Modulation of the cytotoxicity of 3'-azido-3'-deoxythymidine and methotrexate after transduction of folate receptor cDNA into human cervical carcinoma: identification of a correlation between folate receptor expression and thymidine kinase activity.
(1/385)
Cervical carcinoma is an AIDS-defining illness. The expression of folate receptors (FRs) in cervical carcinoma (HeLa-IU1) cells was modulated by stable transduction of FR cDNA encapsidated in recombinant adeno-associated virus-2 in the sense and antisense orientation (sense and antisense cells, respectively). Although sense cells proliferated slower than antisense or untransduced cells in vivo and in vitro in 2% (but not 10%) FCS, [methyl-3H]thymidine incorporation into DNA was significantly increased in sense cells in 10% serum; therefore, the basis for this discrepancy was investigated. The activity of thymidine kinase (TK) was subsequently directly correlated with the extent of FR expression in single cell-derived clones of transduced cells. This elevated TK activity was not a result of recruitment of the salvage pathway based on the presence of adequate dTTP pools, normal thymidylate synthase (TS) activity, persistence of increased thymidine incorporation despite the exogenous provision of excess 5,10-methylene-tetrahydrofolate, and documentation of adequate folates in sense cells. The increase in TK activity conferred significant biological properties to sense cells (but not antisense or untransduced cells) as demonstrated by augmented phosphorylation of 3'-azido-3'-deoxythymidine (AZT) and concomitantly greater sensitivity to the cytotoxic effects of AZT. Conversely, sense cells were highly resistant to methotrexate, but this was reversed by the addition of AZT. The direct correlation of FR expression and TK activity indicates a previously unrecognized consequence of FR overexpression. (+info)
An estimation of the requirement for folic acid in gestating sows: the metabolic utilization of folates as a criterion of measurement.
(2/385)
Sows at their second parity were randomly distributed in five groups of seven animals each to determine the dietary concentration of folic acid that optimizes the metabolic utilization of the vitamin during gestation. The groups differed by dietary supplement of folic acid: 0, 5, 10, 15, or 20 ppm. Sows were fed 2.5 kg of diet each day. The response of serum folates and folate binding capacity to treatments and the excretion of urinary folates after an i.v. injection of folic acid were measured. The total daily excretion of urinary folates was corrected according to the response to one i.v. injection of saline on the day preceding the i.v. injection of folic acid. The decrease of total serum folates throughout gestation was less pronounced in the groups fed 15 and 20 ppm of dietary folic acid (supplement x period interaction, P<.06) than it was in the other three treatments. The proportion of i.v. folic acid not recovered in sow urine (injected - excreted) decreased as the amount of dietary folic acid increased to reach a minimum, which differed according to the period (supplement x period interaction, P<.02); it was 15 ppm during wk 1 of gestation and 10 ppm for the other periods studied. The unrecovered folates increased over a dietary concentration of 15 ppm. These minimum values correspond to the most appropriate feed concentration that covered the whole body utilization (tissue and cell metabolism, catabolism, and storage) of folates by the sows and could be interpreted as a reliable index of the requirement. (+info)
Expression of folate receptor alpha in the mammalian retinol pigmented epithelium and retina.
