Folate Receptor Expression Research Articles

The role of dietary folate in modulation of folate receptor expression, folylpolyglutamate synthetase activity and the efficacy and toxicity of lometrexol

We have studied the molecular effects of a LFD in a murine model in order to better define the biochemical changes associated with folate deficiency. In addition, we have demonstrated the effect of a LFD on the pharmacokinetic profile and therapeutic activity and toxicity of lometrexol. These studies showed increased density of FR in tumors implanted in LFD mice and a decrease in the affinity of these receptors for folic acid. The results suggest that tumors can compensate for low folate bioavailability by up-regulation of a second FR with slightly lower affinity for folic acid. The higher density of this FR would provide greater capacity for garnering serum folate. FPGS activity increased in several tumors and liver and kidney of LFD mice. The increase in this enzyme activity would result in enhanced polyglutamation of folates and classical antifolates and thus increased cellular retention. Consistent with these changes in liver FPGS, mice injected i.v. with a single dose of lometrexol accumulated significantly more drug in liver and tumors of LFD animals compared to SD mice. Also, higher liver concentrations of lometrexol persisted longer in LFD mice. Polyglutamate analysis showed that longer polyglutamate forms appeared earlier in liver of LFD mice. After 7 days, longer polyglutamyl forms were recovered from liver of LFD mice (octa- and hepta-glutamyl lometrexol) compared to those on SD. A comparison of the efficacy and toxicity of lometrexol in C3H mammary tumor-bearing mice showed that in mice on LFD, lometrexol treatment produced a delayed toxicity with an LD 50 of 0.1-0.3 mg/kg, a 3000-fold increase in lethality compared to SD mice. Supplementation of mice with folic acid restored anti-tumor activity and increased the therapeutic dose-range over which efficacy could be assessed. These studies support the use of folic acid supplementation for cancer patients treated with antifolate therapy in order to prevent the biochemical changes in FR and FPGS associated with folate deficiency, prevent delayed toxicity to GARFT inhibitors and enhance the therapeutic potential of this class of drugs.

Rapid Turnover and Impaired Cell-Surface Expression of the Human Folate Receptor in Mouse L(tk-) Fibroblasts, a Cell-Line Defective in Glycosylphosphatidylinositol Tail Synthesis

The human folate receptor (hFR) is a plasma membrane protein that is anchored to the membrane via a glycosylphosphatidylinositol (GPI) tail in some cell types. The KB hFR cDNA sequence predicts a hydrophobic, α-helical 31-residue carboxyl terminus that is thought to be the signal for cleavage and attachment of the GPI tail. Alternatively, this region may serve as a transmembrane domain if GPI attachment is not effcient. In this study, we investigated the latter possibility by expressing the hFR in L(tk-) cells, cells that are unable to synthesize GPI tails for attachment to membrane proteins. We also transfected the same hFR cDNA into Chinese hamster ovary (CHO) cells, cells that can anchor proteins by either a GPI tail or a transmembrane domain. Neither parental cell line expresses detectable levels of folate receptor as determined by folic acid binding assays, Western analysis, or Northern analysis. In L(tk-) cells, we found that the recombinant hFR is not expressed on the cell surface, but is rapidly degraded ( t 1/2 ≤ 4h). Most (>95%) of the recombinant hFR remains Endo H sensitive, suggesting retention in the endoplasmic reticulum. In contrast, transfected CHO cells express functional hFR protein at the cell surface, the half-life of the hFR is long ( t 1/2 ≥ 24 h), and the Endo H glycosylation pattern of the recombinant hFR is consistent with normal processing through the Golgi apparatus. Therefore, in the absence of a GPI tail, the hFR is not sorted to the cell surface and the incompletely processed hFR protein is unstable.

Similarity of Folate Receptor Expression in UMSCC 38 Cells to Squamous Cell Carcinoma Differentiation Markers

