+P2,
RFCP1, and RFCP2 Proteins--
The effects of
the P1 and P2 mutations on
[3H]Mtx were measured for MtxRIIOuaR2-4 and
K500E transfectants expressing wild-type RFC and RFCP1+P2.
By this analysis, clones expressing the double mutant
RFCP1+P2 (RIIP1+P2/13, K43P1+P2/4,
and K43P1+P2/22, respectively) were completely devoid of
[3H]Mtx transport activity (Fig. 4, panels C
and F); i.e. initial rates of
[3H]Mtx uptake over 180 s were identical for the
transfected cells and the untransfected lines from which they were derived.
[3H]Mtx transport was partially restored for
the single mutant RFCP1 and RFCP2 constructs,
expressed in MtxRIIOuaR2-4 cells (i.e.
RIIP1/2, and RIIP2/6A and RIIP2/15A
for RFCP1 and RFCP2, respectively; Fig.
7). For the RIIP2/15A cells,
expressing the highest levels of RFCP2 (8-fold greater than
wild-type RFC in pC43/10), uptake of [3H]Mtx (0.5 µM) was ~50% of that for wild-type RFC-expressing
cells. Although we were able to identify stably expressed
RFCP1 (in RIIP1/2 cells) by screening over 20 G418-resistant colonies, expression was low (Fig. 7, inset).
However, both RIIP1/2 and RFCP2/15A exhibited
sufficient transport activity to calculate kinetic constants for Mtx
uptake (Table II). With both
RFCP1 (RIIP1/2) and RFCP2
(RIIP2/15A), the Kt values for Mtx were
increased from that for wild-type RFC (~11 and ~5-fold,
respectively; Table II). The absolute Vmax
values for Mtx for both RFC mutant constructs were at least 70% of
that for wild-type carrier (i.e. pC43/10 cells; Table II).
When normalized to levels of immunoreactive RFC protein on Western
blots (measured by densitometry; Fig. 7, inset), the relative Vmax for RFCP1
(RIIP1/2 subline) exceeded that for the wild-type carrier
by 3-fold, whereas the relative Vmax for
RFCP2 (RIIP2/15A subline) was only 16% of the
wild-type value (Table II). The Vmax
(normalized)/Kt values were 27 and 2.7%,
respectively, of that for wild-type RFC. In contrast to
RFCP1+P2 (see above), the substitution of arginine for
glycine 44 in the single-mutated RFCP1 construct and
asparagine for serine 127 in RFCP2 had no obvious effect on
the processing to mature, glycosylated (~85 kDa) RFC proteins (Fig.
7, inset).

View larger version (50K):
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|
Fig. 7.
Western blotting and Mtx transport for CHO
transfectants expressing wild-type RFC, and single mutated
RFCP1 and RFCP2 proteins.
MtxRIIOuaR2-4 cells were transfected with the wild-type
RFC, RFCP1, and RFCP2 constructs and positive
transfectants selected with G418. The inset shows the
relative levels of full size (~85 kDa) RFC protein in positive
transfectants expressing wild-type human RFC (pC43/10),
RFCP1 (RIIP1/2), and RFCP2
(RIIP2/6A and RIIP2/15A). No significant
immunoreactive bands were detected for wild-type CHO (Pro-3) and
untransfected (or "mock" transfected; data not shown)
MtxRIIOuaR2-4 cells. The amounts of membrane protein loaded
(in micrograms) are indicated. The bar graph shows the
absolute (not normalized for RFC protein on Western blots) values for
the initial uptake rates of [3H]Mtx (0.5 µM) assayed over 180 s in wild-type Pro-3 and
MtxRIIOuaR2-4 CHO cells, and for the
MtxRIIOuaR2-4 transfectants. Mean uptake data are shown for
duplicate incubations from a single representative experiment.
|
|
View this table:
[in this window]
[in a new window]
|
Table II
Kinetic constants for Mtx influx
Kinetic constants for Mtx transport were calculated by Lineweaver-Burk
analysis of initial rate data from three to four experiments.
|
|
 |
DISCUSSION |
CEM/Mtx-1 cells exhibit only 3% of normal levels of Mtx influx
associated with a 4-fold increased Kt and 6-fold
decreased Vmax (12). This altered substrate
binding extends to a range of folate and antifolate transport
substrates (10-ethyl-10-deazaaminopterin, aminopterin, ZD1694,
GW1843U89, (6R)-5,10-dideaza-5,6,7,8-tetrahydrofolate, folic
acid, and leucovorin) and initially suggested to us that the synthesis
of a structurally altered RFC might be responsible for the
drug-resistant phenotype (12). Although the demonstration of normal
levels of a major 3.1-kb transcript in CEM/Mtx-1 cells lent further
credence to this notion, no significant RFC protein could be detected
by Western blotting or photoaffinity labeling.
