"Contact inhibition" of alpha-fetoprotein synthesis and junctional communication in adult mouse hepatocyte culture

Synthesis of oncofetal serum protein alpha-fetoprotein (AFP) may be reexpressed in adult differentiated mouse hepatocytes both in regenerating liver and in primary monolayer culture of intact adult liver. We have found that appearance of AFP in these cultures was strongly correlated with the loss of junctional communication between hepatocytes as tested by the dye transfer method. When in hepatocyte culture the gradient of cell density was formed, and the cells in the center of the dense monolayer retained an epithelial morphology and junctional communication and were AFP-negative during 5 days of culture. At the periphery of the monolayer hepatocytes lost junctional communication by the third day of cultivation. They acquired fibroblast-like morphology, formed multilayered sheets, and started to produce AFP. These findings suggest that reexpression of AFP synthesis may be regulated through a process related to "contact inhibition" and junctional communication might play an important role in the phenomenon.     

Multiple sequential periods of DNA synthesis and quiescence in primary hepatocyte cultures maintained on the DMSO-EGF on/off protocol

Repeated periods of DNA synthesis activity (each period consisting of two to three cycles) separated by intervals of quiescence in primary rat hepatocytes can be stimulated by sequential addition and removal of 2% dimethyl sulfoxide (DMSO) in the presence of epidermal growth factor (EGF). Hepatocytes can be kept in nonproliferating cultures for 7 days in media supplemented with 2% DMSO and EGF. If DMSO is removed while EGF is maintained, rat and human hepatocytes enter a 3 to 4 day period of DNA synthesis that declines rapidly by days 4 and 5. If DMSO is reintroduced into cultures at that point, kept on for 3 more days and removed again, hepatocytes reenter into proliferation with another self-limited response of 3 to 4 days. Similar phenomena can seen with hepatocytes maintained in the presence of 3 mM phenobarbital. These protocols demonstrate that loss of responsiveness to mitogens in primary hepatocyte cultures is not an irreversible process. They also raise the possibility that signals for termination of DNA synthesis in hepatocytes emanate from hepatocytes themselves. These studies also suggest for the first time the possibility of designing in vitro systems that will allow clonal expansion of differential hepatocytes.     

Coexistence of expressed and non-expressed alpha-fetoprotein genes in somatic cell hybrids

Hybrids have been generated between mouse hepatoma cells, which actively synthesize alpha-fetoprotein (AFP), and adult hepatocytes, where AFP production is shut off. These hybrids maintain an active synthesis of mouse AFP. Using a specific radioimmunoassay, we found that rat AFP production is not activated. Southern blot analysis showed that mouse and rat AFP DNA sequences can be distinguished and that hybrid clones possessing something close to the complete chromosome sets of both parents have retained both parental AFP DNA sequences. Thus expressed and non-expressed AFP genes coexist in these hybrid cells as if their expression were dependent on a cis-acting event.     

Interactions of Polyoma and Mouse DNAs III. Mechanism of Polyoma Pseudovirion Formation

In primary mouse kidney cell cultures infected with polyoma virus, the processes leading to virion and pseudovirion formation were studied. By photometric DNA quantitation, we followed the kinetics of mouse and polyoma DNA synthesis and the formation of low-molecular-weight fragmented mouse DNA (mouse f-DNA). Virus was harvested at different times and analyzed for its proportion of pseudovirions. The following correlations between the intracellular events and the production of virions and pseudovirions were found. (i) Syntheses of cellular and viral DNA were closely linked, both in time and in rates of synthesis. (ii) An increase of mouse f-DNA could only be detected several hours after the onset of mouse and polyoma DNA replication; its formation coincided in time with the appearance of progeny virus. (iii) The proportion of pseudovirions was not dependent on the amount of mouse f-DNA formed, but seemed to be inversely related to the amount of viral DNA synthesized. This was borne out by experiments in which DNA synthesis was partially inhibited by mitomycin C or after a synchronized onset of DNA replication. Under these conditions, virus preparations with a two- to threefold increased proportion of pseudovirions were obtained as compared with those from uninhibited cultures. Virus isolated from the remaining monolayer always had a higher proportion of pseudovirions than virus isolated at the same time from the supernatant medium only; also, the proportion of pseudovirions increased slightly with time after infection. Thus, according to the experimental conditions used, polyoma virus preparations with a low (10 to 20%) or a high (60 to 80%) proportion of pseudovirions can be obtained.     

