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:

assaraf@tx.technion.ac.il

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.

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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

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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

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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?:

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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

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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).

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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

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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.

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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.

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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

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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]

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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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

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