DNA synthesis and mitotic activity in short-term cultures of hepatocytes isolated from regenerating rat liver

Cell suspensions were prepared from normal and regenerating liver of adult rats by perfusion with a calcium-chelating agent (EGTA), collagenase and hyaluronidase, and the cells were incubated in culture medium. In cultures prepared from regenerating liver at 20 h after partial hepatectomy, 23 ± 4% of parenchymal cells initially incorporated [³H]TdR. This incorporation was shown to reflect semiconservative DNA replication. At least some parenchymal cells were able to complete their DNA synthesis and to progress through G2 and mitosis. Numbers of hepatocytes in mitosis increased up to 12 h of culture. On the other hand, no entry of hepatocytes into the S period was detectable in cultures prepared from normal or regenerating liver.     

DNA synthesis and mitotic activity in cells of regenerating rat liver following x-irradiation in stages GO and Gl]

Effect of X-rays on DNA synthesis and mitotic activity of regenerating liver cells has been studied. The irradiation was performed at a dose of 630 rad before hepatectomy and 2.5 and 6 hours after the stimulation of liver. With the stimulated liver being irradiated, the number of cells synthetizing DNA and entering into mitosis was seen reduced almost twice, whereas DNA synthesis and entering into mitosis were delayed, resp., by 4 and 6 hours. Irradiation of liver before the stimulation brings about a delay in DNA synthesis and in start of mitosis by 2 and 4 hours, resp., without reducing the numbers of cells capable to synthesize DNA and to enter mitosis.     

DNA synthesis in the mono- and binuclear cells of regenerating rat liver after x-ray irradiation

The effect of X-irradiation on the dynamics of DNA synthesis during the S-period in bi- and mononucleated of regenerating rat liver was studied autoradiographically and microphotometrically. Rats were treated with X-rays at doses 3.84 X 10(-2), 15.48 X 10(-2), and 30.96 X 10(-2) Kl/kg 23 hours after a partial hepatectomy, and were sacrificed one hour after irradiation. In the control liver the rate of DNA synthesis was the lowest at the beginning of the S-period and the highest at the last quarter of this period in both mono- and binucleated cells. The irradiation results in the inhibition of DNA synthesis mainly at the end of the S-period depending on doses employed. This inhibition was the same in bi- and mononucleated cells. In addition, the increase of correlation of the 3H-thymidine incorporation rate and DNA content was found between nuclei of binucleated cells after irradiation.     

Synchrony between DNA synthesis and thymidine incorporation into DNA in regenerating rat liver

DNA synthesis in regenerating liver was studied to determine whether the onset of stimulated DNA synthesis preceded the onset of increased incorporation of thymidine into DNA. Thymidine incorporation into hepatic DNA was not stimulated 15 h after operation, but was stimulated after 18 h; peak stimulation occurred 30 h after operation. Thymidine kinase activity was stimulated 24 h after operation; highest kinase activity was observed at 36 h. The onset of stimulated DNA synthesis was estimated by following the incorporation of labeled aspartic acid, sodium formate, adenine or orotic acid into appropriate DNA bases, viz., thymine, adenine, adenine or cytosine, respectively. Incorporation of adenine and orotic acid was stimulated between 15 h and 18 h after operation; incorporation of aspartic acid and sodium formate was stimulated between 18 h and 21 h after operation.The incorporation of thymidine into DNA was accelerated by stress stimulus and was inhibited by hydrocortisone. Changes in thymidine kinase activity also were correspondingly accelerated or delayed. Incorporation of labeled thymidine, adenine, formate, orotic acid or thymine into appropriate DNA bases, viz., thymine, adenine, adenine, cytosine or thymine, respectively, was stimulated by stress stimulus or was inhibited by hydrocortisone.It was concluded from these data that stimulation of DNA synthesis and of thymidine incorporation into DNA was essentially synchronized in regenerating rat liver. Results from this study were compared with results from similar studies in 2 other tissues, and the limitations, attendant with using thymidine incorporation into DNA as an indicator of stimulated DNA synthesis, were discussed.     

