Date post: | 06-Sep-2018 |
Category: |
Documents |
Upload: | nguyentuyen |
View: | 215 times |
Download: | 0 times |
1
The reduced folate carrier (RFC) is cytotoxic to cells under conditions of severe folate
deprivation: RFC as a double-edged sword in folate homeostasis
Ilan Ifergan1, Gerrit Jansen
2 and Yehuda G. Assaraf
1
From: 1The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, Technion-Israel
Institute of Technology, Haifa 32000, Israel, and 2 Department of Rheumatology, VU University Medical
Center, Amsterdam, The Netherlands.
Running Title: RFC and detrimental folate efflux.
Address correspondence to: Prof. Yehuda G. Assaraf, The Fred Wyszkowski Cancer Research Laboratory,
Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel. E-mail:
Key Words: Reduced folate carrier; folate efflux; folate deficiency; decreased cellular folate pool;
mathematical bio-modeling.
The abbreviations used are: ABC-ATP-binding cassette; BCRP- Breast cancer resistance protein; CHO -
Chinese hamster ovary; FPGS- Folylpoly-γ-glutamate synthetase; FR- Folate receptor; GGH - γ-glutamate
hydrolase; HF - high folate; HFM - Hereditary folate malabsorption; LCV- Leucovorin; MRP - Multidrug
Resistance Protein; NF- No folate; RFC - Reduced folate carrier; THF- Tetrahydrofolate.
The reduced folate carrier (RFC), a
bidirectional anion transporter, is the major
uptake route of reduced folates essential for a
spectrum of biochemical reactions and thus
cellular proliferation. However, here we show
that ectopic overexpression of the RFC, but not
of folate-receptor α (FRα), a high-affinity
unidirectional folate uptake route serving here
as a negative control, resulted in a ~15-fold
decline in cellular viability in medium lacking
folates, but not in folate-containing medium.
Moreover, in order to explore possible
mechanisms of adaptation to folate deficiency in
various cell lines that express the endogenous
RFC, we first determined the gene expression
status of the following genes: a) RFC, b) ATP-
driven folate exporters (i.e. MRP1, MRP5 and
BCRP), c) Folylpoly-γ-glutamate synthetase
(FPGS) and γ-glutamate hydrolase (GGH),
enzymes catalyzing folate polyglutamylation and
hydrolysis, respectively. Upon 3-7 days of folate
deprivation, semi-quantitative RT-PCR analysis
revealed a specific ~2.5-fold decrease in RFC
mRNA levels in both breast cancer and T-cell
leukemia cell lines which was accompanied with
a consistent fall in methotrexate influx, serving
here as a RFC transport activity assay.
Likewise, a 2.4-fold decrease in GGH mRNA
levels and ~19% decreased GGH activity was
documented for folate deprived breast cancer
cells. These results along with those of a novel
mathematical bio-modeling devised here suggest
that upon severe short term (i.e. up to 7 days)
folate deprivation, RFC transport activity
becomes detrimental as RFC, but not ATP-
driven folate exporters, efficiently extrudes
folate monoglutamates out of cells. Hence, down-
regulation of RFC and GGH may serve as a
novel adaptive response to severe folate
deficiency.
Reduced folate cofactors play an essential role as
one-carbon donors and acceptors in several crucial
intracellular metabolic reactions [1-6]; However,
mammalian cells are devoid of the enzymatic
capacity for folate biosynthesis and thus are
absolutely dependent on folate uptake from
exogenous dietary sources [7]. Therefore, folate
deficiency may impair the de novo biosynthesis of
purines and thymidylate and thereby disrupt DNA
and RNA metabolism, homocysteine remethylation,
methionine biosynthesis and subsequent formation
http://www.jbc.org/cgi/doi/10.1074/jbc.M802812200The latest version is at JBC Papers in Press. Published on May 22, 2008 as Manuscript M802812200
Copyright 2008 by The American Society for Biochemistry and Molecular Biology, Inc.
by guest on September 5, 2018
http://ww
w.jbc.org/
Dow
nloaded from
2
of S-adenosylmethionine, the universal methyl
donor, which in turn may lead to the impairment of
methylation reactions [1-6]. Based on their key
role in cellular metabolism, folate cofactors are
efficiently retained in cells via polyglutamylation,
an ATP-dependent reaction in which up to 9
equivalents of glutamate units are added to the γ-
carboxyl residue of folate cofactors [8, 9] (Fig.
1A). Whereas this reaction is catalyzed by the
enzyme folylpoly-γ-glutamate synthetase (FPGS)
[9], the enzyme γ-glutamyl hydrolase (GGH)
catalyzes the hydrolysis of these terminal γ-
glutamyl residues from polyglutamylated folates
[10]. Importantly, the long chain (n>3) folate
polyglutamylate derivatives can no longer be
extruded via ATP-dependent efflux transporters
such as the multidrug resistance proteins
(MRPs/ABCCs) [11, 12], breast cancer resistance
protein (BCRP/ABCG2) [13] as well as through the
reduced folate carrier (RFC), a bi-directional folate
transporter [14].
The molecular mechanisms underlying adaptation
to folate deficiency are generally associated with
alterations in folate uptake, ATP-driven folate
efflux, intracellular folate retention and folate-
dependent metabolism. These mechanisms include:
a) Altered activity of various folate-dependent
enzymes including dihydrofolate reductase (DHFR)
[15] and thymidylate synthase (TS) [16], b)
Augmented polyglutamylation via increased FPGS
activity [17, 18], c) Overexpression of folate influx
systems including the RFC (SLC19A1) [16, 19]
and the folate receptor (FR) [15, 17, 20], d) Down-
regulation of ATP-dependent folate exporters of the
MRP/ABCC family [21, 22] as well as
BCRP/ABCG2 [18, 23].
RFC, the main focus of this study, serves as the
major uptake route of folates in mammalian cells
[14]. Whereas RFC exhibits a relatively wide
pattern of tissue expression, the expression of the
additional folate uptake systems including the FR
family [19, 24-35] and the proton-coupled folate
transporter (PCFT/SLC46A1 )[36-40], both of
which are unidirectional transport systems, is rather
restricted to a limited number of tissues.
Consistently, in vivo studies revealed that whereas
RFC-null embryos died in utero prior to embryonic
day 9.5 (E9.5), the rescue of the nullizygous
embryonic lethal phenotype was achieved by
supplementation of pregnant monoallelic RFC
dams with 1 mg daily subcutaneous doses of folic
acid [41]. Furthermore, the rescued RFC
nullizygous embryos died within 12 days after birth
due to a failure of hematopoietic organs [41].
RFC functions as a bi-directional anion exchanger
[36, 42] with a high affinity (Km=1 µM) for
reduced folates and hydrophilic antifolates such as
methotrexate (MTX; Km=5-10 µM) but very low
affinity (Km=200-400 µM) for folic acid [42-44].
