Mouse hepatocytes retain the expression of the main differentiation markers during culturing on collagen-chitosan matrices.

Mouse hepatocytes cultured on artificial 3D collagen-chitosan biopolymer matrices retained the expression of hepatocyte markers for 14 days.

[Different sensitivity of inbred mice to hepatocarcinogen ortho-aminoazotoluene may be due to differences in the negative control mechanisms of hepatocyte proliferation]. / Raznaia chuvsvitel'nost' myshei inbrednykh linii k gepatokantserogenu orto-aminoazotoluolu mozhet byt' sviazana s razlichiiami v mekhanizmakh negativnogo kontrolia prolifaratsii gepatotsitov.

A most convenient model to study mechanisms of live organism response to chemical carcinogens is tumor induction in murine liver by aminoazodyes, in particular by ortho-aminoazotoluene (OAT). We studied both early and late stages of hepatocarcinogenesis on several lines of inbred mice differing in sensibility to OAT. By means of autoradiography, we examined proliferative activity of hepatocytes obtained from the liver of sensitive (A/He, DD, SWR) and resistant to OAT AKR, CC57Br, BALB/c lines of mice, which were injected carcinogen. The level of p53, p21Cip1, bax, mdm2, cyclin G, gadd45 genes expression in the liver of mice of different lines given OAT injection was studied by multiplex PCR method. Carcinogen caused a decrease of hepatocyte proliferative activity induced by partial hypatectomy (PHE), and an increase in p53, p21Cip, bax, mdm2, and cyclin G genes within mice of A/He, DD and SWR lines. Cell fusion experiments on hepatocytes obtained from regenerating murine liver sensitive to A/He line carcinogen and given long-time OAT administrations with resting and proliferating fibroblasts of NIH 3T3 mice revealed no obvious suppression of DNA synthesis in heterokaryons. Unlike, in fusion experiments on serum-stimulated fibroblasts with hepatocytes obtained from the liver of BALB/c line mice also given OAT suppression of DNA synthesis in stimulated fibroblasts in heterokaryons was observed 15 days following PHE. These results enable us to conclude that OAT administrations break negative endogenous mechanisms of hepatocyte proliferation control in the liver of mice sensitive to carcinogenes.

[Inhibitors of protein biosynthesis can stimulate proliferation of mouse hepatocytes in vitro]. / Ingibitory biosinteza belka mogut stimulirovat' proliferativnuiu aktivnost' gepatotsitov myshi 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 o 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.

Dobutamine prevents experimental postintoxication liver cirrhosis in mice.

Various chronic inflammatory and necrotic processes in the liver parenchyma are accompanied by pathological morphofunctional changes, which are associated with hepatocyte death and hyperplasia of the connective tissue. Regeneration of the liver parenchyma should include not only prevention of fibrosis, but also stimulation of hepatocyte proliferation. The adrenoceptor agonist dobutamine stimulated proliferative activity of cultured hepatocytes and prevented the development of postintoxication liver cirrhosis in mice produced by chronic poisoning with CCl(4).

[Hepatocytes in heterokaryons can suppress entry into the S-period of fibroblast nuclei from murine NIH 3T3 cells]. / Gepatotsity v geterokarionakh mogut podavliat' vstuplenie v S-period iader fibroblastov myshi linii NIH 3T3.

Mouse liver regeneration after partial hepatectomy can be considered as a spectacular example of controlled tissue increase. In this study serum-deprived (0.2%) resting and serum-stimulated (10%) proliferating NIH 3T3 mouse fibroblasts were fused with primary hepatocytes isolated from normal (intact) and regenerating adult mouse liver at different times after partial hepatectomy (1-15 days) to elucidate mechanisms of liver cell proliferation cessation at the regeneration end. DNA synthesis was investigated in the nuclei of heterokaryons and non-fused cells using radioautography. Hepatocytes isolated from regenerating liver within 1-12 days following operation did not retard the entry of stimulated fibroblast nuclei into the S-period. In contrast, hepatocytes isolated within 15 days after hepatectomy were found to have inhibitory effect on the entry of stimulated fibroblast nuclei into the S-period in heterokaryons. Preincubation of these hepatocytes with cyclocheximide for 2-4 h abolished their ability to suppress DNA synthesis in stimulated fibroblast nuclei in heterokaryons. Possible reasons of inhibitory effect of differentiated cells in heterokaryos are discussed. The data obtained enable us to conclude that the mechanism of proliferative process control in regenerating hepatocytes seems to be stopped being affected by the intracellular growth inhibitors, whose formation depends on protein synthesis.

[The effect of intracellular protease inhibitors on DNA synthesis in fibroblasts from the NIH 3T3 mouse strain]. / Vliianie ingibitorov vnutrikletochnykh proteaz na sintez DNK v fibroblastakh myshi linii NIH 3T3.

