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Sera obtained from rats showing transplantation immunity to syngeneic, malignant epithelial cell line, 2-10-1, were used to identify antigens associated with transformation in vitro. The 2-10-1 cell line was derived from exposure of tracheal explants, in vitro, to the carcinogen MNNG. Tumorigenic passages of the cell line were shown to have common antigenic determinants, shared by independently transformed malignant tracheal epithelial cell lines, as well as antigenic determinants that appear to be limited to the immunizing cell line, 2-10-1. Care was taken to ensure that antibodies were not produced against viral antigens or non-specifically absorbed serum components. Early passages of the 2-10-1 cell line were not tumorigenic in athymic BALB/c (nu/nu) mice, but became malignant with serial passage in vitro. Antigenic specificities recognized by 2-10-1 immune sera were also present on early-passage 2-10-1 cells that had not as yet acquired the malignant phenotype. Such antigen expression must be an early event in neoplastic development.

Cloned hybridoma cell lines were obtained by fusion of murine myeloma cells with spleen lymphocytes of either F344 rats or BALB/c mice immunized against the malignant F344 tracheal cell line, 2-10-1. Monoclonal antibodies were selected for their ability to bind to the immunizing cell line and not to normal tracheal epithelial cells. Results from quantitative binding assays indicated that such monoclonal antibodies recognized six epitopes. Each epitope was detected on four other malignant tracheal epithelial cell lines, and was also expressed during early preneoplastic cell passages (i.e. before the cells acquired the ability to produce carcinomas in vivo). Quantitative differences in epitope expression between non-tumorigenic and tumorigenic passages of individual cell lines could not be detected for five of the epitope groups. However, two monoclonal antibodies, recognizing the same epitope, did show quantitative binding differences between non-tumorigenic and tumorigenic cell passages on three of the five cell lines tested. Our results show that carcinogen-altered tracheal cell populations can be distinguished from non-altered cells (by our current assay methods) by use of monoclonal antibodies. The suggest that the antigen expression is an early event associated with the transformation of rat tracheal epithelial cells in culture.

Flow cytometric histograms frequently consist of several components that show various degrees of overlap. For many types of analysis it is of great importance to decompose the original histogram into its components. To that purpose, we investigated the maximum likelihood approach in detail. It is shown that the iterative method to solve the maximum likelihood equations is well behaved for a variety of initial values. Algorithms to obtain initial values are presented, and the performance of the method is tested when applied to the analysis of DNA measurements from heterogeneous cell populations that differ with respect to DNA content.

In this study we investigated the relationship between binding of anti-tumor antibodies by polyoma-virus-induced SEYF- a tumor cells and the expression of immune-complex receptors on these cells. It was shown that in vivo propagated cells became progressively coated with IgG. The increase in the IgG coating of SEYF-a cells, occurring during the second week of propagation, was directly correlated with an increase in the coating of cells with potentially cytotoxic anti-tumor antibodies. The activity of these antibodies was demonstrated by cell lysis following addition of exogenous complement. Cell populations propagated in vivo for longer periods (3 weeks or more) became less sensitive to exogenous complement although their IgG coating remained high, and although higher titers of anti-tumor antibodies could be eluted from them. This indicated that antibodies coating young tumor cell populations have the capacity to activate complement whereas those coating older tumor cell populations are incapable of complement activation. Previous findings that SEYF-a cell populations are able to bind unrelated immune complexes were confirmed in the present study. We also found that the capacity of these populations to bind such unrelated complexes decreased with propagation time in vivo. The involvement of anti-tumor antibodies in this phenomenon was indicated by the finding that such antibodies inhibited binding of unrelated immune complexes by young cells but not by old cells. Furthermore, treatments causing dissociation of Ig from the cell surface restored, to some extent, the capacity of anti-tumor antibodies to inhibit immune complex binding by old SEYF-a populations.