Folates are water soluble vitamins essential for normal growth and development. Antifolates are antibiotics, antineoplastics, antiinflammatory and immunosuppressive drugs. For example, methotrexate has been a mainstay in the treatment of acute leukemia of childhood for > 40 years and also used in the treatment of autoimmune disease and asthma. Our long-term objectives are to obtain a thorough understanding of folate homeostasis and apply this information to better the use of and stimulate the synthesis of antifolates.
Specific aims i nclude obtaining a more complete understanding of folate accumulation via a specific folate receptor which is a G-PI linked protein. This includes how the receptor translocates folates across the membrane, how transport is related to folate polyglutamate synthesis and how the function and turnover (synthesis and degradation) of the receptor is regulated. Accumulation and metabolism of 5methyl[3H]tetrahydrofolate, radiolabelled ligand of naturally occurring serum folate, by cells grown in physiological folate (~5nM) in vitro is monitored by HPLC. The effects of antifolates, end products of folate mediated reactions and hormones or compounds known to effect glycosylphosphatidylinositol linked proteins as well as inhibitors of folate polyglutamate synthetase are analyzed. The synthesis of the receptor and its mRNA are studied using immunoprecipitation techniques using antibodies against specific moieties of the receptor and RNA blotting using full length cDNA probes for the receptor. In addition to these basic studies, developing a radioimmunoassay for the receptor will allow measurement of plasma concentrations of the released receptor, which may be a marker for persistent or recurrent disease or perhaps even using antibodies as a pharmaceutical agent to localize and/or deliver cytotoxic agents to tumors over-expressing the receptor.
