Some peculiarities of gene expression in inbred and hybrid rats under normal conditions and after partial hepatectomy

The peculiarities of gene expression in line and hybrid rats in the normalcy and after partial hepatectomy were studied. It was shown that the relationship between the RNA synthesis rate in chromatin and RNA transport in the cytoplasm as well as the correlation between the RNA transport rate and the activity of the protein-synthesizing mechanism vary in animals with different genotypes. After partial hepatectomy the hybrids have their S-period of the cell cycle earlier than the line animals. The hybrids do not differ from the parental forms in the rate of RNA chromatin synthesis; however, the newly synthesized RNA of the hybrids in transported into the cytoplasm 5.7 and 5 times faster than that of the lines Vistar and August. The rapidly transported RNA of hybrids is likely to have a more prolonged life as compared to the line animals. After partial hepatectomy an activation of the whole gene expression system occurs. The differences in the gene expression system of hepatocytes of inbred and hybrid animals in the regenerating liver become less pronounced.     

The Ag-NOR proteins and transcription and duplication of ribosomal genes in mammalian cell nucleoli

The relationship between the Ag-NOR (silver-stained Nucleolar Organizer Region) proteins and the functional-structural organization of the nucleolar ribosomal chromatin was studied in regenerating and cortisol-stimulated rat hepatocytes. Statistical analysis of Ag-NOR proteins, carried out with an automated image analyzer, indicated that in regenerating rat hepatocytes the quantity of Ag-NOR proteins mainly increased between the 4th and 12th h of regeneration, reaching a level twice that of resting hepatocytes. Also the synthesis of pre-ribosomal RNA (pre-rRNA) was stimulated after the 4th h of regeneration. Cycloheximide administered to rats at a dose of 0.025 mg/100 g body weight (bw) prevented any increase in Ag-NOR proteins but did not hinder the stimulation of pre-rRNA synthesis. In 8 h cortisol-stimulated hepatocytes no significant change in amount of Ag-NOR protein was observed whereas pre-rRNA synthesis was highly increased as in 12 h regenerating hepatocytes. These results indicated that in rat hepatocytes Ag-NOR proteins and stimulation of pre-rRNA synthesis are not related. The relationship between the Ag-NOR proteins and the distribution of the completely extended intranucleolar ribosomal chromatin was also studied in regenerating rat hepatocytes. At 12 h after partial hepatectomy an increased amount of completely extended ribosomal chromatin was observed, contemporaneously with an increased quantity of Ag-NOR proteins. These ribosomal chromatin changes preceded the beginning of DNA synthesis and were prevented by cycloheximide-induced inhibition of protein synthesis.     

Effect of the activation of macrophages on the course of regeneration of rat liver following partial hepatectomy

Stimulation of the Kupffer cells with E. coli endotoxin (the purified lipopolysaccharide) or with prodigiosan (a polysaccharide from Serratia marcescens) 24 h before partial hepatectomy (resection of 65-70% of the liver) stimulated and intensified the onset of liver regenerative activity (evaluated from changes in liver DNA synthesis, the H5 labelling index and the mitotic activity of the hepatocytes). Liver DNA synthesis increased together with the dose of endotoxin (i.v., from 25 to 1000 micrograms/kg body weight). If E. coli endotoxin was injected during or 3 h after partial hepatectomy, partial inhibition of liver DNA synthesis was observed. In mice stimulated with zymosan (a polysaccharide isolated from yeast), administered 5 days before performing partial hepatectomy, proliferation of the hepatocytes (evaluated from changes in the 3H labelling index and in the mitotic activity of the hepatocytes) was evaluated. The results confirm that proliferation is correlated to the state of reactivity of the Kupffer cells.     

Metabolic responsiveness to phorbol ester and activity of protein kinase C in isolated hepatocytes from partially hepatectomized rats

The ability of isolated rat hepatocytes to respond to phorbol-12-myristate-13-acetate (PMA) with acute stimulation of de novo fatty acid synthesis was markedly depressed at 4, 22 and 48 h after partial hepatectomy (PH). This desensitization was not due to surgical stress as shown by comparison with hepatocytes from sham-operated animals. Moreover, the total activity of protein kinase C (PK-C), the principal phorbol ester receptor, was not down-regulated at 22 h after partial hepatectomy. Partial hepatectomy rather caused a small but distinct shift in subcellular PK-C distribution toward the particulate fraction thereby suggesting a modest activation of PK-C. We conclude that the PH-induced desensitization to PMA occurs at a point beyond PK-C activation.     

