Regulation of DNA synthesis by the higher-order chromatin structure

Mouse erythroleukemia cells were treated with the topoisomerase II poison VP-16, the intrastrand crosslinking agent cis-DDP, and the ribonucleotide reductase inhibitor hydroxyurea. In all cases, the rate of DNA synthesis decreased as a result of the treatment. To study the mechanism of inhibition of DNA chain elongation, we determined DNA synthesis in a cell-free replication system containing isolated nuclei and cytoplasmic extracts. The rate of DNA synthesis in the reactions containing nuclei isolated from untreated cells and extracts from cells treated with the three drugs were slightly reduced and did not show significant differences between the drugs. In the systems containing nuclei from cells treated with cis-DDP, DNA synthesis was again slightly inhibited; synthesis in nuclei treated with hydroxyurea was enhanced, and synthesis in the systems containing nuclei from cells treated with VP-16 was significantly reduced. DNA synthesis was reduced to the same extent in a system containing nuclei isolated from untreated cells that had been briefly sonicated to introduce a limited number of double-strand breaks in the DNA. As VP-16 and sonication mediate changes in chromatin topology, these results suggest that, along with the trans-acting signal transduction pathways, there is a topologic mechanism for regulation of DNA synthesis in the S phase of the cell cycle.     

Target tissue specificity of retinoic acid-induced stress proteins and malformations in mice

Retinoic acid (RA) is teratogenic in rodents and also induces the synthesis of stress proteins in fetal mouse limb buds. To determine if the RA induction of stress proteins is target tissue specific, pregnant CD-1 mice were gavaged with 100 mg/kg RA on day 11 of gestation, and nuclei isolated from tissues susceptible to RA-induced malformations (target tissues) as well as nuclei isolated from nontarget tissues were examined for stress protein synthesis and malformations. Forelimb and hindlimb (target tissues), as well as heart and tail (nontarget tissues), were removed from embryos 2.5 hours after RA treatment (1.5 hr after [3H]leucine labeling). Cell nuclei were isolated, stained with a DNA specific fluorochrome, propidium iodide, and sorted from the G0 + G1 and G2 + M phases of the cell cycle. Forelimb and hindlimb target tissues showed the synthesis in these embryonic nuclear proteins of an 84,000 relative molecular mass (Mr) protein and a 90,000 Mr protein following RA treatment. Two 20,000-25,000 Mr stress proteins were also labeled both in forelimb and hindlimb. Forelimb and hindlimb from untreated dams showed no stress protein labeling. Neither heart nor tail, nontarget tissues, showed any stress protein labeling following RA treatment. Classical teratological evaluation of embryos treated on GD 11 and sacrificed on GD 17 showed that 100% of the fetuses had forelimb and/or hindlimb malformations, while no malformations were observed in either the heart or tail. Based on the correlation of teratological anomalies with the identification of stress proteins in target tissue only, we postulate that stress proteins may be involved in the teratogenic process. Further work is necessary to establish whether a causal relationship exists.     

Mitogen-stimulated phosphorylation of nuclear proteins in Swiss 3T3 cells: evidence for a protein kinase C requirement

When Swiss 3T3 fibroblasts are treated with a combination of IGF-I2 and bombesin at mitogenic concentrations, in vivo phosphorylation of some nuclear proteins occurs within 45-90 min. Among these proteins, histone H1 and a 0.75 M PCA soluble polypeptide with an apparent Mr of 21,000, as revealed by electrophoretic analysis, are phosphorylated in vitro by protein kinase C in isolated nuclei purified from 3T3 cells treated for 90 min with IGF-I and bombesin. Since these phosphorylative events follow the earlier changes, recently demonstrated, in nuclear polyphosphoinositide metabolism induced by the same mitogen combination, it seems possible that these two phenomena are related to each other and trigger the synthetic machinery responsible for replicating DNA.     

