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Clinical Cancer Research 13, 2557-2567, May 1, 2007. doi: 10.1158/1078-0432.CCR-06-1343
© 2007 American Association for Cancer Research
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Human Cancer Biology

The Folate Receptor {alpha} Is Frequently Overexpressed in Osteosarcoma Samples and Plays a Role in the Uptake of the Physiologic Substrate 5-Methyltetrahydrofolate

Rui Yang1, E. Anders Kolb1, Jing Qin2, Alexander Chou3, Rebecca Sowers1, Bang Hoang5, John H. Healey5, Andrew G. Huvos4, Paul A. Meyers3 and Richard Gorlick1

Authors' Affiliations: 1 Department of Pediatrics and Molecular Pharmacology, The Albert Einstein College of Medicine, The Children's Hospital at Montefiore, Bronx, New York and Departments of 2 Epidemiology and Biostatistics, 3 Pediatrics, and 4 Pathology, and 5 Orthopedic Surgery Service, Memorial Sloan-Kettering Cancer Center, New York, New York

Requests for reprints: Richard Gorlick, Department of Pediatrics, The Children's Hospital at Montefiore, 3415 Bainbridge Avenue, Rosenthal 3rd Floor, Bronx, NY 10467. Phone: 718-741-2333, Fax: 718-920-6506; E-mail: rgorlick{at}montefiore.org.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Two major systems exist for folate cell entry: the reduced folate carrier (RFC) and the folate receptor (FR). Although defective RFC-mediated transport was frequently identified as a mechanism of methotrexate (MTX) resistance in osteosarcoma, the status of FR and its role in this disease are unknown.

Experimental Design: mRNA for FR{alpha} was measured in 107 osteosarcoma specimens using quantitative reverse transcription-PCR and was related to RFC expression. The effect of FR{alpha} overexpression on MTX resistance and natural folate uptake was studied using FR{alpha} non-expressing osteosarcoma 143B cells transfected with FR{alpha} cDNA in comparison with those transfected with sense or antisense RFC in the same genetic background.

Results: Eighty-four samples (78.5%) had detectable FR{alpha} mRNA, and 29.9% had higher levels than the ovarian cancer cell line SKOV-3. No correlation was found between mRNA levels of FR{alpha} and RFC (r2 = 0.002). FR{alpha} overexpression had minor effects on the transport of MTX and sensitivity to this drug. Among the transfected 143B sublines, only the 143B-FR{alpha} was able to uptake 5-methyltetrahydrofolate when the extracellular concentration was reduced to 2 nmol/L, which conferred a growth advantage in physiologic folate concentrations compared with vector-only–transfected cells. Importantly, this was not similarly achieved by RFC overexpression.

Conclusions: This study suggests that FR{alpha} plays a role in the uptake of 5-methyltetrahydrofolate when the concentration gradient is insufficient for RFC-mediated transport. FR{alpha} overexpression is unlikely secondary to the decreased RFC expression in osteosarcoma.

In eukaryotic cells, import of folate vitamins is essential for the biosynthesis of thymidine, purine, and some amino acids such as serine and methionine (reviewed in ref. 1). The folate-dependent enzymes are targets of a class of chemotherapeutic agents (antifolates) that include methotrexate (MTX) and trimetrexate (TMTX) among others. At least two major systems exist for folate and antifolate cell entry. The reduced folate carrier (RFC) is the dominant route for reduced folate cofactors and MTX. 5-Methyltetrahydrofolate (5MTHF), which accounts for 99% of folates in circulation, is believed to be the primary substrate for the RFC. Folate receptor {alpha} (FR{alpha}), a glycosylphosphatidylinositol-anchored membrane protein, has a high affinity for folic acid (Kd <1 nmol/L) and, to a less of extent, for 5MTHF (Kd, 1-10 nmol/L). FR{alpha} internalizes folate by receptor-mediated endocytosis (reviewed in ref. 2). Folic acid is rich in green vegetables and is synthesized by bacteria in intestinal tract. Folic acid is also a common supplement in vitamin pills because of its stable chemical properties. Targeted gene ablation of the FR{alpha} is embryonic lethal in mice (3). The autoantibody to FR{alpha} identified in pregnant women was associated with having an infant with neural tube defect (4). Blocking autoantibody against FR{alpha} was identified in patients with cerebral folate deficiency syndrome (5). This indicates a physiologic role of the FR{alpha} in folate uptake even when the RFC is intact. Although RFC is ubiquitously expressed in almost all tissues (6), the biodistribution of FR{alpha} is quite limited (7, 8). Overexpression of FR{alpha} was detected in a spectrum of tumors, especially those of ovarian and uterine origin (9, 10). It was reported that FR{alpha} overexpression gave tumors an advantage in cell proliferation both in vitro and in vivo (11, 12). Furthermore, in ovarian cancer, overexpression of the FR{alpha} was associated with high grade, tumor progression, and poor prognosis (10).

