Methotrexate (MTX) and other classical antifolate compounds used in cancer chemotherapy must be transported across plasma membranes to be effective in killing tumor cells. The influx of MTX via existing transport systems for folate compounds has a major impact on the extent of MTX uptake, but total uptake of MTX is also affected by efflux systems. In L1210 mouse cells total uptake of MTX is determined by composite activities of a single bidirectional influx carrier and two unidirectional efflux routes. The physiological function of the unidirectional efflux routes for MTX is unclear, although a role in general anion extrusion has been suggested since one of these efflux systems also mediates the unidirectional efflux of cholate. The pharmacological implications, physiological function, and biochemical characterization of the two unidirectional efflux routes for MTX in L1210 cells are addressed in this proposal. The working hypotheses which provide the basis for these studies are that the efflux pumps for MTX: exert a significant effect on steady-state levels of free MTX within tumor cells; can accommodate various anion substrates and inhibitors of diverse structure; are components of various cells and tissues; function primarily in cellular detoxification by extruding large and potentially cytotoxic anions; and may be related to the efflux systems which extrude anionic glutathione-S-conjugates. Specific studies are proposed: to utilize intact L1210 cells, efflux variants, reversibly permeabilized cells, and everted plasma membrane vesicles to identify and characterize substrates and inhibitors for the two efflux pumps for MTX; to identify and utilize covalent efflux inhibitors to characterize and isolate efflux pump proteins; to test affinity resins containing covalently attached glutathione-S-conjugates and bromosulfophthalein as a means to purify components of MTX efflux systems; to reconstitute MgATP-dependent transport activity in proteoliposomes with purified proteins and determine substrate and inhibitor kinetics; and to establish the biochemical properties of isolated efflux proteins and obtain antibodies and partial amino acid sequences to proceed towards the isolation and sequencing of the cDNAs for MTX efflux proteins. Studies in human CCRF-CEM lymphoblastoid cells will utilize techniques applied successfully in L1210 cells for the identification and characterization of efflux routes for MTX and for the isolation of efflux proteins. The results will advance our understanding of: anticancer drug action by providing biochemical information on the proteins which mediate the unidirectional efflux of MTX; factors influencing intracellular levels of antifolates and how drug levels might be increased through changes in activity of efflux proteins; and the identification of the primary physiological substrates for anion efflux pumps and their possible role in cellular anion detoxification.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA023970-14
Application #
3166286
Study Section
Experimental Therapeutics Subcommittee 1 (ET)
Project Start
1982-06-01
Project End
1995-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
14
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037