(Received for publication, December 19, 1994; and in revised form, July 25, 1995)
From the
This laboratory previously described an L1210 leukemia cell line
(MTXA) selected for resistance to methotrexate by virtue of
impaired transport due to a functional defect in the translocation
process. We now report on the sequence analysis of cDNAs encoding the
reduced folate carrier from this line and identify a single mutation
that results in the substitution of a proline for an alanine in a
highly conserved transmembrane region of the protein. Transfection of
the parental reduced folate carrier into MTX
A cells
resulted in a cell line which exhibited a complete restoration of
methotrexate uptake and an enhanced sensitivity to methotrexate.
Northern analysis and specific [
H]MTX cell
surface binding indicated that expression of the reduced folate carrier
was elevated
5-fold in the transfectant compared to parental and
MTX
A cells. The MTX influx properties of the transfectant
cell line were identical to those of the well characterized reduced
folate carrier from parental L1210 cells in terms of: 1) patterns of
sensitivity to competing folates, 2) sensitivity to the organic anion
sulfobromophthalein, 3) lack of energy dependence, and 4) capacity for
trans-stimulation. We also provide new data which suggests that the
nucleotide sequence 5` of the predicted ATG initiation codon may encode
additional protein information in the form of a leader sequence.
Finally, we demonstrate that the MTX
A line has both the
mutant and the parental reduced folate carrier alleles but that
expression appears to be restricted to the mutant allele. Thus, the
methotrexate transport phenotype and resultant drug resistance in this
cell line result from genetic/regulatory events at both alleles.
Resistance to methotrexate (MTX) ()is a major
limiting factor in the clinical utility of this agent and can occur by
a variety of mechanisms(1, 2) . Of particular interest
to this and other laboratories has been the resistance associated with
impaired transport of the drug via the reduced folate carrier system.
In murine L1210 leukemia cells, this carrier system mediates the rapid
transport of reduced folates and the 4-amino analogs of folic acid and
its kinetic and thermodynamic properties have been characterized in
detail by several laboratories(3, 4, 5) .
Clones encoding a transmembrane carrier protein have recently been
isolated from murine, hamster, and human cDNA libraries (6, 7, 8, 9) and shown to restore
folate transport in cell lines defective in carrier-mediated transport.
Hydropathy plots of this
60-kDa protein, designated RFC1 in the
murine system, predict 12
-helical transmembrane regions and
suggested that it was likely a member of a superfamily of membrane
spanning transporters. Based upon the relative molecular mass of
membrane components which are labeled when murine cells are treated
with various MTX-affinity analogs (M
=
43-48), the predicted M
of the cloned RFC1
protein is different from that predicted for the reduced folate
carrier(10, 11, 12) . This suggests that, in
the murine system, there is either significant post-translational
modification of RFC1 or that this protein is just one component of the
reduced folate carrier system.
Several mechanisms of
carrier-associated drug resistance have been characterized and shown to
result from an increase in the influx K,
a decrease in the influx V
, or changes in both
parameters(1, 3) . Changes in the latter parameter
usually result from a decrease in the number of carrier sites. This
laboratory previously described a MTX-resistant L1210 murine leukemia
line (MTX
A) which exhibits a unique transport alteration
due to an immobilization of the reduced folate carrier(10) . In
this cell line, the number of apparent carrier binding sites was
slightly decreased, the affinity of these sites for MTX at the cell
surface was unchanged, but the influx V
for MTX
was markedly decreased(10, 13) . These observations
were consistent with a highly specific loss of function, or mobility,
of the reduced folate carrier. The MTX
A line exhibits
multiple membrane protein alterations, but the functional change in
transport is highly specific for the reduced folate carrier as
transport of folic acid, amino acids, nucleosides, and sugars is not
impaired(10) .
In this paper, we describe the isolation and
characterization of cDNAs encoding the RFC1 protein in the
MTXA line and identify a single mutation that appears to be
responsible for the defect in MTX transport. We demonstrate that this
MTX transport phenotype can be complemented by expression of the
parental RFC1 in the MTX
A line and, further, that the
restored transport has properties identical to those of the well
characterized reduced folate carrier present in parental L1210 cells.
MTXA
cells (1
10
cells) were electroporated (250 V, 200
microfarads) with 40 µg of BglII-linearized pPGK-RFC1 in a
final volume of 800 µl of RPMI 1640 folate-free medium without
serum. Cells were brought to 10 ml with RPMI 1640 medium and allowed to
recover 36 h following which they were adjusted to 1
10
cells/ml in RPMI 1640 medium containing G418 (750 µg of
active drug/ml) and distributed into 96-well plates at approximately
20,000 cells per well. Positive (G418 resistant) wells developed within
8-10 days and these cells were expanded for further study.
Figure 1: Alignment of the translated nucleotide sequence upstream of the predicted ATG initiation codon for RFC1 from L1210 and CHO cells. Conserved residues are indicated with a ↕ and dashes represent an alignment adjustment to provide best fit. ``Met'' designates the predicted ATG start codon for RFC1(6, 7) . Numbering is based on the L1210 sequence.