Osteosarcoma is the most common bone malignancy in children and young adults (reviewed in ref. 13). Modern treatment of osteosarcoma includes a combination of chemotherapy and surgical procedures. Effectiveness of preoperative chemotherapy, scored as Huvos Grade, is a determinant of outcome for patients with osteosarcoma (14). The survival of osteosarcoma patients has been improved significantly over the last several decades. However, more than 30% to 40% of them can not be cured at the current time, even after an intensified chemotherapy regimen is administered (15, 16). Studies including identification of mechanisms of drug resistance and more effective therapeutic agents are needed to further improve the outcome of patients with this disease.

High-dose MTX with leucovorin (5-formyl-tetrahydrofolate) rescue is a major component of current protocols for the treatment of patient with osteosarcoma (17, 18). The efficacy of MTX is limited by the presence of intrinsic and/or acquired resistance (reviewed in ref. 19). The mechanisms of MTX resistance include (a) impaired drug transport, (b) reduced drug accumulation because of decreased MTX polyglutamylation by folypolyglutamyl synthetase or increased drug hydrolysis, (c) increased drug efflux, (d) expansion of intracellular folate-cofactor pool, and (e) alterations in the structure or expression of the target enzyme dihydrofolate reductase and some novel mechanisms proposed recently (20). MTX resistance in osteosarcoma is at least in part due to impaired drug transport via the RFC. About 50% osteosarcoma samples had decreased RFC expression at the time of initial diagnosis (21). Sequence alterations were observed in an additional fraction of osteosarcoma samples, and some of them were associated with MTX resistance in vitro (22, 23). Newer antifolates, such as TMTX, a lipophilic analogue of MTX, enter cells by passive diffusion overcoming the RFC-mediated transport defect (24). Some suggestions exist that there may be additional resistance mechanisms to this class of agents in osteosarcoma.

FR{alpha} overexpression is rare in non-epithelial origin malignancies. Frequently observed RFC-mediated transport defects in osteosarcoma raise the question as to the means by which tumor cells obtain sufficient folates for rapid proliferation, given that the RFC is the predominant route for reduced folate cell entry. Intriguingly, in a preliminary screening study, FR{alpha} mRNA levels in osteosarcoma xenografts were observed to be similar to that of an ovarian cancer cell line (SKOV-3). A study was, therefore, initiated to investigate the status of the FR{alpha} in this disease and the possible role it may have in physiologic folate uptake as well as in antifolate resistance.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Compound. [3',5',7,9-3H]MTX (specificity at 30 Ci/mmol, 1.0 mCi/mL), [3',5',7,9-3H]5MTHF (specificity at 30 Ci/mmol, 1.0 mCi/mL), and [3',5',7,9-3H]folic acid (specificity at 69 Ci/mmol, 1.0 mCi/mL) were purchased from Moravek. Nonlabeled folic acid, 5MTHF, leucovorin, and MTX were purchased from Sigma. TMTX was obtained from MedImmune.

Cell culture. Osteosarcoma standard cell lines 143B, SaOS-2, U2OS, and HOS; leukemia cell line CCRF-CEM; and ovarian cancer cell line SKOV-3 were purchased from the American Type Culture Collection. Primary culture of normal human osteoblasts was purchased from Cambrex. Primary culture of osteosarcoma cells were obtained from severe combined immunodeficient mice bearing xenografts M191, M187, M160, and M42 established from osteosarcoma patient sample–derived cell lines and were designated numerically accordingly. Xenografts were examined by a pathologist (A.G.H) to confirm the histologic diagnosis by H&E staining periodically. Primary cultures were used immediately for experiments and were not subjected to further passage. Patient specimen–derived osteosarcoma cell lines OS191CL and OS160CL matched with xenografts M191 and M160, respectively, were included in this study. Cell cultures were regularly monitored to be free of Mycoplasma with a detection kit (American Type Culture Collection).

Osteosarcoma samples. Osteosarcoma samples (n = 107) were obtained from patients treated at the Memorial Sloan-Kettering Cancer Center between 1996 and 2001. All patients provided written informed consent for tissue procurement, and the conduct of the biology study was done in accordance with a protocol approved by the Memorial Hospital Institutional Review Board. All samples were confirmed by a pathologist (A.G.H.) to have a pathologic diagnosis of high-grade osteosarcoma and contained at least 80% of tumor tissue. The treatments of all patients included multiple courses of high-dose MTX. Total RNA was isolated using UltraspecRNA reagent (Biotecx) according to manufacturer's instructions and was reverse-transcribed using a Superscript II RT kit (Invitrogen).

Quantitative real-time reverse transcription-PCR. The measurement of FR{alpha} mRNA expression was based on a methodology described previously (25). Primer design was kindly provided by Dr. Morin (National Institute on Aging, Baltimore, MD) and is as follows: FR1-1, 5'-GCACCACAAGGAAAAGCCAG-3'; FR1-2, 5'-CATTCTTCCTCCAGGGTCGAC-3', which span an intron of 2,933 bp and do not amplify other isoforms of FR. Real-time fluorescent reverse transcription-PCR (RT-PCR) was carried out with a QuantiTect SYBR Green PCR Kit (Qiagen). The housekeeping gene ß-actin was used to normalize the levels of the FR{alpha} mRNA across samples. Thermal cycling was done in a reaction volume of 50 µL in triplicate for FR{alpha} and ß-actin, respectively, followed by melting curve analyses. In a fraction of samples, the PCR products were separated onto 12% neutral polyacrylamide gels and subjected to automated sequencing. An ovarian cancer cell line (SKOV-3) was included in each plate as a positive control. RFC mRNA in osteosarcoma patient samples was determined by real-time fluorescent RT-PCR as described in previous studies, and additional measurement in osteosarcoma cell lines and in xenografts was done with a leukemia cell line (CCRF-CEM) used as a control (26).

