ALTERATIONS IN GENE-EXPRESSION ASSOCIATED WITH STEPWISE ACQUISITION OF MALIGNANCY IN MURINE CYTOTOXIC T-CELL LINES

We have isolated a series of variant cell lines from a murine CD8+ T cell clone representing distinct stages in stepwise acquisition of malignancy. A first type of variant has acquired independency of restimulation with MHC/Ag but has kept dependence on IL-2 for continuous growth in culture. A second type of variant has acquired, in addition, independency of IL-2. A third type of variant was isolated from tumors induced upon injection of IL-2 independent variants into syngeneic mice. Clonal relatedness between the cell lines was ascertained by Southern blot and sequence analyses of their TCRbeta chain genes. The cell lines were analyzed for their expression of genes typical for CD8+ T cells, using Northern blot hybridization, flow cytometry, and functional methods. Concentrating on the transition from IL-2 dependent to IL-2 independent cellular growth, we find the same triad of changes in two independently derived groups of variant cell lines: loss of expression of the CD8alpha gene with concomitant loss of CD8 from the cell surface, a slight but significant overexpression of IL-2Ralpha and beta chains with increased low affinity IL-2 binding sites, and constitutive overexpression of c-myc. Autocrine IL-2 dependent growth could be excluded. Expression of p56lck did not vary between the cell lines. We discuss the possibility that IL-2 independent growth may be associated with intracellular redistribution of p56lck from CD8alpha to IL-2Rbeta, thus generating constitutively active IL-2R. Ex vivo established tumor variants differed from their parental culture cell lines by their constitutive secretion of IFN-gamma. Since tumor variants derived from different organs of several individual mice showed this phenotype, we suggest that constitutive IFN-gamma secretion may be causally related to in vivo tumor formation, for example by induction of endothelial adhesion molecules facilitating tumor cell extravasation. A number of functional molecules did not significantly vary between cell lines, including components of alphabeta CD3 TCR, CD45, LFA-1, and MHC class I.