DNA damage and repair in gamma-glutamyltranspeptidase-positive and negative hepatocytes in primary culture from carcinogen-treated rats.

Chemically induced DNA fragmentation and unscheduled DNA synthesis were determined in gamma-glutamyltranspeptidase (GGT)-positive and GGT-negative hepatocytes isolated from rat livers subjected to a multistage hepatocarcinogenesis regimen (Solt-Farber), which included 0.05% phenobarbital promotion for 6 weeks (early) or 6 months (late). The results indicated that there was DNA damage in untreated GGT-positive and GGT-negative hepatocytes with either period of promotion compared with normal hepatocytes; however, no statistical difference could be seen between GGT-positive and GGT-negative hepatocytes. DNA damage induced in vitro by the activation-dependent carcinogen dimethylnitrosamine was much less in GGT-positive hepatocytes than in GGT-negative hepatocytes or normal hepatocytes. No significant difference in DNA damage was seen in both GGT-positive and GGT-negative cell populations following treatment with the activation-independent carcinogen ethylnitrosourea (ENU), although DNA damage of GGT-positive hepatocytes was less than that of normal hepatocytes. The background of unscheduled DNA synthesis in both GGT-positive and GGT-negative hepatocytes at either time of promotion was higher than that of normal hepatocytes. The capacity for DNA repair in GGT-positive hepatocytes appeared to be lower than that in GGT-negative hepatocytes. GGT-negative hepatocytes exhibited a lower capacity for DNA repair than that of normal hepatocytes in terms of the rate of unscheduled DNA synthesis elicited by dimethylnitrosamine and ethylnitrosourea in vitro.     

Induction by butylated hydroxytoluene of rat liver gamma-glutamyl transpeptidase activity in comparison to expression in carcinogen-induced altered lesions

Butylated hydroxytoluene (BHT) at concentrations of 300-6000 ppm in the diet caused a dose-dependent increase in gamma-glutamyl transpeptidase (GGT) activity in normal F344 male rat liver at 18 weeks. However, the activities of glutathione S-transferases (GSTs) of rat liver cytosol were enhanced only at concentrations of 3000 or 6000 ppm BHT. Histochemically, the enhanced GGT activity was localized to hepatocytes surrounding the portal areas. Autoradiographic measurements of DNA synthesis showed that dietary BHT did not increase the level of cell proliferation and the GGT-positive hepatocytes did not exhibit different rates of DNA synthesis from those of GGT-negative cells. Feeding of the liver carcinogen N-2-fluorenylacetamide (FAA) induced foci and nodules of GGT-positive altered cells which exhibited higher rates of DNA synthesis than those of surrounding GGT-negative hepatocytes. Following iron loading, the periportal GGT-positive hepatocytes produced by BHT accumulated cellular iron, whereas the cells in FAA-induced lesions excluded iron. These results suggest that dietary BHT induces GGT activity in periportal hepatocytes without proliferation of the cells and that induction does not represent fetal expression or a preneoplastic alteration.     

Induction of unscheduled DNA synthesis in the nuclear matrix of rat hepatocytes after whole-body gamma irradiation of the animals

DNA synthesis in hepatocytes was studied by incorporation of [3H]thymidine administered to portal vein of gamma-irradiated (80 Gy) rats. It was shown that the rate of replicative DNA synthesis decreased in hepatocytes of the regenerating liver and unscheduled DNA synthesis was induced at the nuclear matrix of resting cells of the intact liver. In addition to repair synthesis, DNA synthesis resembling replicative one ("aberrant" DNA synthesis) accounts for a considerable fraction of gamma-radiation-induced synthesis of DNA at the nuclear matrix.     

