Ley specific antibody with potent anti-tumor activity is internalized and degraded in lysosomes.
BR96 is a monoclonal antibody (MAb) that recognizes many human carcinomas and can kill antigen-positive tumor cells in vitro. Using both gold and radiolabeled MAb, the distribution and cellular processing of BR96 during cytolysis has been determined. After a brief (< 3 minutes) MAb treatment, cells in suspension are stained by the nuclear viability dye propidium iodide. Whole MAb and F(ab')2 fragments are equally cytotoxic; monovalent F(ab) fragments, however, have no effect on dye uptake unless cross-linked with goat anti-mouse IgG. The level of toxicity is dependent on both MAb dose and on cell surface receptor density. Cell contact may regulate receptor expression. BR96 receptors are more abundant on cells migrating into the open areas of a scratch wounded confluent culture than on the adjacent contact-inhibited cells. BR96 can also inhibit the anchorage-independent growth of tumor cells in soft agar showing that its effects on propidium iodide staining are not due to transient changes in membrane permeability. Immunogold electron microscopy reveals that, after a 1-minute treatment, BR96 induces significant infolding of the plasma membrane and that internalized MAb is localized to these structures. Immediately thereafter, large cell surface and intracellular vesicles form, mitochondria are swollen, and membrane integrity is lost. Therefore, BR96 seems to cause morphological changes characteristic of necrosis rather than apoptosis. When bound to adherent carcinoma cells, BR96 is distributed uniformly on the apical surface of cells labeled at 4 C and is enriched at points of cell substratum contact. Upon warming of the cells to 37 C, BR96 localizes in small perinuclear clusters and the cell margin is now devoid of label. Immunogold electron microscopy reveals that BR96 undergoes receptor mediated internalization and is localized within the same coated pits, endosomes, and lysosomes as the transferrin receptor. Quantitative studies using iodinated BR96 show that after 6 hours of chase, a maximum of 53% of the radiolabel is located within the intracellular pool. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that 84% of this fraction is nondegraded. BR96 probably cycles between the medium and intracellular pools because the remainder of the radiolabel is in the medium as intact MAb. By 24 hours of chase, the intracellular fraction drops to 30%, while the remaining 70% is present in the culture medium, mostly as low molecular weight degradation products.
Garrigues
J
J
Pharmaceutical Research Institute, Bristol-Myers Squibb Company, Seattle, Washington 98121.
Garrigues
U
U
Hellström
I
I
Hellström
K E
KE
D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology; D016923:Cell Death; D002455:Cell Division / Q000187:drug effects; D006801:Humans; D007983:Lewis Blood Group Antigens / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D011990:Receptors, Transferrin / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000473:pathology / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D007983:Lewis Blood Group Antigens; D011990:Receptors, Transferrin