Studies on DNA content of hepatocytes in cirrhosis and hepatoma by means of microspectrophotometry and radioautography

Summary In order to clarify the underlying mechanism of malignant transformation from cirrhosis to hepatoma the cell kinetics of hepatocytes were studied in these two conditions. The content and synthesis of DNA in hepatocyte nuclei were investigated, by means of Feulgen-microspectrophotometry and tritiated thymidine radioautography, in cirrhotic and noncancerous parts of hepatoma with concomitant cirrhosis. The distribution of ploidy patterns was widely spread, from hypodiploid to hyperpolyploid, in the noncancerous parts of a cirrhotic liver containing hepatoma. In normal liver, each paired nuclear DNA content of a binucleate cell recorded almost the same amount, whereas in the noncancerous as well as in hepatoma cells much difference of DNA content was observed between the paired nuclei of the binucleate cells. The ploidy pattern of hepatocytes in patients with liver cirrhosis, who had developed hepatoma during follow-up periods of several months to several years, appeared to resemble that in noncancerous parts of hepatoma cases. On the other hand, the incorporation of tritiated thymidine into hepatocytes was found to be markedly increased in noncancerous parts as well as in cirrhotic liver developing hepatoma during follow-up periods. These results suggest the possibility that the hepatocytes in noncancerous parts of hepatoma have deranged cell-kinetics which might be a driving factor for the development of malignancy.     

Human hepatocyte polyploidization kinetics in the course of life cycle

The processes of polyploidization in normal human liver parenchyma from 155 individuals aged between 1 day and 92 years were investigated by Feulgen-DNA cytophotometry. It was shown that polyploid hepatocytes appear in individuals from 1 to 5 years old. Up to the age of 50 years the accumulation rate of binucleate and polyploid cells is very slow, but subsequently hepatocyte polyploidization is intensified, and in patients aged 86–92 years the relative number of cells with polyploid nuclei is about 27%. Only a few hepatocytes in the normal human liver reach 16C and 8C×2 ploidy levels for mononucleate and binucleate cells respectively. Using a mathematical modeling method, it was shown that during postnatal liver growth the polyploidization process in human liver is similar to that in the rat, and that polyploid cells are formed mainly from binucleate cells. As in rats, prior to an increase in ploidy level, diploid human hepatocytes can pass several times through the usual mitotic cycles maintaining their initial ploidy level. After birth, only one in ten hepatocytes starting DNA synthesis enters the polyploidization process. At maturity about 60% of 2C-hepatocytes starting DNA synthesis divide by conventional mitosis, the rest dividing by acytokinetic mitosis leading to the formation of binucleate cells. During ageing the probability of hepatocyte polyploidization increases and in this period there are two polyploid or binucleate cells for every diploid dividing by conventional mitosis.     

Age-related alterations in the size of human hepatocytes

Age-related alterations in the size of human hepatocytes (both mononuclear and binucleate forms), were studied in histological sections and in separated cells and nuclei using cytophotometrical and microspectrophotometrical methods. The following results were obtained: 1. The volume of nuclear DNA increased in proportion to nuclear size. The increase occurred in a group pattern reflecting nuclear polyploidization. 2. Cell size increased in proportion to nuclear size. Tetraploid cells (4C) were roughly two times greater than diploid cells (2C). 3. In most of the binucleate cells examined, the ploidy class of the two nuclei in a binucleate cell was observed to be equal. Heterogeneity of the ploidy class among the nuclei of a binucleate cell was present in less than 1% of total binucleate cells examined. The nuclear DNA volume of individual nuclei in binucleate cells appeared to be the same as that of mononuclear cells. 4. The cell size of binucleate cells corresponded with that of mononuclear cells whose ploidy class was the same as the sum of the ploidy classes of two nuclei of a binucleate cell. 5. The incidence of binucleate cells in the lobular periphery was about 4 to 6% in the third decade, and increased slightly with age up to 5 to 7% in the tenth decade. 6. The incidence of binucleate cells in the liver at different ages followed a similar pattern to that observed in mononuclear cells whose ploidy class was half of the sum of ploidy classes of the two nuclei of the binucleate cell.     