Pristane induced effects on chromatin of rat lymphoid cells

The effects of pristane on the conformation of chromatin in cells isolated from the lymphoid tissues of pristane-treated Copenhagen rats were examined by flow cytometry, thermal denaturation, sensitivity to enzymatic digestion, and histone protein analyses. Decreases were observed in the fluorescent intensities of propidium iodide (PI) stained nuclei isolated from lymphoid cells of pristane-treated rats when compared with normal rat lymphoid nuclei. Studies to address the possible basis for the pristane-induced changes in the DNA staining characteristics of lymphocytes demonstrated that 1) there were no decreases in the amount of DNA present in the nuclei, 2) nuclei isolated from pristane treated rats were less sensitive to thermal denaturation, as well as DNase I enzymatic digestion, and 3) there were apparent increases in the expression of the H1 histone proteins. Collectively, these results suggest that pristane elicits a conformational change in the chromatin which may be mediated by altered expression of nuclear-associated histone proteins.     

Isolation of chick intestinal nuclei. Effect of vitamin D3 on nuclear metabolism

1. Chick intestinal nuclei were isolated, with practically no contamination from other organelles and whole cells, by centrifugation through 2.4m-sucrose. 2. The proportions of RNA, DNA and protein of the isolated nuclei were unaffected by the vitamin D status of the birds. The RNA/DNA ratio was 0.15. 3. The incorporation of [5-(3)H]orotic acid into the rapidly labelled intestinal nuclear RNA, after a 10min. pulse of the orotic acid, was increased in vitamin D-deficient chicks only 10min. after a 125mug. dose of cholecalciferol. 4. There was no stimulation of the DNA-dependent RNA polymerase activity of the isolated nuclei from birds treated with cholecalciferol. 5. The results are discussed in relation to the changes occurring during the lag period, after administration of cholecalciferol and before Ca(2+) transport is detected, and the function of the vitamin.     

Analysis of the nuclear estrogen receptor from MCF-7 cells by limited proteolysis

The proteolytic fragments of the nuclear estrogen receptor in the MCF-7 cell line were characterized following limited digestion with chymotrypsin and trypsin. Nuclei were isolated from cells previously exposed to 10 nM [3H]estradiol. The proteolytic digestion was performed either on the micrococcal nuclease hydrolysate or on intact nuclei. The molecular weights (Mr) were calculated from the sedimentation coefficients determined on a sucrose gradient and from the Stokes radii estimated by gel filtration. Digestion of the nuclei with micrococcal nuclease solubilized a receptor form of Mr = 151,000. This receptor form was degraded by chymotrypsin to a receptor of Mr = 33,000 and by trypsin to a receptor of Mr = 60,000. Digestion of intact nuclei with chymotrypsin solubilized a receptor form of Mr = 62,000 which dissociated in 0.4 M KCl to a receptor of Mr = 32,000. Digestion of intact nuclei with trypsin followed by micrococcal nuclease solubilized a receptor form of Mr = 75,000 which was further dissociated by 0.4 M KCl to a receptor form of Mr = 60,000. The ability of the receptor forms to bind DNA was tested using DNA-cellulose column chromatography. About 40% of the micrococcal nuclease solubilized receptor form, compared to about 7% of the chymotrypsin degraded receptor and to about 13% of the trypsin degraded receptor forms, all bound to the column and could be eluted by high salt concentrated buffer. We conclude that the nuclear estrogen receptor in the MCF-7 cell line can be partially degraded either in the micrococcal nuclease hydrolysate or in intact nuclei by chymotrypsin or trypsin generating protein moieties, probably receptor fragments of Mr = 33,000 and 60,000 respectively. Both fragments retain their estradiol binding domain and it may be hypothesized that the heavier fragment retains its chromatin binding domain.     

