Shift from polyploidizing to nonpolyploidizing growth in carcinogen-treated rat liver.

Liver growth patterns in normal and carcinogen-treated young Wistar Kyoto rats were analyzed in terms of absolute hepatocyte numbers and ploidy distributions, calculated from DNA measurements made by flow cytometry and microscope counts of binucleated cells. Polyploidizing growth was observed during normal liver development, dominated by progressive polyploidization and a decrease in the number of diploid cells. Nonpolyploidizing growth was seen during liver regeneration and after treatment with 2-acetylaminofluorene (AAF). This mode of growth was characterized by an increase in all mononucleated ploidy classes in the absence of net polyploidization (no increase in binucleated cells). Additional diploid proliferation was detected after initiation with diethylnitrosamine followed by promotion with AAF. This selectively expanding diploid hepatocyte population, which persisted after AAF withdrawal, could represent the AAF-promoted progeny of diethylnitrosamine-altered cells with constitutive nonpolyploidizing growth properties.

Uptake and degradation of asialo-orosomucoid in hepatocytes from carcinogen-treated rats.

The function of the asialo-glycoprotein receptor was studied in hepatocytes from carcinogen-treated rats. The cells were isolated at an early stage of carcinogenesis, when histochemically demonstrable enzyme-altered foci but no neoplastic nodules could be observed. The rate of uptake of a trace concentration of [125I]asialo-orosomucoid in hepatocytes from carcinogen-treated rats was significantly below the rate in normal hepatocytes. In contrast, the rate of degradation of endocytosed protein was not changed. The slower rate of uptake can be ascribed to an approximately 70% lower binding capacity in treated hepatocytes, whereas the association constant of the asialo-glycoprotein receptor is not changed. Hepatocytes from carcinogen-treated rats attached to a substratum of adsorbed asialofetuin at a slower rate than did normal cells, probably because of a lower density of asialofetuin receptors on the cell surface.

Use of glycyl-L-phenylalanine 2-naphthylamide, a lysosome-disrupting cathepsin C substrate, to distinguish between lysosomes and prelysosomal endocytic vacuoles.

Lysosome-disrupting enzyme substrates have been used to distinguish between lysosomal and prelysosomal compartments along the endocytic pathway in isolated rat hepatocytes. The cells were incubated for various periods of time with 125I-labelled tyramine cellobiose (125I-TC) covalently coupled to asialoorosomucoid (AOM) (125I-TC-AOM); this molecule is internalized by receptor-mediated endocytosis and degraded in lysosomes, where the degradation products (acid-soluble, radio-labelled short peptides) accumulate, Glycyl-L-phenylalanine 2-naphthylamide (GPN) and methionine O-methyl ester (MOM), which are hydrolysed by lysosomal cathepsin C and a lysosomal esterase respectively, both diffused into hepatocytic lysosomes after electrodisruption of the cells. Intralysosomal accumulation of the hydrolysis products (amino acids) of these substrates caused osmotic lysis of more than 90% of the lysosomes, as measured by the release of acid-soluble radioactivity derived from 125I-TC-AOM degradation. The acid-soluble radioactivity coincided in sucrose-density gradients with a major peak of the lysosomal marker enzyme acid phosphatase at 1.18 g/ml; in addition a minor, presumably endosomal, acid phosphatase peak was observed around 1.14 g/ml. The major peak of acid phosphatase was almost completely released by GPN (and by MOM), while the minor peak seemed unaffected by GPN. Acid-insoluble radioactivity, presumably in endosomes, banded (after 1 h of 125I-TC-AOM uptake) as a major peak at 1.14 and a minor peak at 1.18 g/ml in sucrose gradients, and was not significantly released by GPN. GPN thus appears to be an excellent tool by which to distinguish between endosomes and lysosomes. MOM, on the other hand, released some radioactivity and acid phosphatase from endosomes as well as from lysosomes.