This application proposes to continue studies on approaches to antifolate chemotherapy based on enzyme targets other than dihydrofolate reductase (DHFR) or thymidylate synthase (TS). The foci of our attention will be the two folate-dependent enzymes folylpolyglutamate synthetase (FPGS) and glycinamide ribonucleotide transformylase (GARTF). Sufficient pure FPGS will be obtained to allow the preparation of antibodies and to obtain partial microsequence of V-8 proteolytic fragments. Pure enzyme will be characterized and difference will be sought between FPGS of different sources at the levels of kinetic behavior. As a major goal of this project, we will isolate and analyze genomic and cDNAs encoding human FPGS. Studies performed during the last grant period have established the feasibility of our approach in this cloning and have furnished us with cell lines that originated from the AUX B1 chinese hamster cell, transfected with human DNA and confirmed to be expressing human FPGS. We now will isolate common human DNA segments from independent genomic libraries which have already been constructed from two of these cell lines and sorted to construct two sub-panels in which all of the genomic clones have human sequences. It appears that some of these sequences include the FPGS gene by their ability to complement the FPGS phenotype of AUX B1 cells. We will confirm these transfection results and extend them by Southern and Northern analysis of appropriate cell lines expressing different levels of human FPGS. In related studies, we will use sequence data on mouse and human GARTF to isolate the cDNA encoding this enzyme and will characterize this gene. During the course of this work, we will purify sufficient human GARTF and compare it to the L1210 cell enzyme already available in bulk in this laboratory. These studies will define the stability of both proteins to proteolysis, their stabilization by 5,10-dideazatetrahydrofolate (DDATHF), and the site(s) of cross-linking of DDATHF to active-site peptides. We will also continue our study of the interaction of DDATHF and its analogs with L1210 and CEM GARTF in vitro and in vivo. In the course of these studies, we will determine what forms of DDATHF are bound to GARTF in vivo, whether DDATHF is bound to or inhibits other enzymes in vivo, and the fine points of this interaction in vivo. A major aim of this work will be to determine why DDATHF is cytotoxic. These studies should improve our understanding of the reasons for the therapeutic activity of inhibitors of GARTF and of the role for FPGS in antifolate chemotherapy.
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