The research initiatives of this section focus on folate and antifolate metabolism with specific emphasis on the molecular, physiologic, and biochemical characterization and regulation of the human folate receptor (hFR) gene family. We have shown that hFRs are critical components of folate/antifolate transport, and that their abundance is a determinant of folate /antifolate uptake, and sensitivity to antifolates. The hFR genes are independently expressed in human tissues. The alpha hFR is overexpressed in > 95% of ovarian tumors and cell lines and in other epithelial tumors, while the beta hFR is commonly expressed in non-epithelial malignancies. The factor(s) modulating expression of hFRs are not well studied. To determine the structure and function of the hFRs and to elucidate mechanisms modulating their expression, we are characterizing the genes and cDNAs encoding the 3 hFRs. We have previously shown that the alpha hFR gene is a complex transcriptional unit that generates heterogeneous transcripts via alternative splicing and/or from 2 promoters (P1 and P4) that are activated in a tissue specific manner. The translational efficiency of P1 and P4 transcripts are different suggesting regulation at the transcriptional and translational level. Based on differences in P1 and P4 transcript abundance, we have hypothesized that these promoters may be activated in a tissue specific manner. We are characterizing the P1 promoter at a functional and structural level to identify tissue specific regulatory elements. Using a reporter gene assay system, we have localized the P1 minimal promoter and identified those cis elements that are essential for P1 promoter activation. On DNase I footprint assays, Hela cell nuclear extracts protected multiple sites within P1 and created a DNase I hypersensitivity site. To identify cis and trans elements, we have synthesized a set of overlapping oligonucleotides from P1 sequence that include potential AP1, AP2, AP4, TF11D, CAAT, Sp1, p300 and NF1 sites for gel shift assays. We observed specific DNA:nuclear protein complexes with nuclear extracts from both non-expressing cells and expressing cells. Several of these cis elements form complexes that are supershifted by Sp1 and Sp3 antibodies while other complexes are supershifted by only SP1 oligonucleotides suggesting that SP1, SP3, or the ratio of these SP1 family nuclear proteins may be important in P1 regulation. Multiple other specific DNA:nuclear protein complexes have been identified and we are studying their nature and contribution to P1 activation. We are currently transiently transfecting FR expressing and non-expressing cells with CAT reporter gene constructs containing various deletions of the P1 promoter to identify cell specific differences. We are also characterizing the binding of nuclear proteins to these elements using an in vivo DNase I assay to determine if structural modifications of the chromosomal DNA may be involved in the P1 promoter expression to identify differences in the P1 chromatin structure. In collaboration with Dr. Tomassetti, we demonstrated that transcripts from the P1 promoter are more abundant than P4 transcripts in ovarian cancer. Furthermore, the ovarian alpha hFR transcripts more frequently initiate from sites near the 3 boundary of exon 1 and commonly include a 66 bp alternatively spliced fragment from exon 3. In reporter gene assays, the P1 and P4 promoters are active in expressing cell lines but inactive in selected non-expressing cells suggesting that tissue or cell specific differences (e.g chromatin structure) may contribute to the expressing of the hFR by ovarian cancer. We have observed a DNA-nuclear protein complex by gel shift assays that is specific for ovarian cancer cells. We are studying the nature of this protein and its role in activation of the alpha hFR in ovarian cancer. We are also studying the transcriptional regulatory elements of the beta folate receptor gene since differences of regulatory elements among the hFR gene family may contribute to our understanding of tissue specific gene expression and to the function of each of these receptors. The beta gene promoter contains one SP1 binding protein site (-77 nt) and a purine rich GA binding protein site (-66 to -40 nt). The latter site contains multiple potential GA binding sites. Using reporter gene assays, we have identified the minimal promoter, as well positive and negative elements in flanking sequences. By mutational anaylysis, we have identified at least 4 GA binding sites, an SP1 site, and have studied the contribution of each of these sites as well as the SP1 site to transcription using reporter gene constructs. Compared to the wild type promoter, transcriptional activity was reduced by mutation of the SP1 and/or all GABP sites. We are determining the activity of these constructs in an expressing cells (MDA231 and CD34+ cells) to determine their effect in vivo. Two gamma cDNA sequences have been reported; a cDNA encoding a full length hFR and a second isoform that contains an internal dinucleotide deletion that introduces a termination codon and a Dde1 restriction site. To determine the molecular basis of the heterogeneity and to study the regulation of this gene, we have characterized the gamma hFR gene. We have confirmed the presence of a promoter upstream from exon 1 and are characterizing the regulatory elements. The gamma hFR isoform represents a polymorphism, is the most common allele, and is commonly expressed. To define the 5 termini of the upstream exons and the gamma hFR transcripts, we have performed RNase protection assays using cDNA and genomic probes which show that expression is restricted and variable in normal human tissue, that expression of the reported cDNA is low, that the most commonly expressed transcript encodes the TA- isoform that contains a unique 5 terminus, and that SKOv3 cells express the TA- isoform that lacks the 3 terminal sequence of exon 2. To further characterize these gamma transcripts, we have performed 5 RACE and cloned the cDNA from a normal lung cDNA library. From these experiments we have identified novel cDNAs characterized by unique 5 termini and alternative splicing, analogous to the structure of the alpha hFR gene. To study the functional relevance of the sequences, we have subcloned the cDNAs into mammalian expression vectors for transfection. To further study folate transport by membrane proteins, we plan to generate knockout mice. We have screened a genomic 129 SvJ mouse Lambda FIX II library with mouse hFR and folate carrier cDNA probes and have isolated homologous clones. We are determining their restriction maps to plan the construction of knockout constructs. In other experiments to study the function of the alpha FR and to determine the feasibility of targeting this hFR vector in ovarian cancer, we have shown that vectors expressing antisense transcripts impair cloning efficiency and decrease hFR translation. We have synthesized 4 hammerhead ribozmes that are specific for the alpha hFR and demonstrated specific cleavage of in vitro synthesized mRNA. We are studying the uptake, stability and effect of these ribozymes following transfection into Hela and KB cells using folate-conjugated polylysine. Preliminary evidence indicates that uptake is specific and that the ribozyme is stable. We are currently investigating their effect on growth and protein synthesis. The role of the beta hFR in folate transport folates has not been well studied. To investigate the function of the beta hFR, we have stably transfected CHO cells with a pCDNA3.1 vector containing the full-length cDNA. Selected clones expressing high levels of the beta hFR have been isolated and their transport and binding of folates properties are being determined and compared to CHO clones expressing the alpha hFR.
