J Thorac Cardiovasc Surg 2002;123:862-868
© 2002 The American Association for Thoracic Surgery
General Thoracic Surgery (GTS) |
Despite some expression of folate receptor 
in human mesothelioma cells, internalization of methotrexate is predominantly carrier mediated
Nushmia Z. Khokhar, MDa,
Alicia F. Y. Lam, BSa,
Valerie W. Rusch, MDb,
F. M. Sirotnak, PhDa
From the Program of Molecular Pharmacology and Experimental Therapeuticsa and Department of Surgery,b Memorial Sloan-Kettering Cancer Center, New York, NY.
Supported in part by National Cancer Institute grants CA08748 and CA55617.
Received for publication July 5, 2001. Revisions requested Aug 24, 2001; revisions received Sept 7, 2001. Accepted for publication Sept 14, 2001.
Address for reprints: F. M. Sirotnak, PhD, Laboratory for Molecular Therapeutics, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021 (E-mail: sirotnaf{at}mskcc.org).
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Abstract
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Objective: As a response to a published report documenting some expression of folate receptor 
in human mesothelioma, studies were carried out examining the role of this receptor versus that of the reduced folate carrier in the internalization of folate analogs in this neoplasm.
Methods: Influx measurements of tritiated methotrexate were carried out in 4 mesothelioma cell lines, and 2 additional cell lines were used as comparators. Relative gene-expression analysis for the carrier and receptor gene was done by using real-time reverse transcriptase-polymerase chain reaction in the above-mentioned cell lines and mesothelioma tumor tissues obtained from patients.
Results: Internalization of tritiated methotrexate in mesothelioma cell lines grown at physiologic folate levels was carrier mediated rather than receptor mediated. Influx of this model permeant by these cells exhibited characteristics of carrier-mediated membrane transport and was only minimally reduced by the addition of 5 µmol/L folic acid, a concentration of this natural folate that would have completely inhibited influx by the folate receptor. Gene-expression analysis revealed prominent expression of the folate receptor in some but not all mesothelioma cell lines, and in only one case was expression of this receptor gene greater than expression of the reduced folate carrier gene. Similar analysis of human mesothelioma tumor tissue showed that, with few exceptions, receptor gene expression was substantially less than that for the carrier gene.
Conclusion: In view of the ongoing reduced folate carrier-mediated internalization that occurs in mesothelioma cells, these results would seem to argue against a role for the folate receptor in the internalization and cellular pharmacokinetics of methotrexate and other classic folate analogs in this neoplasm. Identifying the mediated route for internalization of these agents in tumor cells is a prerequisite for improving their structural design.
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Introduction
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Folate compounds, including folate analogs, are internalized by mammalian cells through either carrier-mediated
1-3 or receptor-mediated 4,5 processes. The former uses binding to a mobile carrier at the plasma membrane surface and mediates bidirectional flux. The latter uses much higher affinity binding at the membrane surface to a receptor protein that more slowly mediates unidirectional flux after internalization of the receptor-folate compound complex in the manner of many other receptor-ligand processes. 6,7 The extent to which receptor-mediated processes contribute to net internalization of folates and their analogs in tumor cells remains controversial 4 but will depend on the level of expression of the corresponding gene vis-à-vis the reduced folate carrier gene (RFC1) in any specific cell type. Translocation through the plasma membrane of folate analogs by the RFC1 carrier-mediated process is what is commonly found in tumor cells. 1-3 Because this process is much more efficient 5,8 than receptor-mediated translocation, the relative level of expression required for the latter to contribute significantly to net intracellular accumulation in tumor cells is proportionally greater, particularly in the case of folate analogs. Net intracellular accumulation of folate compounds in tumor cells also reflects the operation of one or more members of the canalicular multiorganic anion transporter (cMOAT/MRP) family of adenosine triphosphatases, 9,10 which extrude these compounds through the plasma membrane. However, this aspect of the cellular pharmacokinetics of folate analogs is outside the focus of the current presentation.
Folate analogs have shown 11-13 interesting clinical activity against human mesothelioma, a neoplasm found, for the most part, to be relatively unresponsive to most cytotoxic agents. 11,12 In view of these clinical findings and other results 14 showing that the folate receptor gene (FRA) is expressed in human mesothelioma but not in normal pleural mesothelium and the suggestion 14 that this expression may be relevant to the responsiveness of this human neoplasm to folate analogs, it was of interest to determine to what extent receptor-mediated versus carrier-mediated processes might actually account for the internalization of these folate analogs in human mesothelioma. We addressed this question at both the biochemical and the molecular levels. Our results showed that human mesothelioma cell lines predominantly internalized a model permeant (tritiated methotrexate) by means of a carrier-mediated mechanism, ostensibly encoded by the RFC1 gene, with little, if any, receptor-mediated internalization. They also showed that expression of the FRA gene in these cell lines and in patient-derived mesothelioma was almost always substantially less than expression of the RFC1 gene. These results are presented below.
