The initiatives of this section are divided into two broad areas. These include a focus on folate and antifolate metabolism with specific emphasis on the molecular, physiologic, and biochemical characterization of the human folate receptor (hFR) gene family. More recently, we have begun to explore the biology of a secondary leukemia cell line (SAML-1) established in our laboratory that has an abnormality in the 5q region that is commonly deleted in secondary AML. The primary structures of three hFR isoforms, a, b, and g, have been deduced from their respective cDNAs isolated from KB cell, placental, and lung or thymus cDNA libraries, respectively. These homologous proteins are single chain glycoproteins that range in size from 32 to 42 kD and contain a single, high affinity binding site for folates. The a hFR contains at least two important functional domains: the ligand binding site and the membrane anchor. We are probing the a hFR protein using mutant and wild type cDNAs stably transfected into selected cell lines with specific phenotypes to further define the properties of these domains. The mechanism(s) whereby the a hFR internalizes ligand is controversial. In some cells, the a hFR is clustered in caveolae and is thought to internalize folate by potocytosis. In other cells, the a hFR is found in clatharin coated pits and internalizes folate by endocytosis. Our laboratory has recently demonstrated that hFR mediated transport is independent of caveolin, an important protein constituent of caveolae. Furthermore, we have shown that hFR-ligand complex is not internalized by clatherin-mediated endocytosis. Further elucidation of the mechanism(s) involved in the hFR-mediated internalization and of the protein constituents required in this process underway. We have shown that the a, b, and g transcripts are widely expressed in fetal and adult tissues, and that their abundances are variable and independent of each other. To determine the structures of the hFR genes, and to identify the elements regulating transcription of each gene, we have isolated the genes encoding the a, b, and g cDNAs and we are now studying their minimal promoter elements and other regulatory regions. We have cultured a stable cell line, SAML-1, from the bone marrow of patient diagnosed with secondary AML and are completing characterization of its chromosomal abnormalities and growth parameters. We have shown that the SAML-1 cell line contains a balanced translocation of 5q with chromosome 1. A small deletion in the 5q31 region occurred during this translocation that spans the region that is a commonly deleted segment in the 5q- syndrome. The precise location of the breakpoints have not been determined yet but the deleted genes include IL3, IRF-1, IL4 and IL5. This cell line should be especially useful in determining the critical genes lost in leukemias with 5q deletions. We have also been analyzing the response of the leukemic cells to normal regulators of hematopoietic cell growth and differentiation, including the microenvironment of the bone marrow stroma, that could be disrupted during leukemogenesis.