(3/385)
PURPOSE: Folic acid is essential for DNA, RNA, and protein synthesis, and deficiencies in folate can lead to nutritional amblyopia and optic neuropathy. The transport of folate from the choroidal blood supply to the retina is only now beginning to be understood. The reduced-folate transporter was reported recently to be present in cultured human retinal pigment epithelial (RPE) cells and is thought to be localized to the apical region of these cells. The authors hypothesize that the RPE plays a role in the vectorial transport of folate from the choroidal blood to the neural retina and uses not only the reduced-folate transporter but also the folate receptor alpha in mediating this transport. The purpose of the present study was to determine whether the folate receptor alpha was present in the RPE and, if so, whether it was distributed along the basolateral membrane of the RPE, supporting a role for the protein in the initial steps of folate transport into the RPE. METHODS: The expression of the folate receptor alpha in mouse RPE was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), functional assays, in situ hybridization, immunohistochemistry, and laser scanning confocal microscopy. RESULTS: RT-PCR analysis, cloning of the RT-PCR product, and subsequent sequencing established that folate receptor alpha mRNA transcripts are expressed in the mouse RPE/choroid and are expressed also in the neural retina. A heterologous functional expression assay using MTX(R)-ZR-75-1 cells showed that the folate receptor alpha cDNA obtained by RT-PCR from the RPE/choroid complex and the neural retina was functional as assessed by the binding of folic acid and by the uptake of N5-methyltetrahydrofolate. In situ hybridization localized the folate receptor alpha mRNA to the mouse RPE cells and to cells of the neural retina. The folate receptor alpha was detected immunohistochemically in the mouse and rat RPE and in several layers of the neural retina. Laser scanning confocal microscopy revealed the distribution of the folate receptor alpha along the basolateral region of the RPE and not the apical region. CONCLUSIONS: The present work represents the first analysis of the folate receptor alpha expression in intact mammalian retina. The receptor is present and functional in mouse RPE. It is distributed specifically along the basolateral surface of the RPE and is proposed to work in a coordinated manner with the reduced-folate transporter in the vectorial transport of folate from the choroidal blood to the neural retina. (+info)
Complete mapping of divergent amino acids responsible for differential ligand binding of folate receptors alpha and beta.
(4/385)
The folate receptor (FR) type alpha may be distinguished from FR-beta by its higher affinity for the circulating folate coenzyme, (6S)-5-methyltetrahydrofolate (5-CH3H4folate), and its opposite stereospecificity for reduced folate coenzymes. Previous studies showed that a single leucine to alanine substitution at position 49 of the mature protein sequence is responsible for the functional divergence of FR-beta (Shen, F., Zheng, X., Wang, H., and Ratnam, M. (1997) Biochemistry 36, 6157-6163); however, the results also indicated that the minimum requirement for conversion of FR-beta to the functional equivalent of FR-alpha should include amino acid substitution(s) downstream of residue 92 in addition to mutation of L49A. To pinpoint those residues, chimeric FR-betaL49A/FR-alpha constructs including progressively shorter segments of FR-alpha downstream of position 92 as well as selected point mutants were studied. Simultaneous substitution of Leu-49, Phe-104, and Gly-166 in FR-beta with the corresponding FR-alpha residues Ala, Val, and Glu, respectively, reconstituted the ligand binding characteristics of FR-alpha. The results also exclude a role for other residues in FR-alpha in determining its functional divergence. A homology model of FR-alpha based on the three-dimensional structure of the chicken riboflavin-binding protein is used to show the position of residues 49, 104, and 166 in relation to the hydrophobic cleft corresponding to the riboflavin-binding pocket. (+info)
Increased transport of pteridines compensates for mutations in the high affinity folate transporter and contributes to methotrexate resistance in the protozoan parasite Leishmania tarentolae.
(5/385)
Functional cloning led to the isolation of a novel methotrexate (MTX) resistance gene in the protozoan parasite Leishmania. The gene corresponds to orfG, an open reading frame (ORF) of the LD1/CD1 genomic locus that is frequently amplified in several Leishmania stocks. A functional ORF G-green fluorescence protein fusion was localized to the plasma membrane. Transport studies indicated that ORF G is a high affinity biopterin transporter. ORF G also transports folic acid, with a lower affinity, but does not transport the drug analog MTX. Disruption of both alleles of orfG led to a mutant strain that became hypersensitive to MTX and had no measurable biopterin transport. Leishmania tarentolae MTX-resistant cells without their high affinity folate transporters have a rearranged orfG gene and increased orfG RNA levels. Overexpression of orfG leads to increased biopterin uptake and, in folate-rich medium, to increased folate uptake. MTX-resistant cells compensate for mutations in their high affinity folate/MTX transporter by overexpressing ORF G, which increases the uptake of pterins and selectively increases the uptake of folic acid, but not MTX. (+info)
Molecular genetic analysis of human folate receptors in neural tube defects.