Effect of 2% dimethyl sulfoxide on the mitogenic properties of epidermal growth factor and hepatocyte growth factor in primary hepatocyte culture.

Two percent dimethyl sulfoxide (DMSO) reversibly inhibited DNA synthesis in primary rat hepatocyte cultures maintained with epidermal growth factor (EGF) or hepatocyte growth factor (HGF). These data suggest that, in vitro, DMSO is a non-specific inhibitor of hepatocyte proliferation, regardless of the stimulating mitogen. In addition, removal of DMSO from mitogen-free cultures resulted in an increase in DNA synthesis. Protein synthesis gradually but irreversibly declined in all cultures after DMSO removal. The relevance of these findings to regulation of hepatocyte growth is discussed.     

Gap junction expression and cell proliferation in differentiating cultures of Cx43 KO mouse hepatocytes

Primary cultures of adult mouse hepatocytes are shown here to reexpress differentiated hepatocyte features following treatment with 2% DMSO and 10(-7) M glucagon. To examine the roles of gap junctional communication during hepatocyte growth and differentiation, we have compared treated and untreated hepatocytes from connexin (Cx)32-deficient [Cx32 knockout (KO)] and wild-type mice. In untreated cultures, DNA replication of Cx32 KO hepatocytes was markedly higher than of wild types. Although Cx26 mRNA levels remained high at all time points in wild-type and Cx32 KO hepatocytes, Cx32 mRNA and protein in wild-type hepatocytes underwent a marked decline, which recovered in 10-day treated cultures. Increased levels of Cx26 protein and junctional conductance were observed in Cx32 KO hepatocytes at 96 h in culture, a time when cell growth rate was high. Treatment with DMSO/glucagon highly reinduced Cx26 expression in Cx32 KO hepatocytes, and such treatment reinduced expression of both Cx32 and Cx26 expression in wild types. Dye transfer was not observed following Lucifer yellow injection into DMSO/glucagon-treated Cx32 KO hepatocytes, whereas the spread was extensive in wild types. Nevertheless, high junctional conductance values were observed in treated cells from both genotypes. These studies provide a method by which the differentiated phenotype can be obtained in cultured mouse hepatocytes and provide in vitro evidence that expression of gap junctions formed of Cx32 are involved in the regulation of growth of mouse hepatocytes.     

Species differences in cytotoxic and genotoxic effects of phenacetin and paracetamol in primary monolayer cultures of hepatocytes

The cytotoxic and genotoxic effects of phenacetin and paracetamol were examined in monolayer cultures of hepatocytes isolated from the mouse, hamster, rat and guinea pig. No marked increase in unscheduled DNA synthesis (UDS) after exposing hepatocytes from any of the species to phenacetin was observed. At cytotoxic concentrations of paracetamol, an increased UDS in mouse hepatocytes in vitro was observed. Pretreatment of the mice by inducers of drug-metabolizing enzymes, such as 3-methylcholanthrene and Aroclor 1254, lowered the concentration threshold for the toxic responses. With rat hepatocytes only a minor increase in UDS was noted, while with hepatocytes from hamsters and guinea pigs in fact a decrease was seen. The narrow range observed between the cytotoxic and genotoxic effects of paracetamol makes it difficult to predict whether the initial DNA damage could lead to a mutation or whether the cells will die before the mutation is expressed. With respect to the cytotoxic effects, hamster hepatocytes were found to be most susceptible to paracetamol, followed by mouse, while rat and guinea pig were less affected. These data were in accordance with in vivo findings (Davis et al., 1974), indicating the potential value of hepatocyte culture when screening for possible liver toxic substances.     

