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Cytotoxicity of Bacterial Proteases in Various Tumor Cells Mediated through ₂-Macroglobulin Receptor¹

Departments of Microbiology [H. M., A. Mo., A. Mu., Y. M.] and Surgery [K. S., A. Mu.], Kumamoto University Medical School, Kumamoto 860, Japan

The binding and cytotoxicity of a complex of fluorescein isothiocyanate-labeled 56K protease and ₂-macroglobulin (₂M) were determined by using various human and rodent tumor cell lines. The binding was higher at 37°C than at 4°C; a rapid and progressive uptake that was time dependent was noted at 37°C, whereas no uptake was observed at 4°C, which indicated temperature-dependent internalization. The binding was highest in the fibroblastic and adenocarcinoma cells, and lowest in squamous and epidermoid cells. The Scatchard plots for the binding isotherms were linear, with an apparent K_assoc 1.17 to 2.99 x 10^-8 M for those cells with high ₂M receptor. The number of binding sites (₂M receptor) per cell was 1.3 to 4.75 x 10⁶. Values for squamous/epidermoid cells were much lower or undetectable. Fluorescent antibody staining indicated that MCF-7 and other cells with ₂M receptor internalized the protease-₂M complex, whereas B-16 melanoma, which has little ₂M receptor on the cell surface, did not. Furthermore, when the cytotoxicity of this complex was compared with that of different cell lines, the cells with high rates of uptake of the complex required only a low concentration of the protease and vice versa. These results suggest a possible mechanism of cytotoxic action of protease: ₂M receptor-mediated endocytosis of the complex followed by destruction of cellular integrity after regeneration of proteolytic activity. Thus, cells with more ₂M receptor require only a low dose for cytotoxic action when compared with cells with little ₂M receptor.

¹ This work was supported in part by a Grant-in-Aid for Cancer Research from the Japanese Ministry of Education, Science and Culture (for H. M.).

² To whom requests for reprints should be addressed.

Received 6/27/88. Revised 9/30/88. Accepted 10/26/88.

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