Departments of Medicine and Molecular Pharmacology, Albert Einstein College of Medicine, and the Albert Einstein Cancer Research Center, Bronx, New York
Submitted 30 June 2004 ; accepted in final form 17 September 2004
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ABSTRACT |
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folic acid; folate absorption; methotrexate
A major folate transporter in human and murine cells is the reduced folate carrier (RFC). RFC is a bidirectional transporter with properties of an anion exchanger that generates transmembrane gradients by coupling folate transport mediated by RFC to the outward flow of organic phosphates that are present at high concentrations within cells (21). RFC is the major mechanism by which reduced folates and antifolates are transported into human and murine leukemia cells (21). A number of observations have implicated a role for RFC in the transport of folates in intestinal cells. 1) RFC protein is localized to the apical brush-border membrane of murine gastrointestinal cells (33), although some expression in basolateral membrane has been observed as well (7). 2) There is a direct relationship between the level of RFC expression and folic acid influx in jejunal brush-border membrane vesicles prepared from sucking, weaning, and adult rats or rats fed a folate-deficient diet (1, 24). 3) An antibody to RFC was shown to inhibit 5-methyltetrahydrofolate (5-CH3-THF) transport into freshly isolated murine small intestinal cells (4). 4) Transfection of RFC cDNA in intestinal IEC-6 cells produced folate transport activity with a low-pH optimum (19).
There are, however, important unresolved issues regarding the role of this carrier in intestinal transport. First, it is unclear whether this is the sole mechanism by which folates are transported into intestinal epithelial cells. Second, there are unexplained discrepancies between the characteristics of folate transport in intestinal cells and folate transport mediated by RFC in leukemia cells. Hence the following points. 1) The optimum pH for RFC-mediated transport in murine and human leukemia cells is 7.4, with minimal activity at low pH (18, 29). There is no evidence that transport is coupled with proton flow across the cell membrane. However, while RFC is expressed in intestinal cells and a small folate transport component is detected at physiological pH in IEC-6 cells, the major transport component has a low-pH optimum, and transfection of mRFC into these cells produced low-pH activity (19) or low-pH activity together with much more prominent high-pH activity (23). 2) While RFC has a very low affinity for folic acid in leukemia cells that is one or two orders of magnitude below that for 5-CH3-THF, 5-formyltetrahydrofolate (5-CHO-THF), and methotrexate (MTX), the affinity of transport in cells of intestinal origin and in IEC-6 cells at low pH is comparable for all these folates (11, 25, 26). 3) Influx of MTX mediated by RFC in leukemia cells is minimally affected, or increased, by energy inhibitors, while influx in IEC-6 cells is partially suppressed under these conditions (23).
To further characterize the mechanisms underlying the constitutive folate transport activity for folates in IEC-6 cells, studies were undertaken to determine the impact of the loss of endogenous RFC function at low and physiological pH. The approach used was chemical mutagenesis followed by selective pressure with dihydrofolate reductase (DHFR) inhibitors, a technique applied in this and other laboratories (9, 35, 40). The agents used were MTX, a classical antifolate that forms polyglutamate derivatives in cells, and 5,8-dideaza-N-(-4-amino-4-deoxypteroyl)-N
-hemiphthaloyl-1-ornithine (PT632), a novel antifolate with 10-fold higher affinity for DHFR and RFC that does not form polyglutamate derivatives (34) and has very low affinity for low-pH transporters in several tumor cell lines (32). This procedure resulted in the identification of IEC-6 cell lines with mutations in RFC that resulted in a loss of function. This article describes the folate transport properties of these cell lines.
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MATERIALS AND METHODS |
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Cell culture. IEC-6 cells derived from the crypt of normal rat small intestinal epithelium (22) and grown continuously in this laboratory were originally obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained in RPMI 1640 medium containing 2.3 µM folic acid supplemented with 10% fetal calf serum (Hyclone), 2 mM glutamine, 20 µM 2-mercaptoethanol, penicillin (100 U/ml), and streptomycin (100 µg/ml). Wild-type HeLa and RFC-null R5 cells were maintained in cell culture as described previously (38).