RFC can neither bind nor hydrolyze ATP in order
to drive its folate transport activity across the
plasma membrane. Rather, the RFC-dependent
uphill influx of folates is coupled to the downhill
efflux of organic phosphates including thiamine
monophosphate and pyrophosphate [45] that are
readily available in the cytoplasm.
In the current study we hypothesized that under
conditions of severe folate deprivation, the folate
efflux component of RFC transport activity may
result in intracellular folate depletion and
consequently decreased cellular viability. Based
upon experimental results as well as on novel
mathematical bio-simulation data, we show here for
the first time that upon short-term (i.e. up to 7 days)
exposure of cells to folate-free growth conditions,
RFC-dependent folate efflux activity becomes
detrimental as RFC extrudes folate monoglutamates
out of cells, a process facilitated by the GGH-
dependent conversion of folate polyglutamates to
monoglutamates. Moreover, this cytotoxic folate
efflux activity may be abrogated by specific
adaptive down-regulation of both RFC and GGH
via decreased gene expression and subsequently
decreased catalytic activities; indeed, this novel
survival response to folate deprived conditions has
been established here for T-cell leukemia CEM/7A
cells with overexpression of the endogenous RFC
[19] as well as for the breast cancer MCF7/MR
cells which expresses only moderate levels of this
double-edged sword transporter.
Experimental Procedures
RFC-dependent cellular viability in the presence or absence of folates- The Chinese hamster ovary
(CHO) cell line deficient in RFC activity termed C5
]46[ as well as its human RFC- and human FRα-
overexpressing transfectants (i.e. C5/RFC and
C5/FR respectively ]46[ ) were trypsinized and
washed three times with folic acid-free growth
medium. Then, cells (6x104) were seeded in each of
two T25 flasks in 5 ml folic acid-free growth
medium. The sublines in the first set of flasks were
by guest on September 5, 2018
http://ww
w.jbc.org/
Dow
nloaded from
3
termed C5/NF, C5/RFC-NF and C5/FR-NF (i.e. no
folate), whereas the sublines in the second set of
flasks were supplemented with folates derivatives
according to their initial growth conditions ]46[ ,
resulting in the sublines C5-HF (i.e. high folate ;
supplemented with 2.3 µM folic acid), C5/RFC-
3nMLCV (i.e. supplemented with 3nM leucovorin
which was used as a folate source because of its
high affinity to the RFC when compared to folic
acid), C5/FR-3nMFA (i.e. supplemented with 3nM
folic acid which was used as a folate source
because of its high affinity to the FRα when
compared to LCV). All the 6 flasks were
simultaneously incubated for 6 days in a humidified
CO2 incubator without medium replenishment in
each of three independent experiments. Following
these 6 days of incubation, cells were detached by
trypsinization and the number of viable cells was
determined by a haemocytometer counting after
trypan blue staining.
Folate deprivation-The following are the folate
deprivation protocols that we have developed for
the following cell lines in order to explore possible
mechanisms of adaptation to folate deficiency:
A) MCF-7/MR cells (with moderate levels of the
RFC): MCF-7/MR cells were grown as previously
described [18]. Following trypsinization, cells were
washed three times with folic acid-free growth
medium containing 10% dialyzed FCS and
antibiotics. Then, cells (2.3x105) were seeded in
each of two T75 flasks in 15 ml folic acid-free
growth medium; the subline in the first flask was
therefore termed MCF-7/MR-NF whereas the
second flask was supplemented with 2.3 µM folic
acid and was thus termed MCF-7/MR-HF.
Following 7 days of incubation, cells were then
ready for the various analyses including semi-
quantitative RT-PCR and determination of initial
rates uptake of [3H]MTX and GGH activity..
B) CEM/7A cells ( with overexpression of the
RFC)[19]: CEM/7A cells were cultured in growth medium
containing 0.2 nM LCV as has been previously
described [19]. Cells were then washed three times
with folic acid-free growth medium containing 10%
dialyzed FCS and antibiotics and transferred (107
cells) to each of two T75 flasks in 50 ml folic acid-
free growth medium containing 10% dialyzed FCS
and antibiotics; the subline in the first flask was
termed CEM/7A-NF whereas the second T75
flask was supplemented with 0.2 nM leucovorin
(LCV) and was therefore termed CEM/7A-HF.
Following 3 days of, cells were then ready for the
various analyses as mentioned above.
RNA extraction and semi-quantitative RT-PCR- Total RNA extraction followed by cDNA
synthesis
was undertaken as previously described [47]. In
order to evaluate the levels of β-ACTIN,GAPDH,
BCRP, MRP5, MRP1, GGH, FPGS,PCFT,FRα and
RFC gene expression, semi-quantitative RT-PCR
analysis was used. PCR was carried out in a total
volume of 30 µl in the presence of the following
cDNA quantities (using 6-fold serial template
dilutions): 100, 16.7 and 2.8 ng. Each PCR reaction
contained 0.4 µM of the sense and antisense
primers (Table I) and a 1X RedTaqTM
ReadyMixTM
PCR reaction mix solution (Sigma). Following an
initial denaturation at 95°C for 10 min, 24 to 35
cycles each of 1 min denaturation at 95°C, 1 min
annealing at 50–61°C (Table I) and 1 min
elongation at 72°C, as well as a final extension
period of 10 min at 72°C, were carried out. PCR
products were analyzed by electrophoresis on 1%-
2% agarose gels. Representative results of three
independent experiments are shown.
Determination of initial rates of [3H]MTX uptake-
Following the folate deprivation protocols,
[3H]MTX influx was determined as previously
described [46]. The advantage of using MTX rather
than folic acid is its 40-80 fold higher affinity to
RFC when compared to folic acid,[42-44].
GGH activity assay- Catalytic GGH activity assay was measured
according to the original protocol described by
O’Connor et al [48] with some slight modifications
[49]. These protocols are based on the ability of
GGH to convert MTX-Glu2 to MTX [48, 49]. GGH
activity is expressed as nmol MTX formed per
hr/mg protein.
Mathematical bio-modeling of intracellular folate
depletion- Here we devised a mathematical bio-modeling
aimed at evaluating the intracellular folate pool
depleting effect of the RFC as well as of several
representatives of the ABC (ATP-binding cassette)
transporter superfamily (i.e. MRP3 and MRP4)
under folate free growth conditions (Fig. 1B). The
abovementioned folate efflux systems transport
intracellular monoglutamylated folates to the
extracellular compartment which is literally infinite
relative to the intracellular volume. Thus, the influx
by guest on September 5, 2018
http://ww
w.jbc.org/
Dow
nloaded from
4
of the transported folates is negligible and can be
omitted in the model. We applied the Michaelis-
Menten (M-M) equations (Michaelis and Menten,
1913) in order to simulate these efflux transport
activities.
