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Pacific Craniofacial Team and Cleft Prevention Program.

There is no doubt modern genetics have greatly influenced our professional and personal lives during the last decade. Uncovering genetic causes of many medical and dental pathologies is helping to narrow the diagnosis and select a treatment plan that would provide the best outcome. Importantly, having an understanding of multifactorial etiology helps direct our attention toward prevention. We now understand much better our own health problems. In some cases, we can modify our lifestyle and diet in order to prevent "environmental factors" from triggering the mutated genes inherited from our parents. Good examples are diabetes and cardiovascular diseases. If we realize we might have inherited genes for cardiovascular problems from several ancestors who had heart attacks, we already know that these genes will make us only "susceptible" for disease. Those who exercise, watch one's weight, diet, and carefully monitor one's lifestyle will very likely--though possessing "susceptibility genes"--stay healthier and, maybe, will never experience any cardiovascular problems. In principle, the same applies for craniofacial anomalies, especially for nonsyndromic cleft lip and palate. One needs to understand genetic and environmental causes of nonsyndromic orofacial clefts in order to prevent them. With all this in mind, the Pacific Craniofacial Team and Cleft Prevention Program have been established at the Department of Orthodontics, University of the Pacific Arthur A. Dugoni School of Dentistry in San Francisco. A partnership with Rotaplast International, Inc., has made it possible for the faculty, orthodontic residents, and students to participate in 27 multidisciplinary cleft medical missions in underdeveloped and developing countries by donating professional and educational services, and, last but not least, by collecting valuable data and specimens to further research. A significant number of research studies, including 15 master of science theses, have been accomplished in UOP's Craniofacial Genetics Laboratory, with contributions by faculty, undergraduate and graduate students. It has been leading to a better understanding of etiology of nonsyndromic orofacial clefts. It has been learned that genetic factors and environmental factors are ethnicity-specific and, in many places throughout the world, location-specific. Thus, a specific protocol for cleft prevention has to be worked out based on genetic and nutritional studies of each specific population group in order to be effective. This is our ultimate goal.

California↗

Mutations leading to antifolate resistance in Chinese hamster ovary cells after exposure to the alkylating agent ethylmethanesulfonate.

Chinese hamster ovary cells with a single allele for dihydrofolate reductase were used as a model system to study the effect of exposure to an alkylating agent, ethylmethanesulfonate, on rates and types of mutations at the dihydrofolate reductase locus leading to antifolate resistance. After overnight exposure to 400 micrograms/ml ethylmethanesulfonate, cells were allowed to recover for 3 days, and resistant colonies were selected in 8 x 10(-8) M trimetrexate. Trimetrexate, rather than methotrexate, was used as the selecting agent to increase the probability of obtaining mutations in dihydrofolate reductase, rather than in the reduced folate transport carrier protein. Seven of several hundred surviving colonies were selected at random, and cell lines were established. Cell lines 1-3 were maintained in culture in the presence of 8 x 10(-8) M trimetrexate and were 66-170-fold resistant to the drug. Cell lines 4-7 were initially expanded in 8 x 10(-8) M trimetrexate but were then maintained in the absence of the drug. These cell lines were 4.4-26-fold resistant to the drug, compared with the parental cell line. Cell line 1 was found to have an increase in dihydrofolate reductase activity, a corresponding increase in mRNA for dihydrofolate reductase, and amplification of this gene. Cell lines 2 and 6 had a mutated dihydrofolate reductase with altered trimetrexate- and methotrexate-binding properties. Cell line 3 had a 3-fold increase in dihydrofolate reductase activity. In cell lines 4, 5, and 7 the mechanisms of resistance to trimetrexate remain unknown.

Alkylating Agents↗

Thymidylate synthase inhibitors.