Generation of MTX- and PT632-resistant IEC-6 cells.
Two selections were undertaken to obtain cell lines with loss of RFC expression or function. One used MTX and the other PT632, a more potent DHFR inhibitor with a much higher affinity than MTX for RFC (34). IEC-6 cells grown in complete RPMI 1640 medium were treated with 2.4 mM ethylmethanesulfonate for 17 h to achieve 90% cell kill. Cells were then washed to remove the mutagen, allowed to recover for 2 days, and then reseeded in 100-mm dishes in the presence of 180 nM MTX or 20 nM PT 632. Individual resistant clones were detected within 2 wk and picked up by cloning cylinders. One such clonal cell line resistant to MTX, designated IEC-6/A4, was maintained in RPMI 1640 medium with 180 nM MTX, while the PT632 resistant cells IEC-6/PT1 were grown in RPMI 1640 medium containing 20 nM PT632.
Detection of RFC mutations. Total RNA was isolated from cells with the TRIzol reagent (Invitrogen) and was reverse transcribed using oligo(dT) and Superscript II reverse transcriptase (Invitrogen). The entire coding region of rat RFC was amplified by RT-PCR using sense primer 5'-GGAGTGTCATCTTTGTTCACC-3' and antisense primer 5'-ATGAAGCCAGGAAGGTGAAGA-3' derived from the known rat RFC sequence (GenBank accession no. U38180). Both Taq and Pfu DNA polymerases were used in PCR. PCR products were cloned into either pCR-4-ToPo or PCR-Blunt vector (both obtained from Invitrogen) according to the manufacturer's instructions. Initially, inserts (rat RFC coding region) from three or four plasmids derived from the same cell line were fully sequenced. Once a mutation was identified in a plasmid, the PCR product was directly sequenced to determine whether the mutation was homogeneous. The sequencing was conducted in the DNA Sequencing Shared Resource of the Albert Einstein College of Medicine Cancer Research Center.
Generation of constructs for expression of GFP-rat RFC fusion proteins. An upstream primer, 5'-CGGAATTCGGACCTGGCCAACATG-3', containing an EcoRI restriction site, and a downstream primer, 5'-CGGATCCAAGGTGAA GACAGGTCA-3', containing a BamH I restriction site, were used to amplify RFC cDNA with the 420 stop codon by performing PCR. After digestion with these restriction enzymes, the RFC fragment was ligated into the phrGFP-N1 vector (Stratagene) linearized with the same enzymes. The resulting plasmid, named phrGFP-RFC-420 stop, was further sequenced and contained only the one desired mutation. Based on this plasmid, phrGFP-wild-type RFC was generated according to the QuickChange protocol (Stratagene) using two complementary primers, 5'-TCTGGGCCTCCAAGTCCATCA-3' and 5'-TGATGGACTTGGAGGCCCAG-3'. The plasmid phrGFP-RFC-135 deletion was generated by a subsequent QuickChange mutagenesis with phrGFP-wt RFC as the template and the following oligonucleotides used as primers: 5'-CGCATCGCCTACTCCTACATAT-3' and 5'-GAATATGTAGGAGTAGGCGAT-3'. The RFC sequence in the expression vectors was confirmed using automated sequencing.
Transient transfections. The green fluorescent protein (GFP)-wt-RFC, GFP-RFC mutants, and phrGFP control expression vectors were transfected into RFC-null R5 cells IEC-6/A4 or IEC-6/PT1 at 5075% confluence. Transfection was performed using Lipofectamine Plus (Invitrogen) according to the manufacturer's protocol. After 2 days of growth, the cells were examined under a fluorescence microscope and MTX influx was determined.
RNA blot analysis.