Inhibitors of Protein Biosynthesis Can Stimulate Proliferation of Mouse Hepatocytes in Vitro

Hepatocyte proliferation in the liver regenerating after partial hepatectomy ceases when the organ is restored, and the mechanism of this phenomenon is still unclear. In the experiments on fusing hepatocytes from the regenerated mouse liver (15 days after partial hepatectomy) with NIH 3T3 mouse fibroblasts, we revealed no DNA synthesis in the nuclei of stimulated fibroblasts in heterokaryons (in the presence of hepatocyte nuclei), whereas DNA synthesis in nonfused cells was undisturbed. In this work, our purpose was to find out whether the suppression of DNA synthesis in heterokaryons could be due to the appearance in hepatocytes of some endogenous factors having an inhibitory effect on proliferation. To this end, hepatocytes from the mouse liver regenerated after partial hepatectomy were treated with cycloheximide for 1–4 h and were then fused with stimulated fibroblasts. Such a short-term treatment of hepatocytes with cycloheximide proved to result in the loss of their ability to inhibit DNA synthesis in the nuclei of stimulated or quiescent fibroblasts in heterokaryons, but hepatocytes proper actively proliferated in the medium with a low serum content (0.2%). When the mice with the liver regenerated after partial hepatectomy were treated with a single sublethal dose of cycloheximide (3 mg/kg), their hepatocytes taken two days after this treatment had no inhibitory effect. Puromycin, another inhibitor of protein synthesis, had the same effect on hepatocytes. These results may be interpreted as evidence that the final stage of liver regeneration after damage is controlled by the factors having a negative effect on cell proliferation.     

Control of DNA synthesis and ornithine decarboxylase activity by hormones and amino acids in primary cultures of adult rat hepatocytes

The conditions for stimulation of ornithine decarboxylase (ODC) and DNA synthesis in primary monolayer cultures of non-growing, highly differentiated hepatocytes from adult rats were compared. The syntheses of ODC and DNA were not stimulated by hormones on the 1st day of culture, but they were induced markedly by insulin (10⁻⁸ M) and epidermal growth factor (EGF, 0.1 μg/ml) in cells cultured for 40 h. The effects of insulin and EGF were synergistic, and the ODC activity as well as the DNA synthesis in the presence of these hormones was comparable to that of cultured hepatocytes from partially hepatectomized liver. Other factors had different effects on the two processes. Dexamethasone induced ODC slightly, but it inhibited DNA synthesis strongly. Putrescine inhibited ODC activity, but it had no effect on DNA synthesis. Asparagine and glutamine induced ODC activity, but they inhibited DNA synthesis; their inhibitory effects on DNA synthesis were specific to primary cultured liver cells and were not seen in an established rat liver cell line or in mouse L cells. These results show that although there is some correlation between ODC induction and DNA synthesis, the former is not essential for cell growth. There was no indication of cell division under conditions where maximal ODC induction and DNA synthesis were observed. Cytofluorometry of cells treated with insulin and EGF showed that the DNA content increased from 2 N to 4 N, and to 8 N in some cells. Therefore, under the present culture conditions, mature liver cells could enter G2 phase through S phase, but could not enter M phase.     

Differential proliferative response of cultured fetal and regenerating hepatocytes to growth factors and hormones

Upon epidermal growth factor (EGF) stimulation, fetal (20 days of gestation) and regenerating (44-48 h after partial hepatectomy) rat hepatocytes, isolated and cultured under identical conditions, increased DNA synthesis and entered into S-phase and mitosis, measured as [3H]thymidine incorporation and DNA content per nucleus in a flow cytometer, respectively. Fetal hepatocytes consisted of a homogeneous population of diploid (2C) cells. Two different populations of cells were present in regenerating liver, diploid (2C) and tetraploid (4C) cells, that responded to EGF. Glucagon or norepinephrine did not affect EGF stimulation of DNA synthesis in fetal liver cells, but they potentiated EGF response in regenerating hepatocyte cultures. Glucocorticoid hormones (dexamethasone) inhibited DNA synthesis in fetal hepatocyte cultures, an effect potentiated by the presence of glucagon or norepinephrine. In contrast, in regenerating hepatocytes, dexamethasone increased EGF-induced proliferation. EGF-dependent DNA synthesis was inhibited by TGF-beta in both fetal and regenerating cultured hepatocytes. TGF-beta action was partially suppressed by norepinephrine in regenerating hepatocytes, but was without effect in fetal hepatocyte cultures, whereas a synergistic action between TGF-beta and dexamethasone inhibiting growth in fetal but not in regenerating hepatocytes was found. Taken together, these results may suggest that there are significant differences between fetal and regenerating hepatocyte growth in their response to various hormones.     