Hence, the efflux velocity (v) can be derived from
the basic enzymatic reaction kinetics where a
substrate S is converted to a product P as follows:
�1� �� � �� · ����� ��
Where S is the concentration of the
substrate, Vmax is the limiting velocity value
at substrate saturation (i.e. when [S] >>
Km), and Km is the substrate concentration
when v = Vmax/2. In our model: t= The duration of time (in minutes) in which the
cells have been exposed to the folate free growth
conditions.
Vt= The efflux velocity (µmol/l/min which is µM/
minutes) of the examined transporter at time = t.
[S]t= [MonoFP]t= Cytosolic concentration (µM) of
the monoglutamylated folate pool at time=t.
Vmax= the maximum folate efflux velocity of the
examined transporter (µM/ minutes).
Km= the monoglutamylated reduced folate
concentration (µM) of the examined
transporter in which v = Vmax/2.
Hence:
(2) �� � �� · ����������� ��������
Definition: [TFP]t= Intracellular concentration of
the total folate pool (µM ) at time = t; However, the
cytosolic fraction of folates in mammalian cells is
only 38% of the total folate pool size ]50 ,51[ .
Moreover, the cytosolic monoglutamate folate
fraction, which serves as the available folate efflux
fraction for RFC as wells as for several ABC
transporters is only 2% of the total cytosolic folate
pool in mammalian cells ]50 ,51[ . Therefore, the
cytosolic monoglutamylated folate fraction is only
0.76% of the total intracellular folate pool size
under folate replete conditions. Based upon the
essential lack of folates in the extracellular medium
under folate free growth conditions along with the
continuous efflux of cytosolic folate
monoglutamates via the examined transporter as
well as based on the Le Chatelier principle, one
could predict a continuous conversion of
intracellular folate polyglutamates to
monoglutamate congeners via lysosomal GGH
activity (Fig. 1B) in an attempt to retain the original
fraction (i.e. 0.76%) of the monoglutamylated
folate pool (i.e. [MonoFP]t ) relative to the total
intracellular folate pool (i.e. [TFP]t ). Whereas the
kinetic understanding of mitochondrial influx as
well as efflux of folates is limited and based upon
the abovementioned experimental data as well as
on the Le Chatelier principle, we used the
calculated cytosolic monoglutamylated folate
fraction of 0.76% in this theoretical modeling.
Hence:
(3) �� � �.���� · �� ! · "��������.���� · "����
The [TFP]t is equal to the initial total folate pool
(i.e. [TFP]t=0) subtracted from which the amount of
monoglutamylated folates that has been
transported via the examined transporter was
subtracted until time t ( minutes). As such:
�4� $%&�� � $%&�' ( )
*+�
*+''.'',- · �� ! · ./0�!
���'.'',- · ./0�!· 12
34 5 0.0076 · $%&�* ≠ 0: This condition is true
due to the fact that the Km of the examined
transporters has a positive value and [TFP]x ≥ 0 for
t ≥ x ≥0 . Hence, '.'',- · �� ! · ./0�!
���'.'',- · ./0�! is a
continuous function for t ≥ x ≥0 and thus according
to the first part of the fundamental theorem of
calculus we may differentiate both sides of the
equation (with regard to t) as follows:
(5) 9 "����9� � ( �.���� · �� ! · "����
����.���� · "����
This is a separable differential equation, hence:
�6� 1: � ( ���'.'',- · ./0��'.'',- · �� ! · ./0��
· 1 $%&��
Integration of both sides of the equations results in:
(7) : � ( '.'',- · ./0�� � �� · ;< "���� '.'',- · �� !
+ =>?@:A?:
by guest on September 5, 2018
http://ww
w.jbc.org/
Dow
nloaded from
5
The total intracellular folate pool size of cultured
mammalian tumor cells (i.e.[TFP]t=0 ) was
experimentally found to be ~11.3 µM [52].
Hence:
(8) 0 � ( '.'',- · BB.C � �� · ;<DD.E '.'',- · �� !
+ =>?@:A?:
Hence, for each bio-simulation:
(9) =>?@:A?: � '.'FGH � �� · I.JIJF '.'',- · �� !
Hence:
(10) : � ( '.'',- · ./0�� � �� · ;< "���� '.'',- · �� !
+
'.'FGH � �� · I.JIJF '.'',- · �� !
Thus:
�11� : �
'.'FGH � �� · I.JIJFK'.'',- · ./0�� K �� · ;< "���� '.'',- · �� !
This final implicit function (i.e. (11)) demonstrates
the relation between the total intracellular folate
pool (i.e. [TFP]t ) and the duration of folate
deficiency (t in minutes) for an examined
transporter with affinity for reduced folate (i.e. Km
in µM) and the maximum folate efflux velocity (i.e.
Vmax in µM/ minutes). The Km as well as Vmax of
the various folate efflux systems has been derived
in previous publications from cells that overexpress
the examined transporter. These experiments
yielded reliable results for the Km; however, the
calculated Vmax doesn’t represent a physiologic
value. Hence, we used the human leukemia CCRF-
CEM cells that express normal levels of the RFC
along with several members of the ABC
superfamily including substantial levels of MRP1
and MRP4 [21] to derive the total typical capacity
of folate efflux in mammalian cells. Hence, given
the experimental Vmax = 4 pmole/ ( 107cells x min)
for folate based compound [19] and the reported
cell volume for CCRF-CEM cells which is 4 x 10 -
10 ml/cell [36]. We found that the typical maximal
capacity (i.e. Vmax) of folate efflux in these
mammalian cells is exactly 1 µM/min. During
previous studies, a reduction of 60% in the folic
acid efflux rate constant was documented in the
presence of the RFC transport inhibitor, N-
hydroxysuccinimide ester of MTX (NHS-MTX)
[21]. Therefore, the estimated capacity of RFC is
0.6 µM/min whereas the remaining folate efflux
systems have a cellular folate efflux capacity of 0.4
µM/min. These estimated folate efflux capacities
were used to evaluate the folate efflux contribution
of RFC relative to the remaining ATP-driven folate
efflux systems in the current bio-simulation.
However, in order to thoroughly investigate the
folate depleting nature of these efflux transporters,
four hypothetical efflux capacities were used for
several transporters as follows: 1) 1 µM/min 2) 10-1
µM/min 3) 10-2
µM/min 4) 10-3
µM/min. The
theoretical experiments were conducted to
characterize the folate depleting effect of RFC with
a high affinity (Km=1 µM) for reduced folates , that
serve as the main intracellular folate derivatives,
when compared to two representatives of the ABC
transporters including MRP3 with Km = 1.74 mM
for reduced folates ]11 ,53[ and MRP4 with Km =
0.64mM for reduced folates [53, 54]. These
representatives of the ABC transporters have been
chosen because of available kinetic data (i.e. Km)
for the reduced folate derivatives that serve as the
dominant intracellular folate fraction. The various
graphs have been plotted in a single coordinate
system using the Graph 4.3 software.
