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Ly-6E.1 is highly expressed in murine tumor cells with a high malignancy phenotype and may serve as a marker for such a phenotype. In this study, we examined the effects of various growth conditions and stress on the expression levels of Ly-6E.1 by tumor cells. Previous preliminary results have shown that murine DA3 mammary tumor cells expressing high levels of Ly-6E.1 (Ly-6(hi)) are more highly tumorigenic than the same tumor cells expressing low levels of this membrane protein (Ly-6(lo)). In this study, we demonstrate that mice bearing Ly-6(hi) DA3 tumors have a significantly higher burden of spontaneous pulmonary metastasis than mice bearing Ly-6(lo) DA3 tumors. Furthermore, the survival time of the former mice was significantly shorter than that of the latter ones. We further show that certain other members of the Ly-6 gene family such as Ly-6C.1 and Ly-6G.1 are coregulated with Ly-6E.1. This was shown to occur with respect to both DA3 cells as well as A3 tumor cells which are of fibroblast origin. However, these 2 cells differ with respect to regulation of Sca-2 (TSA1, another member of the Ly-6 family) expression on these cells. Levels of Sca-2 on A3 cells appear to be coregulated with Ly-6E.1 (i.e., Ly-6(hi) A3 cells express high levels of Sca-2 and Ly-6(lo) A3 cells express low levels of Sca-2). These 2 Ly-6 proteins were, however, not coregulated on DA3 cells. Both Ly-6(hi) as well as Ly-6(lo) DA3 cells express equal levels of Sca-2. Levels of Thy-1, another glycosylphosphatidylinositol (GPI)-anchored protein expressed by A3 tumor cells, were equally expressed by both Ly-6(hi) and Ly-6(lo) A3 tumor cells. Levels of Ly-6 (but not those of CD44) on A3 tumor cells were upregulated on cells from dense cultures but were not influenced by the position of the cells in the cell cycle. Stress conditions such as serum starvation or heat shock upregulated the expression of Ly-6 by the 2 types of tumor cells but did not induce apoptosis in these cells. The kinetics of the stress-dependent upregulation of Ly-6 expression differed, however, between the epithelial and fibroblastic tumor cells.

We have previously shown that Fc gamma receptor type II B1 (Fc(gamma)RIIB1), when expressed on non-lymphoid tumor cells, significantly enhanced their tumorigenic phenotype. This study elucidates the role of the intracellular domain of Fc(gamma)RIIB1 in the enhancement of the malignant phenotype of polyoma-transformed 3T3 cells. We investigated the tumorigenic potential conferred by different variants of the receptor: Fc(gamma)RIIB1, a full-length receptor (B1) whose intracellular region is encoded by exons 8, 9 and 10; Fc(gamma)RIIB2, a spliced variant (B2) whose cytoplasmic domain comprises exons 9 and 10 and lacks exon 8; and Fc(gamma)RIIB1-CT53, a deleted mutant whose cytoplasmic domain contains the fragment encoded by exon 8 alone. We have investigated various properties of cells transfected with each of the above variants: tumorigenicity in syngeneic mice, formation of colonies in soft agar, growth rate, production of soluble receptor and capping of the ligand-bound receptor. Results show that while the presence of exon 8 did not enhance growth rate in vitro or production of soluble Fc(gamma)R, it did enhance the tumorigenic phenotype of transfected cells (both in vivo and in vitro growth in soft agar). B1-expressing cells exhibited a significantly higher tumorigenic phenotype than B2 cells. The presence of exon 8 alone (CT53 mutant) conferred the transfected cells a higher tumorigenic phenotype than Fc(gamma)R-negative control cells but lower than intact B1 or B2 cells, indicating that the presence of B1-specific exon 8 is not sufficient but that the presence of an intact B1 intracellular domain is essential, for conferring the high tumorigenicity phenotype upon cells. We conclude that the capping, following ligand binding contributed by exon 8, and the function contributed by the specific localization of exons 9 and 10 in B1 cells may determine their malignant phenotype.