While the nucleotide sequence of the translated
region of RFC1 from parental cells was identical to the published
murine sequence, a single mutation (nt G
C
; numbering is based on the published (6) murine
L1210 sequence) was identified in all of the RFC1 clones derived from
the MTX
A line. This mutation resulted in the substitution
of proline for alanine at amino acid residue 130. This residue is also
an alanine in the RFC1 protein isolated from both human and CHO cells
and is located in a highly conserved region in the middle of the
predicted fourth transmembrane domain of the protein.
As shown in Fig. 2, the
transcript encoding the endogenous RFC1 (2300 nt) was present in
all three (L1210 parent, MTX
A, and MTX
A-R16)
cell lines. In contrast, the RFC1-encoding transcript produced by the
pPGK-RFC1 expression vector was diagnostically shorter than the
endogenous message and was present only in the transfected cell line.
Phosphoimage analysis of multiple samples indicated that the steady
state level of total RFC1 message present in the MTX
A-R16
line was
5-fold higher than in the parental line. Conversely,
there was a
30% decrease in the total level of RFC1 message
expressed in the MTX
A line compared with the parental line.
The level of reduced folate carrier expressed on the surface of these
three cell lines was determined by measuring specific cell surface
binding of [
H]MTX at 0 °C. As shown in Table 1, the level of specific [
H]MTX
binding roughly correlated with the total level of RFC1 message present
upon Northern analysis.
Figure 2:
Northern analysis. Total RNA was isolated
from L1210 parent, MTXA, and MTX
A-R16 (R16) cells, fractionated on 1.0% formaldehyde-agarose gels,
and transferred to Nytran. The blots were hybridized successively with
an RFC1-specific probe (
2300 nt) and a probe specific for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The large and small arrowheads designate, respectively,
the endogenous RFC1 transcript and the transcript produced from the
pPGK-RFC1 expression vector. Transcripts were quantitated by
phosphoimage analysis of the radioactive blots and normalized to
GAPDH.
Figure 3:
Transport of [H]MTX
in parental L1210, MTX
A, and MTX
A-R16 cell
lines. Cells were harvested, washed, resuspended in HEPES-buffered
saline, and at time 0, exposed to 1 µM
[
H]MTX (closed symbols) or 0.1
µM [
H] MTX (open symbol).
The graph is representative of four
experiments.
As there is virtually no uptake of MTX in the
MTXA cell line, transport in MTX
A-R16 cells
must be meditated entirely by the transfected RFC1 and should exhibit
properties essentially identical to those of the well characterized
L1210 parental cell line. In parental cells, MTX influx mediated by the
classical reduced folate carrier has the general characteristics of a
carrier mediated process(19) , is unaffected by metabolic
poisons(13, 20, 21) , exhibits sensitivity to
structurally unrelated organic and inorganic
ions(22, 23, 24) , and shows preference for
MTX and reduced folates (k
= 1-5
µM) compared with folic acid (k
= 100-200 µM)(5, 25) .
As shown in Fig. 4, [H]MTX influx in
the transfectant was relatively insensitive to the presence of 5
µM folic acid but was inhibited nearly 60% by 5 µM 5-formyltetrahydrofolate. As in parental L1210 cells, the addition
of the inorganic anion sulfobromophthalein (100 µM) nearly
abolished the influx of [
H]MTX in the
transfectant. In contrast, the presence of 10 mM azide had
essentially no effect on [
H]MTX influx. Finally,
transport by the classical reduced folate carrier in parental L1210
cells shows the characteristic of influx stimulation by the presence
within the intracellular compartment of a similar substrate which
shares this carrier. Accordingly, there was a 1.13-fold
trans-stimulation of [
H]MTX influx when the
transfectant was preloaded with dl-5-formyltetrahydrofolate.
These results demonstrate that the restored (and augmented) MTX influx
mediated by the transfected RFC1 has properties of the well
characterized reduced folate carrier and that RFC1 indeed encodes this
classical murine folate transporter.
Figure 4:
Effect of various treatments on initial
rates of [H]MTX uptake in MTX
A-R16
cells. MTX
A-R16 cells were harvested, washed, resuspended
in HEPES-buffered saline, and at time 0, simultaneously exposed to 0.1
µM [
H]MTX and either 5 µM folic acid or 5 µM 5-formyltetrahydrofolate. Details
of the sulfobromophthalein (BSP), azide, and trans-stimulation
treatments are described under ``Materials and Methods.''
Data represent the mean ± S.E. of four
experiments.
Figure 5:
Genomic amplification and restriction
analysis of the RFC1 alleles. Genomic DNA from L1210 parental and
MTXA cells was isolated and a 520-bp RFC1 fragment
amplified as described under ``Materials and Methods.'' The
fragment was restricted with the indicated enzymes and fractionated on
a 6% TBE polyacrylamide gel. Molecular weight markers are the
1-kilobase ladder (Life Technologies, Inc.). Novel restriction
fragments generated by the loss or gain of a restriction site in the
mutant RFC1 allele (see Table 2) are indicated by bullets to the
right of the fragments.