Intracellular distribution of transglutaminase activity during rat liver regeneration

Transglutaminase and ornithine decarboxylase activities have been assayed at intervals after partial hepatectomy in regenerating liver cells fractionated to obtain nuclear, cytoplasmic-particulate, and cytoplasmic-soluble fractions. Ornithine decarboxylase activity, localized entirely in the cytoplasmic fractions, undergoes a dramatic induction during the first 4 h after partial hepatectomy and remains elevated. This induction is very sensitive to inhibition by cycloheximide and actinomycin D, as previously reported. Transglutaminase activity is localized in both the cytoplasm and the nucleus with the highest specific activity in the nucleus. Nuclear transglutaminase activity approximately doubles in the first 2 h of liver regeneration, apparently as a result of a translocation of enzyme from the cytoplasm to the nucleus. Inhibitor studies indicate that the translocation is not dependent upon protein or RNA synthesis. In the first 2 h, actinomycin D slightly activates transglutaminase activity in the cytoplasmic-particulate and nuclear fractions. Only at 4 h after the onset of regeneration do actinomycin D and cycloheximide show some inhibition of transglutaminase activity indicating de novo synthesis at this time. A broad increase of transglutaminase activity occurs from hours 12–16 to hour 32 after partial hepatectomy in the nuclear and cytoplasmic-particulate fraction. These data suggest the existence of a function for transglutaminase in the nucleus of rat liver cells.     

Intracellular distribution of newly formed hepatocyte RNA in the 1st hours following partial hepatectomy

Two periods in the activity of cell are distinguished during the first three hours after partial hepatectomy. The first period (about 0.5 h) is characterized by the activation of RNA synthesis in all the compartments of the cell, including mitochondria, and by approach of the latter to the nucleus, which is probably induced by the demand of a great quantity of energy for both the present and subsequent intranuclear synthesis. These data have permitted referring the response of hepatocytes in the first period to nonspecific ones. The second period (0.5-3 h) is distinguished by the differentiated response of hepatocytes to the proliferative stimulus.     

Effect of the parenteral administration of energy substrates in different postoperation phases on the initiation and development of liver regeneration in rats subjected to partial hepatectomy

DNA specific activity in the liver, the total DNA content of the liver and the mitotic index of the hepatocytes were studied after the infusion of glucose or lipid emulsions in female laboratory rats with a mean pre-operation weight of 250 +/- 30 g after partial (65-70%) hepatectomy (PH). The infusions were administered in the early prereplication phase (the 1st to 6th hour after the operation), in the late prereplication phase (the 7th to 12th hour after the operation), or continuously from the 1st to the 12th, or the 1st to the 24th, hour after partial hepatectomy. The effect of these parenterally administered energy substrates on the initiation of liver regeneration was evaluated 18 and 24 hours after partial hepatectomy. The results indicate that the infusion of glucose, in any interval after the operation, inhibited the initial phases of liver DNA synthesis (18 h after PH), but not its further development (24 h after PH). Neither the mitotic index of the hepatocytes, nor the total DNA content of the liver differed from the control groups in the case of rats given a glucose infusion. In the experimental groups given lipid emulsions, inhibition of liver DNA synthesis was recorded 18 h after PH only when the infusions were given from the 1st to the 12th or the 1st to the 18th hour after PH. The total DNA content of the liver 18 h after PH was raised in all the experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

The control of nucleic acid and nicotinamide nucleotide synthesis in regenerating rat liver

1. The effect of injecting nicotinamide on the incorporation of [(14)C]orotate into the hepatic nucleic acids of rats after partial hepatectomy was investigated. 2. At 3h after partial hepatectomy the rapid incorporation of [(14)C]orotate into RNA, and at 20h after partial hepatectomy the incorporation of [(14)C]orotate into both RNA and DNA, were inhibited in a dose-dependent fashion by the previous injection of nicotinamide. 3. The injection of nicotinamide at various times before the injection of [(14)C]orotate at 20h after partial hepatectomy revealed an inhibition of the incorporation of orotate into RNA and DNA which was non-linear with respect to the duration of nicotinamide pretreatment. 4. The induction of a hepatic ATP depletion by ethionine demonstrated that the synthesis of hepatic NAD and NADP in partially hepatectomized rats was more susceptible to an ATP deficiency than in control rats. 5. The total hepatic activity of ribose phosphate pyrophosphokinase (EC 2.7.6.1) was assayed at various times after partial hepatectomy and found to be only marginally greater than the maximum rate of hepatic NAD synthesis induced in vivo by nicotinamide injection between 12 and 24h after partial hepatectomy. 6. It is suggested that a competition exists between NAD synthesis and purine and pyrimidine nucleotide synthesis for available ATP and particularly 5-phosphoribosyl 1-pyrophosphate. In regenerating liver the competition is normally in favour of the synthesis of nucleic acid precursors, at the expense of NAD synthesis. This situation may be reversed by the injection of nicotinamide with a subsequent inhibition of nucleic acid synthesis.     