Cellular mechanisms of cirrhotic rat liver regeneration. II. Proliferation, polyploidization and hypertrophy after partial hepatectomy

Using cytofluorimetry and absorptional cytophotometry, hepatocyte DNA and total protein contents were measured in intact and cirrhotic rats in 1, 3 and 6 months after partial hepatectomy (PH). It has been found that within one month of intact rat liver regeneration the level of hepatocyte ploidy rised by 25% to remain elevated for the next 6 months. This was due mainly to reducing the number of cells with diploid nuclei (2c 2-fold, 2c x 2 - 6.6-fold) and to rising the number of octaploid hepatocytes. In cirrhotic animals the ploidy level in hepatocytes increased in 3 months after PH, and decreased by 15% in 6 months. The number of hepatocytes with diploid nuclei (2c and 2c x 2) increased within 3-6 months in both control and cirrhotic rats. The protein content per diploid hepatocyte rised by 30% within 3-6 months of liver regeneration after PH. Special calculations have shown that within 3 months after PH the increase in the liver mass of control and cirrhotic rats was due completely to hepatocyte DNA synthesis, i. e. proliferation and polyploidization. Within the next 3 months of liver regeneration after PH, the contribution of polyploidization to liver mass increase was negative because of depolyploidization of liver parenchyma cell population. At this time hypertrophy was the main process determining the liver mass increase.     

Effect of long-term administration of a high protein or low protein diet on rat liver. Morphological and biochemical findings

An increase in relative liver weight, the total liver DNA content, hepatocyte volume and the total surface area of the membranes of mitochondria and the granular and smooth endoplasmic reticulum of hepatocytes, but a decrease in the size of the nuclei, were found in adult male rats fed three weeks on a high protein diet compared with animals given a standard laboratory diet. Serum transaminase (ALT, AST) and alkaline phosphatase activity was practically the same as the control values. Rats fed three weeks on a low protein diet showed a decrease in relative liver weight, in the total liver DNA content, in hepatocyte and nuclear volume and in ploidy, and also in the surface area of the membranes of the mitochondria and the smooth and granular endoplasmic reticulum; conversely, the number of binucleate hepatocytes rose. Serum ALT, AST and alkaline phosphatase activity was mildly, but statistically significantly elevated.     

Flow cytometric analysis of mouse hepatocyte ploidy

Preparative and mathematical procedures are presented for the investigation of the ploidy pattern of liver cells. The DNA content of enzymatically-isolated liver cells and of nuclei was measured by flow cytometry. The true DNA content could not be measured directly due to super-position of statistical coincidences (demanding "first mode correction") and incomplete separation of the nuclei in binucleate hepatocytes (demanding "second mode correction"). The statistical coincidences (caused by simultaneous measurement of two or more particles or subsequent reaggregation of particles) were corrected by splitting the "unnatural" i.e., aneuploid DNA content, and classifying it with the normal ploidy classes. In addition, the higher normal ploidy classes were reduced by the proportion of the measured coincidences in favour of the lower ones. The second mode correction applied to nuclear distributions only. It is a probability calculation based on counting nuclear pairs on microscope slides, and resulted in a 10% increase of diploid nuclei and a larger standard deviation between the age groups. 8c and 16c values were reduced. The tetraploid values were unchanged.     

Characterization and cell cycle kinetics of hepatocyte populations isolated from adult liver tissue by a nonenzymatic procedure

Suspensions of liver cells enriched in lobular parenchymal hepatocytes were isolated from adult mouse hepatic tissue by nonenzymatic dispersion in chelating buffer and sedimentation of the released cells at unit g. Single cell suspensions so obtained were suitable for flow cytometric measurements of hepatic ploidy class distributions. The more quickly sedimenting cell population consisted of 88% albumin/transferrin-positive epithelial hepatocytes, the nuclei of which were bimodally distributed with respect to RNA content. This dual G1 population was observed in 2C DNA content liver nuclei prepared by several methods and appears to be a general cytochemical characteristic of adult liver parenchymal cells.     

Flow cytometric analysis of mouse hepatocyte ploidy

Summary The development of liver ploidy in mice aged up to 24 months was investigated by flow cytometry in four mouse strains. A mathematical procedure was applied for correction of flow cytometry histograms. In two of the mouse strains, C3H and DBA, both cellular and nuclear ploidy proceed in the same way. The octoploid cell with two tetraploid nuclei is the most numerous cell type in adulthood. On the other hand, strain NZB and the out-bred strain NMRI show at the corresponding age a higher proportion of diploid cells with strikingly low proportions of 4c cells. In addition, high values of 16c cells and nuclei are present in NMRI. In all strains the proportion of binucleate hepatocytes is in the same range (60%). However, the strains differ in ploidy classes of binucleate cells. Development of liver polyploidization does not depend on life span of the specific strain.     