We previously described an L1210 cell line, designated
MTXA, which exhibited a 100-fold increased resistance to
MTX by virtue of a novel, functional defect in the reduced folate
carrier(10, 13) . A cDNA (RFC1) thought to encode the
reduced folate carrier has recently been isolated and, in the present
study, we cloned and characterized cDNAs encoding this protein from the
MTX
A cell line and identified a single mutation in the RFC1
protein that results in the substitution of a proline residue for an
alanine. When the parental RFC1 was transfected into MTX
A
cells, MTX sensitivity was regained and MTX transport restored. The
transport properties of the restored MTX uptake in the transfectant
were identical to those of the well characterized classical reduced
folate carrier present in parental L1210 cells. This confirms that RFC1
does indeed encode this transport protein and that, in the
MTX
A cell line, the mutation identified in this protein is
responsible for the defective MTX transport phenotype.
Based on
hydrophobicity plots, the RFC1 protein is predicted to have 12
-helical transmembrane spanning domains(6) . The alanine
proline substitution identified in the MTX
A line
occurs in one such domain in a highly conserved region of the protein.
Transmembrane
-helices of integral membrane transport proteins,
such as the family of glucose transporters, are believed to form
channels through which substrates pass(26, 27) . While
proline residues frequently occur in such transmembrane helices
(serving both structural and dynamic roles), this imino acid generally
has significant
-helix destabilizing features(28) . Random
insertion of a proline residue into a protein would be expected to have
adverse structural consequences due to the induction of backbone kinks.
In the present case, such a structural alteration in a transmembrane
region of RFC1 could result in a functional change that readily
prevents substrate passage into the cell (i.e. 100-fold
decrease in V
) without having an effect on
substrate binding (i.e. unchanged K
).
The MTXA cell line is essentially unable to transport
MTX and all of the RFC1-encoding cDNAs isolated from this line
contained the alanine
proline mutation. Therefore, it was
apparent that the MTX
A cell line either did not have a
parental (i.e. functional) RFC1 allele or that, if one was
present, it was not expressed. Southern analyses and diagnostic
restrictions of an amplified genomic RFC1 fragment unequivocally
demonstrated that a structurally unaltered parental RFC1 allele was
present in the MTX
A cell line. As such, it appears that the
initial selection of the clonal MTX
A cell line in 50 nM MTX was the result of two events: 1) a G
C transversion
mutation which led to the substitution of a proline for an alanine in
one RFC1 allele, and 2) either a second mutation or epigenetic
mechanism which led to a silencing of the functional RFC1 allele. While
a second mutational event may have occurred, it would likely be present
in the 5`-regulatory region of the RFC1 gene and would not have been
detected in this study. Alternatively, drug induced DNA
hypermethylation and subsequent inactivation of the functional RFC1
allele is also quite possible. DNA hypermethylation has been shown to
occur as a normal cellular response to drugs such as MTX which inhibit
DNA synthesis(29, 30) . Furthermore, as DNA
methylation is often associated with gene silencing, Nyce (29) has suggested that hypermethylation may aid in the
development of drug resistance by inactivating genes whose products are
required for drug toxicity. In fact, a previous study by Hsueh and
Dolnick (31) has shown that decreased expression of a folate
receptor due to hypermethylation of the encoding gene is, in large
part, responsible for the MTX resistance which developed in a human KB
cell line following selection for growth on the drug.
Williams et al.(7) identified several RFC1 clones from a CHO cDNA library that contained internal deletions and were suggestive of alternative splicing. In the present study, a potential alternative RFC1 splice form was identified in a number of RT-PCR generated clones isolated from these L1210 cell lines. While multiple RFC1 transcripts were not visible on Northern analysis, the size of this potential splice form may not differ significantly from the major RFC1 transcript or its steady state level of expression may simply be too low to visualize. The nucleotide sequence of this splice form predicts a protein which contains the first six transmembrane domains of the RFC1 protein followed by a novel hydrophilic carboxyl terminus of 72 amino acid residues. While there is no evidence that this transcript is ultimately translated into a functional protein, it should be noted that other investigators have demonstrated the presence of alternative MTX transport systems with properties distinct from the classical reduced folate carrier(32, 33, 34, 35, 36) . Additionally, Horne et al.(37) have characterized a transporter that mediates the translocation of reduced folates into mitochondria. The potential presence of additional protein information (42 amino acid residues, see Fig. 1) encoded upstream of the putative ATG start codon raises the possibility that putative RFC1 splice forms could be targeted to different membrane systems. Together, these observations raise the possibility that alternative splice forms of the RFC1 gene transcript may give rise to transmembrane carriers which have distinct structural, biochemical, and thermodynamic properties.
Finally, the homologous murine expression system
resulting from a combination of the MTXA cell line and the
pTK-PGK expression vector developed in this study will be a very useful
model for expression and characterization of RFC1 transport proteins
that contain specifically engineered mutations. Such structure-function
studies may aid in the development of additional pharmacologic agents
which utilize this carrier for transport into the cell.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U32469[GenBank].