generated recent excitement as a tumor target because it is vastly overexpressed in malignant tissues and offers other advantages. This application is based upon recent studies in the applicant's laboratory which support the idea that isoforms of FR may be exploited as prognostic or diagnostic tumor/serum markers, as tissue-selective mediators of uptake of potent novel antifolate drugs (in clinical trial) and as potential tissue specific targets for other novel therapies. The general goals of this application are: (1) to identify specific malignancies and their subtypes in which such applications are feasible; (2) to develop and employ sensitive assays for serum folate binding proteins in such cancers; (3) to produce more FR isoform specific monoclonal antibodies for investigations of diagnostic/prognostic/therapeutic applications; (4) to identify antifolate substructures that will distinguish between FR isoforms; and (5) to examine the cause-effect relationship between FU expression, cell proliferation, and cytotoxicity of novel antifolates. The applicant has established strong collaborations with prominent clinicians and a synthetic folate chemist to support the above studies. A wide range of normal and malignant tissues will be examined by in situ hybridization for the cell type specificities and relative levels of FRs alpha, beta, and gamma. Both immunological and sensitive fluorescent ligand binding assays will be developed and employed to screen for serum FRs. The results will be correlated with malignancy characteristics. Based on the applicant's observation of FR-beta upregulation in myeloid leukemias, the level and differentiation stage specificity of this receptor isoform in a large number of myeloid leukemias will be examined by mRNA analysis, immunostaining of bone marrow smears, and FACS analysis. Similarly, subtypes of lymphoid, and possibly other hematopoietic cells, that express the constitutively secreted FR-gamma or the truncated FR-gamma' will be examined. In addition to previously produced monoclonal rat antibodies that recognize FRs-alpha and -beta, he will produce rat antibodies specific for either FR-beta or FR-gamma. Antifolates from Dr. John Hynes will be screened for differential binding to FR-alpha and FR-beta. Antisense FR mRNA will be expressed both constitutively and under the control of an inducible promoter in selected malignant cell types expressing FR for evaluation of the role of FR in supporting cell growth and in mediating the cytotoxicity of antifolates.