Total RNA was isolated from cells with the TRIzol reagent (Invitrogen) according to the manufacturer's protocol, and 25 µg were resolved by electrophoresis on 1.2% agarose gels containing formaldehyde. RNA was transferred to Nytran N-membranes (Schleicher & Schuell, Keene, NH) and fixed with a Stratalinker UC cross-linker (Stratagene, La Jolla, CA). The blot was first probed with rat RFC cDNA and then stripped and reprobed with -actin cDNA. The radioactive signals in blots were quantitated by performing PhosphorImager analysis.
Transport studies. Cells were grown in monolayer culture adherent to the bottom of glass scintillation vials in which influx measurements were obtained (28). Before experiments, the growth medium was aspirated and the cells were washed three times with 3 ml of ice-cold transport buffer. Experiments at pH 5.5 were performed in MES-buffered saline (MBS) (in mM: 20 MES, 140 NaCl, 5 KCl, 2 MgCl2, and 5 glucose, pH 5.5) and at pH 7.4 in HEPES-buffered saline (HBS) (in mM: 20 HEPES, 140 NaCl, 5 KCl, 2 MgCl2, and 5 glucose, pH 7.4). For some MTX influx studies, an anion-free buffer (HEPES-sucrose buffer) (20 mM HEPES and 225 mM sucrose, pH adjusted to 7.4 with MgO) was also used. Before influx determinations, 1 ml of the desired buffer was added to the vials, and the cells were equilibrated for 20 min at 37°C. The buffer was then aspirated, and uptake was initiated by the addition of 0.5 ml of prewarmed buffer containing tritiated folate or antifolate. Uptake was terminated by the addition of 10 ml of HBS at 0°C to the vials. The cells were then washed three times with 3 ml of HBS and lysed by the addition of 0.5 ml of 0.2N NaOH, and radioactivity was determined on 0.4 ml of cell lysate. Protein concentration in the lysate was measured with the bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL). Cell folate or antifolate is expressed as nanomoles per gram of protein.
Folate binding assay. Cells grown in scintillation vials were washed twice with acid buffer (10 mM sodium acetate and 150 mM NaCl, pH 3.5) and then once with HBS, all at 0°C. Cells were then incubated with 0.5 ml of HBS containing 5 nM [3H]folic acid at 0°C for 15 min. The cells were then washed three times with 0°C HBS at pH 7.4, followed by the addition of 0.5 ml of the acid buffer to release bound [3H] folic acid; radioactivity in 0.5 ml of the acid supernatant was then measured. The cells were digested with 0.2 M NaOH, and protein concentration was determined (see Transport studies). The data, expressed as nanomoles per gram of protein, are the average of two separate experiments.
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RESULTS |
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MTX influx was assessed in wild-type and IEC-6/A4 RFC-null cells. Influx at pH 7.4 in IEC-6/A4 cells was <10% the rate in wild-type cells (Fig. 1A). However, at pH 5.5, while MTX influx was decreased in IEC-6/A4 cells, 45% of wild-type activity was retained (Fig. 1B). Initial rates were also obtained under these conditions for 5-CH3-THF. Here, too, influx in the IEC-6/A4 cells was virtually abolished (<2%) at pH 7.4, but 30% of influx activity was retained at pH 5.5 compared with wild-type cells (based on two separate experiments; data not shown). Folic acid transport in these cells was negligible in both cell lines at pH 7.4 as expected because this folate has a very low affinity for RFC. However, 40% of activity was retained at pH 5.5 in IEC-6/A4 cells (average of three separate experiments; data not shown).