The enhancement of catabolic reactions of cell macromolecules, primarily proteins, raises the question about the role of active proteolysis and particular kinds of proteases in the process leading to cell proliferation retardation. The application of highly specific inhibitors of intracellular proteases helps to answer this question even in part. It has been shown that a short-term (2-6 h) treatment of resting cells of line NIH 3T3 with inhibitors of lysosomal cysteinous proteases--cystatine or leupeptin, as well as with inhibitors of non-lysosomal Ca(2+)-activated cystein proteases--L-cystamine and L-cystin--enhances the cell proliferative response on the serum and even stimulates tritiated thymidine incorporation in the cells cultured in the medium with a low (0.2%) serum content. Unlike, the treatment of resting cells with the lysosomotropic agent chloroquin, known to shut down all lysosomal hydrolases, reduces the cell proliferative response of the serum. In the experiments of fusing the resting and stimulated cells, it was found that the pretreatment of the former with inhibitors of lysosomal proteases enhanced the index of labeled nuclei in the latter, while observed in dikaryons, compared to the control, although failed to remove totally the effect of DNA synthesis inhibition in heterokaryons (Setkov et al., 1984; Setkov, Kazakov, 1989). A conclusion is made that the short-term metabolic shift in resting cells towards anabolism may result in that resting cells commit a cycle, and that an enhanced activity of cystein proteases is one of specific metabolic processes in resting cells leading to proliferation retardation.

Brief exposures of resting fibroblasts to okadaic acid stimulate DNA synthesis.

To study further the factors providing for cellular quiescence, we used okadaic acid (OA) at concentrations (0.1, 1, 10 or 100 nM) inhibiting type 1 and/or type 2A protein phosphatases in mammalian cell cultures. Brief (2 h) exposure of resting (0.2% serum for 72 h) NIH 3T3 mouse fibroblasts to OA with subsequent incubation of cells in a medium with 0.2% serum, stimulated DNA synthesis at all concentrations studied. Maximal stimulation was observed following pre-incubation of resting cells with 10 nM OA. Treatment of cycling cells (10% serum) with OA (2 h pulses at 12 h intervals for 72 h) prevented their exit to the resting state on transfer to a medium with 0.2% serum. Brief exposures of resting cells to OA did not affect the rate of protein synthesis. OA pulses in the late pre-replicative period had no effect on the entry of serum-stimulated cells into the S phase. Cell fusion experiments with resting (serum-deprived) and proliferating (serum-stimulated) NIH 3T3 cells, using radioautography with a double-labelling technique, revealed that pre-incubation of resting cells with OA for 2 h before and after fusion abrogates their ability to suppress the onset of DNA synthesis in the nuclei of proliferating cells in heterodikaryons. The results indicate that protein phosphatases of type 1 and/or 2A may be involved in the growth-arrest machinery that provides for cellular quiescence.

[DNA synthesis in heterodikaryons obtained by the fusion of long-term stimulated cells of the NIH 3T3 line with short-term stimulated cells]. / Sintez DNK v geterodikarionakh, poluchennykh pri sliianii dlitel'no stimulirovannykh kletok linii NIH 3T3 s kratkovremenno stimulirovannymi kletkami.

Serum-deprived (0.2%) resting NIH 3T3 mouse fibroblasts, stimulated for 0.5-4.0 h by serum (10%) or PDGF (beta-beta homodimer) (early cells), were fused with stimulated cells taken 10 h after changing the medium to one containing 10% serum DNA synthesis was investigated in the nuclei of monokaryons, homodikaryons and heterodikaryons, using radioautography with the double-labeling technique. Preincubation of resting cells with serum for 3 h or with the competence factor PDGF for 1.0-1.5 h abolished their ability to suppress DNA synthesis in late stimulated nuclei in heterodikaryons. Actomycin D restores the inhibitory capacity of resting cells stimulated by PDGF but not by serum. The results give evidence that the acquirement by resting cells of competence for DNA replication includes as a necessary step the down-regulation of intracellular growth inhibitors, and thus serum can overcome the influence of endogenous growth inhibitors, at any rate, by two ways.

Transient inhibition of protein synthesis induces expression of proto-oncogenes and stimulates resting cells to enter the cell cycle.

There is evidence that resting cells are able to produce molecules with antiproliferative activity, some of which behave as short-lived repressor proteins. We suggest that transient inhibition of protein synthesis in resting cells would lead to a decrease in the levels of these negative growth regulators and might, therefore, promote mitogenic responses. We report that treatment of resting (serum-deprived) NIH 3T3 cells with cyclocheximide (CH) or puromycin induces expression of c-fos, c-jun and c-myc proto-oncogenes in a manner similar to that of platelet-derived growth factor (PDGF). Actinomycin D (Act D) abrogates the induction of proto-oncogene expression. Transient inhibition of protein synthesis by CH or puromycin also induces the resting NIH 3T3 and C3H 1OT1/2 cells to enter the cell cycle. Inhibition of new RNA or protein synthesis abolishes the proliferative response. These findings show that control mechanisms at both transcriptional and translational levels are operative in the resting cells treated with protein synthesis inhibitors. Cell fusion experiments with resting and serum-stimulated NIH 3T3 cells revealed that brief pre-incubation of resting cells with either PDGF, CH or puromycin abrogates their ability to suppress the onset of DNA synthesis in the nuclei of stimulated cells in heterodikaryons. However, the abrogative effect of PDGF disappeared in the presence of Act D, whereas the effects of protein synthesis inhibitors did not, indicating their independence of the induction of transcription. The data suggest that the observed effects of protein synthesis inhibitors are connected with elimination of some short-lived negative growth regulators, since a brief translational arrest is sufficient for the resumption of DNA synthesis in the nuclei of stimulated cells blocked by resting cells in heterodikaryons.