Separation of intact rat hepatocytes and rat liver nuclei into ploidy classes by velocity sedimentation at unit gravity

A system is described which permits the separation of isolated hepatocytes and isolated rat liver nuclei belonging to different ploidy classes by velocity sedimentation at unit gravity.The problem of obtaining single cells suspensions is discussed and preparations were obtained that contained 96% single hepatocytes.By improving the sedimentation method, it took 2.5 h to separate rat liver nuclei on sucrose gradients into diploid and tetraploid ploidy classes. Recoveries were generally over 95%. The diploid band was 99% pure. DNA and protein content of the ploidy classes were measured. After partial hepatectomy and [³H]thymidine injection it was found that the label moved largely into the tetraploid compartment.Isolated hepatocytes were fractionated in 1 h on Ficoll gradients. Erythrocytes were separated from small nucleated cells and the population of hepatocytes was clearly separated from these two cell populations. Diploid hepatocytes were 80% and tetraploid hepatocytes were 99% pure. Viability was about 80% after fractionation.The gene dosage of NADPH cytochrome c reductase, succinate dehydrogenase and lactate dehydrogenase was estimated in diploid and tetraploid hepatocytes. Gene dosage was equal in diploid and tetraploid hepatocytes for succinate dehydrogenase and NADPH cytochrome c reductase. It is suggested, after correcting for non-viable tetraploid hepatocytes, that the gene dosage of lactate dehydrogenase was significantly lower in diploid than in tetraploid hepatocytes.     

Cytofluorimetric study of the glycogen content in hepatocytes of varying ploidy in adult rats]

A combined method, that allows measuring glycogen and DNA contents in one of the same cell, was applied for quantitative determination of these in mono- and binucleate hepatocytes with different ploidy obtained from adult rats. The mean glycogen content was shown to increase proportionally to the genome number within the changes of the hepatocyte ploidy from 2 to 8c.     

Effect of "vilon" on cirrhotically changed rat liver. Liver regeneration, and status of glycogen-forming function of hepatocytes

Effects of a dipeptide preparation "Vilon" on rehabilitation of functional activity of hepatocytes and regeneration of the cirrhotically altered rat liver were studied. The liver cirrhosis was produced by poisoning of rats for 4 months with carbon tetrachloride (CCl4). On the end of the poisoning with CCl4, one group of animals was not submitted to any further actions, whereas animals of the other group were injected "Vilon" (1.7 micrograms/kg) daily for 5 days. On smears of isolated hepatocytes, contents of total glycogen (TG), and its labile and stable fractions (LF and SF) were determined in addition to cell ploidy levels and the total protein content. In liver homogenates, activities of glucose-6-phosphatase (G6P), glycogen synthase (GS), and glycogen phosphorylase (GP) were measured. In 2 weeks after the drug application, G6P activity being reduced in cirrhosis 1.2 times, elevated under effect of "Vilon". In non-treated rats the contents of TG and its fractions and of G6P activity remained at the level characteristic of the cirrhotic liver prior to "Vilon" administration. In both groups of rats, GP and GS activities in the cirrhotically altered liver did not differ from their control values throughout the entire experiment. "Vilon" has been shown to exert a weak stimulating effect on regeneration of the cirrotically altered rat liver: in hepatocytes of the second group of rats the total protein content and ploidy levels were higher than those in the first group by 4.7 and 11.5%, respectively.     

The influence of hepatoprotector 2-ethylthiobenzimidazole hydrobromide (bemithyl) on the content of glycogen in cirrhotic rat liver hepatocytes located in various microenvironments

Using absorption and fluorescent cytophotometry methods, glycogen contents were studied in hepatocytes located in liver lobules and in hepatocytes, which make the general population of these cells in normal and cirrhotic rat liver. In cirrhosis, the content of glycogen in hepatocytes located in lobules obviously rises in comparison with the norm, but to a lesser degree, than in hepatocytes making the general population of these cells in cirrhotic liver. The content of glycogen in hepatocytes, located in lobules of pathologically changed liver in bemithyl treated rats, did not differ from the norm. At the same time, the glycogen content in hepatocytes, representing the general population of these cells in cirrhotically altered bemithyl injected rat liver, remained higher than in the norm. The data obtained indicate that distinctions in particular cell microinvironment, obviously present in cirrhotic liver, render essential influence on hepatocyte functional activity.     

