Differential spontaneous transformation in vitro of newly established mouse fibroblast lines carrying or lacking the viable yellow mutation (A^vy) of the mouse agouti locus

The pleiotropic effects of the viable yellow mutation (A^vy), an allele of the mouse agouti coat-color locus, include increased susceptibility to spontaneous and chemically induced tumors that affect a wide variety of tissues. As a first step toward understanding the molecular basis of this phenomenon, we established permanent fibroblast-like cell lines from newborn A^vy/a and control congenic a/a mice and compared their growth characteristics in vitro. From the VY/WffC3Hf/Nctr and YS/WffCH3f/Nctr-A^vy inbred strains, each of which carries the A^vy allele on a congenic background, 38 clonal A^vy/a and 16 clonal a/a lines were established. Regardless of inbred strain, all A^vy/a cell lines exhibited a significant degree of spontaneous transformation, as assessed by focus formation in monolayer culture, whereas none of the a/a cell lines formed foci in prolonged cultures. To test whether changes in dosage of the A^vy- or a-bearing chromosomes were related to these events, we analyzed each cell line with a closely linked molecular probe from the Emv-15 locus, which in the VY strain detects a restriction fragment length variant (RFLV) informative for the A^vy- and a-bearing chromosomes. Most of the transformed foci maintained heterozygosity for RFLVs detected by the probe, but two of the transformants lost the a-associated RFLV, and at least one of the transformants exhibited amplification of the A^vy-associated RFLV. When the transformants were analyzed with 5′ sequences derived from the recently cloned agouti gene, three of eight transformants lost the a-associated RFLV, and two of the transformants showed amplification of the A^vy-associated RFLV. Reverse transcriptase-polymerase chain reaction assays indicated that agouti RNA was detected in A^vy/a, not a/a cell lines. Surprisingly, some of the A^vy/a transformants lacked agouti RNA. These results suggest that deregulated expression of the A^vy allete is required for the initiation but not for the maintenance of transformation of the A^vy/a cell cultures. These cell lines may provide an in vitro culture system for studying the effect of the agouti gene on tumorigenicity as well as to potentially study other pleiotropic phenotypes. © 1996 Wiley-Liss, Inc