Inhibition of alpha-fetoprotein synthesis in hepatocytes of adult mice by dextran sulfate in vivo and in vitro

Embryospecific serum protein alpha-fetoprotein (AFP) is known to be synthesized in the adult liver only during regeneration and development of hepatocellular carcinomas. It was shown that collagenase digestion of hepatic tissue followed by monolayer cell cultivation was a powerful inducer of AFP synthesis, more potent than the liver regeneration in vivo. The treatment of hepatocytes in culture with 50-100 micrograms/ml of dextran sulphate caused a remarkable inhibition of cell proliferation, formation of cord-like multicellular structures and reduction of AFP synthesis. Mouse liver regeneration after CCL4 poisoning was accompanied by a 1000-fold increase in blood AFP levels. Blood AFP levels and the content of AFP-positive cells in the liver tissue were maximum on the 3rd-4th day after poisoning. Injections of 50 micrograms of dextran sulphate per g body weight 3-5 h after poisoning and 24 and 48 h later caused nearly tenfold reduction in AFP blood level and a decrease in the content of AFP-positive cells in the liver on the 3rd day of regeneration.     

Synthesis of alpha-fetoprotein in primary cultures of hepatocytes from adult mice

Alpha-fetoprotein (AFP) and albumin synthesis in primary hepatocyte cultures of adult mice was studied by the immunoperoxidase techniques. Albumin was detectable in all hepatocytes during the cultivation period (from day 1 to day 8). AFP could be found regularly beginning from days 3-4 up to day 8. Attempts to obtain hepatocytes surviving for a longer period of time ended in failure. The number of AFP-containing hepatocytes in the culture ranged within single cells to about half of all the hepatocytes. The proliferating hepatocytes that contained both AFP and albumin were found in some of the experiments.     

Acetaminophen-induced hepatotoxicity of gel entrapped rat hepatocytes in hollow fibers

An important application of primary hepatocyte cultures is for hepatotoxicity research. In this paper, gel entrapment culture of rat hepatocytes in miniaturized BAL system were evaluated as a potential in vitro model for hepatotoxicity studies in comparison to monolayer cultures. After exposure for 24 and 48 h to acetaminophen (2.5 mM), gel entrapped hepatocytes were more severely damaged than hepatocyte monolayer detected by methyl thiazolyl tetrazolium (MTT) reduction, intracellular glutathione (GSH) content, reactive oxygen species (ROS) levels, urea genesis and albumin synthesis. CYP 2E1 activities detected by 4-nitrocatechol (4-NC) formation were higher in gel entrapped hepatocytes than in hepatocyte monolayers while the addition of CYP 2E1 inhibitor, diethyl-dithiocarbamate (DDC), more significantly reduced acetaminophen-induced toxicity in gel entrapped hepatocytes. In addition, protective effects of GSH, liquorice extract and glycyrrhizic acid against acetaminophen hepatotoxicity were clearly observed in gel entrapped hepatocytes but not in hepatocyte monolayer at an incubation time of 48 h. Overall, gel entrapped hepatocytes showed higher sensitivities to acetaminophen-induced hepatotoxicity than hepatocyte monolayer by a mechanism that higher CYP 2E1 activities of gel entrapped hepatocytes could induce more severe acetaminophen toxicity. This indicates that gel entrapped hepatocytes in hollow fiber system could be a promising model for toxicological study in vitro.     

Induction of unscheduled DNA synthesis in primary cultures of rat, mouse, hamster, monkey, and human hepatocytes

Variation in hepatic metabolism between species may be an important factor in the differences observed in chemical carcinogenesis. We examined 6 chemicals representative of 4 chemical classes in the in vitro hepatocyte DNA repair assay using cells isolated from the Fischer-344 rat, B6C3F1 mouse, Syrian golden hamster, cynomolgus monkey and from human liver. Hepatocytes were isolated by in situ or biopsy liver perfusion and incubated with [3H]-thymidine and the test chemical. Unscheduled DNA synthesis (UDS) was measured as net grains/nucleus (NG) by quantitative autoradiography. Qualitative and quantitative differences in UDS responses were observed for every chemical. Liver cultures isolated from the rat, mouse, hamster, human, and monkey and treated with aflatoxin B1 or dimethylnitrosamine all yielded dose-related increases in NG. Human, rat, and hamster hepatocyte cultures yielded positive responses following exposure to the aromatic amines 2-acetylaminofluorene, 4-aminobiphenyl, and benzidine, whereas cultures isolated from the monkey and mouse yielded less than 0 NG. Treatment with benzo[a]pyrene (BAP) produced strong positive responses in monkey and human hepatocyte cultures, weak positive responses in hamster cultures, and equivocal or negative responses in rat and mouse hepatocyte cultures. Hepatocyte function was assessed by measurement of DNA content, glutathione content, BAP hydroxylase activity, p-nitroanisole-O-demethylase activity, p-nitrophenol conjugation, and urea synthesis rates. The functional capabilities of isolated hamster, monkey, and human hepatocyte cultures do not appear to correlate with UDS responses observed for any compound; however, they indicate that the cultures were metabolically competent at the time of chemical exposure. These studies suggest that rat hepatocytes are a suitable model for human hepatocytes, whereas mouse and male monkey hepatocytes may be insensitive to aromatic amines.     