Delays in prophase induced by adenosine 2-deoxyriboside and their relation with DNA synthesis

The rate of DNA synthesis in the course of the division cycle in root meristem ofAllium cepa growing under constant temperature and aeration conditions has been studied by means of treatment with AdR, as a specific inhibitor of the synthesis, as well as by the incorporation of tritiated thymidine. The one-hour treatment with AdR or tritiated thymidine was given at various hours in the course of the interphase of a synchronous population of binucleate cells induced by caffeine. In the case of AdR, sensitivity to the inhibition of DNA synthesis was studied by recording the delays produced by the treatment in the appearance of biprophases and bitelophases. The selection by the use of caffeine, of spontaneously synchronous populations of cells going through the telophase and becoming binucleate and the detection of the first biprophases in the subsequent mitosis provide a highly synchronized system with which to study the incorporation of tritiated thymidine during the interphase. The curves representing sensitivity to the inhibition of DNA synthesis by AdR and the rate of tritiated thymidine incorporation coincide, so that we can regard the delays, under our conditions, as proportional to the rate of DNA synthesis at the moment of the AdR treatment. This rate, in the S period, was found to be variable by both methods, being higher in the first and the last thirds of the S period (S₁ and S₃) and lower in the middle third (S₂).     

Effect of partial hepatectomy on the glycogen level in hepatocytes in the portal and central lobule zones in cirrhotically-changed rat liver

Using cytophotometric method, the content of glycogen was studied in hepatocytes of the portal and central zones of a liver lobule in norm, in cirrhosis, and 1, 3, and 6 months after a partial hepatectomy of the normal and cirrhotic rat liver. As we showed earlier, glycogen content in cirrhotic liver hepatocytes rose 2-3-fold, along with obvious impairment of glycogen metabolic heterogeneity in these. In cirrhotic liver glycogen dominates in the central zone, whereas in norm more glycogen is observed in the portal one. The objective of this study was to find out to what degree a partial hepatectomy of cirrhotic liver may promote recovery of the metabolic glycogen heterogeneity in hepatocytes. Glycogen was determined in hepatocytes, using a quantitative variant of PAS-reaction on sections of the material obtained from serial supravital punctate liver biopsies. Glycogen amount in hepatocytes of different liver lobule zones was determined by an image analyzer technique that allows to bring together the cytophotometric analysis of the substance with its localization in a particular liver lobule. Results of these studies have shown that a partial hepatectomy of cirrhotic liver promotes restoration of the hepatocyte metabolic heterogeneity in the liver lobule.     

Liver regenerative potential of the lake frog Rana ridibunda after partial hepatectomy

A histomorphological study of the regenerating liver of Rana ridibunda, within 2 months after partial hepatectomy, shows that regenerative processes on the wound surface are slowly proceeding. Processes of reticular fiber reconstruction occurred in the composition of the basal membrane of liver sinusoids. A cytophotometric study shows that glandular cells in R. ridibunda liver are commonly tetraploid. The post-traumatic regeneration of the liver after partial hepatectomy involves activation of DNA synthesis in hepatocytes, leading to increase in their ploidy. Within the 1st month of regeneration, the mitotic index of hepatocytes substantially increased. Regeneration of glandular parenchyma of the liver is accompanied by a quantitative increase in binucleate hepatocytes, which is most highly expressed within 5-20 days after partial hepatectomy.     

Cytophotometric analysis of amitosis in nuclear endosperm of Vicia faba.

DNA content in amitotically dividing nuclei of endosperm of Vicia faba subsp. major cv. Hangdown was cytophotometrically measured after Feulgen reaction. Measurements of DNA content indicate that nuclei with lower ploidy level divide into equal parts. Divisions of nuclei with higher ploidy level result both in nuclei with equal and nuclei with different DNA content. Nuclei with higher ploidy level can still divide mitotically that lead to the equalising of DNA content in endosperm nuclei.     

CHROMOSOME PULVERIZATION IN HUMAN BINUCLEATE CELLS FOLLOWING COLCEMID TREATMENT

Under the influence of Colcemid, a substantial number of binucleate human cells from a line infected with herpes-like virus was found to possess pulverized chromosomes. Although this abnormality was also detected in untreated binucleate cells, the increase in the number of pulverized cells after the addition of Colcemid was too striking to be explained by accumulation of spontaneously occurring cells in response to the mitotic inhibition by Colcemid. Furthermore, the induction of pulverization may be dependent upon Colcemid concentration. These findings imply an involvement of Colcemid in the mechanism of pulverization induction in the system studied. When tritiated thymidine was added to the culture medium simultaneously with Colcemid, the majority of binucleate cells with an intact and a pulverized chromosome set incorporated this isotope into the pulverized set only. This obviously suggests that the nuclei in the binucleate cell are asynchronous in DNA synthesis, and that this asynchrony is intimately related to the induction of the pulverization phenomenon. It seems very probable that the late S phase in the late synthesizing nuclei represents a critical stage at which damage to the chromosomes most readily occurs.     

