Distribution and subcellular localization of a growth inhibitory factor in hamster liver and its intracellular partner(s)

The subcellular, intralobular distributions and intracellular partner(s) of a factor which inhibits the proliferation of cell growth (Hashimoto C. et al. (1994) Biochim. Biophys. Acta 1221, 107-117) were determined in hamster livers, using a combination of immunological and biochemical techniques. The IgG fraction from an antiserum raised against the growth inhibitory factor with 37 kDa was shown to be highly specific for the antigen. The nuclear and cytosolic fractions demonstrated inhibitory effects on cell growth and Western blot analysis revealed that both fractions contained the immunoreactive 37 kDa protein with the anti-inhibitory factor IgG but microsomal and mitochondrial fractions did not. The nuclear and cytoplasmic localization of the inhibitory factor were further confirmed by immunochemical staining mediated through the immune IgG and an avidin-biotinylated horseradish peroxidase complex, the parenchymal liver cells were clearly stained, but endothelial and connective tissue cells were not. Although some staining was evident throughout the liver parenchyma, the hepatocytes with most intensively stained nuclei were located in the periportal region. In the liver from hamsters 6 days old or the regenerating hamster livers 3 days after partial hepatectomy, the staining intensity was low and the number of hepatocytes with the inhibitory factor positive nuclei was very few compared with the adult hamster livers. In primary cultures of the isolated hepatocytes from adult hamster the inhibitory factor disappeared from nuclei after incubation for 24-48 h. The extracts of hepatic nuclei from adult hamsters were immunoprecipitated with either the anti-growth inhibitory factor IgG or a monoclonal antibody to the RM protein. The growth inhibitory factor and the RB protein coprecipitated in each case, implying that the proteins were complexed with each other in the nuclei. The RB protein family is composed of two sets of species, an un- or underphosphorylated species and a hyperphosphorylated one. It was suggested that the factor bound preferentially to the un- or underphosphorylated member of the family.