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Materials and methods
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Cell lines and transport measurements
Mesothelioma cell lines were cultured in RPMI medium in accordance with procedures published earlier. 15,16 The VAMT-1 and JMN cell lines were generously provided by Dr B. Gerwin in the laboratory of Dr Curtis Harris at the National Cancer Institute (Bethesda, Md). The MESO 9 and MESO 10 cell lines were generously provided by Dr S. Jhanwar and the SKOV-3 (ovarian cancer) by Dr D. Spriggs (both at Memorial Sloan-Kettering Cancer Center, New York, NY). CCRF-CEM (acute lymphoblastic leukemia) and TSU-PR1 (prostate cancer) cells were obtained from the American Type Culture Collection and cultured in RPMI medium, as described earlier. 15,16 Measurement of 3H methotrexate influx was carried out by procedures described in detail previously. 1,2,15,16 Before the measurements of influx were carried out, the cells were grown to late logarithmic phase either in 2.2 µmol/L folic acid or for 2 transfer generations in 20 nmol/L dIL5-formyl tetrahydrofolate. The cells were resuspended in 0.2 mL of HMO buffer with 7 mmol/L D-glucose in 1.5-mL Eppendorf tubes, and transport experiments were carried out by incubating cells with 2 µmol/L radioactive folate analog 3H methotrexate at 37°C for varying time intervals. Parallel experiments were conducted in the presence and absence of 5 or 100 µmol/L folic acid.
Quantitative reverse transcriptase-polymerase chain reaction analysis
Total RNA was prepared with Trizol reagent (Gibco BRL; Life Technologies, Rockville, Md) from all the tumor cell lines. First-strand complementary DNA was prepared by using the Superscript System (Gibco BRL). Mesothelioma tumor samples were harvested from patients at the time of surgical resection and snap-frozen in liquid nitrogen. Messenger RNA was purified from these tissue samples by using the Quick Prep mRNA Purification Kit (Amersham Pharmacia Biotech Inc, Piscataway, NJ) and then reverse transcribed by using M-MLV RT and random hexamers (Gibco BRL). RFC1 and FRA gene expression was quantified with the aid of an ABI Prism 7700 Sequence Detection System (TaqMan; PE Biosystems, Foster City, Calif). With the nuclease activity of Taq Polymerase, this method is based on the displacement and cleavage of a labeled fluorogenic probe specific to the target sequence flanked by polymerase chain reaction (PCR) primers. This allows the quantitation of target genes while PCR is in the log phase of amplification. A detailed description of this methodology has been previously provided. 17 The primer and probe sets were designed with Primer Express Software (Applied Biosystems, Foster City, Calif), and the sequences are as follows: RFC1, forward 5'-CCGCGGCTCCTACCAGTT-3', reverse 5'-AAGACCAGGGCACAGAGCTCTT-3', probe 6FAM-ATTCTGAACACCGTCGCTTGGAAGACACT-TAMRA; FRA, forward 5'-GAGCAATGGTGGGAAGATTGTC-3', reverse 5'-CTCCCACTGCGCACTTGTTA-3', probe 6FAM-AACCCTGAAGTCCAGTTCCAGCCCTTGT-TAMRA; and ß-actin, forward 5'-CTGGCACCCAGCACAATG-3', reverse 5'-GCCGATCCACACGGAGTACT-3', probe 6FAM-TCAAGATCATTGCTCCTCCTGAGCGC-TAMRA.
Relative quantitation was done by using the comparative threshold cycle (CT) method. The CT indicates the fractional cycle number at which the amplified target reaches a fixed threshold. The amount of target gene (RFC1 and FRA) normalized to an endogenous reference (ß-actin) is given as follows:
2-
CT,18-20
where CT is CT for the target gene minus CT for the reference gene. Serial dilutions of a control cDNA were run in triplicate, amplifying ß-actin to generate a standard curve (Figure 1).
The slope of this curve is used for calculating the run efficiency. The relation is given as follows:
E = 10-1/s - 1,
where E is the efficiency and S is the slope. In our hands the efficiency of all runs was greater than 0.975, with a correlation coefficient of 0.998 or greater.
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Fig. 1. Standard curve of CT value against log of starting quantity of ß-actin. Dots represent standard samples plotted in triplicate. The slope is -3.37, and the correlation coefficient is 0.998.