Folate analogues that inhibit thymidylate synthase (TS) selectively were developed based on TS and folate molecular structures and properties. The structure-activity relationship, preclinical and clinical development, and issues of potential importance in the future success of these TS inhibitors are reviewed herein. Properties of these new compounds depend mainly on the use of the reduced folate carrier (RFC) proteins for cellular entry and on intracellular polyglutamation, which augments TS inhibitory function and cellular retention. CB3717 [Zeneca (formerly ICI Pharmaceuticals), Macclesfield, United Kingdom], the first selective TS inhibitor developed, demonstrated antineoplastic activity in Phase I trials, but its development was abandoned due to nephrotoxicity. ZD1694 (Tomudex; Zeneca), a water-soluble, nonnephrotoxic quinazoline, demonstrated activity in colorectal, breast, and pancreatic cancer. Phase III trials of Tomudex in advanced colorectal cancer were completed recently. LY231514 (Eli Lilly Research Labs, Indianapolis, IN) uses the RFC and polyglutamation to exert its selective TS inhibitory action. Phase I trials of this compound have been completed. ZD9331 (Zeneca), currently in preclinical studies, was designed to obviate the use of the RFC for cellular entry. 1843U89 (Glaxo-Wellcome, Research Triangle Park, NC), currently in Phase I trial, does not require the RFC; polyglutamation of this potent TS inhibitor leads to its higher cellular retention without augmenting its TS inhibitory activity. Currently in clinical trials, AG337 (p.o. and i.v. forms) and AG331 (both by Agouron, La Jolla, CA) are lipophilic potent TS inhibitors with action independent of the RFC and polyglutamation. Multiple mechanisms through which cells may overcome TS inhibition have been described. Combining TS inhibitors with other agents that affect TS, interfere with TS gene and mRNA regulation, or inhibit salvage mechanisms of thymidylate depletion may potentially enable greater clinical utility of this class of compounds.

Animals↗

Roles of USF, Ikaros and Sp proteins in the transcriptional regulation of the human reduced folate carrier B promoter.

The hRFC (human reduced folate carrier) is ubiquitously but differentially expressed in human tissues and its levels are regulated by up to seven non-coding regions (A1, A2, A, B, C, D and E) and at least four promoters. For the hRFC-B basal promoter, regulation involves binding of Sp (specificity protein) transcription factors to a critical GC-box. By transiently transfecting HT1080 cells with 5'- and 3'-deletion constructs spanning 1057 bp of upstream sequence, a transcriptionally important region was localized to 158 bp flanking the transcriptional start sites. By gel shift and chromatin immunoprecipitation assays, USF (upstream stimulatory factor), Sp1 and Ikaros-related proteins were bound to consensus elements (one E-box, two GC-box and three Ikaros) within this region. The functional importance of these elements was confirmed by transient tranfections of HT1080 cells with hRFC-B reporter constructs in which they were mutated, and by co-transfections of Drosophila Mel-2 cells with wild-type hRFC-B promoter and expression constructs for USF1, USF2a, Sp1 and Ikaros 2 and 8. Both USF1 and Sp1 proteins transactivated the hRFC-B promoter. Sp1 combined with USF1 resulted in a synergistic transactivation. Identical results were obtained with USF2a. Ikaros 2 was a repressor of hRFC-B promoter activity whose effects were partly reversed by the dominant-negative Ikaros 8. In HT1080 cells, transfection with Ikaros 2 decreased endogenous hRFC-B transcripts, whereas USF1 and Sp1 increased transcript levels. Ikaros 2 also decreased reporter gene activity and levels of acetylated chromatin associated with the endogenous promoter. Collectively, these results identify transcriptionally important regions in the hRFC-B promoter that include multiple GC-box, Ikaros and E-box elements. Our results also suggest that co-operative interactions between transcription factors Sp1 and USF are essential for high-level hRFC-B transactivation and imply that these effects are modulated by the family of Ikaros proteins and by histone acetylation.

Acetylation↗

Decreased expression of the reduced folate carrier and folypolyglutamate synthetase is the basis for acquired resistance to the pemetrexed antifolate (LY231514) in an L1210 murine leukemia cell line.