Cloning of FR{alpha} and stable transfection. mRNA extracted from a primary culture of M160 was reverse-transcribed using Superscript II RT (Invitrogen). The entire coding region of human FR{alpha} cDNA was PCR amplified with 2.5 units of Platinum Taq (Invitrogen) in 50 µL of 1x manufacturer buffer using primers described previously (27). The purified 810-bp PCR product was subcloned into the expression vector pcDNA3.0 (Invitrogen), and its sequence was confirmed by automated sequencing. An osteosarcoma cell line (143B) was used for transfection because a negligible level of endogenous FR{alpha} mRNA expression was detected. Stable transfection was obtained by G418 (Life Technologies) selection at 800 µg/mL for 2 weeks.

Stable transfection of RFC. Full-length hRFC cDNA was subcloned into the expression vector pcDNA4.0 (Invitrogen) in the sense strand and antisense directions at the BamHI site and transfected into 143B cells. Stable transfection was established by zeocin (Invitrogen) selection at 200 µg/mL for 2 weeks.

Cell surface [3H]folic acid–specific binding. Cells were plated in 12-well plate and grew for 3 days to reach about 90% confluence. [3H]folic acid was purified by high-pressure liquid chromatography before use, and binding assay was done as described previously with modifications (28). In brief, cells were incubated with 5 pmol [3H]folic acid in 1 mL of HBSS buffer for 60 min at 4°C and were then washed twice with cold PBS. Membrane-bound [3H]folic acid was extracted with 0.5 mL acid buffer [10 mmol/L sodium acetate, 150 mmol/L sodium chloride (pH 3.5)] and subjected to scintillation counting with a Beckman LS6500 Scintillation Counter. Nonspecific binding was determined in the presence of 1,000-fold excess nonlabeled folic acid. The binding was calculated by subtraction of the nonspecific binding.

Transport assay. Transport assays were done as described previously (27). Briefly, exponentially growing cells were incubated with transport media (folic acid–free RPMI 1640 with 10 mmol/L HEPES) for at least 30 min at 37°C. Transport was started with the addition of 300 µL transport media containing [3H]MTX to a final concentration of 1 µmol/L, or [3H]folic acid of 0.25 µmol/L, or [3H]5MTHF of indicated concentrations, respectively. Cells were incubated with [3H]MTX or [3H]folic acid for indicated durations, or with [3H]5MTHF for 30 min. Nonspecific (background) uptake was determined by including 1,000-fold excess nonlabeled respective substrates. For [3H]MTX competition assay, cells were first washed with acid buffer to remove the surface-bound folate before transferred into assay buffer.

MTX and folate retention assay. MTX accumulation was carried out as described previously (28). In brief, exponentially growing cells were incubated with [3H]MTX or [3H]5MTHF at a concentration of 20 nmol/L in either complete media or indicated conditions. After 72 h, drug-containing media were aspirated, and cells were then washed thrice. Cells were lysed, and the retained [3H]MTX or [3H]5MTHF was measured by scintillation counting.

Minimal folate requirement and cell proliferation in low-folate media. Exponentially growing cells were first grown in folate-free RPMI 1640 supplemented with 5% dialyzed FCS (Hyclone) containing 200 µmol/L glycine, 100 µmol/L adenosine, and 10 µmol/L thymidine (GAT) for 1 to 2 weeks to deplete endogenous folates. Cells were washed twice with serum-free RPMI 1640 lacking folic acid to eliminate GAT. Cells were then seeded into 96-well plates (Costar) at a density of 2 x 103 per 0.2 mL PER well in folate-free media with 5% dialyzed FCS containing a spectrum concentration of 5MTHF, leucovorin, or folic acid, respectively, as the sole folate source. For minimal growth requirement, cell numbers were measured at 72 h by the Alamar Blue assay (Biosource) on a CytoFluor4000 plate reader (PerSeptive Biosystems). EC50 is defined as the folate concentration required producing 50% of the maximal cell growth. For proliferation assay, cells were seeded in folate-free media with 5% dialyzed FCS containing 20 nmol/L of indicated folates, and cell number was determined at indicated time point.

Cell growth inhibition. Cell growth inhibition by MTX and TMTX were done after cells were pregrown for 1 week in folic acid–free media supplemented with 5% dialyzed FCS containing GAT and 20 nmol/L folic acid, or 5MTHF, or leucovorin, respectively. Cells were exposed continuously to drug for 72 h at a range of concentrations of drugs in the indicated media, respectively. IC50 is defined as the drug dose at which cell growth was inhibited by 50% relative to untreated controls.