The search for mechanisms that regulate tumor progression has motivated the authors' laboratory to establish a unique murine model system, consisting of two lines of DA3 mammary adenocarcinoma cells that were derived originally from a common ancestor but differed in their malignant potential. Studies indicated that the highly malignant phenotype manifested by one of the cell lines (termed Ly-6hi DA3 cells) was associated with high expression of the Ly-6E.1 antigen. To characterize the mechanisms controlling the high malignancy phenotype expressed by Ly-6hi DA3 cells, the study was focussed on the potential contribution of tumor-derived factors to the high malignancy phenotype expressed by these cells. To this end, the expression of CC chemokines, major chemoattractants of monocytes and T cells, by the highly malignant Ly-6hi DA3 cells as compared to the low malignancy Ly-6lo DA3 cells was evaluated. The results indicate that the highly malignant cells express higher levels of factors that induce monocyte migration than the low malignancy cells. Two CC chemokines were shown to be highly produced by Ly-6hi DA3 cells, MIP-1alpha and MCP-1, of which only the latter was shown to contribute to the high migratory activity expressed by the high malignancy Ly-6hi DA3 cells. Since MCP-1 may attract monocytes to tumor sites, these findings suggest that monocyte-derived mediators, such as growth factors or angiogenic cytokines, have pro-malignancy effects that contribute to the high malignancy phenotype expressed by Ly-6hi DA3 cells.

Angiogenic and poorly angiogenic tumor variants were obtained by an intraperitoneal inoculation of cells from clones of polyoma-virus transformed BALB c 3T3 cells into syngeneic mice. The angiogenic tumor cells expressed a higher tumorigenicity phenotype and a higher capacity to produce artificial pulmonary metastases than cells from the poorly angiogenic tumors. The former cells expressed also significantly higher levels of the lymphocyte activation protein Ly-6E.1 than the former cells. The two types of cells did not differ in expression levels of CD44 and of a polyoma-virus specific membrane antigen. These results raise the possibility that the angiogenic phenotype is coregulated with Ly-6. The effect on Ly-6 expression of signal transduction through TNF receptors, functioning as pivotal regulators of angiogenesis was therefore studied. It was found that TNFα and more so antibodies against Fas down-regulate expression levels of Ly-6. This down-regulation seemed to be selective as expression levels of CD44 were not affected by this treatment.

Nonadherent Fc gamma receptors (Fc gamma R) expressing cells from SEYF-a tumors that form rosettes with sheep erythrocytes coated with IgG antibodies (EA) were isolated by Percoll density gradients. The EA-IgG rosette-forming cells were characterized by the parameters of 1) binding of IgG immune complexes; 2) binding of purified monoclonal antibodies against mouse FcR; 3) sensitivity to complement-dependent lysis mediated by syngeneic anti-SEYF-a antibodies; 4) expression of parental H-2 antigen when grown in F1 hybrids; 5) incorporation of [125I]5-iodo-2'-deoxyuridine; and 6) growth in syngeneic mice. The nonadherent EA-IgG rosette-forming cell population was found to be composed of both host lymphocytes as well as of tumor cells. Tumor-seeking lymphocytes then were removed from SEYF-a tumors by velocity sedimentation on Percoll. The remaining cell population was tumorigenic and expressed FcR, as well as tumor antigens. These tumor EA-IgG rosette-forming cells exhibited a very low rate of DNA synthesis compared with that of non-rosetting tumor cells.

Making use of 125 I-labelled monoclonal rat antibodies against mouse Fc receptor we have developed a solid-phase radioimmunoassay for cell-free antigenic mouse Fc receptor (FcR). An 80% acetone solution was used for fixation of relatively large amounts of soluble proteins on PVC microtiter plates. As a result of this treatment FcR practically lost its activity to bind the Fc portion of an IgG molecule but retained its antigenicity, the binding of specific anti-FcR (aFcR) antibody being increased following acetone fixation. Concentrations of cell-free FcR in NP-40 extracts of FcR-expressing cells were calculated from the linear part of a standard curve and expressed in units of antigenic activity, 1 unit being the amount of antigenic FcR capable of binding 1 μg of 125 I-aFcR. The method may be used for detecting cell-free FcR as a minor constituent in a mixture of proteins.

Serum from young normal BALB/c mice was found to contain IgM antibodies able tomediate complement-dependent lysis of certain syngeneic or allogeneic tumor target cells. The titer of such naturally occurring antitumor antibodies (NATA) was found to increase with aging. A longitudinal serological study comparing the cytotoxicity potential of NATA fromnormal and from urethan-treated BALB/c mice was performed. It was found that urethan-treated mice that did not develop primary lung-adenomas within the duration of the experi-ment had significantly lower NATA titers, against one out of 4 target cells assayed, than urethan-treated animals that developed lung adenomas. This difference was evident in two independent experiments. The results suggested that the lower NATA activity of the urethan-treated mice that did not develop tumors existed even before exposure to the carcinogenic insult. This raises the possibility that certain populations could be segregated according to their natural antibody profile into those individuals which will develop primary tumors within a certain period if exposed to a subthreshold amount of carcinogen, and those which will not.