Transduction of folate receptor cDNA into cervical carcinoma cells using recombinant adeno-associated virions delays cell proliferation in vitro and in vivo.

Although folate receptors (FRs) mediate folate uptake into cells, the independent role of FRs in cell proliferation remains unclear. We tested the hypothesis that transduction of FR cDNA in sense or antisense orientation using recombinant adeno-associated virus modulated FR expression and altered proliferation of cervical carcinoma cells (which constitutively overexpress FR genes). We determined that the integration of recombinant adeno-associated virions was not site specific. When compared with untransduced cells, sense and antisense FR cDNA-transduced cells exhibited an increase and decrease in FR mRNA and FR expression on the cell surface, respectively. However, when compared with antisense FR cDNA-transduced and untransduced cells, sense FR cDNA-transduced cells exhibited statistically significant (a) increased in total FRs, (b) smaller colonies, (c) lowered cell proliferation in vitro, and (d) less tumor volume with dramatic prolongation of tumor doubling times (225.6 h vs. 96 h) after transplantation into nude mice. Finally, (f) using single cell-derived transduced clones, an inverse relationship between cell proliferation and FR expression was established (r = 0.90, P < 0.001). Thus, transduction of sense/antisense FR cDNA into cervical carcinoma cells modulated expression of FRs and had an impact on cell proliferation in vitro and in vivo.

Maternal-to-fetal transfer of 5-methyltetrahydrofolate by the perfused human placental cotyledon: evidence for a concentrative role by placental folate receptors in fetal folate delivery.

Folates play a vital role in cellular processes that are essential for fetal growth and viability. Thus the human placenta, which contains high-affinity membrane-associated placental folate receptors (PFRs), maintains a concentrative maternal-to-fetal flux of the vitamin under conditions of minimal dependence on variations of maternal dietary intake. To define transplacental folate transport and the role of PFRs in this mechanism, we utilized the isolated perfused human placental cotyledon. In closed system perfusions with 10 nmol/L 5-methyltetrahydrofolate, placental binding was rapid and extensive (47%), with a gradual maternal-to-fetal transfer of 5-methyltetrahydrofolate. Although hydrophilic PFRs were released into the fetal perfusate, PFR-bound folates constituted only a fraction of net transplacental folate transport. Transfer was bidirectional, not saturable, not inhibited by anion channel blockers, and dependent on perfusate levels. Placental binding far exceeded transfer, and pulsing the maternal circuit with tritiated 5-methyltetrahydrofolate, followed by washout of unbound radiolabel and rechallenge with unlabeled 5-methyltetrahydrofolate or folate, led to release of bound tritiated 5-methyltetrahydrofolate, illustrating reversible binding. Perfusion with the N-hydroxysuccinimide ester of folic acid eliminated essentially all 5-methyltetrahydrofolate binding to PFRs, while increasing net maternal-to-fetal transfer of the vitamin. Finally, because it has been suggested that impaired placental transport of folate may be linked to the fetotoxic effects of ethanol, the effect of this compound on the above processes was examined. An acute 6-hour exposure to ethanol (2.5 to 3.1 mg/ml) had no effect (p > 0.05) on net maternal-to-fetal transfer of 5-methyltetrahydrofolate. These studies suggest that net maternal-to-fetal transfer is a process consisting of two steps. First is the concentrative component in which circulating 5-methyltetrahydrofolate is bound to (captured by) PFRs on the maternally facing chorionic surface. Although kinetics favor binding, there is a dynamic state wherein a gradual release of 5-methyltetrahydrofolate from this pool can add to incoming circulating folates to generate an intervillous blood level approximately 3 times that in the maternal blood. In the second step, folates are passively transferred to the fetal circulation along a downhill concentration gradient. This unique mechanism for transplacental folate transport may be applicable to other small relative molecular mass ligand nutrients that bind to high-affinity placental receptors.

Isolation and characterization of a folate receptor-directed metalloprotease from human placenta.

Glycosyl-phosphatidylinositol-anchored hydrophobic placental folate receptors (PFRs), which have an important functional role in maternal-to-fetal transplacental folate transport, can be converted to soluble hydrophilic forms by a placental metalloprotease. Using a Triton X-114 temperature-induced phase separation assay to monitor enzyme-mediated conversion of radiolabeled hydrophobic PFR into hydrophilic PFR, a metalloenzyme was isolated to apparent homogeneity from Triton X-114-solubilized human placenta using concanavalin A-Sepharose and reverse-phase high performance liquid chromatography (HPLC) as major purification steps. The purified hydrophobic enzyme eluted as a single protein peak on reverse-phase HPLC and SDS-polyacrylamide gel electrophoresis revealed a single 63,000 M(r) species, which was reduced to 58,000 M(r) following deglycosylation, findings comparable with amino acid analysis (M(r) approximately 59,000). The metalloenzyme was activated by Mg2+, Zn2+, Mn2+, and Ca2+, optimally at physiologic pH; it also exhibited EDTA-sensitive endoproteolytic cleavage of [3H]leucine-labeled full-length nascent PFR polypeptide generated in vitro in the absence of microsomes. Rabbit polyclonal anti-metalloprotease antiserum specifically immunoprecipitated 125I-metalloprotease and recognized cross-reacting moieties on plasma membranes of normal human hematopoietic progenitor cells and human cervical carcinoma cells, both of which also express FR.

Isolation and characterization of a folate receptor mRNA-binding trans-factor from human placenta. Evidence favoring identity with heterogeneous nuclear ribonucleoprotein E1.

The interaction of an 18-base cis-element in the 5'-untranslated region of human folate receptor (FR)-alpha mRNA with a cytosolic trans-factor protein is critical for the translation of FR (Sun, X.-L., and Antony, A. C. (1996) J. Biol. Chem. 271, 25539-25547). This trans-factor was isolated to apparent homogeneity as a 43- and 38-kDa doublet from human placenta using poly(U)-Sepharose, followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-elution as major purification steps. Amino acid microsequencing of two cyanogen bromide-generated peptide fragments of the 43-kDa trans-factor revealed complete identity with 43-kDa heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1). Purified specific rabbit anti-hnRNP E1 peptide antibodies (generated against a synthetic oligopeptide that was not represented in microsequenced peptides of the trans-factor) also recognized the purified trans-factor on Western blots. Conversely, the 18-base FR RNA cis-element also bound hnRNP E1 protein on Northwestern blots. Moreover, a 19-base RNA cis-element in the 3'-untranslated region of 15-lipoxygenase mRNA that is known to bind hnRNP E1 also interacted with placental 43-kDa trans-factor. In addition, several murine tissues containing a hnRNP E1-related protein (also known as alphaCP-1) readily interacted with the 18-base FR RNA cis-element. Finally, anti-hnRNP E1 antibodies specifically inhibited translation of FR in vitro in a dose-dependent manner, and the antibody effect could be reversed in a dose-dependent manner by either purified trans-factor or hnRNP E1. Collectively, the data favor identity of the FR mRNA-binding trans-factor and hnRNP E1, confirm its critical role in the translation of FR, and highlight yet another role of multifunctional hnRNP E1 in eukaryotic mRNA regulation.