Statistical analysis. Statistical analysis was done by the Department of Epidemiology and Biostatistics (J.Q.) at the Memorial Sloan-Kettering Cancer Center. The association between FR{alpha} mRNA levels with RFC mRNA levels in the osteosarcoma samples, as well as with clinical information, including age, gender, specimen type (primary or metastatic), tumor location, histologic subtype, the histologic response to preoperative chemotherapy (Huvos Grade), and patient outcome, were assessed by Spearman's correlation coefficient statistic (continuous variables versus continuous variables) and Wilcoxon rank sum or Kruskal-Wallis statistic (continuous variable versus discrete variable). Student's t test was used for analyses of all the in vitro experiments. All statistic analyses were two tailed, and P < 0.05 was regarded as statistically significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
FR{alpha} mRNA was overexpressed in osteosarcoma xenografts. In a preliminary screen, osteosarcoma xenografts showed similar levels of FR{alpha} mRNA to that of an ovarian carcinoma cell line (SKOV-3) as shown in Fig. 1A . In contrast, primary culture of normal osteoblasts had low levels of FR{alpha} expression. It was noted that osteosarcoma xenografts had a statistically significantly (P < 0.05) higher expression compared with osteosarcoma grown in culture, whether standard or patient derived, including two pair-matched samples. Consistent with a prior report (8), 143B cells had nearly negligible level of FR{alpha} mRNA. In a fraction of samples, PCR products were visualized by resolving onto 12% polyacrylamide gels, and the sequence was confirmed by sequencing (data not shown) to ensure that the fluorescent intensity of SYBR Green detected by the real-time RT-PCR reflected the unique gene amplification as shown in Fig. 1B. To validate these results at the protein level, surface specific binding to [3H]folic acid was done. Primary cultures from FR{alpha}-expressing osteosarcoma xenografts M187 and M160 showed similar levels of binding capacity to that of the SKOV-3, and an almost negligible binding was detected in FR{alpha} non-expressing 143B cells as shown in Table 1 . This is in accordance with the results obtained by real-time RT-PCR.


Figure 1
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  		Fig. 1. FR{alpha} mRNA expression in osteosarcoma xenografts and cell lines. A, real-time fluorescent RT-PCR was used to detect the FR{alpha} mRNA levels in osteosarcoma (OS) xenografts, standard cell lines, patient specimen–derived cell lines, and primary culture of normal osteoblasts (OB). Relative FR{alpha} mRNA levels for each sample were shown after normalization to the housekeeping gene ß-actin. B, PCR products were visualized on a 12% polyacrylamide gel to ensure that the fluorescent intensity of SYBR Green detected in real-time RT-PCR reflected the unique gene amplification. Predicted PCR fragments of 68 bp for FR{alpha} (NCBI accession no. NM_016731.2, nucleotides 133-200) and 59 bp for ß-actin were confirmed by comparing with the migration pattern of 25- and 100-bp DNA ladders, respectively.

 

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  		Table 1. Specific surface binding capacities to [3H]folic acid

 
FR{alpha} mRNA was overexpressed in a subset of osteosarcoma samples. Prompted by the results of preliminary screening, a cohort of osteosarcoma patient specimen was further studied. Detectable levels of FR{alpha} mRNA were found in 84 of 107 (78.5%) osteosarcoma samples, with a median level of 2.8 (arbitrary unit) compared with 8.1 for SKOV-3 cells; 29.9% (n = 32) had higher levels (median, 20.3; mean, 90.4) of FR{alpha} mRNA than SKOV-3 cells. The mRNA level of the RFC was measured by real-time RT-PCR as reported in a previous study (26) and was related to that of FR{alpha} in these samples. No correlation between the mRNA levels of these two genes was found (r2 = 0.002). FR{alpha} mRNA level was also related to the specimen clinical information, including age, gender, location, subtype, presence of metastases, response to preoperative chemotherapy, and survival. However, no significant correlation was found in this group of osteosarcoma samples with these clinical features (data not shown).

A stable FR{alpha}-expressing 143B cell line was established. Because FR{alpha} mRNA level was low in all the osteosarcoma standard cell lines tested, the construction of an FR{alpha}-overexpressing osteosarcoma cell line was desirable to facilitate subsequent in vitro studies. Full-length human FR{alpha} cDNA was transfected into 143B cells. One of the single-cell derived colonies among 24 screened, which displayed a similar level of FR{alpha} mRNA (63.1) to that of M187 (55.7; Fig. 1A), confirmed by binding capacity to [3H]folic acid (0.7897 pmol/106 cells), was selected (named as 143B-FR{alpha}) because this level is relevant to that observed in the fraction of osteosarcoma samples with higher FR{alpha} mRNA than SKOV-3. Vector-only–transfected 143B cells (named as 143B-Neo) had nearly negligible levels of FR{alpha} mRNA and [3H]folic acid binding (0.0122 pmol/106 cells) similar to the parental cells. To make the comparison with the RFC system, the sense and antisense strand cDNA of the RFC were stably transfected into 143B cells so they would be in the same genetic background. A 33-fold increase of RFC mRNA expression was obtained in 1 of 12 single-cell derived colonies transfected with the sense strand (named 143B-RFC) compared with the vector-only–transfected zeocin-resistant cells (named 143B-Zeo), and a 61% decrease was achieved in one (named 143B-RFC-AS) of the 12 colonies transfected with the antisense RFC cDNA.