Primmorphs from archaeocytes-dominant cell population of the sponge hymeniacidon perleve: improved cell proliferation and spiculogenesis

Marine sponges (Porifera) possess an extraordinary diversity of bioactive metabolites for new drug discovery and development. In vitro cultivation of sponge cells in a bioreactor system is very attractive for the sustainable production of sponge-derived bioactive metabolites; however, it is still a challenging task. The recent establishment of sponge primmorphs, multicellular aggregates from dissociated mixed-cell population (MCP), has been widely acknowledged to hold great promise for cultivation in vitro. Here we present a new method to establish an in vitro sponge primmorph culture from archaeocyte-dominant cell population (ADCP) enriched by a Ficoll gradient, rather than a mixed-cell population (MCP). Our rationale is based upon the totipotency (the ability of a cell to differentiate into other cell types) of archaeocyte cells and the different biological functions of various sponge cell types. A sponge, Hymeniacidon perleve collected from the China Yellow Sea was used as a model system for this investigation. Distinct dynamics of primmorph formation were observed while significant increases in DNA synthesis, cell proliferation (up to threefold), and cell growth (up to fourfold) were achieved. Furthermore, a time-dependent spiculogenesis was clearly demonstrated in our longterm culture, indicating high metabolic activity of primmorphs from the ADCP. This new method represents an important step forward to advance sponge cell culture in vitro that may lead to commercial exploitation of sponge-derived drugs.     

The control of DNA synthesis in primary cultures of hepatocytes from adult and young rats: interactions of extracellular matrix components, epidermal growth factor, and the cell cycle

Hepatocytes from adult and 4-week-old rats cultured on one of several extracellular matrix components were stimulated to replicate by epidermal growth factor (EGF). DNA synthesis was increased at 44-48 hr in adult hepatocytes and at 24, 48, and 72 hr in hepatocytes from young rats when EGF was added 2 hr after explantation. When EGF was added at 24 hr, maximal DNA synthesis of adult hepatocytes was observed at 48 hr, whereas that of 4-week-old hepatocytes was seen at 48 and 72 hr. Ten ng EGF per ml was the optimal concentration for maximal DNA synthesis in both adult and young cells. DNA synthesis decreased with increasing cell density, but this effect was less in hepatocytes from young than in those from adults. When hepatocytes were cultured on substrata consisting of individual extracellular matrix components, neither the time that adult cells needed to respond to EGF nor the time from stimulation by EGF to the peak of maximal DNA synthesis was altered in either adult or young cells. The optimal EGF concentration for maximal DNA synthesis and the cell density control of replication were also not altered by the substrata used. Substrata made from each of the extracellular matrix components studied enhanced DNA synthesis of adult and young hepatocytes stimulated by EGF in the following decreasing order: fibronectin, type IV collagen, type I collagen, and laminin. In both adult and young hepatocytes the enhancement of DNA synthesis was greatest when cultured on fibronectin. Thus the initiation and magnitude of DNA synthesis in primary cultures of rat hepatocytes were altered both by the age of the donor and the substratum on which the cells were explanted.     

Developmental time,cell lineage,and environment regulate the newly synthesized proteins in sea urchin embryos

Strongylocentrotus purpuratus embryos were fractionated into two cell populations of defined lineages at times corresponding to two critical developmental events: determination (16-cell stage) and early differentiation (mesenchyme blastula). The 16-cell stage blastomeres, labeled with [³⁵S]methionine, exhibited identical protein synthesis patterns by fluorography, and this pattern was not significantly altered by cell separation. In comparing the proteins of the mesenchyme blastula to the 16-cell stage, differences (increases and decreases) were seen by fluorography of newly synthesized proteins. The synthesis of 2.9% of the mesenchyme blastula proteins is specific to or enriched in primary mesenchyme cells and 8.2% is specific to or enriched in endoderm/ectoderm cells. Additionally, in contrast to the earlier stage, the pattern of protein synthesis in the mesenchyme blastula embryos is substantially altered by cell separation. The ability to alter protein synthesis in response to environmental factors may be a further demonstration of the differentiation of these cells.     

Cell culture density dependent toxicity and chromatin changes upon cadmium treatment in murine pre-B-cells