Role of endogenous TNF-alpha and sphingosine in induced DNA synthesis in regenerating rat liver after partial hepatectomy.

Cytokine-stimulated metabolism of sphingomyelin results in the accumulation of ceramide and sphingosine which play a part in the regulation of cell proliferation, differentiation, and reception, as well as in oncogenesis. Formation of TNF-alpha (a member of the cytokine family), accumulation of sphingosine, and DNA synthesis (measured by immunoblotting, HPLC, and [3H]thymidine incorporation, respectively) were studied in rat liver after partial hepatectomy. The content of TNF-alpha was found to increase during 12 h following hepatectomy. The maximum of sphingomyelinase activity and accumulation of sphingosine precede the maximum of DNA synthesis. Sphingosine is known to inhibit protein kinase C. On the other hand, it stimulates the metabolism of phosphatidylinositol, thus causing accumulation of diacylglycerol and inositol-1,4,5-triphosphate, which in turn activate protein kinase C. Hence, the release of TNF-alpha in regenerating liver may modulate DNA synthesis through the accumulation of sphingosine which is involved in regulation of protein kinase C activity and of phosphatidylinositol turnover.     

Effect of colchicine and vincristine on DNA synthesis in regenerating rat liver

The increases in the activities of hepatic thymidylate synthetase and thymidine kinase were significantly suppressed at 24 h after 70% partial hepatectomy in rats which had been administered a microtubule disrupter, colchicine or vincristine. The decrease of these enzymic activities was accompanied by a reduction of DNA content in 24 h regenerating liver. The immunoblotting assay showed that the depression of the thymidylate synthetase activity by the injection of colchicine or vincristine was due to the decrease of the enzyme protein. These results indicate that colchicine and vincristine inhibit the DNA synthesis during liver regeneration by inhibiting the induction of the key enzyme in DNA synthesis.     

Influence of cyclic AMP on DNA synthesis in slices of regenerating rat liver.

DNA synthesis in slices of regenerating rat liver is inhibited by adenosine cyclic 3',5'-monophosphate [cAMP]. The number of cells synthesizing DNA as assayed by 2-14C-thymidine incorporation is reduced by 65% in the presence of 10(-3) M cAMP. The inhibition of cAMP is not specific; other adenosine compounds, N6,O2,-dibutyryl adenosine 3',5'-monophosphate, 5'AMP and adenosine have the same effect. Moreover, the concentration of cAMP in the cell required for this inhibition is much higher than the normal levels of cAMP in liver cells.     

Nonrepetitive DNA transcription in normal and regenerating rat liver.

Initial RNA excess hybridization experiments employing total cell RNA and the complete complement of nonrepetitive DNA sequences showed no differences between normal and regenerating rat liver. However, when the DNA from the RNA-DNA hybrids was isolated and then reacted with homologous and heterologous RNAs the sensitivity of the assay was sufficiently improved to reveal that some of the nonrepetitive DNA transcrips present in normal liver are missing at 24 h and 48 h after a 70% partial hepatectomy. Additional experiments showed that while some of the missing sequences were common to both stages of regeneration, some were also different. The data thus suggest both quantitative and qualitative changes during liver regeneration in the population of RNA molecules transcribed from nonrepetitive DNA.     

Effect of urethan on polyamine and DNA synthesis in the regenerating rat liver

When a single dose of urethan was injected into the peritoneal cavity of rats immediately after partial hepatectomy, DNA synthesis was delayed by 12 h. The induction of ornithine decarboxylase which was induced biphasically following partial hepatectomy was also reduced and delayed by 14–15 h by the administration of urethan. S-Adenosylmethionine decarboxylase activity in urethan-treated rat liver at 20 h and 29 h after operation was significantly lower than that of untreated animals. This enzyme activity was shown to increase thereafter, reaching a higher level than in untreated rats at 37–42 h. Hepatic spermidine content changed biphasically in a manner similar to DNA synthesis. These results suggest that the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase may correlate with DNA synthesis and that an increase of spermidine concentration is necessary to DNA synthesis.     