The folate receptor is a 38 to 39 kd glycoprotein attached to the cell membrane via a glycosylphosphatidylinositol anchor. It serves as the initiation point for receptor-coupled transport of folate in a process known as potocytosis. The receptor is overproduced by a number of malignant cell lines in vitro and in a large percentage of ovarian and uterine malignancies. Immunohistochemistry has shown the receptor to be expressed primarily in normal differentiated tissues such as choroid plexus, placenta, thyroid, and kidney. The receptor gene(s) has been mapped to the human chromosomal locus 11q13. In order to better understand regulation of the synthesis of the receptor, we studied the expression and accumulation of the folate receptor in UMSCC 38 cells, a human malignant squamous cell carcinoma line with a karyotype that is amplified at the folate receptor gene locus. Western blot analysis of octylglucoside-solubilized cell membranes was used to analyze expression of several proteins by UMSCC 38 cells grown in culture under varied conditions. Bands on autoradiographs, representing electrophoresed proteins detected by indirect antibody labeling and an enhanced chemiluminescence reaction, were quantitated by densitometry. The expression of the folate receptor was found to increase approximately fourfold as UMSCC 38 cells were grown to higher cell densities over increasing lengths of time, ranging from 3 to 9 days, in culture. The expression of K1 keratin protein, a known marker of differentiation in squamous cell carcinomas, was elevated to a similar degree (3.2-fold) between day 5 and day 9 in cultures of these cells. Expression of folate-receptor protein in UMSCC 38 cells was also found to be reduced approximately 10-fold when cells were exposed to 1 microM retinoic acid for 48 hours and increased approximately eightfold after a 5-day exposure to 3 microM hydrocortisone. We present evidence that synthesis of the folate receptor in UMSCC 38 cells is affected by the same pathway that changes the expression of some markers of differentiation in squamous cell carcinomas. The fact that folate receptor expression in these malignant cells can be manipulated using retinoids and steroids suggests that these hormones could modulate the efficiency of folate and antifolate uptake via the folate receptor and may enhance the usefulness of the receptor as a target for immunotherapy.

Differential regulation of folate receptor isoforms in normal and malignant tissues in vivo and in established cell lines. Physiologic and clinical implications.

Despite significant differences in ligand binding between the two known isoforms of the human membrane folate receptor (FR), designated herein as FR-beta (placenta) and FR-alpha (placenta, KB cells), little is known about their tissue specificities, and there is no report on the relative expression of FR-beta in any tissue other than in placenta. The mRNA for each FR isoform in a wide variety of normal fetal and adult tissue explants, primary normal cell cultures, malignant tumor explants, and established tumor cell lines was estimated by a polymerase chain reaction assay. Total receptor levels were estimated by a [3H] folic acid binding assay. Both the FR isoforms were expressed in fetal as well as adult tissues. Normal tissues generally expressed low to moderate amounts of FR-beta. FR-alpha alone was expressed in normal epithelial cells and was frequently strikingly elevated in a variety of carcinomas, with the exception of squamous cell carcinomas of the head and neck. In contrast, a variety of malignant tissues of nonepithelial origin generally expressed elevated levels of FR-beta alone. Established tumor cell lines expressed FR-alpha virtually alone and did not reflect FR expression patterns in vivo. KB cells and JEG-3 cells grown at low folate concentrations further up-regulated FR-alpha but not FR-beta. Although FR-beta is the more common isoform, FR-alpha and FR-beta are differentially regulated in normal tissues, carcinomas, nonepithelial malignancies, and immortalized cells or in response to changes in extracellular folate concentrations. The tissue specificity of FR isoforms and their elevation in malignant tissues may be a significant factor in FR-mediated folate uptake, in tissue responsiveness to promising novel antifolates, and in FR-related immunodiagnosis/immunotherapy.

Identification of a novel folate receptor, a truncated receptor, and receptor type beta in hematopoietic cells: cDNA cloning, expression, immunoreactivity, and tissue specificity.

The expression of a membrane-associated folate receptor (FR) was elevated in spleen samples from patients with chronic (CML) and acute (AML) myelogenous leukemias compared with normal spleen. Contrary to earlier reports, antibodies to a purified FR from placenta cross-reacted quantitatively with this protein in solution radioimmunoassays. Similar to FR-alpha (KB cells) and FR-beta (placenta), the protein was released from the membrane by phosphatidylinositol-specific phospholipase C, indicating a glycosylphosphatidylinositol (GPI) membrane anchor. Screening of a cDNA library from CML spleen with a heterologous murine FR cDNA and also amplification of FR cDNAs from spleen and bone marrow in CML, AML, chronic lymphocytic leukemia (CLL), and acute lymphocytic leukemia (ALL) by polymerase chain reaction (PCR) using degenerate oligonucleotides yielded cDNA clones representing FR-beta, a novel FR (type gamma), and an aberrant transcript of FR-gamma with a 2 base pair deletion resulting in a truncated 104-residue polypeptide; FR-alpha was not detected in these tissues. The cDNA for FR-gamma predicts a 243-residue polypeptide with an amino acid sequence homology of 71% and 79% with FR-alpha and FR-beta, respectively, a 23-residue amino-terminal signal peptide, and 3 potential sites for N-linked glycosylation. Transfection of COS-1 cells with the cDNA for FR-gamma resulted in low expression of a [3H]folic acid binding protein on the cell surface that was GPI-anchored. PCR analysis of total RNA from a number of normal and malignant tissues and cell lines indicated a limited tissue specificity of FR-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased expression of the human folate receptor mediates transport-defective methotrexate resistance in KB cells.