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Tissues and clinical database
Surgical specimens were obtained from consecutive patients undergoing thoracotomy and surgical resection. They were divided into small samples (3 x 3 x 3 mm), snap-frozen, and stored at -140°C. Histologic sections were reviewed by a pathologist to identify those tumor samples that had predominantly tumor cells among nucleated cells on the slide. Relevant clinical data were collected for all the patients and linked to the specimens, and the link-up to patient identifiers was destroyed by the Tumor Bank before distribution to this project to protect patient confidentiality. Tissue collection and these experiments were performed in accordance with the guidelines of Memorial Sloan-Kettering Cancer Center.
Materials
Samples of 3H methotrexate were obtained from Moravek Biochemicals, Inc (Brea, Calif). This and all other folate compounds were purified by means of high-performance liquid chromatography to greater than 98% purity. 21 All other chemicals were reagent or molecular biology grade.
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Results
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The results of experiments measuring the mediated internalization of 3H methotrexate by 4 human mesothelioma cell lines are given in Figure 2 and Table 1.
The results obtained with VAMT-1 cells are compared in Figure 1
with those obtained with CCRF-CEM and SKOV-3 cells. In each case the time course for internalization obtained with 2 µmol/L 3H methotrexate was consistent with that expected for an RFC1-mediated process. 1,2 Internalization was initially rapid and constant, with time followed by an exponential approach to steady state (influx = efflux). Moreover, the addition of 5 µmol/L folic acid to the cell suspension, an extremely effective inhibitor of FRA-mediated, but not RFC1-mediated, internalization, 1-5 had only a minimal effect on these time courses. However, influx could be inhibited by a much higher concentration (100 µmol/L) of folic acid, a result consistent with a role for RFC1 in the internalization of 3H methotrexate in these cells. These results were similar to those obtained (Figure 2 and Table 1) with the other mesothelioma cell lines and the CCRF-CEM and SKOV-3 cell lines, which were used as comparators. Again, the data in Table 1 show only a slight effect of 5 µmol/L folic acid on influx of 3H methotrexate. The same results after folic acid addition were obtained when the cells were grown in 2.2 µmol/L folic acid or 20 nmol/L dIL5-formyl tetrahydrofolate, a concentration approximating the physiologic level of folate in human blood. Because FRA has an extremely high affinity (Km = 1 ± 0.2 mmol/L) for this natural folate, whereas RFC1 has extremely low affinity (Km = 100 ± 20 µmol/L), these results clearly implicate RFC1 as the predominant, if not exclusive, route for mediated internalization of 3H methotrexate in VAMT-1 cells, as is the case for CCRF-CEM 1 and SKOV-3 cells.
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Fig. 2. Time course for internalization of 3H methotrexate in various human tumor cells. Cells were incubated with 2 µmol/L 3H methotrexate (MTX) with or without 5 µmol/L folic acid in 50 mmol/L HEPES and 180 mmol/L sucrose brought to pH 7.4 with MgO. Aliquots of cell suspensions were removed at the indicated times for processing. Additional details are provided in the text. Average of 3 experiments with SEM ± 20% or less.
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Table 1. Internalization of 3H methotrexate by human mesothelioma, CCRF-CEM, SKOV-3, and TSU-PR1 cells in the presence and absence of 5 µmol/L folic acid
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As an adjunct to the studies described above, we also examined, for the purpose of comparison, these human mesothelioma cell lines, as well as CCRF-CEM and SKOV-3 cells, for the expression of both FRA and RFC1 genes by using real-time quantitative reverse transcriptase-PCR (RT-PCR), with ß-actin as the reference comparator gene (Figure 3).
The data in Figure 4 show that expression of these genes was extremely variable over a wide range.
In only one case (MESO 10) was expression of FRA equal to or higher than that of RFC1. Moreover, expression of RFC1 appeared to show (Table 1) a general correlation with influx of 3H methotrexate in these cells. Influx was lowest in the case of MESO 9 and MESO 10 and highest in the case of VAMT-1, the cell lines with the least and greatest expression of the RFC1 gene, respectively. The single discrepancy between the rate of internalization and RFC1 gene expression occurred with SKOV-3 cells. However, in this case, and to some extent with the other tumor cell lines, functional expression of the RFC1 gene could also reflect differences in the rate of translation of RFC1 mRNA. The results with SKOV-3 cells, showing that FRA gene expression was far greater than for RFC1, was of interest given the finding that 3H methotrexate internalization in these cells was relatively low and apparently RFC1 mediated. The level of expression of FRA in SKOV-3 cells was even higher than that found in all the other tumor cells included here in a parallel analysis. It is known that high expression of FRA is a common occurrence in ovarian cancer cells. 9 The results obtained with SKOV-3 cells, for which there is companion transport data (Figure 2), are strikingly consistent with the accepted notion that receptor-mediated internalization of 3H methotrexate is much less efficient than carrier-mediated internalization.