Pemetrexed (LY231514) is a new-generation antifolate that, in its polyglutamyl forms, is a potent inhibitor of thymidylate synthase and glycinamide ribonucleotide formyltransferase (GAR transformylase). This study explored the mechanisms of resistance to pemetrexed in L1210 murine leukemia cells using chemical mutagenesis with 5-formyltetrahydrofolate (5-formylTHF) as the growth substrate. A cell line, MTA-13, was identified that was 8.5-fold resistant to pemetrexed with comparable cross-resistance to ZD1694 (Tomudex) and lesser cross-resistance (5-fold) to ZD9331 [(2S)-2-(O-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydro-quinazolin-6-ylmethyl)-N-(prop-2-ynyl)amino]benzamido)-4-(tetrazol-5-yl)-butyric acid], DDATHF (dideazatetrahydrofolate) (3.5-fold), and methotrexate (MTX) (2.7-fold) but comparable sensitivity to trimetrexate. Influx of pemetrexed, MTX, and 5-formylTHF into MTA-13 cells was decreased by 56, 47, and 38% compared to wild-type cells. Folate receptor expression was negligible in both cell lines. Net drug uptake declined within 15min to a slower, constant rate over the next 45min, reflecting the rate of accumulation of pemetrexed polyglutamate derivatives. This rate in the MTA-13 line was half that of the wild-type cells. Accumulation of 50nM [3H]pemetrexed, 25nM [3H]5-formylTHF, or 50nM [3H]DDATHF after 3 days was decreased to 35, 46, and 56% the level of L1210 cells. The reduced folate carrier (RFC) message and protein were decreased by 50%, and folypolyglutamate synthetase (FPGS) message was decreased by 65% in MTA-13 cells. No mutations were detected in either protein by DNA sequence analysis. There was a slight decrease (approximately 25%) in thymidylate synthase mRNA, without mutations in the protein, and there was no change in GAR transformylase message. The data indicate that resistance to pemetrexed in the MTA-13 cell line was due to changes in both RFC and FPGS expression, two proteins that act in tandem to regulate polyglutamation of folates and antifolates in cells, resulting in cellular depletion of these active pemetrexed congeners.

Animals↗

Functional interactions between arginine-133 and aspartate-88 in the human reduced folate carrier: evidence for a charge-pair association.

The human reduced folate carrier (hRFC) is an integral membrane protein that mediates cellular uptake of reduced folates and antifolates. hRFC contains several highly conserved charged residues predicted to lie in the transmembrane domains (TMDs). To explore the possible roles of the conserved arginine-133, located in TMD 4, in hRFC structure and function, this residue was systematically mutagenized to histidine, leucine, lysine and glutamate. When transfected into transport-impaired K562 cells, the mutant hRFC constructs were expressed at high levels; however, only lysine-133 hRFC was able to transport methotrexate and (6S)-5-formyl tetrahydrofolate. Substitution of aspartate-453 (in hRFC TMD 12) by valine largely preserved transport activity for both substrates. Although mutagenesis of aspartate-88 (in TMD 2) to leucine completely abolished transport activity in transfected cells, substitution with a glutamate preserved low levels ( approximately 12%) of transport. To assess the possibility that arginine-133 and aspartate-88 may form a charge-pair to stabilize hRFC tertiary structure, both charges were neutralized (by substituting leucine and valine, respectively) in the same construct. In contrast to the singly mutated hRFCs, the double mutant exhibited high levels of transport with both methotrexate and 5-formyl tetrahydrofolate. These results strongly suggest that arginine-133 and aspartate-88 form a charge-pair and that TMD 4 lies next to TMD 2 in the hRFC tertiary structure.

Amino Acid Sequence↗

Structural organization of the reduced folate carrier gene in Chinese hamster ovary cells.

The reduced folate carrier gene (rfc) encodes a putative protein that is involved in the intracellular accumulation of folates. In this report, we describe the organization of the rfc gene from Chinese hamster ovary cells. The hamster rfc gene contains 7 exons and 6 introns, which span 15.3 kilobases. It codes for two alternatively spliced messenger RNAs, one that contains all 7 exons and one that lacks exon 2 but contains the remaining 6 exons. The transcriptional start of the gene has been mapped to six sites approximately 200 base pairs upstream of the putative ATG initiation codon. The promoter region has no TATA box-like sequence but contains a consensus Sp1 binding site. This is the first report of the genomic structure of the reduced folate carrier gene from any species.