Statistical Analysis - We used a paired student’s T-
test to examine the significance of the difference
between two populations for a certain variable. A
difference between the averages of two populations
was considered significant if the P-value obtained
was < 0.05.
RESULTS AND DISCUSSION
In mammalian cells, the transport of THF cofactors
(i.e. tetrahydrofolate , a reduced folate derivative)
proceeds primarily via the RFC, a high affinity
transporter of naturally occurring reduced folates
(e.g. Km=1µM for 5-methylTHF). RFC is a non-
concentrative, facilitative transporter with the
characteristics of a bi-directional anion exchanger
that equally displays influx and efflux of reduced
folates. We hence postulated here that the high
affinity folate efflux activity component of the RFC
may be detrimental to cells subjected to folate-free
by guest on September 5, 2018
http://ww
w.jbc.org/
Dow
nloaded from
6
conditions. The rationale behind this hypothesis
was that under these conditions of folate-
deprivation, RFC would extrude reduced folate
monoglutamates out of cells. Given the lack of
folates in the extracellular medium under
conditions of severe folate deprivation along with
the high affinity RFC-dependent efflux of folate
monoglutamates as well as based upon the Le
Chatelier principle, one could predict a continuous
conversion of intracellular folate polyglutamates to
monoglutamate congeners via lysosomal GGH
activity (Fig. 1B). This should result in the
continuous efflux of folate monoglutamates via
RFC thereby leading to decreased intracellular
folate pool and consequent loss of cellular viability
(Fig. 1B and Fig. 2). Furthermore, the folate influx
component of the RFC becomes useless when
folates are absent from the growth medium, hence
rendering RFC a high affinity unidirectional folate
exporter (Fig. 1B). To explore this hypothesis that
RFC exerts a cytotoxic effect under conditions of
folate deficiency, we first compared the viability of
three sublines in medium containing or lacking
folates; these cell lines included: RFC-null CHO
C5 cells, their stable C5/RFC transfectants
overexpressing the RFC [46] as well as C5/FR
transfectants overexpressing FRα, the latter of
which lacks folate efflux activity and thus serves as
a negative control to the efflux component of the
RFC (Fig. 2). The principal advantage of using this
particular panel of cell lines is that they are devoid
of endogenous RFC transport activity [46].
Moreover, in order to investigate the possible
cytotoxic effect of the RFC we preferred to use
C5/RFC cells with ectopic RFC overexpression
driven by a dominant CMV promoter [46] rather
than an endogenously overexpressed RFC that may
be down-regulated via a protective mechanism and
thereby may compromise this cytotoxic effect. The
results revealed a similar viability of C5, C5/RFC
and C5/FR cells in folate-replete growth medium
(i.e. C5-HF, C5/RFC-3nMLCV and C5/FR-
3nMFA, respectively) (Fig. 2). In contrast, in
folate-free medium, the viability of C5/RFC cells
(i.e. C5/RFC-NF), but not C5/FR-NF cells was
14.8-fold decreased (P= 0.025), relative to RFC-
null C5 cells (i.e. C5-NF) (Fig. 2). Therefore, these
results strongly suggest that RFC-mediated folate
efflux activity is responsible for the deleterious
effect on cellular viability under conditions of
severe folate deprivation. Whereas RFC
transfectant C5/RFC cells were instrumental in
demonstrating the cytotoxic effect of RFC under
folate deficiency conditions, we further explored
possible mechanisms of adaptation to folate
deficiency in various cell lines displaying
endogenous RFC expression, thus enabling us to
identify possible programmed protective
response(s) to folate deficiency. Toward this end,
we used T-cell leukemia CEM/7A cells with
overexpression of the endogenous RFC [19] as well
as breast cancer MCF7/MR cells expressesing only
moderate RFC levels. The latter cell line was
specifically chosen as it co-expresses various folate
influx and efflux transporters; we therefore
determined which of these folate transporters may
be down- or up-regulated upon short-term folate
deprivation. Semi-quantitative RT-PCR analysis
revealed a specific 2.4-fold (P= 0.01) and 2.6-fold
(P= 0.005) decrease in RFC and GGH mRNA
levels, respectively, upon 7 days of folate
deprivation in breast cancer MCF-7/MR cells (i.e.
MCF-7/MR-NF versus MCF-7/MR-HF); 7 days
were the minimal duration time of folate deficiency
that enabled us to detect a statistically significant
difference in the expression of one or more genes in
this cell line (Fig. 3). Similarly, RFC
overexpressing CCRF-CEM-7A leukemia cells [19]
displayed a specific 2.5-fold decrease (P= 0.009) in
RFC mRNA levels after 3 days of incubation in
folate-free medium (i.e. CEM/7A-NF versus
CEM7A-HF; Fig. 3); in these cells, 3 days were the
minimal duration time of folate deficiency that
yielded a statistically significant difference in RFC
gene expression. The stable gene expression status
of the additional folate uptake systems including
FRα [19, 24-35] and PCFT [36-40] (Fig. 3)
strongly suggests that the folate efflux component
of RFC was detrimental to cells upon folate
deficiency rather than its folate influx component.
This ~2.5-fold decrease in RFC mRNA levels in
both breast cancer cells and T-cell leukemia lines
under folate deficiency, was then examined at the
transport activity level (Fig. 4); the initial rates of
[3H]MTX uptake was determined in the two folate
deprived cell lines. Consistent with the decreased
RFC transcript levels in folate-deprived cells, both
MCF-7/MR (expressing low levels of RFC) as well
as CCRF-CEM-7A cells (overexpressing the RFC)
showed a 49% (P= 0.03) and 44% (P=0.004 )
decrease in the influx of [3H]MTX under conditions
of folate-deprivation, respectively (Fig. 4A-B).
by guest on September 5, 2018
http://ww
w.jbc.org/
Dow
nloaded from
7
Thus, the cytotoxic effect of the RFC upon folate
deficient conditions was probably minimized due to
the decreased RFC transport activity. Likewise, the
2.4-fold decrease in GGH mRNA levels was
accompanied with ~19% decreased GGH activity
(P=0.025; Fig. 5A) for folate deprived MCF-7/MR-
NF cells, relative to their folate-replete
counterparts. This specific down-regulation of both
RFC and GGH gene expression and activity may
serve a as novel cellular adaptive-protective
response under folate deficient conditions aimed at
counteracting the detrimental conversion of folate
polyglutamates to monoglutamates and their
subsequent high affinity extrusion via the RFC.
