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Extraction of a Murine Tumor-specific Antigen from Cells and Plasma Membranes Using 1-Butanol: Augmentation of Antigen Yield by Colchicine¹

Departments of Surgery [S.J.L., B.D.K.] and of Biochemistry and Molecular Biology [S.J.L.], The University of Texas Medical School at Houston, Houston, Texas 77030

The purpose of this investigation was to examine the ability of single-phase aqueous solutions of 1-butanol to release immunoprotective tumor antigen activity from partially purified plasma membranes of the methylcholanthrene-induced fibrosarcoma, MCA-F. Tumor antigen activity was assessed by s.c. immunization of syngeneic C3H/HeJ mice 10 days prior to supralethal challenge. Although brief incubation of intact MCA-F cells in 2.5% butanol releases potent immunoprotective activity, application of this protocol to plasma membranes did not result in antigen extraction. Modification of the extraction protocol using higher concentrations of butanol and longer extraction times did release measurable tumor antigen activity. However, a significant amount of the membrane-associated activity remained with the insoluble membrane fraction, as demonstrated by the immunoprotective capacity of the extracted membranes. The dramatic difference in the extractability of antigen from intact cells and plasma membranes suggested that membrane architecture may influence antigen release. To investigate this possibility, we extracted with butanol MCA-F cells that had been preincubated in colchicine. Treatment of cells with colchicine significantly potentiated the extraction of tumor antigen activity. Augmentation of antigen yield was also observed when plasma membranes were pretreated with colchicine prior to 2.5% butanol extraction. These results suggest that the tumor-specific transplantation antigen may be directly or indirectly associated with the cytoskeleton underlying the plasma membrane.

¹ Supported by Grants CA-29814 and CA-38500 awarded by the National Cancer Institute, Department of Health and Human Services (S.J.L.) and a Biomedical Research Support Grant awarded by the University of Texas Medical School at Houston (S.J.L.).

² Present address: Department of Immunology, The University of Texas M. D. Anderson Hospital and Tumor Institute, HMB Box 178, Houston, TX 77030. To whom requests for reprints should be addressed.

³ Recipient of Grant IM62J awarded by the American Cancer Society.

Received 8/30/84. Revised 12/28/84. Accepted 1/23/85.