FR{alpha} overexpression had a minor effect on [3H]MTX uptake. The effect of FR{alpha} on MTX transport was first assessed using [3H]MTX transport competition assay by comparing the FR{alpha} high-expressing M187, the FR{alpha} medium-expressing M160, and the FR{alpha} non-expressing 143B cells. In the absence of cold folic acid, variable [3H]MTX initial uptake rates were measured in M187 (Fig. 2A ), M160 (Fig. 2B), and 143B cells (Fig. 2C), roughly proportional to their levels of RFC expression (2.6-fold for M187, 1.4-fold for M160, and 1.9-fold for 143B relative to that of CCRF-CEM, respectively). In the presence of 20-fold nonlabeled folic acid, similar levels of inhibition on [3H]MTX uptake were observed among M187, M160, and 143B cells (Fig. 2), regardless of their levels of FR{alpha} expression. Furthermore, MTX uptake was measured in stably transfected 143B cells. For the 143B-FR{alpha} cells, the increase in [3H]MTX uptake (Fig. 3A ) is minor (0.34 pmol/106 cells at 5 min) compared with 143B-Neo cells (0.31 pmol/106 cells at 5 min). In contrast, 143B-RFC cells had an apparent increase of [3H]MTX uptake (0.43 pmol/106 cells at 5 min) compared with the 143B-Zeo cells (0.28 pmol/106 cells at 5 min) and the 143B-RFC-AS cells (0.15 pmol/106 cells at 5 min).


Figure 2
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  		Fig. 2. [3H]MTX uptake competition assay. Tritium-labeled MTX uptake was measured in FR{alpha} high-expressing M187 (A), FR{alpha} medium-expressing M160 (B), and FR{alpha} non-expressing 143B cells (C), at an extracellular concentration of 1 µmol/L. Competition assay was done in the presence of 20-fold excess nonlabeled folic acid (FA). Point, mean from three experiments; bars, SD. Similar levels of inhibition by folic acid were observed among M187, M160, and 143B cells.

 

Figure 3
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  		Fig. 3. [3H]MTX transport and [3H]5MTHF uptake assay. A, tritium-labeled MTX transport was measured in 143B-FR{alpha}, 143B-RFC, 143B-RFC-AS, and vector-only–transfected 143B sublines at an extracellular concentration of 1 µmol/L. Points, mean derived from three experiments; bars, SE. The trend line was drawn to best fit the data at all time point for each sample. B, uptake of tritium-labeled 5MTHF was measured in 143B-FR{alpha}, 143B-Neo, and 143B-RFC cells at indicated extracellular concentrations for 30 min. Nonspecific background was determined by including 1,000-fold excess cold 5MTHF in transport media. Points, mean derived from three experiments; bars, SE. *, P < 0.05; **, P < 0.005, significant difference above the respective nonspecific background examined by two-tailed t test.

 
143B-FR{alpha} cells were able to uptake 5MTHF at low concentration. [3H]5MTHF uptake was measured in 143B-FR{alpha}, 143B-RFC, and 143B-Neo cells. At an extracellular concentration of 50 nmol/L, all sublines showed detectable uptake of [3H]5MTHF above the nonspecific background (Fig. 3B), and a <2-fold increase was measured in 143B-FR{alpha} cells compared with 143B-RFC. At an extracellular concentration of 10 nmol/L, a 5.7-fold increase was measured in 143B-FR{alpha} cells over 143B-RFC, whereas the [3H]5MTHF uptake was not detectable in 143B-Neo. When the extracellular concentration was further reduced to 2 nmol/L, only 143B-FR{alpha} cells displayed a distinguishable uptake above the background. Marked increase of [3H]folic acid was also detected in 143B-FR{alpha} cells compared with the vector-only–transfected cell lines (data not shown).

