DIFFERENTIAL SPONTANEOUS TRANSFORMATION IN-VITRO OF NEWLY ESTABLISHEDMOUSE 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 alle le of the mouse agouti coat-color locus, include increased susceptibil ity to spontaneous and chemically induced tumors that affect a wide va riety of tissues. As a first step toward understanding the molecular b asis of this phenomenon, we established permanent fibroblast-like cell lines from newborn A(vy)/a and control congenic ala mice and compared their growth characteristics in vitro. From the VY/WffC3Hf/Nctr and Y S/WffCH3f/Nctr-A(vy) inbred strains, each of which carries the A(vy) a llele on a congenic background, 38 clonal A(vy)/a and 16 clonal ala li nes were established. Regardless of inbred strain, all A(vy)/a cell li nes exhibited a significant degree of spontaneous transformation, as a ssessed by focus formation in monolayer culture, whereas none of the a la cell lines formed foci in prolonged cultures. To test whether chang es in dosage of the A(vy)- Or a-bearing chromosomes were related to th ese events, we analyzed each cell line with a closely linked molecular probe from the Emv-15 locus, which in the VY strain detects a restric tion fragment length variant (RFLV) informative for the A(vy)- and a-b earing chromosomes. Most of the transformed foci maintained heterozygo sity for RFLVs detected by the probe, but two of the transformants los t the a-associated RFLV, and at least one of the transformants exhibit ed amplification of the A(vy)-associated RFLV. When the transformants were analyzed with 5' sequences derived from the recently cloned agout i gene, three of eight transformants lost the a-associated RFLV, and t wo of the transformants showed amplification of the A(vy)-associated R FLV. Reverse transcriptase-polymerase chain reaction assays indicated that agouti RNA was detected in A(vy)/a, not ala cell lines. Surprisin gly, some of the A(vy)/a transformants lacked agouti RNA. These result s suggest that deregulated expression of the A(vy) allele is required for the initiation but not for the maintenance of transformation of th e A(vy)/a cell cultures. These cell lines may provide an in vitro cult ure system for studying the effect of the agouti gene on tumorigenicit y as well as to potentially study other pleiotropic phenotypes, (C) 19 96 Wiley-Liss, Inc.