The human membrane-associated folate binding protein, or folate receptor (hFR), is a necessary component of folate and methotrexate (MTX) transport in some cell lines. To investigate the role of hFR in acquired MTX resistance in human cells, we characterized nine MTX-resistant clones selected from human nasopharyngeal epidermoid carcinoma (KB) cells (cells which transport folates/anti-folates via the hFR) cultured in media containing low folate concentrations. Compared with wild type KB cells, the level of resistance of the clones ranges from 2- to 80-fold higher and the resistant phenotypes of the clones are characterized as follows. 1) DHFR levels are increased (3-13-fold) in four of nine clones; 2) MTX polyglutamation is not detectably different; 3) the extents of MTX efflux are similar; 4) initial rates of MTX efflux are similar except for two clones which exhibit slightly faster efflux rates (approximately 2-fold); and 5) the Vmax for specific MTX and 5-methyltetrahydrofolate transport are decreased (2-18-fold) in all mutants. The Kt values for MTX transport of each mutant are similar to the Kt of KB cells. These results indicate that all nine MTX-resistant clones exhibit defective MTX transport and that four clones also have increased DHFR levels. Based on folic acid binding assays, the hFR is reduced by 1.8-24-fold in these clones relative to KB cell hFR expression. Western, Northern, and Southern analyses are consistent with decreased hFR expression in these clones rather than mutations, resulting in alterations in the size or ligand binding affinities of the hFR. The decrement in hFR expression correlates closely with the degree of reduction in MTX transport Vmax for each clone. Since folate and MTX influx proceed via hFR in KB cells and in these mutants, the correlation (R2 = 0.90) between hFR expression and the MTX transport Vmax of each clone indicates that hFR expression is an important determinant of acquired MTX resistance in this human tumor cell line. These studies demonstrate that defective transport (manifested by decreased Vmax) resulting from decreased expression of the hFR is frequent in KB cells cultured under these conditions and suggest that modulation of hFR may be relevant to MTX cytotoxicity or resistance in tissues or cells expressing functionally significant levels of hFR.

Effects of folate receptor expression following stable transfection into wild type and methotrexate transport-deficient ZR-75-1 human breast cancer cells.

Two biochemically distinct systems, the high affinity folate receptor and the lower affinity reduced-folate carrier, have each been implicated in mediating the transport of folates and antifolates into cells. Previous studies from our laboratory have shown that methotrexate accumulation into wild type (WT) ZR-75-1 human breast cancer cells involves a system with characteristics of the reduced-folate carrier, that this system is deficient in methotrexate resistant (MTXR) ZR-75-1 cells in which methotrexate transport is undetectable and that neither breast cancer cell line expresses folate receptors. In this report we examined the possible interaction of the reduced-folate carrier with folate receptors by stably transfecting both WT ZR-75-1 and MTXR ZR-75-1 cells with an expression vector containing a folate receptor cDNA. Clones of stably transfected MTXR ZR-75-1 and WT ZR-75-1 cells expressing comparable levels of folate receptors were studied and compared to the nontransfected cell lines. Although nontransfected WT and MTXR ZR-75-1 cell lines require concentrations > or = 100 nM folic acid for growth, the expression of folate receptors in transfected WT and MTXR ZR-75-1 cells permitted the growth of both cell lines in low concentrations (1 nM) of folic acid. While the defect in the reduced-folate carrier system in MTXR ZR-75-1 cells inhibits their growth in medium containing low concentrations of folinic acid (< or = 1 microM), MTXR ZR-75-1 cells expressing folate receptors display uninhibited growth in 1 nM folinic acid. The accumulation of folic acid, folinic acid, and methotrexate is enhanced in folate receptor-transfected WT ZR-75-1 cells and MTXR ZR-75-1 cells. Furthermore, the accumulation of folates and antifolate was similar in both transfected WT and MTXR ZR-75-1 cell lines that expressed folate receptors. This suggests that alterations in the reduced-folate carrier do not affect folate receptor function. We also examined the effect of folate receptor expression on the sensitivity of WT and MTXR ZR-75-1 cells to methotrexate and to the lipophillic antifolate trimetrexate. Increased folate receptor expression decreased the sensitivity of WT ZR-75-1 cells toward the antifolate trimetrexate, presumably through increased uptake of reduced folates. Although the expression of the folate receptor enhanced the growth of both cell lines in low folate concentrations, it did not affect the sensitivity of either WT or MTXR ZR-75-1 cells to methotrexate.