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.     

Role of cell-surface modulator of DNA synthesis in liver regeneration

A cell-surface modulator of DNA synthesis by cultured rat hepatocytes was studied in relation to the liver regeneration process. When rat hepatocytes isolated 24 h after partial hepatectomy were cultured, the first burst of DNA synthesis peaked at 5-8 h and declined until 24 h, followed by the second burst. Rat liver plasma membranes added 2 h after plating inhibited only the second burst, while in the case of the normal hepatocytes where the DNA synthesis began to increase after 5 h, this inhibition was observed at 16 h but not at 8 h. The inhibition did not differ when the membranes obtained from regenerating livers 12 h after partial hepatectomy were used. Epidermal growth factor binding to the cultured hepatocytes was not hindered by the membranes. These results suggest that the modulator inhibits hepatocyte proliferation at the early G1-phase of the cell cycle and that its action might be controlled by other factors in the process of liver regeneration.     

Specific inhibition of the synthesis of putrescine and spermidine by 1,3-diaminopropane in rat liver in vivo.

Chronic administration of 1,3-diaminopropane, a compound inhibiting mammalian ornithine decarboxylase (EC 4.1.1.17) in vivo, effectively prevented the large increases in the concentration of putrescine that normally occur during rat liver regeneration. Furthermore, repeated injections of diaminopropane depressed by more than 85% ornithine decarboxylase activity in rat kidney. Administration of diaminopropane 60 min before partial hepatectomy only marginally inhibited ornithine decarboxylase activity at 4 h after the operation. However, when the compound was given at the time of the operation (4 h before death), or any time thereafter, it virtually abolished the enhancement in ornithine decarboxylase activity in regenerating rat liver remnant. An injection of diaminopropane given 30 to 60 min after operation, but not earlier or later, depressed S-adenosyl-L-methionine decarboxylase activity (EC 4.1.1.50) 4 h after partial hepatectomy. Diaminopropane likewise inhibited ornithine decarboxylase activity during later periods of liver regeneration. In contrast to early regeneration, a total inhibition of the enzyme activity was only achieved when the injection was given not earlier than 2 to 3 h before the death of the animals. Diaminopropane also exerted an acute inhibitory effect on adenosylmethionine decarboxylase activity in 28-h regenerating liver whereas it invariably enhanced the activity of tyrosine aminotransferase (EC 2.6.1.5), used as a standard enzyme of short half-life. Treatment of the rats with diaminopropane entirely abolished the stimulation of spermidien synthesis in vivo from [14C]methionine 4 h after partial hepatectomy or after administration of porcine growth hormone. Both partial hepatectomy and the treatment with growth hormone produced a clear stimulation of hepatic RNA synthesis, the extent of which was not altered by injections of diaminopropane in doses sufficient to prevent any enhancement of ornithine decarboxylase activity and spermidine synthesis.     

Hepatocytes in heterokaryons can suppress entry into the S-period of fibroblast nuclei from murine NIH 3T3 cells

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.     

Expression of hepatocyte growth factor mRNA in regenerating rat liver after partial hepatectomy.

Hepatocyte Growth Factor (HGF) is a potent complete mitogen for primary cultures of hepatocytes in vitro. There is strong evidence that this novel growth factor may mediate hepatocyte regeneration after liver damage. We have shown previously that the amount of immunoreactive HGF markedly increases in the serum of rats soon after partial hepatectomy or CCl4 administration. In the present paper, we demonstrate that the level of HGF mRNA in rat liver also dramatically increases from 3 to 6 hours post hepatectomy, peaks at 12 hr and gradually returns to undetectable levels by 72 to 96 hours post hepatectomy. In separate experiments, DNA synthesis (in vivo) was determined in rat liver remnants after partial hepatectomy. DNA synthesis peaked 24 hr after hepatectomy, 12 hr after the peak of HGF mRNA expression. These results suggest that HGF may be one of the major early signals that triggers hepatocyte proliferation during liver regeneration.     