Murine pre-B-cells grown in the presence of lower (1 μM) or higher (5 μM) concentration of cadmium chloride were separated into 13 fractions by centrifugal elutriation. The rate of DNA synthesis after cadmium treatment determined in permeable cells was dependent on cell culture density during cadmium treatment. Cell cycle analysis revealed a shift in the profile of DNA synthesis from replicative to repair DNA synthesis upon cadmium treatment. The study of the relationship between cell culture density and cell diameter at lower and higher cell densities in the presence of 1 μM cadmium chloride concentration showed that a. at 5×10⁵ cell/ml or lower densities cells were shrinking indicating apoptotic changes, b. at higher cell culture densities the average cell size increased, c. the treatment of cells with low CdCl₂ concentration (1 μM) at higher cell culture density (>5×10⁵ cell/ml) did not change significantly the average cell diameter. At 5 μM cadmium concentration and higher cell culture densities (>5×10⁵ cell/ml) the average cell size decreased in each elutriated fraction. Most significant inhibition of cell growth took place in early S phase (2.0–2.5 C value). Apoptotic chromatin changes in chromatin structure after cadmium treatment were seen as large extensive disruptions, holes in the nuclear membrane and stickiness of incompletely folded chromosomes.     

Protein synthesis in different populations of rat hepatocytes separated according to density

Hepatocytes were isolated from fasted rats by a two-step Ca⁺⁺-free/collagenase perfusion method. The cells were subjected to centrifugation under mild conditions at 12°C in a linear metrizamide gradient (1.075–1.12 gm/cm³). The cells were distributed in the gradient a bell-shaped manner. According to their position in the gradient the cells were divided in five different population. The heaviest population was omitted from the subsequent evaluation because it contained a high proportion of dead cells. The activity of alanine aminotransferase increased with increasing cell density indicating that the lightest cell population was enriched in perivenous cells, whereas the heaviest cell population had an excess of periportal cells. Protein synthesis was more rapid in the light (perivenous) cell population than in the heavy (periportal) cell population as measured by means of incoporation of radioactively labeled valine into protein. The distribution measured in vitro indicated approximately 80% higher rates in perivenous cells. On the other hand, the synthesis and secretion of export proteins were similar in all cell populations regardless of their density. Protein degradation measured as appearance of free valine in cell media was higher in the light (perivenous) cell population than in the other populations. Thus protein metabolism seemed to be faster in the light cell population.     

Bidirectional effects of Kupffer cells on hepatocyte proliferation in vitro.

The control of rat hepatocyte DNA synthesis in vitro by Kupffer cells and transformed perisinusoidal lipocytes, i.e. myofibroblast-like cells was studied. Conditioned media from Kupffer cells inhibit the replicative (hydroxyurea-sensitive) DNA synthesis dose-dependently in primary cultures of hepatocytes stimulated by epidermal growth factor (EGF). The cytokine responsible for the inhibition was identified as transforming growth factor beta (TGF beta). After neutralization of activated TGF beta in these media, DNA synthesis is stimulated in quiescent hepatocytes via transforming growth factor alpha (TGF alpha) demonstrated by competitive TGF alpha/EGF-receptor blocking on hepatocytes. Results similar to those obtained with Kupffer cells were found with conditioned media of myofibroblast-like cells. Northern blot hybridization confirms the expression of both TGF beta and TGF alpha in Kupffer cells and myofibroblast-like cells, respectively. These findings support the notion that Kupffer cells and myofibroblast-like cells might regulate in both directions liver regeneration depending on the proportion of secreted TGF alpha and TGF beta and on the activation status of TGF beta, of which a significant fraction is secreted in the latent form.     

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.     

DNA synthesis inEscherichia coli B/r after UV-irradiation

When studying the kinetics of DNA synthesis, growth and cell division inEscherichia coli B/r after irradiation with different doses of UV-radiation (254 nm) we could demonstrate, by means of pulse incorporation of³H-thymidine, a lag in DNA synthesis after the irradiation. The relative rate of the restored DNA synthesis (related to the number of viable cells) was higher than in the non-irradiated culture. After 3 h the rate of DNA synthesis settled at a constant value, which was identical with the control rate up to the “critical dose” of 20 J/m². The irradiated cell population is heterogenous and contains basically two categories of cells — surviving and non-surviving. Cells of both types contribute to DNA synthesis restored after the lag period to a different extent. During the first hour after the irradiation even the nonviable portion of the population,i.e. cells that do not form colonies but are still penicillin-sensitive, is involved in the DNA synthesis.     

