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

The pleiotropic effects of the viable yellow mutation (Avy), 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 Avy/a and control congenic a/a mice and compared their growth characteristics in vitro. From the VY/WffC3Hf/Nctr and YS/WffCH3f/Nctr-Avy inbre strains, each of which carries the Avy allele on a congenic background, 38 clonal Avy/a and 16 clonal a/a lines were established. Regardless of inbred strain, all Avy/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 Avy- 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 Avy- 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 Avy-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 Avy-associated RFLV. Reverse transcriptase-polymerase chain reaction assays indicated that agouti RNA was detected in Avy/a, not a/a cell lines. Surprisingly, some of the Avy/a transformants lacked agouti RNA. These results suggest that deregulated expression of the Avy allele is required for the initiation but not for the maintenance of transformation of the Avy/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.

Synthesis and disposition of 14C-labelled gentian violet in F344 rats and B6C3F1 mice.

The synthesis of [phenyl-U-14C]gentian violet from [U-14C]benzene is described. The 14C-labelled dye was administered by gavage to groups of male and female F344 rats which were killed at 2, 4, 14, 24 or 36 hr after the single dose. Radioactivity was measured in urine, and determined in faeces, liver, kidney, fatty tissue, gonads and muscle by combustion analysis. Residues were maximal at 4 hr in liver, kidney, muscle and gonads, and in fat they reached a plateau after 24 hr. Depletion half-lives for male and female livers were 14.5 and 17.0 hr, respectively. The 14C-labelled dye was also administered in multiple doses by gavage to both sexes of F344 rats and B6C3F1 hybrid mice for 7 days. The highest residue level was found in fatty tissue of females of both species, with a highly significant sex difference (P less than 0.01). Significant sex differences were also noted for residue levels in kidney and muscle tissue from both species and in mouse liver. Bile collected from cannulated rats contained 5.7-6.4% of a single oral dose of the dye. The results suggest that gentian violet is absorbed from the gastro-intestinal tract to a greater extent than has been reported for other triphenylmethane dyes.

The effect of isoproterenol on nuclear protein synthesis in electrostatically sorted rat hepatocytes.

Rat hepatocyte nuclei were electrostatically sorted and collected from specific regions of the cell cycle. Proteins were extracted from homogeneous populations of nuclei with cetyltrimethylammonium bromide and chromatographed by one- and two-dimensional acid-urea polyacrylamide gel electrophoresis. Small quantitative differences were observed in amido black stained two-dimensional patterns of the nuclear proteins of the G0 + G1 and G2 + M populations. However, comparison of the two-dimensional autoradiographs of 3H-leucine labeled nuclear proteins in these populations revealed dramatic differences. After isoproterenol administration, animals showed enhanced labeling of certain nuclear proteins in G2 + M populations, but no noticeable effect in protein labeling was observed in the G0 + G1 populations. Four days after the administration of isoproterenol, fluorescent histograms exhibited increased amounts of DNA in the G2 + M populations. Comparison of the total amino acid composition of two proteins from control animals revealed the distinct chemical contrast of these proteins. Both proteins possessed higher ratios of acidic to basic residues than anticipated for basic proteins. These procedures and findings should prove useful for further understanding of mechanisms of toxicant effects upon nuclear protein metabolism.