In MOLT3/Mtx10,000 cells, the absence of immunoreactive RFC
was previously attributed to the presence of mutations in the RFC
coding sequence which resulted in early translation termination and the
synthesis of severely truncated RFCs (8). Since an analogous mechanism
could occur in the CEM/Mtx-1 subline, we sequenced partial cDNAs
amplified from CEM/Mtx-1 transcripts. Indeed, a 4-bp (CATG) insertion
was identified at position 191 of the RFC coding sequence which
resulted in a frameshift and the use of a new stop codon at position
1176. Although this would generate a predicted ~48-kDa protein with
only 11% of recognizable RFC sequence and unlikely to react with
GST-RFC antibody on Western blots, the low frequency at which the 4-bp
insertion was detected (19-30% of cDNA clones) suggested, at
most, its minor contribution to the lack of RFC expression in these cells.
Wild-type RFC sequence was also detected in a small number of CEM/Mtx-1
cDNAs. However, its low frequency, combined with the lack of a
signal on Western blots (even at high protein loading; data not shown),
indicated that an insignificant amount of wild-type RFC protein was
actually synthesized in these cells. The variable frequencies at which
wild-type cDNA and genomic sequences were detected in these
analyses may reflect the localization of RFC to chromosome 21 (21q22.2-22.3; Ref. 33) and presence of a third (and possibly
wild-type) RFC allele due to a random trisomy 21 in the CEM/Mtx-1
subline (1 of 8 karyotypes).2
Rather, the majority of RFC transcripts in the CEM/Mtx-1 subline
contained G to A substitutions at both nucleotide positions 130 and 380 (in general, without the 4-bp insertion at position 191) which result
in replacements of amino acids 44 and 127. The lack of detectable
mutated RFC proteins in these cells could result from impaired
translation of mutant RFC transcripts, and/or an accelerated
degradation or inefficient plasma membrane targeting of mutant
proteins. These possibilities could not be evaluated in CEM/Mtx-1
cells. Consequently, we expressed mutant RFC cDNAs in
transport-defective CHO and human cells to better correlate levels of
RFC transcripts and immunoreactive protein for comparison with
wild-type RFC. As with the wild-type RFC transfectants (6, 21),
double-mutated RFCP1+P2 transcripts were observed on
Northern blots at high frequencies for both CHO and K500E
transfectants. However, only for the cells transfected with wild-type
RFC constructs were significant accumulations of immunoreactive RFC
protein detected. For the three clones which expressed sufficient
mutant RFCP1+P2 protein for immunoblot detection, the
carrier migrated as a ~70-kDa band, distinguishable from both native
wild-type RFC (~85 kDa) and enzymatically deglycosylated RFC (65 kDa).
Hence, the presence of the P1 and P2 mutations
appears to alter processing to the mature N-glycosylated
carrier and, likewise, results in markedly decreased levels of membrane
RFCP1+P2 protein. This, in part, reflects the dramatically
accelerated turnover of the mutant carrier, likely due to altered
secondary and tertiary structures, however, differences in translation
efficiencies between wild-type and RFCP1+P2 cannot be
discounted as a contributing factor. Increased turnover rates of mutant
proteins are well established as a mechanism for maintaining cellular
homeostasis (36, 37). For other integral membrane proteins, including
cystic fibrosis transmembrane regulatory protein (38) and
p-glycoprotein (39), increased rates of mutant protein
degradation accompany incomplete glycosylation and impaired membrane
targeting due to retention in the endoplasmic reticulum. However, for
RFCP1+P2, there was no evidence for endoplasmic reticulum
retention and degradation since both wild-type RFC and mutant
RFCP1+P2 co-localized with 5'-nucleotidase activity in
particulate and sucrose gradient-purified plasma membrane fractions
(data not shown). Furthermore, there were no significant differences in the levels of wild-type RFC or RFCP1+P2 between sucrose
gradient-purified and crude particulate membrane fractions, differing
~3-fold in NADPH-cytochrome c reductase (an endoplasmic
reticulum marker enzyme) activity. Thus, both carrier forms are
primarily targeted to the cell surface.
