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Anti-G_M2 Monoclonal Antibodies Induce Necrosis in G_M2-rich Cultures of a Human Glioma Cell Line¹

Department of Pathology, The Gade Institute, University of Bergen, N-5021 Haukeland Hospital, Bergen, Norway [R. B., O. E., O. D. L.]; Department of Psychiatry and Neurochemistry, Gothenburg University, St. Jrgen Hospital, S-43303, Hisings Backa, Gothenburg, Sweden [P. F., L. S.]; and Department of Pathology [F. D. V., C. J. W.] and Preuss Laboratory for Brain Tumor Research [D. D. B.], Duke University Medical Center, Durham, North Carolina 27710

The effects of four anti-G_M2 monoclonal antibodies (DMAb-1, DMAb-2, DMAb-3, and DMAb-5) were studied on spheroid cultures from a human glioma cell line (D-54 MG) that is known to express high levels of G_M2. The spheroids developed central necrosis 48 h after antibody exposures at concentrations >6 µg/ml. No necrosis was found with antibodies that had been absorbed with G_M2 prior to exposure or with unrelated cytotoxic antibodies. Immunohistochemistry showed that the necrosis started shortly after the antibodies were evenly distributed throughout the spheroids. Light and transmission electron microscopy revealed that a small portion of the cells, mainly in the periphery of the spheroids, was unaffected by antibody exposure. New monolayer cultures established from antibody-treated cells expressed a 50% lower G_M2 content as shown by flow cytometry and determination of ganglioside content throughout at least 12 passages. Thus, the G_M2-rich D-54 MG cell line has subpopulations of cells with lower G_M2 content. Spheroids obtained from this subpopulation developed only minor necrosis after antibody treatment. These results show that G_M2 antibodies cause severe necrosis of G_M2-containing glioma cells in vitro, but the effect depends on the concentration of antigen, and a threshold number of G_M2 molecules is required.

¹ Supported by the Norwegian Cancer Society, the Swedish Medical Research Council (Project 03X-637), and by NIH Grants NS 20023, CA 11898, and CA 32672.

² To whom requests for reprints should be addressed, at The Gade Institute, Department of Pathology, University of Bergen, N-5021 Haukeland Hospital, Bergen, Norway.

Received 3/ 4/91. Accepted 6/17/91.

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