Synthesis of alpha-fetoprotein (AFP) and cell proliferation in regenerating livers of NMRI mice after partial hepatectomy. An immunohistochemical and autoradiographic study with 3H-thymidine

To get more information concerning the relation between cell proliferation and the synthesis of alpha-fetoprotein (AFP), liver regeneration and AFP production was followed simultaneously in short intervals in NMRI mice after two-thirds hepatectomy using autoradiographic and immunohistochemical methods. The results indicate that AFP synthesis is not linked closely to cell proliferation in the sense that each proliferating hepatocyte synthesizes AFP during a definite phase of the cell cycle. However, AFP production, as measured by the index of anti-AFP positive hepatocytes, occurs in a very small population of parenchymal cells when the bulk of the hepatocytes has just passed the S-phase and is considered to be in the G2-, M or early G1-phase of the cell cycle when the cells are still in an undifferentiate state.     

The early effects of chemical carcinogens on adult rat hepatocytes in primary culture. II. Effects on unscheduled DNA synthesis, cell division and alpha-fetoprotein production.

The effects of exposure of rat hepatocytes in primary maintenance culture to chemical carcinogens has been studied with respect cytotoxicity and alterations in mitotic index, unscheduled DNA synthesis and alpha-fetoprotein (AFP) production. All compounds tested produced cytotoxicity. Increases in mitotic index and unscheduled DNA synthesis and the production of AFP were observed after treatment of the cells with the carcinogens but not after treatment with the non-carcinogenic isomers. These increases were dose-dependent and depended on the time of exposure and the time incubated postexposure. The patterns of the increase in mitotic index and AFP production after cessation of carcinogen exposure were very similar, with the increase in mitotic index occurring slightly before that for the AFP production and it is suggested from this and other data that the production of AFP is dependent on the generation of a cell species functionally distinct from the non-dividing hepatocytes. It is also suggested that measurement of unscheduled DNA synthesis in conjunction with that of AFP production in cultured hepatocytes may be useful as part of a screening programme for chemical carcinogens.     

Magnesium and phosphate enrichment of culture medium stimulates the proliferation of epidermal cells from newborn and adult mice

The proliferation and differentiation of mouse epidermal cells can be sequentially analyzed by modification of extracellular calcium. Newborn cells cultured in low calcium medium (less than 0.1 mM) proliferate as a monolayer and maintain a typical basal cell phenotype in culture but have a limited proliferative capacity and short lifespan. Elevation of the magnesium content of the culture medium from 1 to 5 mM stimulated the proliferation of newborn mouse (1-3 days old) keratinocytes. Maximal DNA synthesis rates, as determined on day 5 of culture, were up to 2-3-fold higher in the magnesium-enriched cultures. Exposure to high magnesium caused 3-4-fold increases in the DNA content of newborn keratinocyte cultures, and extended the confluent phase of epidermal cell growth to over 10 days. Other divalent cations (strontium, copper, zinc, nickel, beryllium, and barium) did not improve keratinocyte growth in culture. Keratinocytes from the tail skin of adult (3 months old) mice displayed an absolute requirement for high phosphate in the culture medium. The medium containing an optimal (10 mM) phosphate concentration prevented the cell detachment caused by the standard low (1 mM) phosphate medium, and in combination with an elevated magnesium content (10-15 mM) it markedly increased both DNA synthesis rates and DNA content of the adult cell cultures. Optimally growing, newborn or adult cultures contained less cells in the G1 phase of the cell cycle and more cells in S and G2 +M. The addition of phosphate and magnesium per se did not induce keratinocyte differentiation and did not interfere with the high calcium (1 mM)-induced differentiation.