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Folate/antifolate influx in a PT632-resistant IEC-6 cell subline due to deletion of an amino acid in RFC. Because suppression of MTX transport activity at low pH was detected when cells were selected and maintained continuously in the presence of MTX (albeit a reversible phenomenon), another selection was undertaken with PT632, a novel new-generation DHFR inhibitor (34), to determine whether a carrier-deficient cell line could be identified in which this phenomenon does not occur. Unlike MTX, PT632 has a very low affinity for the RFC-independent low-pH transporter in HeLa cells (32) and does not inhibit MTX influx at pH 5.5 in IEC-6 cells (data not shown). Using PT632 at a concentration (20 nM) fivefold greater than its IC50 in IEC-6 cells, 10 surviving clones were obtained. Initial screening indicated that MTX influx at pH 7.4 was decreased, whereas MTX influx at pH 5.5 was similar in all of these clones compared with wild-type cells. Further studies focused on one of the clones, IEC-6/PT1, which was maintained in the presence of 20 nM PT632.
Influx properties of the PT632-resistant IEC-6/PT1 cell line were assessed using [3H]MTX and [3H]folic acid as transport substrates. An anion-free (HEPES-sucrose) buffer was used for studies at pH 7.4 to amplify any possible RFC-mediated component by the removal of Cl because RFC transport is suppressed by this and other anions (13). As shown in Fig. 4, influx of MTX was virtually abolished in IEC-6/PT1 cells at pH 7.4 but was unchanged at pH 5.5 compared with wild-type cells. Influx of folic acid into IEC-6/PT1 cells was also the same as that in wild-type IEC-6 cells at pH 5.5. This folate has a very low affinity for RFC, with negligible transport activity at pH 7.4 but a high affinity for low-pH transporter in HeLa and IEC-6 cells (23, 32). Hence, unlike what was observed in the IEC-6/A4 cells maintained in the presence MTX, influx of MTX or folic acid in IEC-6/PT1 cells maintained in the presence of 20 nM PT632 was the same as that in the wild-type cells at pH 5.5.
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Assessment of the consequence of RFC mutations by transient transfection into RFC-null HeLa cells.
Expression vectors for GFP-RFC fusion proteins were constructed with the GFP tag linked to the NH2 terminus. The expression vectors were transiently transfected into a HeLa subline, R5, that lacks genomic RFC. As shown in Fig. 5, MTX influx in R5 cells transfected with wild-type RFC was 10 times greater than in R5 cells transfected with the GFP control vector. Hence, rat RFC expressed as a GFP fusion protein was fully active. There was, however, no transport function detected in R5 cells transfected with either RFC-420 stop or RFC-135 deletion constructs.
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The very same transient transfectants used for assessment of MTX influx (Fig. 5) were also monitored using fluorescence microscopy (Fig. 6). In GFP vector control transfected cells, green fluorescence was visualized diffusely within R5 (Fig. 6A) or IEC-6/A4 cells (Fig. 6E) but not on the plasma membrane. R5 cells were significantly larger than IEC-6/A4 cells, and the percentage of green R5 transfectants was greater than that of green IEC-6/A4 transfectants, probably because of higher transfection efficiency in R5 cells, a HeLa subline. In cells transfected with wild-type RFC, the GFP fusion protein was localized largely to plasma membranes, with some fluorescence detected in intracellular organelles (Fig. 6, B and F). No fluorescence at all was seen in cells transfected with the RFC-420 stop construct, indicating that the protein was not stable (Fig. 6, C and G). In R5 cells transfected with the RFC-135 deletion construct, fluorescence was not observed on the plasma membrane but was restricted to some cytoplasmic organelles (Fig. 6, D and H), indicating that the mutated RFC, although apparently stable, does not reach the plasma membrane.
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DISCUSSION |
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There was a decrease in low-pH activity in the IEC-6/A4 cells when 180 nM MTX was included in the growth medium. This decrease was not related to loss of RFC function in these cells, because growth in the absence of MTX increased the low-pH transport activity to the levels of wild-type cells, while RFC function at pH 7.4 remained defective. There are two factors that might account for why MTX in the growth medium suppresses transport activity at pH 5.5. 1) The medium of confluent cultures is acidic, and because the low-pH transport activity has high affinity for MTX, the presence of MTX in growth medium could select those cell subpopulations that have a lower level of the low-pH folate transport activity. 2) Residual MTX influx at physiological pH in IEC-6/A4 cells may be attributed, at least in part, to residual activity of the low-pH transporter as recently suggested in HeLa cells (36). The presence of MTX would therefore select cells that have lower levels of MTX influx in both neutral and acidic growth media.