Further studies are warranted to pin-point the
putative RFC and GGH promoter elements that
may respond to folate deprivation and thereby
result in repression of gene expression in medium
lacking folates. The fact that neither of the ATP-
driven folate exporters including MRP1, MRP5 and
BCRP [11, 12, 53, 55] underwent down-regulation
under these folate-deplete conditions (Fig. 3),
suggests an augmented detrimental role for RFC-
dependent high affinity folate efflux activity,
relative to the above ATP-driven, low affinity (e.g.
transport Km values for folic in the millimolar
range) efflux transporters of the ABC superfamily
[53](Fig 1B and Fig. 6). However, in an attempt to
provide a comparative quantification of the
intracellular folate depleting effect of RFC with
those of ATP-driven folate exporters, we devised a
novel mathematical bio-modeling (Fig. 6). The in-
silico experiments showed 10- and 100-fold
decrease in the intracellular folate pool within 8.7
and 17.1 hours under folate free growth conditions,
respectively, as a result of the estimated cellular
efflux activity of RFC (i.e. 0.6 µM/min, as was
calculated in the Experimental Procedures; Fig. 6).
Furthermore, the activity of RFC resulted in a
dramatic contraction in the intracellular folate pool
within days (i.e. 10- and 100-fold decrease in the
intracellular folate pool within 2.2 and 4.3 days,
respectively) if only 10% of the cellular efflux
capacity (i.e. 0.1 µM/min) was attributed to this
transporter (Fig. 6). In contrast to RFC, the folate
efflux activity of MRP3 and MRP4 resulted in the
retention of the vast majority of the intracellular
folate pool (i.e. 96% and 89% retention,
respectively) after 7 days of incubation in folate
free medium (Fig. 6). This lack of a substantial
folate depleting effect was observed even when the
complete cellular folate efflux capacity (i.e. 1
µM/min) was attributed to each of the two ABC
transporters (Fig. 6). Hence, these results provide a
mechanistic basis for the highly specific down-
regulation of the cytotoxic efflux activity of the
RFC, but not of folate exporters of the ABC
superfamily (Fig. 3) that failed to cause any major
decrease in the intracellular folate pool (Fig. 6),
upon short term (up to 7 days) folate deprivation.
However, the medium term and long term (i.e.
weeks and months, respectively) folate depleting
effect of several transporters of the ABC
superfamily has been previously suggested as
detrimental to cells under folate deficient
conditions [18, 21, 23]. Indeed, our mathematical
bio-modeling reveals that the folate efflux activity
of MRP4, as a representative of folate exporters of
the ABC superfamily, may be responsible for a
30% decrease in the intracellular folate pool within
3 weeks of exposure to folate free growth
conditions; this moderate depleting effect of this
ABC transporter may contribute to the detrimental
effect of medium term and long term folate
deprivation. Collectively, our findings strongly
suggest that the bidirectional folate transport
activity of RFC is responsible for its double-edged
sword impact on folate homeostasis. Moreover, it is
possible that the ability to down-regulate RFC and
GGH gene expression under states of severe folate
deprivation stems from evolutionary roots
originating in unicellular organisms and perhaps
metazoic ancestral organisms undergoing transient
yet frequent states of starvation and severe folate
deprivation. One important emerging question from
the current study is what physiological-pathological
conditions and syndromes could match the transient
folate-deprivation conditions used in the current
paper. The first pathological syndrome,
hereditary folate malabsorption (HFM) [56], is
caused by loss of function mutations in the proton-
coupled folate transporter (PCFT/HCP-
1/SLC46A1) normally responsible for the high
affinity intestinal influx of naturally occurring
folates at the acidic microclimate of the upper
intestinal mucosal epithelium. Hence, HFM
patients suffer from extremely low folate levels (≤
0.2 nM) in the blood and cerebrospinal fluid (CSF)
[40, 56]. Another physiological-pathological
condition that may match such severe folate
deprivation state includes nutritional folate
deficiency or insufficiency. Indeed, folate
by guest on September 5, 2018
http://ww
w.jbc.org/
Dow
nloaded from
8
deficiency affects approximately 10% of the
population as well as more than 50% of the
children and elderly that live in poverty in the
United States, Thailand and rural areas of India
[38, 57, 58]. Hence, under such physiological-
pathological conditions of severe folate deficiency,
RFC and GGH may possibly undergo a significant
down-regulation in gene expression and activity in
order to protect cells from further loss of
intracellular folates due to folate efflux via the
RFC.
The current study may have profound implications
relating to various disciplines in biology and
medicine including: a) Developmental biology and
embryonic development- The central
developmental role that mammalian RFC plays has
been revealed in RFC knockout mice studies [41];
in contrast to this vital role of the RFC under
conditions in which folates are available for the
pregnant dams, our findings suggest that under
conditions of severe nutritional folate deficiency or
insufficiency of mammalian pregnant dams, the
RFC may in fact exacerbate the embryos
pathological status caused by the folate deficiency
by further extruding folates. Thus, RFC may play a
key role in inducing early abortions during early
stages of mammalian pregnancy when folates are
severely limited in the growth environment and
thereby may confer an evolutionary advantage by
eliminating embryos that are certain to fail normal
cell proliferation, differentiation and proper
development. b) Molecular medicine- RFC is an
important route for the uptake of various antifolates
including for example MTX and Pemetrexed
(Alimta) [59, 60]; hence, our findings suggest that
the down-regulation of the RFC as a result of folate
deficient conditions may decrease antifolate uptake
and thus compromise the pharmacological efficacy
of antifolates currently used in chemotherapy of
various cancers. Moreover, down-regulation and
mutational inactivation of the RFC serve as major
mechanisms of antifolate drug resistance practiced
by cancer cells [47, 59, 61, 62]. Hence, antifolate-
resistant cancer cells may readily survive not only
the exposure to antifolates but also transient folate
deficiency due to the markedly decreased folate
efflux via the RFC.
by guest on September 5, 2018
http://ww
w.jbc.org/
Dow
nloaded from
9
REFERENCES
1. Kim, Y.I., Folate and DNA methylation: a mechanistic link between folate deficiency and
colorectal cancer? Cancer Epidemiol Biomarkers Prev, 2004. 13(4): p. 511-9.
2. Kim, Y.I., Nutritional epigenetics: impact of folate deficiency on DNA methylation and
colon cancer susceptibility. J Nutr, 2005. 135(11): p. 2703-9.
3. Qureshi, A.A., D.S. Rosenblatt, and B.A. Cooper, Inherited disorders of cobalamin
metabolism. Crit Rev Oncol Hematol, 1994. 17(2): p. 133-51.
4. Scott, J.M., Folate and vitamin B12. Proc Nutr Soc, 1999. 58(2): p. 441-8.
5. Carmel, R., et al., Update on cobalamin, folate, and homocysteine. Hematology Am Soc
Hematol Educ Program, 2003: p. 62-81.
6. Stokstad, E.L.R. Folic acid metabolism in health and disease, M.F. Picciano, E.L.R.
Stokstad (eds), Wiley-Liss: New York, pp.1-21.
7. Birn, H., The kidney in vitamin B12 and folate homeostasis: characterization of receptors
for tubular uptake of vitamins and carrier proteins. Am J Physiol Renal Physiol, 2006.