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Fig. 3. Real-time RT-PCR determinations of FRA and RFC1 gene expression in the VAMT-1 human mesothelioma cell line. Rn represents the normalized fluorescence signal minus the baseline signal established in the first few cycles of PCR. CT represents the fractional cycle number at which an increase in the reporter fluorescence above a set threshold (horizontal black line) is first detected. ß-Actin gene expression was used as a control to normalize these PCR reactions. These data are representative of replicate runs. Additional details are given in the text.
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Fig. 4. Relative levels of RFC1 and FRA mRNA expression in various cell lines. The y axis scale is in relative units (specific target mRNA relative to ß-actin mRNA expression).
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The same RT-PCR analysis was carried out (Figure 5) with a large number of mesothelioma specimens obtained from patients during surgical resection of tumors.
These data also revealed highly variable expression of both RFC1 and FRA genes. However, with few exceptions, levels of expression for FRA were very substantially lower than those for RFC1. The data also show that there were no appreciable differences observed (Figure 5) in the level of expression of either gene among tumors with epitheloid or mixed (epitheloid-sarcomatoid) histologic features. Only one of the available samples was pure sarcomatoid in origin and was included because RFC1 gene expression in this sample was exceptionally high compared with expression of FRA.
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Fig. 5. Relative levels of RFC1 and FRA expression in mesothelioma tumor samples on the basis of their histologic subtypes. The y axis scale is in relative units (specific target mRNA relative to ß-actin mRNA expression).
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Discussion
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FRA- and carrier-mediated transport of folate compounds in mammalian cells are readily distinguishable by the avidity of the receptor- or carrier-permeant interaction. 1-5 Binding of these compounds by FRA occurs with an affinity several orders of magnitude greater than binding by the RFC1 folate carrier. Moreover, in the case of folic acid itself, binding to RFC1 is approximately 2 orders of magnitude less than for substituted reduced folates or folate analogs, 3 which have influx Km or Ki values in the low micromolar range. For all other transporters described, 1-4 the affinity for all these folate compounds is similar and in the low micromolar range. Thus the relative inability of folic acid to inhibit internalization of competing, structurally similar permeants in tumor cells is a defining characteristic of RFC1-mediated transport. We have taken advantage of these differences to delineate FRA- versus RFC1- mediated internalization of 3H methotrexate in these mesothelioma cell lines.
The physiologic or pharmacologic significance of FRA gene expression in mesothelioma is not understood nor is it understood in ovarian carcinoma, the only example of high-level expression in patient-derived material. 22 Despite this high level of expression of FRA in ovarian cancer cells, earlier studies from our laboratory 16 and, more recently, elsewhere, 23 as well as our studies described here, have documented internalization of folate compounds exclusively as a carrier-mediated process in these cells in culture, as well as for patient-derived ovarian cancer cells. Our findings showed that internalization of 3H methotrexate in the 4 mesothelioma cell lines examined was also predominantly RFC1 mediated. Because of the relative inefficiency of receptor-mediated versus RFC1-mediated internalization, 1-6 the low to modest level of expression of this receptor observed here in mesothelioma cells has no effect on total intracellular accumulation of this folate analog, as shown by the actual measurements of 3H methotrexate influx. The situation with regard to gene expression was similar in those mesothelioma cells derived directly from patients. Thus, the lack of a role for FRA in the mediated internalization of folate analogs in human mesothelioma cells relates to the fact that both gene expression and transport efficiency of FRA is considerably less than that of RFC1 in this neoplasm.
The documentation of overexpression of the FRA gene in mesothelioma compared with in normal adjacent tissue is not sufficient to conclude that FRA has a role in making this tumor susceptible to antifolates. The previous study 14 making this observation failed to look at the corresponding RFC1 level of expression. Our studies, on the other hand, compared the level of expression of both genes and also carried out transport experiments in mesothelioma cell lines, clearly showing primarily carrier-mediated internalization of 3H methotrexate. This information is of importance if RFC1 gene expression is to be used as a molecular marker for clinical response to folate analogs in this human neoplasm and as a focus for new efforts at designing more efficacious analogs. Prospective studies focusing on the former question will need to be carried out in the context of clinical trials of newer folate analogs to establish the relevance of RFC1 gene expression. Such a trial examining the efficacy of a new 10-deazaaminopterin analog (10-propargyl-10-deazaaminopterin) is currently in progress. 24
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Abstract
Introduction
