In an extension of the applicant's ongoing program on the therapeutics, cellular pharmacology and biochemistry of folates and their analogs, studies are proposed which pursue what the applicant believes are new biological insights that have emerged from his studies of folate compound transport in mammalian cells. Evidence has been generated in these studies as to the probable oncofetal nature of a specific folate transport property expressed only in embryonic and tumor cells. Studies proposed in this application would seek proof of the oncofetal nature of this putative tumor-specific property and will examine, as well, various mechanistic aspects of the membrane transport of folate compounds in methotrexate sensitive and resistant tumor cells and normal proliferative cells as a concommitant to other studies proposed on the genetic regulation and biology of this process in tumor cells. Four individual aims are proposed (1). Additional physiological characterization of folate and folate analog transport will be sought in L1210 and human HL-60 promyelocytic leukemia cells and their drug-resistant variants and normal proliferative murine intestinal epithelial cells. These new studies will examine transport multiplicity, kinetics and energetics of influx and efflux routes and identify their normal physiological substrates. (2) The comparative biochemistry of folate compound transport will be pursued in normal proliferative and tumor cells including the physical basis of functional compartmentation of transport and physical components of these systems in the tumor cell plasma membrane; (3) using cDNA probes and monoclonal antibodies raised against the tumor cell folate transporter, regulation of specific """"""""folate"""""""" transporter gene expression will be studied during cell cycle traverse in tumor cells following stimulation of density-inhibited growth of embryonic cells and following neoplastic transformation and induction of terminal maturation in tumor cells. (4) Cytogenic and ontogenetic aspects of folate transporter gene expression will be sought in tumor cells and drug-resistant variants with increases or decreases in specific folate transport. Our ultimate purpose is to obtain information that will allow selective manipulation of antifolate transport or targeting of this transport property in tumor cells for therapeutic gain and to circumvent transport related resistance.
| Schlemmer, S R; Sirotnak, F M (1993) Retentiveness of methotrexate polyglutamates in cultured L1210 cells. Evidence against a role for mediated plasma membrane transport outward. Biochem Pharmacol 45:1261-6 |
| Schlemmer, S R; Sirotnak, F M (1992) Energy-dependent efflux of methotrexate in L1210 leukemia cells. Evidence for the role of an ATPase obtained with inside-out plasma membrane vesicles. J Biol Chem 267:14746-52 |
| Yang, C H; Pain, J; Sirotnak, F M (1992) Alteration of folate analogue transport inward after induced maturation of HL-60 leukemia cells. Molecular properties of the transporter in an overproducing variant and evidence for down-regulation of its synthesis in maturating cells. J Biol Chem 267:6628-34 |
| Sirotnak, F M; O'Leary, D F (1991) The issues of transport multiplicity and energetics pertaining to methotrexate efflux in L1210 cells addressed by an analysis of cis and trans effects of inhibitors. Cancer Res 51:1412-7 |