291(1): p. F22-36.
8. Shane, B., Folylpolyglutamate synthesis and role in the regulation of one-carbon
metabolism. Vitam Horm, 1989. 45: p. 263-335.
9. McGuire, J.J., C.A. Russell, and M. Balinska, Human cytosolic and mitochondrial
folylpolyglutamate synthetase are electrophoretically distinct. Expression in antifolate-
sensitive and -resistant human cell lines. J Biol Chem, 2000. 275(17): p. 13012-6.
10. B., S., Folate chemistry and metabolism. In: Bailey LB, editor. Folate in health and
disease. New York: Marcel Dekker; . p. 1-22. 1995.
11. Zeng, H., et al., Transport of methotrexate (MTX) and folates by multidrug resistance
protein (MRP) 3 and MRP1: effect of polyglutamylation on MTX transport. Cancer Res,
2001. 61(19): p. 7225-32.
12. Wielinga, P., et al., The human multidrug resistance protein MRP5 transports folates and
can mediate cellular resistance against antifolates. Cancer Res, 2005. 65(10): p. 4425-30.
13. Volk, E.L. and E. Schneider, Wild-type breast cancer resistance protein (BCRP/ABCG2) is
a methotrexate polyglutamate transporter. Cancer Res, 2003. 63(17): p. 5538-43.
14. Matherly, L.H. and D.I. Goldman, Membrane transport of folates. Vitam Horm, 2003. 66:
p. 403-56.
15. Zhu, W.Y., et al., Severe folate restriction results in depletion of and alteration in the
composition of the intracellular folate pool, moderate sensitization to methotrexate and
trimetrexate, upregulation of endogenous DHFR activity, and overexpression of
metallothionein II and folate receptor alpha that, upon folate repletion, confer drug
resistance to CHL cells. J Exp Ther Oncol, 2002. 2(5): p. 264-77.
16. Backus, H.H., et al., Folate depletion increases sensitivity of solid tumor cell lines to 5-
fluorouracil and antifolates. Int J Cancer, 2000. 87(6): p. 771-8.
17. Gates, S.B., et al., Characterization of folate receptor from normal and neoplastic murine
tissue: influence of dietary folate on folate receptor expression. Clin Cancer Res, 1996.
2(7): p. 1135-41.
18. Ifergan, I., et al., Folate deprivation results in the loss of breast cancer resistance protein
(BCRP/ABCG2) expression. A role for BCRP in cellular folate homeostasis. J Biol Chem,
2004. 279(24): p. 25527-34.
19. Jansen, G., et al., Methotrexate transport in variant human CCRF-CEM leukemia cells with
elevated levels of the reduced folate carrier. Selective effect on carrier-mediated transport
by guest on September 5, 2018
http://ww
w.jbc.org/
Dow
nloaded from
10
of physiological concentrations of reduced folates. J Biol Chem, 1990. 265(30): p. 18272-
7.
20. Lamers, Y., et al., Red blood cell folate concentrations increase more after
supplementation with [6S]-5-methyltetrahydrofolate than with folic acid in women of
childbearing age. Am J Clin Nutr, 2006. 84(1): p. 156-61.
21. Assaraf, Y.G., et al., Loss of multidrug resistance protein 1 expression and folate efflux
activity results in a highly concentrative folate transport in human leukemia cells. J Biol
Chem, 2003. 278(9): p. 6680-6.
22. Hooijberg, J.H., et al., Folate concentration dependent transport activity of the Multidrug
Resistance Protein 1 (ABCC1). Biochem Pharmacol, 2004. 67(8): p. 1541-8.
23. Ifergan, I., G. Jansen, and Y.G. Assaraf, Cytoplasmic confinement of breast cancer
resistance protein (BCRP/ABCG2) as a novel mechanism of adaptation to short-term folate
deprivation. Mol Pharmacol, 2005. 67(4): p. 1349-59.
24. Ratnam, M., et al., Homologous membrane folate binding proteins in human placenta:
cloning and sequence of a cDNA. Biochemistry, 1989. 28(20): p. 8249-54.
25. Lacey, S.W., et al., Complementary DNA for the folate binding protein correctly predicts
anchoring to the membrane by glycosyl-phosphatidylinositol. J Clin Invest, 1989. 84(2): p.
715-20.
26. Shen, F., et al., Folate receptor type gamma is primarily a secretory protein due to lack of
an efficient signal for glycosylphosphatidylinositol modification: protein characterization
and cell type specificity. Biochemistry, 1995. 34(16): p. 5660-5.
27. Shen, F., et al., Identification of a novel folate receptor, a truncated receptor, and receptor
type beta in hematopoietic cells: cDNA cloning, expression, immunoreactivity, and tissue
specificity. Biochemistry, 1994. 33(5): p. 1209-15.
28. Sadasivan, E. and S.P. Rothenberg, The complete amino acid sequence of a human folate
binding protein from KB cells determined from the cDNA. J Biol Chem, 1989. 264(10): p.
5806-11.
29. Elwood, P.C., Molecular cloning and characterization of the human folate-binding protein
cDNA from placenta and malignant tissue culture (KB) cells. J Biol Chem, 1989. 264(25):
p. 14893-901.
30. Elnakat, H. and M. Ratnam, Distribution, functionality and gene regulation of folate
receptor isoforms: implications in targeted therapy. Adv Drug Deliv Rev, 2004. 56(8): p.
1067-84.
31. Elnakat, H. and M. Ratnam, Role of folate receptor genes in reproduction and related
cancers. Front Biosci, 2006. 11: p. 506-19.
32. Spiegelstein, O., J.D. Eudy, and R.H. Finnell, Identification of two putative novel folate
receptor genes in humans and mouse. Gene, 2000. 258(1-2): p. 117-25.
33. Wang, X., et al., Differential stereospecificities and affinities of folate receptor isoforms for
folate compounds and antifolates. Biochem Pharmacol, 1992. 44(9): p. 1898-901.
34. Antony, A.C., The biological chemistry of folate receptors. Blood, 1992. 79(11): p. 2807-
20.
35. Brigle, K.E., et al., Increased expression and characterization of two distinct folate binding
proteins in murine erythroleukemia cells. Biochem Pharmacol, 1994. 47(2): p. 337-45.
36. Rubin, R.C., et al., Uptake of methotrexate-3H by rabbit kidney slices. Cancer Res, 1967.
27(3): p. 553-7.
by guest on September 5, 2018
http://ww
w.jbc.org/
Dow
nloaded from
11
37. Sierra, E.E., et al., pH dependence of methotrexate transport by the reduced folate carrier
and the folate receptor in L1210 leukemia cells. Further evidence for a third route
mediated at low pH. Biochem Pharmacol, 1997. 53(2): p. 223-31.