Effect of polyamines on ADP-ribosylation by chick-embryo-liver nuclei

Effects of polyamines on poly(ADP-ribose) formation and DNA synthesis in the chick-embryo-liver nuclei were investigated. When 14-day chick-embryo-liver nuclei were incubated with [3H]NAD in the presence of 1 mM spermine, 2.5 mM spermidine, or 3.5 mM putrescine, a 9-fold increase in poly)ADP-ribose) formation was observed. Nuclei treated with nuclease showed high poly(ADP-ribose) synthetase activity as spermine-treated nuclei. However, no further increase in the polymer formation by polyamines was detected in the nuclease-treated nuclei. We found that an increase in the polymer formation by spermine was the result of an increase in both chain length and chain number of the polymer at 2.3- and 6-fold, respectively. The major ADP-ribosylated proteins were determined as two non-histone proteins of Mr 130 000 and 70 000. The experiment of DNA synthesis with nuclei ADP-ribosylated in the presence of spermine showed a 7-fold increase in [3H]dTMP incorporation into the acid-inaoluble fraction. A similar stimulation was also found with nuclei treated with other polysmines, spermidine and putrescine, in the presence of NAD. These results indicate that DNA synthesis in growing tissues containing polyamines at high levels, such as is the case with tumors and the fetus, is stimulated by polyamine-mediated ADP-ribosylation of the nuclear proteins.     

STRUCTURAL ALTERATION IN ISOLATED RAT LIVER NUCLEI AFTER REMOVAL OF TEMPLATE RESTRICTION BY POLYANIONS

Specific polyanions release DNA template restrictions for DNA synthesis in isolated rat liver nuclei. The degree to which DNA synthesis is enhanced can be correlated with a spectrum of changes in nuclear structure Each polyanion which is effective in the release of template restriction produces a characteristic alteration in nuclear ultrastructure. Polyanions which have no effect on DNA synthesis do not appear to cause any change in nuclear organization or ultrastructure. Parallel measurements of nuclear DNA release and nuclear volume changes also indicate that template-activating polyanions cause remarkable changes in the structural organization of the treated nuclei. These results indicate that DNA template activation involves direct interactions between polyanions and nuclear constituents and suggest the possibility that naturally occurring polyanions might have a role in the control of gene activity     

Thymocyte apoptosis induced by phosphorylation of histones is associated with the change in chromatin structure to allow easy accessibility of DNase

The inhibitors of protein phosphatase such as calyculin A and okadaic acid induce the apoptotic cell death in rat thymocytes. To clarify the molecular mechanism of these inhibitor-induced apoptosis, the effect of calyculin A on DNA fragmentation in the isolated nuclei were studied. A significant increase in DNA fragmentation was observed in the nuclei prepared from the cells treated with calyculin A that caused histone hyperphosphorylation. No changes of the activities of caspase-8 and -3 were observed in the extract from the cells treated with calyculin A. The circular dichroism analysis of soluble chromatin from calyculin A-treated thymocyte nuclei indicated that phosphorylation of histones decreased its alpha-helical content. Thus, the change in the chromatin structure may be due to the chemical modification of histones. Moreover, the structural change in chromatin preceded DNA fragmentation in the nuclei. Therefore, these results suggest that the change of chromatin structure allow easy accessibility of nuclear DNase to chromosomal DNA.     

The relationship between DNA strand-scission and DNA synthesis inhibition in HeLa cells treated with neocarzinostatin.