The role of short-lived lesions in the induction of micronuclei in rat liver by ethylnitrosourea and methyl methanesulphonate: the importance of experimental design

Rats received single injections of ethylnitrosourea (ENU) or methyl methanesulphonate (MMS) at the peak of DNA synthesis after partial hepatectomy. Hepatocytes were isolated 1–4 days later, and analysed for presence of micronuclei. With both chemicals, frequencies of micronucleated hepatocytes were increased in a dose-dependent manner, but ENU proved to be both more effective (6 times; based on molar dose) and more efficient (18 times; based on total DNA alkylation) than MMS.

In general, the micronucleus frequency was relatively low at 1 day after injection, then increased to reach a maximum at days 2 or 3 (depending on the dose), after which it decreased strongly in the case of MMS or remained stable in the case of ENU. The result with ENU is interpreted as a balance between loss and/or dilution of micronucleated hepatocytes and simultaneous formation of new ones. The present observations are in line with our earlier conclusion that ENU, in contrast with MMS, is able to induce persistent preclastogenic lesions in rat hepatocytes.

ENU also proved to be more effective and efficient than MMS with respect to the formation of micronuclei in bone marrow cells. Our results with ENU and MMS indicate that administration of the genotoxin at the peak of DNA synthesis after partial hepatectomy, instead of before hepatectomy, increases the sensitivity of the liver micronucleus assay at least in the case of directly acting chemicals.     

Re-utilization of pyrimidine nucleotides during rat liver regeneration.

The changes in the specific radioactivities of the pool of total acid-soluble uridine nucleotides and of uridine and cytidine components of total cellular and nuclear RNA were monitored in regenerating rat liver for 12 days after partial hepatectomy. Evidence is presented for the re-utilization of pyrimidine nucleotides derived from cytoplasmic RNA degradation for the synthesis of new RNA. The extent of recycling was assessed and the true rate of rRNA turnover determined more accurately. The reutilization of the uridine components of RNA was 7.0%/day during the proliferative and 3.2%/day during the post-proliferative phase, whereas that of the cytidine nucleotides was more pronounced (9.6%/day and 18.1%/day respectively). The results reveal the existence of partial compartmentalization of pyrimidine ribonucleoside triphosphate pools in the nucleus and cytoplasm of rat liver cells.     

Cytophotometric analysis of the protein and nucleic acid content of the nucleus and cytoplasm of resting and proliferation-activated hepatocytes in the mouse

Mouse hepatocytes, quiescent and activated to proliferation after hepatectomy, were investigated using cytophotometry in the ultraviolet region of selective absorbtion of nucleic acids, and then were examined in the visible region of spectrum after cytochemical reaction for total protein. Some features of RNA and protein accumulation were revealed in the nucleus and cytoplasm of activated cells of different ploidy. The activation resulted in the reduction of histone content and in the increase in the nucleus: cytoplasm ratio in cells of all the ploidy classes.     

Perichromatin fibrils and chromatin ultrastructural pattern

The relationship between the synthesis of acidic nuclear proteins, phosphoproteins, RNA and chromatin ultrastructural pattern was studied in regenerating rat hepatocytes after partial hepatectomy. α-Amanitin induced, as early as 30 min after injection, a reduction of RNA synthesis to about 50% of the control level; the degree of inhibition had remained the same at 2 h after poisoning. No change was detected either in acidic nuclear protein synthesis or in phosphorylation for the whole time examined. The DNA-containing structures, demonstrated by the Gautier staining procedure, were in a dispersed pattern either in untreated regenerating hepatocytes or 30 min after α-amanitin administration to rats; but they did appear in a condensed form 1 h and more especially 2 h after toxin injection. In untreated regenerating hepatocytes, the regressive EDTA staining method for RNP revealed a large quantity of perichromatin fibrils which remained unchanged 30 min after α-amanitin treatment and were diminished at 1 h and strongly reduced 2 h thereafter.Cycloheximide treatment promptly reduced the synthesis of nuclear acidic proteins while leaving unchanged the synthesis of RNA; the quantity of perichromatin fibrils and the loosened appearance of DNA-containing structures were the same as in the control rat nuclei.Our results showed that the ultrastructural pattern of chromatin was not directly related either to the synthesis of RNA or to acidic nuclear proteins or to the phosphorylation of phosphoproteins; on the contrary, a strict relationship with the quantity of perichromatin fibrils was demonstrated. The possible interaction of perichromatin fibrils with other chromatin components was discussed as a possible regulatory mechanism of chromatin pattern.