38. Pathak, P., et al., Prevalence of multiple micronutrient deficiencies amongst pregnant
women in a rural area of Haryana. Indian J Pediatr, 2004. 71(11): p. 1007-14.
39. Assaraf, Y.G., S. Babani, and I.D. Goldman, Increased activity of a novel low pH folate
transporter associated with lipophilic antifolate resistance in chinese hamster ovary cells.
J Biol Chem, 1998. 273(14): p. 8106-11.
40. Zhao, R., et al., The spectrum of mutations in the PCFT gene, coding for an intestinal
folate transporter, that are the basis for hereditary folate malabsorption. Blood, 2007.
41. Zhao, R., et al., Rescue of embryonic lethality in reduced folate carrier-deficient mice by
maternal folic acid supplementation reveals early neonatal failure of hematopoietic
organs. J Biol Chem, 2001. 276(13): p. 10224-8.
42. Goldman, I.D., The characteristics of the membrane transport of amethopterin and the
naturally occurring folates. Ann N Y Acad Sci, 1971. 186: p. 400-22.
43. Sirotnak, F.M., Obligate genetic expression in tumor cells of a fetal membrane property
mediating "folate" transport: biological significance and implications for improved
therapy of human cancer. Cancer Res, 1985. 45(9): p. 3992-4000.
44. Sirotnak, F.M., Determinants of resistance to antifolates: biochemical phenotypes, their
frequency of occurrence and circumvention. NCI Monogr, 1987(5): p. 27-35.
45. Zhao, R., F. Gao, and I.D. Goldman, Reduced folate carrier transports thiamine
monophosphate: an alternative route for thiamine delivery into mammalian cells. Am J
Physiol Cell Physiol, 2002. 282(6): p. C1512-7.
46. Rothem, L., et al., The reduced folate carrier gene is a novel selectable marker for
recombinant protein overexpression. Mol Pharmacol, 2005. 68(3): p. 616-24.
47. Rothem, L., M. Stark, and Y.G. Assaraf, Impaired CREB-1 phosphorylation in antifolate-
resistant cell lines with down-regulation of the reduced folate carrier gene. Mol
Pharmacol, 2004. 66(6): p. 1536-43.
48. O'Connor, B.M., et al., Secretion of gamma-glutamyl hydrolase in vitro. Cancer Res, 1991.
51(15): p. 3874-81.
49. Rots, M.G., et al., Role of folylpolyglutamate synthetase and folylpolyglutamate hydrolase
in methotrexate accumulation and polyglutamylation in childhood leukemia. Blood, 1999.
93(5): p. 1677-83.
50. Lin, B.F., R.F. Huang, and B. Shane, Regulation of folate and one-carbon metabolism in
mammalian cells. III. Role of mitochondrial folylpoly-gamma-glutamate synthetase. J Biol
Chem, 1993. 268(29): p. 21674-9.
51. Osborne, C.B., K.E. Lowe, and B. Shane, Regulation of folate and one-carbon metabolism
in mammalian cells. I. Folate metabolism in Chinese hamster ovary cells expressing
Escherichia coli or human folylpoly-gamma-glutamate synthetase activity. J Biol Chem,
1993. 268(29): p. 21657-64.
52. Assaraf, Y.G., et al., Computer modelling of antifolate inhibition of folate metabolism
using hybrid functional petri nets. J Theor Biol, 2006. 240(4): p. 637-47.
53. Assaraf, Y.G., The role of multidrug resistance efflux transporters in antifolate resistance
and folate homeostasis. Drug Resist Updat, 2006. 9(4-5): p. 227-46.
54. Chen, Z.S., et al., Analysis of methotrexate and folate transport by multidrug resistance
protein 4 (ABCC4): MRP4 is a component of the methotrexate efflux system. Cancer Res,
2002. 62(11): p. 3144-50.
by guest on September 5, 2018
http://ww
w.jbc.org/
Dow
nloaded from
12
55. Chen, Z.S., et al., Transport of methotrexate, methotrexate polyglutamates, and 17beta-
estradiol 17-(beta-D-glucuronide) by ABCG2: effects of acquired mutations at R482 on
methotrexate transport. Cancer Res, 2003. 63(14): p. 4048-54.
56. Qiu, A., et al., Identification of an intestinal folate transporter and the molecular basis for
hereditary folate malabsorption. Cell, 2006. 127(5): p. 917-28.
57. Bailey, L.B., et al., Folacin and iron status and hematological findings in black and
Spanish-American adolescents from urban low-income households. Am J Clin Nutr, 1982.
35(5): p. 1023-32.
58. Assantachai, P. and S. Lekhakula, Epidemiological survey of vitamin deficiencies in older
Thai adults: implications for national policy planning. Public Health Nutr, 2007. 10(1): p.
65-70.
59. Assaraf, Y.G., Molecular basis of antifolate resistance. Cancer Metastasis Rev, 2007.
26(1): p. 153-81.
60. Walling, J., From methotrexate to pemetrexed and beyond. A review of the
pharmacodynamic and clinical properties of antifolates. Invest New Drugs, 2006. 24(1): p.
37-77.
61. Rothem, L., et al., Resistance to multiple novel antifolates is mediated via defective drug
transport resulting from clustered mutations in the reduced folate carrier gene in human
leukaemia cell lines. Biochem J, 2002. 367(Pt 3): p. 741-50.
62. Worm, J., et al., Methylation-dependent silencing of the reduced folate carrier gene in
inherently methotrexate-resistant human breast cancer cells. J Biol Chem, 2001. 276(43):
p. 39990-40000.
63. Rothem, L., A. Aronheim, and Y.G. Assaraf, Alterations in the expression of transcription
factors and the reduced folate carrier as a novel mechanism of antifolate resistance in
human leukemia cells. J Biol Chem, 2003. 278(11): p. 8935-41.
64. Cole, P.D., et al., Effects of overexpression of gamma-Glutamyl hydrolase on methotrexate
metabolism and resistance. Cancer Res, 2001. 61(11): p. 4599-604.
65. Jordheim, L.P., et al., Characterization of a gemcitabine-resistant murine leukemic cell
line: reversion of in vitro resistance by a mononucleotide prodrug. Clin Cancer Res, 2004.
10(16): p. 5614-21.
66. Kanzaki, A., et al., Expression of multidrug resistance-related transporters in human
breast carcinoma. Jpn J Cancer Res, 2001. 92(4): p. 452-8.
Acknowledgements
Grant support: This study was supported by a research grant from the Fred Wyszkowski Cancer Research
Fund (#2007346) to YGA as well as by a grant from the Dutch Arthritis Association (Grant NRF-030-I-40)
to GJ.
We wish to thank Prof. Godefridus J. Peters for his critical assistance with the GGH activity assay.
by guest on September 5, 2018
http://ww
w.jbc.org/
Dow
nloaded from
13
FIGURE LEGENDS
Fig. 1: Model of intracellular folate metabolism under replete (A) and deplete conditions (B).
