Drug-carrying proteins transported into cells by endocytosis can have marked pharmacologic effects, provided active drug is released inside cells. They are excellent tools, therefore, to study the intracellular processing of macromolecules. This project will compare drug conjugates using the same drug (methotrexate, MTX) but differing either in their carrier or in their linkage of drug to carrier. Two non-degradable drug carriers will be used, i.e., poly(D-lysine) internalized by non-specific endocytosis, and anti-TNP:MTX-TNP-poly(D-homocitrulline) immune complexes internalized by Fc-receptor-mediated endocytosis. The linkages can be tailored so as to cleave in three different cellular environments. They will include a triglycine linkage cleaved by lysosomal proteases, an acid-sensitive cis-aconityl linkage hydrolysed in acidic intracellular organelles, and a disulfide linkage cleaved wherever reducing conditions prevail along the endocytotic pathway. The conjugates will be used to characterize these three intracellular compartments, especially the third, about which least is known. The compartments will be identified by subcellular fractionation following exposure of cells to conjugates containing 3H-MTX and 14C-labeled carriers. Since released drugs will diffuse out of organelles, a decrease in the 3H/14C-ratio will identify the fractions in which cleavage is taking place. Factors known to influence these intracellular functions (inhibitor of proteases, lysosomotropic amines, precursors and inhibitors of glutathion synthesis) will be tested for their effect on both the intracellular distribution of 3H-MTX and the cytotoxicity of the conjugate. The same MTX-linkages will be inserted in two further carriers which follow different endocytotic pathways, namely transferrin, and a monoclonal antibody against the SSEA-1 tumor antigen. The fate of tumor antigen-antibody complexes is largely unknown and will be clarified by comparison with that of known ligands. Defects in endocytotic functions have been associated with important diseases (atherosclerosis, storage diseases). This research will provide new information on the intracellular processing of macromolecules in endocytosis. It will also yield basic information on mechanisms of drug release from macromolecular carriers, including carriers that are selectively targeted to tumor cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA034798-08
Application #
3172627
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1987-07-01
Project End
1991-11-30
Budget Start
1989-12-01
Budget End
1991-11-30
Support Year
8
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Southern California
Department
Type
Schools of Pharmacy
DUNS #
041544081
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Taub, M E; Shen, W C (1992) Polarity in the transcytotic processing of apical and basal membrane-bound peroxidase-polylysine conjugates in MDCK cells. J Cell Physiol 150:283-90
Wan, J; Taub, M E; Shah, D et al. (1992) Brefeldin A enhances receptor-mediated transcytosis of transferrin in filter-grown Madin-Darby canine kidney cells. J Biol Chem 267:13446-50
Mandel, R; Ryser, H J; Niaki, B et al. (1991) Isolation of variants of Chinese hamster ovary cells with abnormally low levels of GSH: decreased ability to cleave endocytosed disulfide bonds. J Cell Physiol 149:60-5
Wan, J S; Persiani, S; Shen, W C (1990) Transcellular processing of disulfide- and thioether-linked peroxidase--polylysine conjugates in cultured MDCK epithelial cells. J Cell Physiol 145:9-15
Feener, E P; Shen, W C; Ryser, H J (1990) Cleavage of disulfide bonds in endocytosed macromolecules. A processing not associated with lysosomes or endosomes. J Biol Chem 265:18780-5
Shen, W C (1990) Acid-sensitive dissociation between poly(lysine) and histamine-modified poly(glutamate) as a model for drug-releasing from carriers in endosomes. Biochim Biophys Acta 1034:122-4
Shen, W C; Wan, J S; Shen, D (1990) Proteolytic processing in a non-lysosomal compartment is required for transcytosis of protein-polylysine conjugates in cultured Madin-Darby canine kidney cells. Biochem Biophys Res Commun 166:316-23
Persiani, S; Ballou, B; Shen, W C et al. (1989) In vivo antitumor effect of methotrexate conjugated to a monoclonal IgM antibody specific for stage-specific embryonic antigen-1, on MH-15 mouse teratocarcinoma. Cancer Immunol Immunother 29:167-70
Persiani, S; Shen, W C (1989) Increase of poly(L-lysine) uptake but not fluid phase endocytosis in neuraminidase pretreated Madin-Darby canine kidney (MDCK) cells. Life Sci 45:2605-10
Ryser, H J; Li, W; Mandel, R et al. (1988) Stable variant of LM fibroblast defective in fluid-phase but competent in receptor-mediated endocytosis. J Cell Physiol 137:490-6

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