Our research focuses on the study of a fundamental membrane function of mammalian cells, namely the transport of macromolecules into cells through endocytosis. Because poly(lysine) is avidly taken up by endocytosis, it can be used as a carrier for other macromolecules or for drugs that are otherwise poorly transported. By conjugating a fragment of poly(lysine) of 9,000 Mr to horseradish peroxidase at a ratio of 1:1, the cellular uptake of active enzyme by cultured fibroblasts has been increased 1,000-fold. By conjugating methotrexate (MTX) to a poly(lysine) of 70,000 Mr at a ratio of 13:1, the cellular uptake of MTX is increased 20- to 40-fold in normal CHO cells, and 200- to 400-fold in CHO mutants defective in drug transport. In these cells, resistance due to defective MTX transport can be overcome by using MTX-poly(lysine), but not by using MTX-poly(D-lysine). This difference is due to the fact that D-isomer is not degraded and does not release active drug inside cells. These two conjugates are now used in two further lines of investigation, namely: (1) the isolation and characterization of mutants which are sensitive to MTX, but resistant to MTX-poly(lysine), and which are therefore defective in their ability to process a foreign macromolecule. Several such mutants have been found to be pleiotropically defective in endocytosis, and to show interesting patterns of cross resistance to plant and bacteriol toxins; and (2) the development of specific linkages between drugs and poly(D-lysine). Three different types of linkages were shown to restore the cytotoxicity of drug-X-poly(D-lysine), namely a tripeptide that is cleaved by proteolysis, an acid-sensitive linkage that cleaves in acidic endosomes-lysosomes, and a disulfide linkage that is reduced in a yet undefined compartment. Two other macromolecular carriers are being examined for their potential ability to target drugs to tumor cells. A methotrexate-carrying human serum albumin, complexed with anti-human serum albumin IgG, has been used to kill selectively two Fc-Receptor positive tumor cells lines in vitro. A MTX-carrying monoclonal anti-tumor antibody (anti-SSBA-1) is being used to kill tumor cells that carry the SSEA-1 antigen. (A)

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
3R01CA014551-15S2
Application #
3163956
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1978-12-01
Project End
1989-01-31
Budget Start
1987-08-01
Budget End
1989-01-31
Support Year
15
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Boston University
Department
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Gallina, Angelo; Hanley, Timothy M; Mandel, Richard et al. (2002) Inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent HIV-1 entry. J Biol Chem 277:50579-88
Merkel, B J; Mandel, R; Ryser, H J et al. (1995) Characterization of fibroblasts with a unique defect in processing antigens with disulfide bonds. J Immunol 154:128-36
Ryser, H J; Levy, E M; Mandel, R et al. (1994) Inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction. Proc Natl Acad Sci U S A 91:4559-63
Mandel, R; Ryser, H J; Ghani, F et al. (1993) Inhibition of a reductive function of the plasma membrane by bacitracin and antibodies against protein disulfide-isomerase. Proc Natl Acad Sci U S A 90:4112-6
Ryser, H J; Mandel, R; Ghani, F (1991) Cell surface sulfhydryls are required for the cytotoxicity of diphtheria toxin but not of ricin in Chinese hamster ovary cells. J Biol Chem 266:18439-42
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
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
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
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
Ryser, H J; Mandel, R; Hacobian, A et al. (1988) Methotrexate-poly(lysine) as a selective agent for mutants of Chinese hamster ovary cells defective in endocytosis. J Cell Physiol 135:277-84

Showing the most recent 10 out of 13 publications