(6/385)
Neural tube defects (NTDs) are the most common congenital malformations and are considered to have a multifactorial origin, having both genetic and environmental components. Periconceptional folate administration reduces the recurrence and occurrence risk by 70-100%. Recently we discovered the first genetic risk factors for NTDs: the 677 C-->T and the 1298 A-->C mutations in the methylenetetrahydrofolate reductase gene explaining at the most 35-50% of the protective effect of folate. In this study we further explored the genetic component of NTDs by analysing the coding region, including the intron-exon boundaries and signal sequences of the folate receptor genes by SSCP analysis. Among 39 patients with spina bifida (SB), 47 mothers with a child with SB, and 10 controls, no polymorphism was present in the folate receptor alpha (FR-alpha) gene or in the folate receptor beta (FR-beta) gene. (+info)
Expression and functional characterization of the beta-isoform of the folate receptor on CD34(+) cells.
(7/385)
We have investigated the expression and functional competence of folate receptor (FR) isoforms on human hematopoietic cells. Using immunofluorescence and reverse transcriptase-polymerase chain reaction (RT-PCR) methodology, we find that a substantial fraction of low-density mononuclear and CD34(+) cells express both the beta and gamma isoforms of FR. The alpha isoform of FR (the form most commonly found on cancer cells) was surprisingly absent from all hematopoietic cells examined. Compared with KB cells (a human cell line known for its elevated expression of FR-alpha), the abundance of FR-beta on CD34(+) cell surfaces was relatively low (approximately 8% of KB cell levels). Because many antifolates and folic acid-linked chemotherapeutic agents enter malignant cells at least partially via FR endocytosis, it was important to evaluate the ability of FR on CD34(+) cells to bind folic acid (FA). Based on three FR binding assays, freshly isolated CD34(+) cells were found to display no affinity for FA. Thus, regardless of whether steps were taken to remove endogenous folates before receptor binding assays, FR on primitive hematopoietic cells failed to bind 3H-FA, fluorescein isothiocyanate (FITC)-linked FA, or FA-derivatized liposomes. In contrast, analogous studies on KB cells showed high levels of receptor binding for all three FR probes. These studies show that although multipotent hematopoietic progenitor cells express FR, the receptor does not transport significant amounts of FA. Consequently, antifolates and FA-linked chemotherapeutic agents that can be engineered to enter malignant cells exclusively through the FR should not harm progenitor/stem cell function. (+info)
A radioimmunoassay for porcine intrauterine folate binding protein.
(8/385)
A RIA was developed for porcine intrauterine folate binding protein (FBP). Displacement of [125I]FBP caused by increasing dilutions of uterine flushings collected from either d-15 pregnant or nonpregnant gilts or media from culture of endometrial tissue from d-15 pregnant or nonpregnant gilts was parallel to the displacement caused by the standard curve. Addition of known amounts of purified allantoic fluid FBP to dilutions of either intrauterine flushings or endometrial culture medium were measured accurately with the RIA. To test specificity, 2-mL samples of uterine flushings collected from d-15 pregnant and nonpregnant gilts were preincubated with 10 microCi of [3H]folic acid and then chromatographed using Sephadex G-100 (Sigma Chemical Co., St. Louis, MO). The fractions were subsequently assayed for radioactivity by liquid scintillation spectrophotometry and for FBP by RIA. The [3H]folic acid and FBP peaks coincided, indicating that the RIA is specific for FBP. Uterine flushings were collected on d 10, 11, 12, 13, 14, and 15 of the cycle or pregnancy from 1) White crossbred, 2) progesterone-treated White crossbred (200 mg of progesterone at 48 and 72 h after estrus), and 3) Meishan gilts and assayed for FBP. Total FBP increased 140-fold from d 10 to 15, and the pattern of change across day did not differ between pregnant and nonpregnant gilts. Progesterone treatment increased intrauterine FBP content on d 10 and 11. No difference in FBP concentrations was detected between White crossbred and Meishan gilts. These results indicate that the RIA for FBP is valid, allowing measurement of this protein in uterine flushings and endometrial culture medium. The onset of FBP secretion by the uterus between d 10 and 15 of the cycle or pregnancy is influenced by the timing of onset of progesterone influence in a manner similar to the endometrial proteins uteroferrin and retinol binding protein. In contrast to these endometrial proteins, FBP concentrations are similar in Meishan and White crossbred gilts. (+info)