Alternative Splicing↗

Methotrexate transport and resistance.

Methotrexate (MTX), the antifolate drug widely used as both an anticancer chemotherapeutic drug and as an immunosuppressive agent, mimics natural folates to inhibit critical cellular biosynthetic pathways. One of the most important determinants of cellular sensitivity to MTX is the degree to which this drug is internalized by cancer cells, and one of the major pathways of folate uptake results from the activity of the reduced folate carrier (RFC). Decreased RFC activity has been associated with several models of transport-mediated MTX resistance. Recently, the rodent and human genes which encode this protein have been isolated (RFC1), and defects in the expression of RFC1 genes have been identified in transport-deficient, MTX-resistant cell lines. Therefore, these studies have demonstrated the importance of RFC1 expression in transport-mediated antifolate drug resistance. In addition, however, studies of both MTX uptake in cancer cells and of folate transport in physiologic systems indicate that there are other proteins with uptake characteristics similar to RFC, and which maybe encoded by genes other than RFC1.

Antimetabolites, Antineoplastic↗

Structural organization of the human reduced folate carrier gene: evidence for 5' heterogeneity in lymphoblast mRNA.

The reduced folate carrier (rfc1) gene encodes a protein that is involved in the intracellular accumulation of folates. Point mutations in this gene and alterations resulting in the down regulation of its message are major factors involved in the resistance to antifolate chemotherapeutic compounds. As a framework for understanding the significance of such changes in relation to gene expression and function, in this report we describe the organization of the rfc gene from human lymphoblasts. The gene contains 5 exons (2 to 6) coding for protein. At least four 5' exons, used in a mutually exclusive manner in the production of the rfc message from lymphoblast cells, are spliced to exon 2, which contains the translational start site. "Semi-quantitative" PCR indicates that exon 1 is preferentially used. The major transcriptional start site has been mapped by RACE and RNase protection to a region 109 to 135 base pairs 5' to the start of exon 1. The 5' region of the gene has no TATA box-like sequence but contains several consensus binding sites for transcriptional factors such as SP-1, MZF1, CREB, AP-1, ETS, GATA-1 and GATA-2. The overall organization of the human gene is similar to that of the hamster and mouse genes.

Alternative Splicing↗

Impaired membrane transport in methotrexate-resistant CCRF-CEM cells involves early translation termination and increased turnover of a mutant reduced folate carrier.

The basis for impaired reduced folate carrier (RFC) activity in methotrexate-resistant CCRF-CEM (CEM/Mtx-1) cells was examined. Parental and CEM/Mtx-1 cells expressed identical levels of the 3. 1-kilobase RFC transcript. A approximately 85-kDa RFC protein was detected in parental cells by photoaffinity labeling and on Western blots with RFC-specific antiserum. In CEM/Mtx-1 cells, RFC protein was undetectable. By reverse transcriptase-polymerase chain reaction and sequence analysis, G to A point mutations were identified in CEM/Mtx-1 transcripts at positions 130 (P1; changes glycine 44 --> arginine) and 380 (P2; changes serine 127 --> asparagine). A 4-base pair (CATG) insertion detected at position 191 (in 19-30% of cDNA clones) resulted in a frameshift and early translation termination. Wild-type RFC was also detected (0-9% of clones). Wild-type RFC and double-mutated RFC (RFCP1+P2) cDNAs were transfected into transport-impaired K562 and Chinese hamster ovary cells. Although RFC transcripts paralleled wild-type protein, for the RFCP1+P2 transfectants, disproportionately low RFCP1+P2 protein was detected. This reflected an increased turnover of RFCP1+P2 over wild-type RFC. RFCP1+P2 did not restore methotrexate transport; however, uptake was partially restored by constructs with single mutations at the P1 or P2 loci. Cumulatively, our results show that loss of transport function in CEM/Mtx-1 cells results from complete loss of RFC protein due to early translation termination and increased turnover of a mutant RFC protein.

