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 (Hashim oto C, et al. (1994) Biochim. Biophys. Acta 1221, 107-117) were determ ined in hamster livers, using a combination of immunological and bioch emical techniques. The IgG fraction from an antiserum raised against t he growth inhibitory factor with 37 kDa was shown to be highly specifi c for the antigen. The nuclear and cytosolic fractions demonstrated in hibitory effects on tell growth and Western blot analysis revealed tha t 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 fa ctor were further confirmed by immunochemical staining mediated throug h the immune IgG and an avidin-biotinylated horseradish peroxidase com plex, the parenchymal liver cells were clearly stained, but endothelia l and connective tissue cells were not. Although some staining was evi dent throughout the liver parenchyma, the hepatocytes with most intens ively stained nuclei were located in the periportal region. In the liv er from hamsters 6 days old or the regenerating hamster livers 3 days after partial hepatectomy, the staining intensity was low and the numb er 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 disappe ared from nuclei after incubation for 24 similar to 48 h. The extracts of hepatic nuclei from adult hamsters were immunoprecipitated with ei ther the anti-growth inhibitory factor IgG or a monoclonal antibody to the RE protein. The growth inhibitory factor and the RE protein copre cipitated in each case, implying that the proteins were complexed with each other in the nuclei. The RE protein family is composed of two se ts of species, an un- or underphosphorylated species and a hyperphosph orylated one. It was suggested that the factor bound preferentially to the un- or underphosphorylated member of the family.