Overexpression of the FR{alpha} in 143B cells had a minor effect on MTX sensitivity. The effect of FR{alpha} overexpression on the sensitivity to antifolates in 143B cells was further assessed in comparison with those with varying levels of RFC expression. When tested in complete media (containing 2.3 µmol/L folic acid), no apparent difference in the sensitivity to MTX or TMTX was observed between 143B-FR{alpha} and 143B-Neo cells (data not shown). When tested in folate-free media supplemented with 20 nmol/L folic acid (Fig. 4A ), 143B-FR{alpha} cells (IC50 = 65.4 nmol/L) were 3.6-fold resistant to MTX compared with 143B-Neo cells (IC50 = 18.2 nmol/L). When tested in media containing 20 nmol/L 5MTHF, a minor resistance was observed in 143B-FR{alpha} cells (IC50 = 77.7 nmol/L) compared with 143B-Neo cells (IC50 = 56.8 nmol/L). When tested in media with 20 nmol/L leucovorin, no apparent difference in sensitivity to MTX was observed between these two sublines, although both lines displayed an 8- to 10-fold increase in their IC50 values (508.7 nmol/L for 143B-FR{alpha} and 443.3 nmol/L for 143B-Neo, respectively) compared with those measured in media containing 20 nmol/L folic acid or 5MTHF. For TMTX, 143B-FR{alpha} (IC50 = 0.85 nmol/L) was 2.4-fold more resistant than 143B-Neo (IC50 = 0.35 nmol/L) in media containing 20 nmol/L 5MTHF (Fig. 4B) but displayed similar IC50 values as 143B-Neo in media containing 20 nmol/L folic acid (0.65 and 0.50 nmol/L, respectively), and both became resistant in media with 20 nmol/L leucovorin (53.6 and 39.5 nmol/L, respectively) as shown in Fig. 4B. Interestingly, in 143B cells with manipulated RFC expression, 143B-RFC (IC50 = 68.3 nmol/L) was nearly equally sensitive to MTX as 143B-Zeo (IC50 = 61.3 nmol/L), and a 1.9-fold resistance was observed in 143B-RFC-AS cells (IC50 = 116.2 nmol/L) when tested in complete media (Fig. 4C). No difference in their sensitivity to TMTX was observed (data not shown).


Figure 4
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  		Fig. 4. Growth inhibition assay. A and B, 143B-FR{alpha} (squares) and 143B-Neo(triangles) cells were exposed to MTX (A) and TMTX (B) continuously for 72 h at indicated concentrations in folate-free media containing 20 nmol/L folic acid (blank), 5MTHF (gray), or leucovorin (LV, black). Cell number was determined by the Alamar Blue assay and was plotted as survival percentage over untreated controls. Points, mean derived from three independent experiments; bars, SE. C, 143B-RFC (filled cycle), 143B-Zeo (line), and 143B-RFC-AS (blank cycle) were exposed to MTX continuously for 72 h at the indicated concentrations in complete media containing 2.3 µmol/L folic acid. Cell number was determined by the Alamar Blue assay and was plotted as survival percentage over untreated controls. Points, mean derived from three independent experiments; bars, SE.

 
143B-FR{alpha} cells had decreased MTX accumulation but increased 5MTHF retention. Because the initial uptake of MTX was not affected in 143B-FR{alpha} compared with the 143B-Neo cells, [3H]MTX 72-h accumulation and intracellular 5MTHF retention were measured. In complete media and in folate-free media containing 20 nmol/L leucovorin, or 20 nmol/L 5MTHF, similar levels of [3H]MTX accumulation were observed in these two sublines (Fig. 5A ). When measured in folate-free media supplemented with 20 nmol/L folic acid, [3H]MTX accumulation was 2.1-fold lower in 143B-FR{alpha} than that in 143B-Neo cells. In contrast, for the retention of [3H]5MTHF, as folic acid concentration in growth media was reduced from 2.3 µmol/L to 20 nmol/L and to 0 nmol/L (20 nmol/L leucovorin), there was an apparent augmentation of [3H]5MTHF retention in 143B-FR{alpha} but not in 143B-Neo cells (Fig. 5B). For the RFC-transfected sublines, although the initial uptake rate of [3H]MTX was 1.6-fold greater in the 143B-RFC, the 72-h accumulation (0.47 ± 0.03 pmol/106 cells) was similar to that of 143B-Zeo cells (0.44 ± 0.02 pmol/106 cells) in complete media (Fig. 5A, inset). Only in 143B-RFC-AS was there a significant decrease of [3H]MTX accumulation (0.34 ± 0.004 pmol/106 cells) compared with 143B-Zeo cells or 143B-RFC cells.


Figure 5
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  		Fig. 5. [3H]MTX 72-h accumulation and [3H]5MTHF 72-h retention. Cells were pregrown in folate-free media containing GAT for 1 to 2 wks and were exposed to 20 nmol/L [3H]MTX (A) or [3H]MTHF (B) in folate-free media supplemented with 20 nmol/L folic acid, 5MTHF, or leucovorin, or complete media (2.3 µmol/L folic acid) as indicated. After 72 h, the amount of [3H]MTX or [3H]MTHF was measured. Inset, [3H]MTX 72-h accumulation in RFC-transfected 143B sublines measured in complete media. **, P < 0.005, significant difference between transfected cells by two-tailed t test.

 
Overexpression of FR{alpha}, but not RFC, gave 143B cells a growth advantage in physiologic concentrations of folate. The cell growth requirement for different folate cofactors was measured in folate-free media supplemented with a range of concentrations of folic acid, 5MTHF, or leucovorin as the sole folate source. Although only a 2-fold lower EC50 of leucovorin was measured in 143B-FR{alpha} compared with 143B-Neo cells (Fig. 6A ), the growth requirements for folic acid and 5MTHF were 107- and 780-fold, respectively, lower than those detected in 143B-Neo cells. However, for the RFC-transfected sublines, the growth requirements for these folate cofactors were observed to be similar and close to those measured in 143B-Neo cells. No significant difference of growth requirement for any of the cofactors was observed between 143B-RFC and 143B-RFC-AS cells (Fig. 6B). Cell proliferation assays were further done in media supplemented with physiologic concentrations of folate cofactors. In media containing 20 nmol/L leucovorin, these 143B sublines proliferated at a comparable rate (Fig. 7, top ). However, in media containing 20 nmol/L folic acid (Fig. 7, middle), or 5MTHF (Fig. 7, bottom), all sublines, except 143B-FR{alpha}, had a reduced proliferation rate over a period of 3 days. Importantly, 143B-RFC cells did not show a proliferation advantage in any of the conditions tested.