Animals↗

Mechanism and regulation of folate uptake by human pancreatic epithelial MIA PaCa-2 cells.

After the liver, the pancreas contains the second highest level of folate among human tissues, and folate deficiency adversely affects its physiological function. Despite that, nothing is currently known about the cellular mechanisms involved in folate uptake by cells of this important exocrine organ or about folate uptake regulation. We have begun to address these issues, and in this report we present the results of our findings on the mechanism of folate uptake by the human-derived pancreatic MIA PaCa-2 cells. Our results show folic acid uptake to be 1). temperature and energy dependent; 2). pH dependent, with a markedly higher uptake at acidic pH compared with neutral or alkaline pH; 3). Na(+) independent; 4). saturable as a function of substrate concentration (apparent K(m) = 0.762 +/- 0.10 microM); 5). inhibited (with similar affinity) by reduced, substituted, and oxidized folate derivatives; and 6). sensitive to the inhibitory effect of anion transport inhibitors. RT-PCR and Western blot analysis showed expression of the human reduced folate carrier (hRFC) at the RNA and protein levels, respectively. The functional contribution of hRFC in carrier-mediated folate uptake was confirmed by gene silencing using gene-specific small interfering RNA. Evidence also was found suggesting that the folate uptake process by MIA PaCa-2 cells is regulated by cAMP- and protein tyrosine kinase (PTK)-mediated pathways. These studies demonstrate for the first time the involvement of a specialized, acidic pH-dependent, carrier-mediated mechanism for folate uptake by human pancreatic MIA PaCa-2 cells. The results also show the involvement of hRFC in the uptake process and suggest the possible involvement of intracellular cAMP- and PTK-mediated pathways in the regulation of folate uptake.

Carrier Proteins↗

The human reduced folate carrier gene is regulated by the AP2 and sp1 transcription factor families and a functional 61-base pair polymorphism.

Recently, our laboratory reported an intricate regulation of the human reduced folate carrier (hRFC) gene, involving multiple promoters and noncoding exons. We localized promoter activity to a 452-bp GC-rich region upstream of noncoding exon A, including a 47-bp basal promoter with a CRE/AP-1-like consensus element that bound the bZip family of DNA-binding proteins (e.g. CREB-1 and c-Jun). We now report that three nearly identical tandem repeats (49-61 bp) in the hRFC-A upstream region are involved in regulating promoter activity. By in vitro binding assays, multiple transcription factors (e.g. AP2 and Sp1/Sp3) bound this region. When AP2 was cotransfected with the hRFC-A reporter construct into HT1080 cells, promoter activity increased 3-fold. In Drosophila SL2 cells, Sp1 transactivated promoter A and showed synergism with CREB-1. However, c-Jun was antagonistic to the effects of Sp1. A sequence variant in the hRFC-A repeated region was identified, involving an exact duplication of a 61-bp sequence. This variant had an allelic frequency of 78% in 72 genomic DNAs and resulted in a 63% increase in promoter activity. These results identify important regions in the hRFC-A promoter and critical roles for AP2 and Sp1, in combination with the bZip transcription factors. Moreover, they document a functionally novel polymorphism that increases promoter activity and may contribute to interpatient variations in hRFC expression and effects on tissue folate homeostasis and antitumor response to antifolates.

Animals↗

Reduced folate carrier: biochemistry and molecular biology of the normal and methotrexate-resistant cell.

The cytotoxic drug methotrexate uses the reduced folate carrier for transport into the cell, where it inhibits key enzymes in nucleotide biosynthesis. Resistance to methotrexate can be achieved by altering the genetic code of the reduced folate carrier gene and thus change the structure and function of the protein. Our understanding of RFC structure and function is based on the information gained from studying the uptake of folates and antifolates in living cells and the application of molecular techniques to determine gene expression and genetic mutations. The aim of this essay is to explain the structure and function of the reduced folate carrier, review the molecular biology of the reduced folate carrier gene and the mutations and polymorphisms that can result in methotrexate resistance.