Neocarzinostatin inhibits DNA synthesis in HeLa S3 cells and induces the rapid limited breakage of cellular DNA. The fragmentation of cellular DNA appears to precede the inhibition of DNA synthesis. Cells treated with drug at 37 degrees C for 10 min and then washed free of drug show similar levels of inhibition of DNA synthesis or cell growth, or of strand-scission of DNA as when cells were not washed. If cells are preincubated with neocarzinostatin at 0 degrees C before washing, the subsequent incubation of 37 degrees C results in no inhibition of DNA synthesis or cell growth, or cutting of DNA. Isolated nuclei or cell lysates derived from neocarzinostatin-treated HeLa S3 cells are inhibited in DNA synthesis but this can be overcome in cell lysates by adding activated DNA. A cytoplasmic fraction from drug-treated cells can stimulate DNA synthesis by nuclei isolated from untreated cells, whereas nuclei from drug-treated cells are not stimulated by the cytoplasmic fraction from untreated cells. By contrast, neocarzinostatin does not inhibit DNA synthesis when incubated with isolated nuclei, but it can be shown that under these conditions the DNA is already degraded and is not further fragmented by the drug. These data suggest that the drug's ability to induce breakage of cellular DNA in HeLa S3 cells is an essential aspect of its inhibition of DNA replication and may be responsible for the cytotoxic and growth-inhibiting actions of neocarzinostatin.     

Mycoplasma nucleases able to induce internucleosomal DNA degradation in cultured cells possess many characteristics of eukaryotic apoptotic nucleases

It was previously shown (Paddenberg et al (1996) Eur J Cell Biol 69, 105 - 119) that cells of established lines like NIH3T3 fibroblasts and the human pancreatic adenocarcinoma PaTu 8902 line only degrade their chromatin at internucleosomal sites after an apoptotic stimulus when infected with Mycoplasma hyorhinis. In order to distinguish mycoplasma nucleases (Mr 47 - 54 kDa) from already described eukaryotic apoptotic enzymes, the mycoplasma nucleases were partially purified from serum-free culture supernatants and further characterized. Here we demonstrate directly that the enriched mycoplasma nucleases were able to fragment the DNA of nuclease-negative substrate nuclei at internucleosomal sites. The DNA degradation was accompanied by morphological changes typical of apoptosis like chromatin condensation and margination followed by shrinkage of the nuclei. The biochemical characterization revealed that the mycoplasma nucleases had a neutral to weakly basic pH-optimum. They required both calcium and magnesium in the mM range for maximal activation and were inhibited by zinc chloride, EGTA and EDTA. In two dimensional zymograms they migrated as three spots with isoelectic points between 8.1 and 9.5. They were not inhibited by monomeric actin. Our data also demonstrate that nuclear extracts prepared from nuclei isolated from Mycoplasma hyorhinis infected cells contained the mycoplasma nuclease activities leading to their internucleosomal DNA-degradation after incubation in the presence of calcium and magnesium.     

DNA damage induced by hydrogen peroxide in cultured tobacco cells is dependent on the cell growth stage

The level of hydrogen peroxide (H(2)O(2))-induced genomic DNA damage measured by the Comet assay in tobacco suspension cells (TX1) increased as a function of the age of the culture. After treatment of TX1 cells with 15 mM H(2)O(2), the average (+/-S.E.) median tail moment value was only 4.85+/-1.00 microm in nuclei isolated from 2-day-old cells compared to 72.33+/-1.40 microm in nuclei isolated from 12-day-old cells. By contrast, nuclei first isolated from 2 and 12-day-old cells and then treated with H(2)O(2), expressed the same level of DNA damage. The activity of catalases was markedly higher in 2-day-old TX1 cells compared to 12-day-old cells. The results indicate that the reaction of the H(2)O(2) with nuclear DNA is modified by the presence of the plant cell wall, and enzymes and macromolecules present in the cytosol, and is not connected with changes in the nuclear DNA sensitivity during cell suspension growth.     

Analysis of human cytomegalovirus nucleoprotein complexes

When chromatin was isolated from cells infected with human cytomegalovirus, the virus DNA remained with the chromatin fraction. If deproteinized virus DNA was added to either isolated nuclei or chromatin, the DNA was lost during the chromatin isolation. When isolated chromatin from cytomegalovirus-infected cells was banded in isopycnic metrizamide gradients, a single peak with a density of 1.18 g/cm3 was present. Analysis of this peak in isopycnic neutral CsCl gradients indicated that it contained both human cytomegalovirus and human embryonic lung cell DNAs. When infected nuclei were treated with micrococcal nuclease, 11S subunit particles which cosedimented with cell nucleosomes and contained virus DNA were isolated.     