It was of interest that both point mutations identified in CEM/Mtx-1
RFC resulted in replacement of highly conserved amino acids (glycine 44 and serine 127) and, together, they completely abolished transport
activity. When expressed individually in transport-impaired CHO cells,
both P1 and P2 mutant constructs exhibited low
levels of transport activity. Although the presence of arginine 44 in the mutant RFCP1 actually increased the Mtx
Vmax over wild-type RFC in RIIP1/2
cells, this was accompanied by an increased Kt so that net transport was appreciably impaired. For RFCP2, Mtx
uptake was low due to effects on both Kt and
Vmax and was only detected in the
RIIP2/15A transfectant expressing extremely high levels of
the mutant carrier. Thus, both glycine 44 and serine 127, or the
protein domains including these residues, are likely important for RFC function. Important functional roles were also implied for glutamate 45 (11) and serine 46 (10), and for alanine 132 (alanine 130 in the murine
RFC; Ref. 7) from studies in transport-impaired L1210 cells.
Hence, our results with the double-mutated RFCP1+P2
construct in transfected CHO and K562 cells approximate those for the
mutant RFC proteins and transcripts in CEM/Mtx-1 cells. The complete loss of RFC protein in the CEM/Mtx-1 subline appears to result from
early translation termination and rapid turnover of the
RFCP1+P2 protein, although inefficient translation from
mutant RFC transcripts may also be a contributing factor. It is of
interest that a very recent report by Jansen et al. (40)
described a CEM/Mtx subline of apparently identical origin to the
CEM/Mtx-1 clonal line described herein yet exhibiting distinctly
different characteristics. These relate to relative levels of
folate-dependent enzymes (folylpolyglutamate synthetase and
thymidylate synthase; Ref. 12), 5-fold decreased levels of RFC
transcripts compared with parental CCRF-CEM cells, and the presence of
a point mutation at position 133 of the RFC coding sequence which
results in the synthesis of a mutated carrier with a lysine
substitution for glutamate 45. These discrepancies with our results
cannot be explained simply by differences in experimental methodologies
or data interpretation. Rather, the most likely explanation is that the
CEM/Mtx cells (18) somehow changed during long-term culture and/or
different clonal variants of CEM/Mtx were studied in the different laboratories.
In our studies, the complete absence of RFC protein and the total lack
of [3H]Mtx transport activity for RFCP1+P2
strongly implies that the residual uptake and anomalous substrate binding properties in CEM/Mtx-1 cells (12) are not due to
the RFCP1+P2. While the transport kinetics observed for
this resistant subline are clearly incompatible with those of the
wild-type carrier, the small amounts of wild-type RFC activity may be
modulated by unknown endogenous factors (6, 21, 41, 42). Alternatively, non-RFC modes of uptake (13-15) may also contribute to the CEM/Mtx-1 transport phenotype. Studies are underway to further explore these possibilities.
 |
ACKNOWLEDGEMENT |
We thank Dr. Wayne Flintoff for providing the
Mtx transport defective MtxRIIOuaR2-4 CHO cells and Dr.
Andre Rosowsky for providing the CCRF-CEM and CEM/Mtx sublines. We
thank Daryel Taliaferro for secretarial assistance in preparing this manuscript.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant CA 53535 and a grant from the United Way of Michigan (Detroit, MI).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: Experimental and
Clinical Therapeutics Program, Karmanos Cancer Institute, 110 E. Warren
Ave., Detroit, MI 48201. Tel.: 313-833-0715 (ext. 2407); Fax:
313-832-7294; E-mail: matherly{at}kci.wayne.edu.
2
B. Hukku, unpublished observation.
 |
ABBREVIATIONS |
The abbreviations used are:
Mtx, methotrexate;
APA-[125I]ASA-Lys, N
-(4-amino-4-deoxy-10-methylpteroyl)-N
-(4-azido-5-[125I]iodosalicylyl)-L-lysine;
CHO, Chinese hamster ovary;
GST, glutathione
S-transferase;
RFC, reduced folate carrier;
RT-PCR, reverse
transcriptase-polymerase chain reaction;
kb, kilobase pair(s);
bp, base pair(s);
RFC/ps, peptide-specific RFC.
 |
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