DNA synthesis in Hela cells at low temperature

It has been proved that ³H-thymidine is incorporated into DNA of HeLa cells cultured at 4 °C and its labelling distribution in DNA is homogeneous. This incorporation of ³H-thymidine increased with the duration of incubation and only 30% of the cell population was labelled after 12 h. When synchronous cell populations were used, the rate and extent of DNA synthesis at 4 °C was proportional to the relative number of cells in S phase at that temperature. Thus, cellular labelling at 4 °C does not result from a non-specific absorption phenomenon, but indicates a DNA synthesis process.     

Effect of tryptophan on isolated hepatocytes of rats

The addition of tryptophan to adult rat hepatocyte cultures stimulated DNA synthesis. The increase in DNA synthesis as measured by 3H-thymidine incorporation into DNA was observed on treatment of the cultures with tryptophan for 48 h but also as short as for 6 h in comparison with control cultures. An increase was also apparent at 30 h which was maintained for up to 48 h post treatment with tryptophan. The increase in DNA synthesis by tryptophan cannot be attributed to cell injury or to increased DNA degradation. Of the degradative enzymes added after harvesting the hepatocytes, only DNase decreased incorporation of 3H-thymidine. The observed effect was specific for tryptophan since treatment with kynurenine, isoleucine, methionine or serine failed to show a significant effect. Pretreatment of cultured hepatocytes with hydroxyurea prevented the tryptophan stimulated increase in DNA synthesis suggesting that the latter was due to replicative and not to reparative DNA synthesis. Experiments performed with the addition of diethylnitrosamine also alluded to tryptophan's role in replicative DNA synthesis. The mechanism of tryptophan-induced DNA synthesis is discussed.     

Further characterization of a cell-free system for measuring replicative and repair DNA synthesis with cultured human fibroblasts and evidence for the involvement of DNA polymerase alpha in DNA repair.

DNA repair synthesis can be specifically measured in osmotically opened, confluent cultured human fibroblasts after exposure to DNA damaging agents such that both induction and mediation of DNA repair synthesis can take place in this cell-free system. Alternatively, by utilizing osmotically shocked, log phase cells and altering the DNA precursors, pH and ionic strength, replicative DNA synthesis can be specifically monitored. Autoradiographic studies show that virtually all of the nuclei from the lysates of the confluent, UV-iradiated cells are lightly labeled in the fashion characteristic of DNA repair. By contrast, only a fraction of nuclei is labeled in a population of unperturbed, opened log phase cells and the labeling is heavy and characteristic of replicative synthesis. Furthermore, equilibrium density gradient sedimentation shows that DNA synthesis in lysates of log-phase cells is semiconservative, whereas that with UV-irradiated cells is repair synthesis. This open cell system has been used to study the enzymology of DNA repair. Thus, dideoxythymidine triphosphate, a specific inhibitor of DNA polymerases beta and gamma, does not inhibit either replicative or repair synthesis. By contrast, aphidicolin, a specific inhibitor of DNA polymerase alpha, inhibits DNA repair and replicative synthesis in both intact and permeabilized cells. Finally, phage T4 UV-exonuclease stimulates repair synthesis, but only when phage T4 UV-endonuclease is also added to the UV-irradiated nuclei.     

DNA synthesis in vivo and in vitro in Escherichia coli irradiated with ultraviolet light

DNA synthesis was followed in vivo and in permeable Escherichia coli after ultraviolet light irradiation, irradiation and incubation in a growth medium containing chloramphenicol and in unirradiated cells. In vitro, replicative type DNA synthesis was partially restored after incubation of cells in medium containing chloramphenicol, but not in vivo. The DNA was pulse-labeled in permeable cells in the presence of deoxyribonucleoside triphosphates and ribonucleoside triphosphates. dCTP was replaced by 5-Hg-dCTP as a substrate for DNA synthesis. Hg-DNA was separated from cellular nucleic acids on thiol-agarose affinity columns. The 5' termini of newly synthesized DNA were analyzed after treatment with alkaline phosphatase and rephosphorylation with polynucleotide kinase and [gamma-32P]ATP. DNA synthesis in unirradiated permeable E. coli represents a replicative process dependent on ATP and inhibited by novobiocin. About 70% of the nascent DNA carried terminally labeled RNA moiety at its 5' end. In vitro DNA synthesis in irradiated cells was suppressed and hardly influenced by the presence of ATP or novobiocin. The 5'-RNA content of this cell population was less than 5%.     