Amino Acid Sequence↗

Association of deletions and translocation of the reduced folate carrier gene with profound loss of gene expression in methotrexate-resistant K562 human erythroleukemia cells.

Severe impairment of methotrexate membrane transport in methotrexate-resistant K562 (K500E) cells was characterized by a nearly complete loss of reduced folate carrier (RFC) transcripts and RFC protein. As determined by 5'-rapid amplification of cDNA ends (5'-RACE), approximately 93% of the RFC transcripts in wild-type cells contained the KS43 5'-untranslated region transcribed from the RFC-B promoter. KS43 transcripts decreased > 90% in K500E cells. The basal and full-length RFC-B promoters were more active (3- and 2-fold, respectively) in directing transcription of a luciferase reporter gene in K500E than in wild-type cells. Treatment with a demethylating agent, 5-aza-2'-deoxycytidine, or with a histone deacetylase inhibitor, trichostatin A, did not increase the levels of RFC transcripts in K500E cells. No differences in RFC gene structure were detected between the lines on Southern blots; however, the RFC signals were decreased approximately 60% in K500E cells. DNA sequences were identical between the lines for the RFC coding region and the two 5'-non-coding exons and their respective promoters. Spectral karyotype analysis and fluorescence in situ hybridization in wild-type cells showed two normal chromosome 21 copies and one or two marker chromosomes, each with an RFC signal. In K500E cells, the RFC gene locus was no longer localized to a normal chromosome 21 (at 21q22.2), and a single RFC signal was associated with a small metacentric chromosome, characterized by a 21/22 translocation. Our results suggest that loss of RFC transcripts in K500E cells is unrelated to changes in the levels of critical transcription factors, or to differences in the extent of RFC promoter methylation or core histone deacetylation. Rather, this phenotype is due to the loss of one or more RFC alleles, and to a translocation of the remaining RFC allele with the formation of a 21/22 fusion chromosome.

5' Untranslated Regions↗

Placental folate transport and binding are not impaired in pregnancies complicated by fetal growth restriction.

Maternal folate deficiency is associated with fetal growth restriction, however, transfer of folate across placentae of pregnancies complicated by fetal growth restriction has never been investigated. We studied whether maternal to fetal 5-methyltetrahydrofolate (5MTF) transport in the ex vivo dually perfused isolated cotyledon, binding of [(3)H] folate (PteGlu) to the syncytial microvillous membrane, and protein expression of folate receptor alpha (FR-alpha) and reduced folate carrier (RFC) in these placentae are disturbed. Placental clearance of 5MTF from the maternal perfusate appeared to be non-saturable over a range of 50 to 500 nm, independent of albumin and flow-independent. No statistically significant differences between placentae complicated with fetal growth restriction and uncomplicated pregnancies were observed. Binding characteristics of [(3)H-]PteGlu to microvillous membranes of fetal growth restriction versus control placentae were similar: B(max)of 3.9+/-2.0 (mean+/-s.d.) versus 4.0+/-1.6 pmol/mg protein and a K(d)of 0.037+/-0.010 versus 0.040+/-0.018 nm. Expression of FR-alpha and RFC were not different in placentae of both groups studied. In conclusion, fetal growth restriction appears not to be associated with impaired maternal to fetal placental folate transport, placental receptor binding, or expression of FR-alpha and RFC.

Adult↗

Expression of resistance markers to methotrexate predicts clinical improvement in patients with rheumatoid arthritis.