Effect of realimentation after several days' pure carbohydrate intake on DNA synthesis in regenerating rat liver

The authors studied the effect of realimentation after several days' isolated glucose or fructose intake on DNA synthesis in liver regenerating after partial hepatectomy (PH) (65-70%) or after carbon tetrachloride (CCl4) poisoning 1.5 ml/kg. Two days before PH or the administration of CCl4 and two days after, the experimental rats were given glucose (50% solution) of fructose (50% solution) as the only source of energy. Rats with PH were then fed for one day on a standard laboratory diet (25 cal% protein) or a high protein diet (81 cal% protein). Rats with CCl4 liver damage were fed for one day on the standard laboratory diet only. In the rats given glucose, liver DNA synthesis and the total amount of these nucleic acids in the liver 48 hours after CCl4 administration was lower than in the controls or the rats given fructose. In all the experimental groups (PH and CCl4), stimulation of liver DNA synthesis was observed after one day's realimentation. The total DNA content of the liver of rats with PH rose markedly during realimentation. The experiments indicate that the regenerative activity of damaged liver can be influenced by the nutritional regimen.     

Synthesis and accumulation of polyamines in rat liver regenerating after treatment with carbon tetrachloride

A single intraperitoneal injection of carbon tetrachloride into rats resulted within 12 hours in a marked accumulation of putrescine in liver with a concomitant decrease in the concentration of spermidine. The accumulation of putrescine apparently was partly due to an immense stimulation of ornithine decarboxylase activity occurring at the same time. However, in addition it was found that during the maximal accumulation of putrescine there was a marked incorporation of radioactivity from labelled spermidine to liver putrescine $\text{in vivo}$. The conversion of spermidine to liver putrescine was hardly detectable in control animals. Besides the treatment with carbon tetrachloride, increased conversion of radioactive spermidine to liver putrescine $\text{in vivo}$ also occurred after treatment with growth hormone, after partial hepatectomy and after treatment with thioacetamide, $\text{i. e.}$ under circumstances characterized by a stimulation of ornithine decarboxylase activity and an increased accumulation of putrescine.     

Degenerative changes, DNA synthesis and mitotic activity in rat liver following single exposure to diethylnitrosamine

Degenerative and regenerative changes induced in rat liver by single exposure to diethylnitrosamine (DEN) were examined by morphological and biochemical approaches. Apoptotic changes were observed in livers of rats exposed to a 'subnecrogenic' dose of DEN (10 mg/kg) as well as in liver parenchyma of those receiving a necrogenic dose (100 mg/kg). Zonal centrilobular necrosis was observed exclusively in the latter group. Regenerative changes, i.e., increases in DNA synthesis, labeling index and mitotic activity, occurred only in animals exposed to the higher dose. The mitogenic effect obtained in these conditions was about half that induced by two-thirds hepatectomy and the maximum response occurred about 24 h later than in partially hepatectomized rats.     

Enhanced uridine kinase and RNA synthesis in regenerating rat liver after 5-azacytidine administration

The administration of 5-azacytidine to partially hepatectomized rats results in the increase of uridine kinase activity in cell-free liver extracts 24–72 hr after drug administration. At the same time the activity of uridine phosphorylase and of uridine 5′-nucleotidase is decreased, while uridinemonophosphate kinase and uridine 5′-triphosphatase are not affected. The repeated administration of 5-azacytidine leads to a further enhancement of uridine kinase which is 6- to 8-fold higher in 96-hr regenerating livers than in untreated controls. Simultaneously the enhanced incorporation of uridine into liver ribonucleic acids was observed. The metabolic alterations occurring in the liver at later phases after 5-azacytidine in vivo administration are discussed.