生物体衰老与复制衰老--体内与体外研究

体外连续培养的细胞在有人数的细胞分裂后,更新换代合成ＤＮＡ及分裂的能力,最后导致增殖能力的丧失,但基本代谢过程仍能维持,这种现象称为复制衰老。本文讨论了复制衰老现象存在的普遍性,描述了衰老细胞伯特征,对复制衰老和生物体衰老之间的联系进行了重点分析。现有的研究虽然还不完全,但都提示复制衰老是生物体衰老在细胞水平上的反映,并充分肯定了复制衰老是一个较好的研究机体衰老的模型。     

DNA content and synthesis in the nuclei of rat cerebellar cells in organotypic cultures

Using cytophotometric and autoradiographic methods, it has been shown for the first time that in condition of an organotypic culture the replicative synthesis of DNA is induced in the Purkinje neurons of the cerebellum of newborn rats completing their terminal differentiation. This synthesis is accompanied by polyploidization of the initially diploid population of these cells (4c, much more rarely 8c, and a single 16c cell appear) rather than by cell division. In constant, the granular cells mostly retain their diploid state and only a few of them synthesize DNA to H2c values. The glial cells divide actively. Hence, evidence is presented that neurons, at least those of cerebellum, retain their potential of replicative synthesis of DNA in the organotypic culture. The important point is that DNA synthesis in their nuclei proceeds simultaneously with processes of differentiation.     

Mechanism of hepatic ornithine decarboxylase induction by the ornithine decarboxylase-inducing factor isolated from tumor ascites fluid: determination of target cells for the factor in the liver

The effect of the ODC-factor, which was partially purified from ascites fluid of mice bearing Ehrlich ascites hepatoma, on DNA synthesis in the normal mouse liver and spleen was studied, and target cells for the factor in the liver were examined. DNA synthesis in the liver increased about 4-fold over the basal level 39-42 h after the increase of ODC activity induced by injection of the factor into normal mice. This increase of DNA synthesis was inhibited by repeated injection of DAPol. The inhibition was completely reversed by the administration of an appropriate amount of putrescine at about the same time. TK activity also increased in parallel with DNA synthesis. Normal mice with and without treatment with the factor were used to examine which cell population in the liver is the real target for the factor. The livers were dispersed and three cell populations (heavy, medium, and light) were separated by centrifugation. The heavy and light cell populations were characterized as mature hepatocytes and a cell population consisting mainly of immature hepatocytes and non-hepatocytes by analysis of marker enzymes, pyruvate kinase isozymes, L and M2, respectively. The factor stimulated ODC induction, with concomitant increases in TK and DNA poly activities and DNA synthesis, most effectively in the light cell fraction followed in order by the medium and heavy fractions. A nutritional factor (a high protein diet), which is a potent inducer of liver ODC, appeared to act on liver in a different way from the ODC-factor, judging from the results of studies of both whole liver and the fractionated cell system described above. Autoradiography of [3H]thymidine incorporation into liver cells showed that DNA synthesis in mature hepatocytes as well as nonhepatocytes was enhanced by injection of the factor. Stimulation of non-hepatocytes seems to be suggestive evidence that an immunologic response of mice might be developed by the factor. In fact, ODC activity, DNA synthesis, and DNA poly activity (but not TK) in the spleen significantly increased in response to the factor and their increments were suppressed by DAPol, though less sensitively than those in the light cell fraction of the liver.     

Testing of hydralazine in in vivo-in vitro hepatocyte assays for UDS and stimulation of replicative DNA synthesis

The vasodilator hydralazine was tested for induction of DNA-repair synthesis and stimulation of replicative DNA synthesis in rat hepatocytes after administration in vivo, either once or repetitively. No increase in unscheduled or replicative DNA synthesis was observed. By contrast, positive controls clearly induced DNA-repair synthesis, either after a single treatment (4-aminobiphenyl, dimethylnitrosamine and methyl methanesulphonate) or after repetitive treatment (benzo[a]pyrene), or stimulated replicative DNA synthesis (carbon tetrachloride and dimethylnitrosamine). Thus, hydralazine displayed no genotoxic and no tumour-promoting activity in these in vivo-in vitro test systems.