While RFC does not contribute to folate transport activity at very low pH, there is a continuum of transport mediated by the low-pH process and RFC as the pH is increased from 5.5 to 7.4, with the contribution by the low-pH transporter decreasing and the contribution by RFC increasing. Hence, at pH levels in the microclimate that surrounds the jejunal villi (pH 66.5) (17, 27), RFC plays a role in transport of physiological folates and MTX. The properties of folate transport at low pH are different from those observed for RFC-mediated transport in RFC-competent cells at physiological pH. This cannot be attributed to pH-dependent changes in RFC-mediated transport, because recent studies at our laboratory (32) have indicated that there are only minimal changes in the affinity of RFC for MTX or the inhibitory properties of folic acid during transport mediated by RFC at pH 5.5 vs. 7.4.
There are some studies suggesting that the low-pH activity in intestinal cells is related in some way to RFC function. For instance, low-pH transport activity is inhibited in freshly isolated intestinal epithelial cells by an antibody to the NH2 and COOH termini of RFC (4). It is unclear, however, how this antibody could block the function of this carrier in intact cells when it is directed to domains located in the cytoplasm (8). Transfection of RFC into IEC-6 cells resulted in the induction of a low-pH transport activity (19). However, in another case, this change was relatively small compared with much more prominent increases in transport activity at pH 7.4 (23). It is not possible at this point to reconcile these observations with the results of the present study, which exclude a role for constitutive RFC activity in IEC-6 cells in the transport of folates at low pH.
The present study does not clarify whether this RFC-independent, low-pH transport activity is present to the same extent in intact mature intestinal epithelium. It is possible, for instance, that in these replicating cells of intestinal crypt origin, there is a low-pH activity not present in cells of the mature intestinal villus. This point could be clarified in studies with intestine obtained from RFC-null mice. However, homozygous deletion of the RFC gene in the mouse is embryonic lethal, and while some RFC / mice can be brought to term and live up to 12 days when the pregnant (or later, lactating) female is administered high doses of folic acid subcutaneously, these animals die as a result of severe atrophy of hematopoietic tissues (39). There is, however, essentially no intestinal pathology, suggesting the presence of an RFC-independent transport pathway in this tissue (39). The intestine of these pups is too small to permit acquisition of absorptive cells for transport studies.
Folate transport activity with a low-pH optimum has also been identified in murine leukemic cells in which RFC function is impaired. In one case, the carrier was present but mutated; in the other case, the basis for impaired carrier function was not clear (12, 30). The transport activity identified in these cells has a pattern of structural specificity and energy requirement similar to findings in IEC-6 cells (23, 26). Low-pH folate transport activity has also been detected in freshly isolated hepatocytes, primary cultured rat astrocytes, and primary cultured cerebellar granule cells, as well as in a few cancer cell lines (2, 3, 1416). Recently, >30 human solid tumor cell lines derived from a variety of tissue origins were evaluated for the presence of physiological and low-pH MTX transport activities. In 90% of these cell lines, transport activity at pH 5.5 was equal to or greater than activity at pH 7.4 (37). A HeLa subline, R5, was obtained in which RFC was deleted from the genome, but there was no decrease in the transport activity at low pH, and MTX transport by this route was markedly suppressed by folic acid (37). Hence, the low-pH folate transport activity in R5 cells was completely independent of RFC. It is unclear whether the low-pH folate transport activities in IEC-6 and HeLa R5 cells represent the same process or whether all of the low-pH transport activities in primary cultures and other tumor cell lines described above are also independent of RFC.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* Y. Wang and A. Rajgopal contributed equally to this work.
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