Growth state-dependent phenotypes of adult hepatocytes in primary monolayer culture.

Abstract

A proliferation-competent adult rat liver cell monolayer system has been analyzed for tissue-specific functions during its growth cycle. High levels of the adult (L type) form of pyruvate kinase (EC 2.7.1.40) and glutathione S-transferase B ("ligandin," EC 2.5.1.18) are observed during the early lag phase; they decline markedly during the logarithmic phase and reappear during the stationary phase. By contrast, elevated levels ofthe fetal (K type) form of pyruvate kinase and ai-fetoprotein production appear only after proliferation begins; this pattern diminishes slightly during stationary phase as the adult phenotype is restored. Albumin production continues throughout the entire growth cycle. These in vitro findings simulate those observed during hepatoproliferative transitions in the intact animal and, as such, constitute a developmental program for normal epithelial cells in primary culture. Many specialized animal cell functions are related to proliferative and developmental states (for review, see ref. 1). Much of this evidence comes from physiological studies of the liver (2-5). For example, the L-isozymic form of pyruvate kinase (EC 2.7.1.40) (6) and glutathione S-transferase B ("ligandin," EC 2.5.1.18) (7) are prominent hepatocyte properties associated with quiescent or adult states whereas the production of alfetoprotein (8) and the K-isozymic form of pyruvate kinase (6) are properties associated with proliferating or fetal states. Temporal aspects of the appearance or disappearance of differentiated properties during transitional states imply that distinct regulatory "programs" exist. However, the molecular and cellular bases of such programs are poorly understood and difficult to study in the intact animal. Hepatocyte tissue cultures offer a more direct approach (9) (15,16). Additional functional and proliferative properties of this system have been reported (9,15,17).Pyruvate Kinase Isozymes. At indicated times, culture media were removed and monolayers (10-20 dishes per point) were washed three times with 2 ml of ice-cold 10 mM phosphate-buffered saline, pH 7.0. Attached cells were scraped with a rubber policeman into 10 mM potassium phosphate, pH 7.0/5 mM magnesium sulfate/0.5 mM dithiothreitol/10% (vol/vol) glycerol and sonicated twice for 30 sec (Branson sonifier set at 50 W). Particulate material was removed by centrifugation at 100,000 X g at 40 for 1 hr. Total activity in high-speed supernatants was measured by using the coupled lactate dehydrogenase assay (18). The two different isozyme forms were measured by: (i) differential kinetic assay, based upon greater sensitivity of K-isozyme to inhibition by tryptophan (19) and (if) separation on DEAE-cellulose columns that, under the conditions used, bind only the L-isozymic form (20). Supernatant aliquots were applied to columns (10 X 0.6 cm), the K form was eluted with additional buffer, and the bound L form was eluted with buffer containing either 0.5 M KCl or with a 0-0.5 M KCl gradient. Enzyme activity and protein concentrations in co...

Tissue-specific gene expression in mouse hepatocytes cultured in growth-restricting medium.

“…Several of these transcriptional effects were similar to those observed following partial hepatectomy (17; note that clones 3 and 6 of references 17 and 34 correspond to AAT and Tf, respectively [11]) and also mimicked the transcription pattern of fetal liver (34). An increase in the expression of AFP and other fetal functions has been observed in cultured rat hepatocytes (29,37). However, a major difference is that SA transcription occurs in fetal liver and is sustained following partial hepatectomy, whereas it declined rapidly in the plated cells.…”

Thyroid hormone metabolism in primary cultured rat hepatocytes. Effects of glucose, glucagon, and insulin.

“…Our preliminary data suggest that rT3 degrading enzymes are steadily decreasing in the cultured cells (unpublished observation), which accounts at least in part for the accumulation of rT3 in the medium. In view of the fact that some enzymes undergo alteration from adult to fetal type in primary cultured hepatocytes (53,54), phenotypic changes might also occur in the deiodinases in primary cultured rat hepatocytes as seen in monkey hepatocarcinoma cells (22). Although one of the greatest disadvantages of the primary culture of adult rat hepatocytes is the difficulty in obtaining nonproliferating hepatocytes for a number ofdays, methods have been reported extending the cell survival for up to 10-14 d (54).…”

Fetal phenotypic expression by adult rat hepatocytes on collagen gel/nylon meshes.

“…In this respect, the changes in y-glutamyl transpeptidase activity, a1-fetoprotein production, and aldolase substrate specificity simulated those exhibited by hepatocytes in vivo during various hepatoproliferative conditions. Recently, Leffert et al (38) have independently described a liver cell culture system that, like the mesh system, displayed adult-to-fetal cell-like changes in apparent association with hepatoproliferative transitions. In comparison, the mesh/gel culture system exhibited kinetics for al-fetoprotein production similar to those found with this other system, and the alteration in the aldolase isozyme pattern of the hepatocytes on the mesh occurred at a time in culture similar to that shown by Leffert …”