Characterization of the CB antigen, a DNA-binding protein recognized by autoantibodies and which has a differential expression in lymphoid cells

We have analyzed the characteristics of the CB Ag, a nuclear protein recognized by autoantibodies. Approximately 4% (12 out of 280) of the antinuclear-positive sera examined contained anti-CB antibodies. By immunofluorescence, these sera brightly stained the nuclei of most cells analyzed, including peripheral lymphocytes, but only dull or no staining was observed in thymocytes or B cells of the bursa of Fabricius. The CB Ag has been characterized as a DNA-binding protein, dissociable from DNA at 1.5 M NaCl, and with a Mr of 40,000 Da. Moreover, the ability of the extracted Ag to bind back to DNA has enabled us to design an ELISA system for its detection.     

Interactions of acetylated histones with DNA as revealed by UV laser induced histone-DNA crosslinking

The interaction of acetylated histones with DNA in chromatin has been studied by UV laser-induced crosslinking histones to DNA. After irradiation of the nuclei, the covalently linked protein-DNA complexes were isolated and the presence of histones in them demonstrated immunochemically. When chromatin from irradiated nuclei was treated with clostripain, which selectively cleaved the N-terminal tails of core histones, no one of them was found covalently linked to DNA, thus showing that crosslinking proceeded solely via the N-terminal regions. However, the crosslinking ability of the laser was preserved both upon physiological acetylation of histones, known to be restricted to the N-terminal tails, and with chemically acetylated chromatin. This finding is direct evidence that the postsynthetic histone acetylation does not release the N-terminal tails from interaction with DNA.     

Heat-induced alterations in DNA polymerase activity of HeLa cells and of isolated nuclei. Relation to cell survival

The activity of DNA polymerase alpha and beta was assayed in heated HeLa S3 cells as well as in nuclei isolated from these cells. The enzyme activity as measured in cells and in nuclei has been compared with the extent of cell survival after the different hyperthermic doses. It was found that although the activity of the cellular DNA polymerases was related to cell survival after single heat doses, no correlation was found when thermotolerant cells were heated. When the activity of the DNA polymerases was determined in nuclei isolated from non-heated and heated cells, more polymerase activity was found in the nuclei of the heated cells. However, the heat sensitivity of DNA polymerase activity was the same for nuclei isolated from control, pre-heated and thermotolerant cells. Heat protection of polymerase activity by erythritol and sensitization by procaine was found when cells, but not when nuclei, were heated in the presence of these modifiers. It is concluded that (the nuclear bound) DNA polymerases are not to be considered as key enzymes in cellular heat sensitivity of HeLa S3 cells.     

Two-dimensional electrophoresis of proteins of citric acid nuclei prepared with aid of a Tissumizer

Nuclei prepared from normal rat liver and Novikoff hepatoma ascites cells with the aid of a Tissumizer® in media containing 0.5 and 5 % citric acid were compared on the basis of electron microscopic appearance, DNA, RNA and protein content. Electron microscopy revealed better preservation of the nucleolar and nuclear morphology in the nuclei isolated in 0.5 % citric acid than in nuclei isolated in 5 % citric acid. Moreover, losses of protein and DNA from liver nuclei prepared by the sucrose-Ca²⁺ procedure were significantly less in nuclei treated with 0.5 % citric acid than in nuclei treated with 5 % citric acid. The preservation of nuclear morphology and the retention of the majority of types of nuclear protein were significantly better with the procedure using 0.5 % citric acid than with the procedure using 5 % citric acid. The 5 % citric acid treatment was found to alter nuclear morphology and extract specific nuclear proteins, as demonstrated by two-dimensional polyacrylamide gel electrophoresis of the proteins.