BACKGROUND: Methotrexate is transported into the cell by the reduced folate carrier (RFC) and out of the cell by members of the multidrug resistance protein family (MRP). Transport proteins may affect the therapeutic efficacy of this drug in patients with rheumatoid arthritis. OBJECTIVE: To investigate the potential benefit of the presence of RFC and the absence of functional MRP for the efficacy of methotrexate treatment. METHODS: The study involved 163 patients (116 female, 47 male; mean age 59.5 years) on methotrexate (mean weekly dose 12.2 mg). RFC was determined using reverse transcriptase polymerase chain reaction, and MRP function by flow cytometry, using a calcein acetoxymethylesther/probenecid assay. Clinical response to methotrexate was evaluated by the EULAR response criteria and the ACR 20% improvement criteria. The clinical data were obtained at the beginning of methotrexate treatment and at the time of blood sampling during ongoing therapy. Patients were divided into four groups according to the presence (+) or absence (-) of RFC and functional (f) MRP. RESULTS: fMRP+/RFC+ and fMRP-/RFC- patients more often had good EULAR response rates (60%, p = 0.014, and 53%, p = 0.035, respectively) in comparison with the fMRP-/RFC+ group (29%); fMRP+/RFC- patients had a low frequency of good disease activity responses. CONCLUSIONS: Absence of fMRP plus presence of RFC did not prove to be related to beneficial effects of methotrexate, but the lack or the presence of both fMRP and RFC led to a significantly better therapeutic outcome. Determination of these markers may predict responsiveness to methotrexate.

Antirheumatic Agents↗

Functional role of arginine 373 in substrate translocation by the reduced folate carrier.

The reduced folate carrier (RFC) plays a critical role in the cellular uptake of folates. However, little is known regarding the mechanism used to transport substrates or the tertiary structure of the protein. Through the analysis of a Chinese hamster ovary cell line deficient in folate uptake, we have identified a single residue in TM10 (Arg-373) of RFC that appears to play a critical role in the translocation of substrate. Replacement of this position with various amino acids (KHQNA) diminished the rate of translocation by 16-50-fold, although substrate binding, protein stability, and localization were unaffected. Furthermore, the translocation capabilities of an R373C mutant in a cysteine-less form of the reduced folate carrier were enhanced 2.5-fold by the positively charged methanethiosulfonate reagent, confirming the essential role of a positive charge at this position. When considering the membrane-impermeable nature of this reagent, the data further suggest that the Arg-373 residue is located within the substrate translocation pathway of the RFC protein. Moreover, cross-linking analysis of the Arg-373 residue demonstrates that it is within 6 A of residue Glu-394 (TM11), providing the first definitive tertiary structural information for this protein.

Animals↗

Reduced-folate carrier (RFC) is expressed in placenta and yolk sac, as well as in cells of the developing forebrain, hindbrain, neural tube, craniofacial region, eye, limb buds and heart.

BACKGROUND: Folate is essential for cellular proliferation and tissue regeneration. As mammalian cells cannot synthesize folates de novo, tightly regulated cellular uptake processes have evolved to sustain sufficient levels of intracellular tetrahydrofolate cofactors to support biosynthesis of purines, pyrimidines, and some amino acids (serine, methionine). Though reduced-folate carrier (RFC) is one of the major proteins mediating folate transport, knowledge of the developmental expression of RFC is lacking. We utilized in situ hybridization and immunolocalization to determine the developmental distribution of RFC message and protein, respectively. RESULTS: In the mouse, RFC transcripts and protein are expressed in the E10.0 placenta and yolk sac. In the E9.0 to E11.5 mouse embryo RFC is widely detectable, with intense signal localized to cell populations in the neural tube, craniofacial region, limb buds and heart. During early development, RFC is expressed throughout the eye, but by E12.5, RFC protein becomes localized to the retinal pigment epithelium (RPE). CONCLUSIONS: Clinical studies show a statistical decrease in the number of neural tube defects, craniofacial abnormalities, cardiovascular defects and limb abnormalities detected in offspring of female patients given supplementary folate during pregnancy. The mechanism, however, by which folate supplementation ameliorates the occurrence of developmental defects is unclear. The present work demonstrates that RFC is present in placenta and yolk sac and provides the first evidence that it is expressed in the neural tube, craniofacial region, limb buds and heart during organogenesis. These findings suggest that rapidly dividing cells in the developing neural tube, craniofacial region, limb buds and heart may be particularly susceptible to folate deficiency.

Animals↗