Biochemical and morphological changes in the nuclear matrix prepared from apoptotic HL‐60 cells: Effect of different stabilizing procedures

Apoptotic cell death is characterized by deep morphological changes that take place in the nucleus. It is unclear whether modifications also occur in the nuclear matrix, a mainly proteinaceous structure that conceivably acts as a nuclear framework. We have investigated whether biochemical and morphological alterations of the nuclear matrix prepared from apoptotic HL‐60 cells were dependent on the manipulations to which isolated nuclei were subjected before DNase I digestion and 2 M NaCl extraction. Our results showed that the stabilizing procedures employed to preserve the inner fibrogranular network and nucleolar remnants of the matrix (i.e., a 37°C incubation; exposure to sodium tetrathionate at 4°C; exposure to sodium tetrathionate at 37°C) had no effect on the protein recovery of apoptotic nuclear matrices, which was always approximately two‐ to fivefold less than in control matrices. Moreover, one‐ and two‐dimensional gel analysis of nuclear matrix proteins showed that, in apoptotic samples, striking quantitative changes were present, as compared with controls. Once again, these changes were seen irrespective of the stabilizing procedures employed. Also, transmission electron microscope analysis showed similar morphological alterations in all types of apoptotic nuclear matrices. By contrast, the immunofluorescent distribution of the 240‐kDa NuMA protein seen in apoptotic samples was more sensitive to the stabilizing treatments. Our results indicate that the biochemical and morphological changes of the apoptotic nuclear matrix are largely independent of the isolation protocols and strengthen the contention that destruction of the nuclear matrix network is one of the key events leading to apoptotic nuclear destruction. J. Cell. Biochem. 74:99–110, 1999. © 1999 Wiley‐Liss, Inc.     

No discrete complexes containing DNA polymerase α activity can be solubilized from the heat-stabilized nuclear matrix prepared from HeLa S3 cells

Most of the DNA polymerase α activity, bound to the heat-stabilized nuclear matrix prepared from HeLa S3 cells, was released as a matrix extract by sonication. When the extract was centrifuged in a 5–20 per cent linear sucrose gradient no definite peaks of activity could be identified. Most of the activity sedimented to the bottom of the tube under all the conditions tested, whilst the remaining activity was associated with matrix fragments of various and irregular size. No 10 S complexes, containing polymerase activity, were seen after incubation of the extract for 16 h before centrifugation. Other solubilization procedures (i.e. treatment of the matrix with chelating agents, high pH associated with reducing agents, ionic and nonionic detergents) failed to produce release of matrix-bound DNA polymerase α activity. In contrast, we released 10 S complexes, containing polymerase activity, from the matrix prepared from nuclei not exposed to heat. We conclude that a 37°C incubation of isolated nuclei before extraction with 2 M NaCl and DNase I digestion causes DNA polymerase α to bind to the nuclear matrix in a form that cannot subsequently be released as discrete components, at variance with previous results obtained with the matrix prepared from regenerating rat liver.     

Catalytic properties of DNA polymerase α activity associated with the heat-stabilized nuclear matrix prepared from HeLa S3 cells

We have investigated whether or not ATP or other nucleoside di- and trisphosphates (including some nonhydrolysable ATP analogues) can stimulate the activity and/or the processivity of DNA polymerase α associated with the nuclear matrix obtained from HeLa S3 cell nuclei that had been stabilized at 37°C prior to subfractionation, as has been reported previously for DNA polymerase α bound to the nuclear matrix prepared from 22-h regenerating rat liver. We have found that HeLa cell matrix-associated DNA polymerase α activity could not be stimulated at all by ATP or other nucleotides, a behaviour which was shared also by DNA polymerase α activity that solubilizes from cells during the isolation of nuclei and that is thought to be a form of the enzyme not actively engaged in DNA replication. Moreover, the processivity of matrix-bound DNA polymerase α activity was low (< 10 nucleotides). These results were obtained with the matrix prepared with either 2M NaCl or 0·25 M (NH₄)₂SO₄ and led us to consider that a 37° incubation of isolated nuclei renders resistant to high-salt extraction a form of DNA polymerase α which is unlikely to be involved in DNA replication in vivo.     