Fig. 2: Viability of RFC overexpressing cells versus RFC null cells under short-term folate deficiency. The CHO cell line deficient in RFC transport activity termed C5 as well as its RFC- and FR-
overexpressing transfectants were trypsinized and washed three times with folic acid-free growth medium.
Then, cells (6x104) were seeded in each of two T25 flasks in 5 ml folic acid-free growth medium. The
sublines in the first set of flasks were termed C5/NF, C5/FR-NF and C5/RFC-NF (i.e. no folate). The
sublines in the second set of flasks were supplemented with folates derivatives according to their initial
growth conditions (i.e. 2.3 µM folic acid, 3 nM folic acid and 3 nM LCV) resulting in the sublines C5-HF,
C5/FR-3nMFA and C5/RFC-3nMLCV, respectively. All 6 flasks were simultaneously incubated for 6 days
in a humidified CO2 incubator at 37oC. Following these 6 days of incubation, cells were detached by
trypsinization and the number of viable cells was determined by a haemocytometer counting after trypan
blue staining. The asterisk denotes a statistically significant difference.
Fig. 3: Gene expression status of folate influx and efflux transporters as well as folate-dependent
enzymes under folate deplete- and replete conditions. Total cellular RNA was extracted from the folate-
supplemented cell lines MCF7/MR-HF and CEM/7A-HF as well as from their folate-deprived counterparts
MCF7/MR-NF and CEM/7A-NF cells, respectively. Then, the transcript levels of β-ACTIN, GAPDH,
BCRP, MRP5, MRP1,GGH, FPGS, PCFT, FRα and RFC in the various sublines were quantified by semi-
quantitative RT-PCR analysis. Signal intensity was quantified using the densitometric program TINA
(version 2.10g). After normalizing versus GAPDH and β-ACTIN levels, average fold decrease (as depicted
within the figure and located adjacent to the relevant gene) was determined. Each experiment was repeated
three times. Moreover, each semi-quantitative RT-PCR experiment was done in a titration manner (i.e. 1,
1/16, 1/36 using 6-fold serial template dilutions) in order to identify and exclude signals originating from
the plateau phase. Note that in contrast to MCF7/MR, the gene expression levels of FR, PCFT, MRP5 and
BCRP in CCRF-CEM cells is negligible.
Fig. 4: [3H]MTX transport in folate supplemented and deprived sublines. Initial rates of [
3H]MTX
uptake were determined for the folate-supplemented cell lines MCF7/MR-HF and CEM/7A-HF as well as
for their folate-deprived counterparts MCF7/MR-NF and CEM/7A-NF sublines, respectively. Note that the
asterisk denotes a statistically significant difference whereas the average percent decrease of initial rates of
[3H]MTX uptake is depicted within the figure and shown adjacent to the relevant column.
Fig. 5: GGH activity in folate supplemented and folate deprived sublines. GGH activity was determined
in the folate-supplemented cell lines MCF7/MR-HF and CEM/7A-HF as well as for their folate-deprived
counterparts MCF7/MR-NF and CEM/7A-NF, respectively. Note that the asterisk denotes a statistically
significant difference whereas the average percentage decrease of GGH activity is depicted within the figure
and shown adjacent to the relevant column.
Fig. 6: Mathematical bio-modeling of intracellular folate pool depletion by folate efflux transporters. Mathematical modeling aimed at evaluating the residual intracellular folate pool (Y axis) in the presence of
the following folate efflux transporters: RFC or MRP3 or MRP4; the different maximal folate efflux
capacities for each experiment are depicted in a close proximity to its plotted graph or via arrows. The
theoretical experiments simulate the cellular folate pool status for duration of up to 7 days (X axis) upon
folate free conditions in the presence of one examined folate efflux transporter. Note that the folate
depleting effect of the RFC was evaluated in four different efflux capacities including the hypothetical
by guest on September 5, 2018
http://ww
w.jbc.org/
Dow
nloaded from
14
negligible capacities of 1% and 0.1% (i.e. 0.01µM/min and 0.001µM/min, respectively) of the total cellular
folate efflux capacity.
Table I: Oligonucleotides used for semi-quantitative RT-PCR.
Gene
Primers:
Sense primer (S)
Antisense primer (AS)
Annealing
temperature
( °C )
Cycles'
Number
Product's
Length
(bp)
Reference
RFC 5’-AGCCTCCCTGGAGCAGAGAC-3’ (S)
5’-ACTCCTGTGGGGCCAGTGTC-3’ (AS)
58
24 -30
619
-----
FRα
5’-CCAGCAGGTGGATCAGAGCTG-3’ (S)
5’-CGACCATGGAGCAGGAACC-3’ (AS)
61
34
510
-----
PCFT 5’-ATGCAGCTTTCTGCTTTGGT-3’ (S)
5’-GGAGCCACATAGAGCTGGAC-3’ (AS)
55
35
100
[56]
FPGS 5’-CCGGCTGAACATCATCCA-3’ (S)
5’-CTTTCTGCCATGCGATCTTCT-3’ (AS)
60
28
449
[63]
GGH 5’-GCTTATTAACTGCCACAGATACGTTG-3’ (S)
5’-GAACATTCTGCTGTGCAATGAC-3’ (AS)
50
30
79
[64]
MRP1 5’-CAGGAGCAGGATGCAGAGGA-3’ (S)
5’-TGTAGTCCCAGTACACGGAAAGC-3’ (AS)
60
28
280
[63]
MRP5 5’-CCCAGGCAACAGAGTCTAACC-3’ (S)
5’-CGGTAATTCAATGCCCAAGTC-3’ (AS)
58
30
112
[65]
BCRP 5’-TGCCCAAGGACTCAATGCAACA-3’ (S)
5’-ACAATTTCAGGTAGGCAATTGTG-3’ (AS)
60
27
172
[66]
GAPDH 5’-AGGGGGGAGCCAAAAGGG-3’ (S)
5’-GAGGAGTGGGTGTCGCTGTTG-3’ (AS)
60
35
514
[63]
β-ACTIN 5’-CCGTCTTCCCCTCCATCGTG-3’ (S)
5’-GGGCGACGTAGCACAGCTTCT-3’ (AS)
56
28
576
[63]
by guest on September 5, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Ilan Ifergan, Gerrit Jansen and Yehuda G. Assaraffolate deprivation: RFC as a double-edged sword in folate homeostasis
The reduced folate carrier (RFC) is cytotoxic to cells under conditions of severe
published online May 22, 2008J. Biol. Chem.
10.1074/jbc.M802812200Access the most updated version of this article at doi:
Alerts:
When a correction for this article is posted•
When this article is cited•
to choose from all of JBC's e-mail alertsClick here
by guest on September 5, 2018
http://ww
w.jbc.org/
Dow
nloaded from