Figure 6
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  		Fig. 6. Cell growth requirements for folate cofactors. FR{alpha}-transfected 143B cells (A) and RFC-transfected 143B sublines (B) were pregrown in folate-free media containing GAT for 1 to 2 wks and were transferred to folate-free media supplemented with a range of concentrations of folic acid, 5MTHF, and leucovorin, respectively, as the sole folate source for cell growth. Cell number was determined after 72 h. EC50 is defined as the folate concentration producing 50% maximal growth. Columns, mean derived from three independent experiments; bars, SD. **, P < 0.005 by two tailed t test.

 

Figure 7
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  		Fig. 7. Proliferation assay. FR{alpha}- and RFC-transfected 143B sublines were pregrown in folate-free media containing GAT for 1 to 2 wks and were seeded in folate-free media supplemented with 20 nmol/L folic acid, 5MTHF, or leucovorin, respectively, as the sole folate source for cell growth. Cell number was determined at day 0 to day 3, respectively, and relative cell numbers were plotted.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
FR{alpha} overexpression was found in osteosarcoma xenografts but not in cultured osteosarcoma cell lines. This discrepancy between tumor samples and cell lines has been observed previously and was partly explained by the excess folic acid in the culture media, which may inhibit expression of FR{alpha} (8). An up-regulation of FR{alpha} expression was observed after cells were transferred to low-folate media (29). There is also evidence suggesting that FR{alpha} mRNA is more stable in folate-depleted media than in folate-replete conditions (30, 31). A high level of FR{alpha} expression was further detected in 29.9% of 107 osteosarcoma patient samples. Overexpression of FR in osteosarcoma was first described in a screening study on normal tissues and malignancies (8); however, no subsequent study was reported. FR{alpha} overexpression was frequently observed in ovarian cancer as well as in some other epithelial-derived malignancies (7–10) and was reported to be associated with malignant transformation, tumor progression, and poor prognosis (10). In the current study, no association was found between FR{alpha} mRNA level and specimen histologic type, location, response to preoperative chemotherapy, metastases, or survival. This study was initially designed to determine the status of the FR{alpha} in osteosarcoma samples; hence, a variety of specimens were included. The majority of the samples (n = 56) were specimens from definitive surgery after multiagent chemotherapy, 20 of them were biopsy specimens, and the remainder were metastatic/relapsed samples. For assessing a prognostic factor, a focus on biopsy specimens would be more appropriate, and this is a limitation of this assessment, but a relationship was not evident.

Lack of correlation between the mRNA levels of FR{alpha} and RFC in this cohort of osteosarcoma samples suggests that the transcriptional regulations of these two systems are independent. Therefore, FR{alpha} expression is unlikely secondary to the defective RFC-mediated transport frequently observed in osteosarcoma (21, 22). By manipulating the expression levels of FR{alpha} and RFC, in the same genetic background of 143B cells, we provided further evidence that these two systems play distinct roles in the uptake of physiologic folates and the antifolate MTX in particular (32–34). The current study suggests that FR{alpha} is critical in the uptake and retention of the physiologic 5MTHF, consistent with previous studies (11, 33, 34). Moreover, we showed that only FR{alpha}-expressing 143B cells were able to uptake 5MTHF at low concentration. Importantly, overexpression of RFC was not capable of replacing the role of FR{alpha} in this condition. Similarly, folate deficiency was identified in heterozygous mice with FR{alpha}-targeted gene ablation and in human diseases with FR{alpha}-directed autoantibodies, despite RFC being intact (3–5). Together, these studies reflect the profound differences between these two systems. RFC displayed low affinities (micromolar range) to its natural substrate 5MTHF, or to synthetic substrates leucovorin and MTX, indicating that the interaction between the carrier and its substrates is weak (1). The carrier may merely provide an aqueous path with its 12 trans-membrane domains for folate entry driven by the concentration gradient across the membrane, consistent with its high transport capacity. Concentrative transport by RFC was only shown for MTX, which binds to the target enzyme dihydrofolate reductase, irreversibly; thus, the intracellular-free drug concentration is inferred indirectly by the current methodology (35). The concentration-dependent nature of RFC is also supported by the fact that the fetus of heterozygous FR{alpha}-null mice, and receptor-associated folate deficiency in human diseases could be rescued or relieved by high-dose leucovorin administration (3–5). It is possible that the primary physiologic function of RFC is tissue re-distribution of folates. In contrast, FR{alpha} has a very high affinity to 5MTHF (1-10 nmol/L) or folic acid (1 nmol/L), indicating a strong interaction with the substrate. FR{alpha} internalizes substrate through energy-dependent receptor-mediated endocytosis, thus overcoming the concentration gradient (36–38). This active transport via the FR{alpha} is apparently important in cerebrospinal fluid, where 4-fold higher concentration of 5MTHF was measured compared with plasma, and in trans-placental folate delivery, where higher concentrations of 5MTHF in fetus than the matched maternal plasma were detected (39–41). Rapidly proliferating cells, such as tumor cells, often show overexpression of the FR{alpha}, but not RFC, to meet the increased requirement for folate cofactors. In vitro, up-regulation of the FR{alpha} is often induced by stepwise folate depletion in growth media. These data further suggest the physiologic role of the FR{alpha} in folate uptake.