Novel nuclear targeting coiled-coil protein of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> showing Ca<Superscript>2+</Superscript>-independent,Mg<Superscript>2+</Superscript>-dependent DNase I activity

HP0059, an uncharacterized gene of Helicobacter pylori, encodes a 284-aa-long protein containing a nuclear localization sequence (NLS) and multiple leucine-rich heptad repeats. Effects of HP0059 proteins in human stomach cells were assessed by incubation of recombinant HP0059 proteins with the AGS human gastric carcinoma cell line. Wild-type HP0059 proteins showed cytotoxicity in AGS cells in a concentration-dependent manner, whereas NLS mutant protein showed no effect, suggesting that the cytotoxicity is attributed to host nuclear localization. AGS cells transfected with pEGFP-HP0059 plasmid showed strong GFP signal merged to the chromosomal DNA region. The chromosome was fragmented into multiple distinct dots merged with the GFP signal after 12 h of incubation. The chromosome fragmentation was further explored by incubation of AGS chromosomal DNA with recombinant HP0059 proteins, which leaded to complete degradation of the chromosomal DNA. HP0059 protein also degraded circular plasmid DNA without consensus, being an indication of DNase I activity. The DNase was activated by MgCl₂, but not by CaCl₂. The activity was completely blocked by EDTA. The optimal pH and temperature for DNase activity were 7.0–8.0 and 55°C, respectively. These results indicate that HP0059 possesses a novel DNase I activity along with a role in the genomic instability of human gastric cells, which may result in the transformation of gastric cells.     

Calmodulin and calmodulin-binding proteins in liver cell nuclei

Three nuclear subfractions were prepared from isolated hepatocytes nuclei. The calmodulin content in whole nuclei was 79 ng/mg of protein. The soluble fraction obtained after digestion of the nuclei with DNase I and RNase A (S1 fraction) contained 252 ng of calmodulin/mg of protein. The pellet obtained after the digestion with nucleases was treated with 1.6 M NaCl, and the soluble fraction and the residual structures obtained after the treatment were called S2 fraction and nuclear matrix, respectively. The calmodulin contents of the S2 fraction and of the nuclear matrix were 68 and 190 ng/mg of protein, respectively. If nuclei were digested only with DNase I, the calmodulin content in the soluble fraction increased to 703 ng/mg of protein, indicating that part of the nuclear calmodulin is associated with active DNA. Five nuclear calmodulin-binding proteins were identified. Two, having apparent molecular masses of 240 and 150 kDa were only found in the nuclear matrix, whereas the other three, having molecular masses of 120, 65, and 40 kDa were found in different proportions in all nuclear subfractions. A calmodulin-dependent inhibition of protein phosphorylation in the S1 fraction was discovered. Purification attempts on the calmodulin-binding proteins of the S1 subfraction by calmodulin affinity chromatography yielded four major polypeptides with apparent molecular masses of about 41, 46, and 120 (two products) kDa. These polypeptides retained the ability to inhibit protein phosphorylation but not the sensitivity to calmodulin.     

Organization of the thyroid hormone receptor in the chromatin of C6 glial cells: Evidence that changes in receptor levels are not associated with changes in receptor distribution

The association of [¹²⁵I]T3-receptor complexes with C6 cell chromatin was analysed after a limited digestion with micrococcal nuclease (MN) or DNase I. Both nucleases solubilized up to 60–70% of receptor and 0.4 M KCl extracted 70%, of the non-digested receptor, thus showing that only a residual fraction of receptor is associated with the nuclear matrix. With DNase I the receptor was released 2–3-fold faster than the bulk of chromatin, whereas a preferential release of receptor over total chromatin was not observed with MN. The digestion of receptor with DNase I and MN occurred 14- and 6-fold faster, respectively, than the appearance of PCA-soluble chromatin. Preincubation for 48 h with 4 nM T3 of 2 mM butyrate significantly altered receptor levels but did not change sensitivity to the nucleases. These results suggest that the thyroid hormone receptor is associated with chromatin highly sensitive to nuclease digestion, and that changes in receptor number are not associated with changes in its distribution in chromatin.     

DETERMINATION OF RNA AND DNA CONCENTRATIONS IN NATURAL PLANKTON SAMPLES USING THIAZOLE ORANGE IN COMBINATION WITH DNASE AND RNASE DIGESTIONS1

A fluorometric technique, based on the combination of RNase and DNase incubation with the use of thiazole orange (RNase/DNase method), was investigated to determine DNA and RNA concentrations in marine plankton. Tests were performed to optimize both RNase and DNase assay conditions. The RNase assay should be conducted at 37° C for 20 min with 0.5 μg·mL^?1 of DNase-free RNase. An incubation at 25° C for 20 min with 10 units ·mL^-1 of RNase-free DNase were the optimal conditions required for DNA digestion by DNase. The detection limits in terms of minimum biomass for reliable measurements of DNA and RNA were 7.5 and 10 μg of protein · (mL assay)^?1, respectively. RNA and DNA concentration were estimated in oligotrophic water samples using the RNase/DNase and other available methods (e.g. a double fluorochrome method). The different techniques provided similar DNA estimations. However, the RNase/DNase method provided the highest sensitivity and a low variability for the estimation of RNA.     

