The research initiatives of this section focus on folate and antifolate metabolism with specific emphasis on the molecular, physiologic, and biochemical characterization of the human folate receptor (hFR) gene family, including the alpha, beta, and gamma isoforms. Folates are essential vitamins required for DNA and protein synthesis, and serve as the sole methyl group donor in all intracellular reactions. Folate deficiency is a common cause of megaloblastic anemia and more recently has been implicated in neural tube birth defects, mental disorders, dysplasia, and vascular disorders. Our laboratory has demonstrated that hFRs are critical components of folate and antifolate transport, and are an important determinant of antifolate sensitivity. The hFR genes are independently expressed in normal and malignant human tissues. The alpha hFR is overexpressed in > 95% of ovarian tumors and cell lines. The factor(s) modulating expression of hFR are not well studied; however, the alpha folate receptor expression is inversely related to folate concentrations. We are examining the function of the hFRs by mutagenesis and using antisense oligonucleotides and hFR-targeted ribozymes to modulate expression. To determine the sequence and structure of the hFRs, we are characterizing the genes and cDNAs encoding the 3 hFRs and their transcriptional regulatory elements. In the past year, we have reported the structure of the alpha hFR, the identification of two tissue-specific alpha hFR promoters, the effect of the divergent 5' terminal sequences of the alpha hFR on translational efficiency, the role of N-linked glycosylation on the function of the alpha hFR, the results of a phase 1 trial of 9-aminocamptothecin in relapsed lymphomas, and the the CT findings of a chylous pleural effusion in Hodgkins disease. MAJOR FINDINGS: We have previously shown that the alpha hFR gene is a complex transcriptional unit that generates heterogeneous transcripts via alternative splicing and/or from two independent promoters, designated P1 and P4, that are activated in a tissue specific manner. We have shown that the P1 sequence contains sequences capable of forming stable secondary structures and demonstrated that the P4 transcripts are translated more efficiently in vivo and in vitro than the P1 transcripts. We have previously shown that the P4 minimal promoter contains an INR sequence preceded by an array of 3 SP1 binding sites. Using a reporter gene assay system, we have delineated the boundaries of the P1 minimal promoter (between nucleotides +50 and +256 relative to the 5' termini of exon 1) and have demonstrated that sequences spanning the exon 1 ? intron A boundary are absolute requirements for transcription activation. RNase protection assays show that P1 transcription initiates from multiple sites within exon 1 and demonstrate tissue restricted expression. Using Dnase 1 footprint assays and gel shift assays, we have identified multiple sequences that form specific DNA-nuclear protein complexes. At least 4 of these complexes include SP1-like proteins. The nature of the proteins involved in formation of the other complexes and the contribution of each factor relative to transcritional activation is under study. The alpha hFR is relatively over-expressed in ovarian carcinomas. In collaboration with Dr. Tomassetti, we demonstrated that transcripts from the P1 promoter are more abundant than those driven by the P4 promoter in ovarian cancer. Furthermore, in contrast to other human cell lines and tissues, the ovarian alpha hFR transcripts frequently inititate from sites near the 3' boundary of exon 1 and typically include a 66 bp alternatively spliced fragment from exon 3. Following transient transfection of reporter gene constructs, both promoters are active in expressing cell lines whereas transcription from these promoters is undetectable in non-expressing cell lines. These results suggest that tissue or cell specific differences perhaps involving the chromatin structure contribute to the activation of the alpha hFR gene in ovarian cancer. By gel shift assays, we have recently identified a DNA-nuclear protein complex by gel shift assays that appears to be specific for expressing ovarian cancer cell lines. Studies are ongoing to define the nature of this nuclear protein and its role in activation of the alpha hFR in ovarian cancer. We are also characterizing regulatory elements of the beta hFR gene. We have previously described the structure of beta hFR gene and identified a promoter that contains sequences capable of forming SP1 or GA binding (GABP) protein - DNA complexes by gel shift assays. Analysis of 5' flanking sequences shows multiple sequences capable of binding GABP. We are investigating the role of each of these proteins and elements in transcriptional activation and tissue specific expression of the beta hFR promoter. Based on reporter gene assays, the minimal promoter is contained within 189 bp upstream of exon 1, and intron A contains both positive elements. The minimal promoter sequence contains a single SP1 and 4 or 5 GABP elements. We have introduced mutations abrogating protein binding to each of the sites to examine the role of each site in transcription activation. Preliminary results using reporter gene assays suggest that both nuclear proteins are required for optimal activation of the promoter (although their relative contribution may vary among cell lines), that the positive element within intron A is more active in selected cell lines, and that the negative intron A element is active in all cell lines studied. 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 identified a promoter upstream from exon 1 and are characterizing its cis and trans elements. By GSA we have demonstrated the formation of specific DNA-nuclear protein complexes from extracts prepared from expressing cell lines. We have demonstrated that the gamma hFR' isoform represents a polymorphism, is the most common allele, and is commonly expressed.