MTX uptake is the determinant factor for drug efficacy with a pulse exposure; however, for longer durations, drug accumulation and intracellular folate pool become more important (19). Although FR{alpha} expression had minor effects on MTX uptake and drug sensitivity in this study, RFC levels correlated well with initial uptake rates, and lower RFC level was associated with defective transport, long-term accumulation, and drug resistance. This again suggests its importance for the effectiveness of MTX. Interestingly, although overexpression of the carrier increased the initial uptake of MTX, no effect on the long-term accumulation, or the efficacy of this drug, was seen in 143B cells. MTX is a good substrate for folypolyglutamyl synthetase, which adds glutamyl residues onto the drug. This modification results in retention of (anti)folates because the polyglutamyl derivatives are not substrate for the efflux systems. Conceivably, folypolyglutamyl synthetase may be saturated by the increased drug influx via RFC in 143B-RFC cells; therefore, polyglutamylation becomes the rate-limiting step instead of impaired drug transport as in the RFC low-expressing 143B-RFC-AS cells. The importance of polyglutamylation to the efficacy of MTX is also revealed in FR{alpha}-transfected 143B cells by experimentally replacing 20 nmol/L folic acid in growth media containing MTX. A 2.1-fold decrease of MTX 72-h accumulation in 143B-FR{alpha} cells is likely due to a competitive inhibition of folypolyglutamyl synthetase by increased uptake of folic acid, a better substrate than 5MTHF for folypolyglutamyl synthetase, whereas the reduction of folic acid is blocked by MTX. Indeed, this conferred a 3.6-fold resistance to MTX. This hypothesis is further supported by the fact that under the same conditions, almost equal sensitivity to TMTX was seen in 143B-FR{alpha} and 143B-Neo. TMTX, a lipophilic analogue of MTX, is not capable of being polyglutamylated. Overexpression of the FR{alpha} may increase the folate pool as indicated by the increasing 72-h 5MTHF retention in 143B-FR{alpha} cells when the concentration of competing folic acid was reduced. It has been suggested that the slow process of receptor-folate complex cycling was the underlying mechanism (42). It is noted that 5MTHF, although physiologic, is not stable. The absolute value, therefore, might not be accurate as measured in this study. However, the relative difference between 143B sublines reflected by this measurement (Fig. 5B) may still be of merit. In the current study, transfection of FR{alpha} into 143B cells had a greater effect on sensitivity to TMTX (2.4-fold resistance) than to MTX (1.4-fold). This can be explained by the fact that folate pools have greater effects on the efficacy of TMTX than that of MTX (19). This is also supported by the evidence that when folic acid or 5MTHF in media was experimentally replaced by the same concentrations of leucovorin, a substrate directly repleting the folate pool, nearly 100-fold resistance to TMTX was observed in both sublines. In contrast, only 8- to 10-fold resistance to MTX was observed by doing so. The effect of FR{alpha} overexpression on folate pool as well as on the efficacies of antifolates needs to be further investigated in future study.

It is noteworthy that FR{alpha} expression was not detected in >20% of samples in this study. Possible contamination by surrounding tissue can not be excluded in these specimens because normal osteoblasts had very low levels of FR{alpha} (Fig. 1A). The status of other isoforms, such as FRß, may also be relevant but were not investigated in this initial study (8). An alternative folate transport pathway was reported recently (28). It is important to note that the frequently expressed FR{alpha} in osteosarcoma may offer a potential means of obtaining targeted delivery of diagnostic and therapeutic agents (reviewed in ref. 43). Newer antifolates have been developed using FR{alpha} for cell entry (44). Folic acid–conjugated liposomes have been tested successfully for FR{alpha}-targeted delivery for a variety of chemotherapeutic drugs, including doxorubicin (45). This study provides rationale for possible studies of these approaches in osteosarcoma.


   Footnotes
 
Grant support: National Cancer Institute grant R01 CA-83132, Foster Foundation, and Cure Search Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: B. Hoang and J.H. Healey are affiliated with Weill Medical College of Cornell University.

Current address for J. Qin: Department of Health and Human Services, Public Health Service, NIH, Bethesda, Maryland.

Current address for B. Hoang: Department of Orthopedic Surgery, University of California, Irvine, California.

Received 6/ 1/06; revised 2/ 1/07; accepted 2/19/07.


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 Materials and Methods
 Results
 Discussion
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