肝硬化患者肝脏表皮生长因子受体的表达及其意义

作者采用免疫组织化学方法研究８例肝硬化患者及６例非肝病患者肝脏表皮生长因子（ＥＧＦ）受体的表达及意义。结果显示肝硬化患者肝细胞核出现ＥＧＦ受体阳性反应;增生的小胆管上皮细胞呈ＥＧＦ受体强阳性反应并出现细胞核ＥＧＦ受体。提示肝硬化患者肝脏ＥＧＦ受体分布与对照组肝脏明显不同。肝硬化患者肝细胞核内ＥＧＦ受体可能与维持肝实质细胞数量有关;ＥＧＦ与胆管上皮细胞增生有关     

The inhibitory effect of 4-hydroxy-nonenal on DNA-polymerases alpha and beta from rat liver and rapidly dividing Yoshida ascites hepatoma

The purpose of this study was to determine firstly whether the isolated enzyme DNA polymerase alpha, which functions within the DNA replicase system, exhibits different sensitivity against the thiol-blocking agent 4-hydroxy-nonenal (HNE) when adult rat liver and the rapidly dividing Yoshida ascites hepatoma were used as enzyme sources and, secondly, whether the reaction catalysed by DNA polymerase is the most sensitive step of the DNA replicase system of native cells. DNA polymerase alpha as well as the non-replicative DNA polymerase beta, isolated from both sources, were remarkably similar with regard to their sensitivity against HNE, as indicated by the incorporation of radioactive label from [3H]deoxy-thymidine-triphosphate into DNA. The transport of [14C]thymidine through the plasma membrane and the incorporation of this precursor into DNA were studied with neonatal hepatocytes and with hepatoma cells. The incorporation of thymidine was inhibited at lower concentrations of HNE in both cell lines than the transport process and the reaction catalysed by DNA polymerase alpha. It was concluded that in the DNA replicase system of native liver and hepatoma cells another process different from the reaction catalysed by DNA polymerase alpha is more sensitive to HNE.     

Light and electron microscopic radioautographic studies on macromolecular synthesis in amitotic hepatocytes of aging mice.

The problem on cell divisions whether cells proliferate by mitosis or amitosis has been the heated controversy among many biologists since the late 19th century. We confirmed by extensive experiments since the mid 20th century that all the cells proliferated by mitosis not by amitosis but that amitosis actually existed in some glandular cells such as hepatocytes or pancreatic acinar cells which showed only amitotic nuclear divisions without cytoplasmic division producing binucleate cells in several kinds of experimental animals. We further verified that such amitotic cells did not synthesize macromolecular compounds incorporating macromolecular precursors such as 3H-thymidine for DNA, 3H-uridine for RNA or 3H-leucine for proteins. Recent experiments at the end of 20th century using many groups of aging mice, from fetal day 19 to postnatal month 24, injected with such precursors, amitotic cells and resulting binucleate cells in the hepatocytes were detected by electron microscopic radioautography and compared to mononucleate cells. The results demonstrated that only a few hepatocytes showing amitotic nuclear division were found labelled with the 3 precursors demonstrating DNA, RNA and protein synthesis. However, the numbers of silver grains showing incorporations of labelled precursors in respective amitotic cells were very few. It was clarified that the amitotic cells did not synthesize such macromolecules as mononucleate hepatocytes did. On the other hand, more binucleate cells were found than the amitotic cells. DNA synthesis of mononucleate and binucleate hepatocyte nuclei was observed at perinatal stage and disappeared at adult stage. The labeling index of mononucleate hepatocytes was greater than that of binucleate hepatocytes. RNA and protein syntheses in karyoplasm and cytoplasm in both mononucleate and binucleate cells increased from perinatal stage, reaching the maxima at adult stage then decreased to senescent stage. Grain counts revealed that synthesized RNA and proteins were more in binucleate cells than mononucleate cells at respective aging stages.