The effect of in vitro heat exposure on the recovery of nuclear matrix-bound DNA polymerase alpha activity during the different phases of the cell cycle in synchronized HeLa S3 cells.

HeLa S3 cells were synchronized by a double thymidine block or aphidicolin treatment and the levels of nuclear matrix-bound DNA polymerase alpha activity were then measured using activated calf thymus DNA as template. The nuclear matrix was obtained by 2 M NaCl extraction and DNase I digestion of isolated nuclei incubated at 37 degrees C for 45 min prior to subfractionation. In all phases of the cell cycle 25-30% of nuclear DNA polymerase alpha activity remained matrix-bound, even when cells were in the G1 phase. No dynamic association of DNA polymerase alpha activity with the matrix was seen, at variance with previous results obtained in regenerating rat liver. The variations measured in matrix-bound activity closely followed those detected in isolated nuclei throughout the cell cycle. If nuclei were not heat-stabilized very low levels of DNA polymerase alpha activity were measured in the matrix (1-2% of total nuclear activity). Heat incubation of nuclei failed to produce any enrichment in matrix-associated newly replicated DNA, whereas the sulfhydryl cross-linking chemical sodium tetrathionate did. Therefore the results obtained after the heat stabilization procedure do not completely fit with the model that envisions the nuclear matrix as the active site where eucaryotic DNA replication takes place.     

On the association of DNA primase activity with the nuclear matrix in HeLa S3 cells

We have reinvestigated the association of DNA primase activity with the nuclear matrix prepared from exponentially growing HeLa S3 cells. We have found that 25–30 per cent of the nuclear primase activity resists extraction with 2 M NaCl and digestion with Dnase I. Unlike previous investigations, done with the same cell line, the results showed that nuclear matrix-bound DNA primase activity represented less than 10 per cent of the total cell activity. Association of high levels of primase activity with the nuclear matrix was strictly dependent on a 37°C incubation of isolated nuclei prior to subfractionation. Evidence was obtained that the method used for preparing nuclei can have a dramatic effect on the amount of primase activity which is recovered both in the postnuclear supernatant and in isolated nuclei, thus seriously affecting the interpretation of the results about the quantity of DNA primase activity bound to the nuclear matrix.     

Absence of high levels of DNA polymerase α activity in the nuclear matrix prepared from mouse erythroleukemia cells

We have examined the association of DNA polymerase α activity with the nuclear matrix prepared by different techniques from mouse erythroleukemia cells. At variance with the data obtained using other cell types we have found that only a small amount (less than 2%) of nuclear DNA polymerase α activity resisted extraction with high-ionic strength buffers, even if nuclei were heat-stabilized by incubation at 37°C for 45 min prior to subfractionation. The recovery of DNA polymerase α activity bound to the matrix was unaffected by the type of extracting agent used (NaCl or (NH₄)₂ SO₄), by the extraction sequence or by the method employed for obtaining nuclei. These results could indicate that in some types of cells the nuclear matrix is not involved in DNA replication.     

Exchange assays using sodium thiocyanate to measure total nuclear content of estrogen receptors in the hypothalamic pituitary axis of mice

We studied the effect of sodium thiocyanate (NaSCN) in the determination of specific nuclear estrogen receptors from the hypothalamic-pituitary axis (HPA) of mice. We compared the results with those of the exchange assay using either suspensions of whole nuclei or KC1-nuclear extracts. Our findings demonstrated that 0.5 M NaSCN was more efficient than 0.5 M KC1 in extracting [³H] E₂ nuclear content from HPA (91% vs 79.3%). Nuclear fractions extracted with 0.5 M NaSCN revealed the presence of a single class of low-capacity-high affinity binding sites that sedimented in the 4.0 S area. Nuclear binding was T° dependent and reached maximum levels of 450 ± 156 (S.E.) fmol/mg DNA after an overnight incubation at 4°C. Such levels were comparable to those observed in whole nuclei suspensions after 1 hour incubation at 37°C (618 ± 71 fmol/mg DNA, p > 0.05) but two-fold higher (p <0.01) than the concentration of binding sites measured in KC1-extracted nuclear fractions under similar experimental conditions. We conclude that NaSCN extracted the total content of nuclear estrogen receptors in HPA of mature mice.     

The selective inhibitory effect of NH4Cl on estrogen-stimulated rat uterine in vitro RNA synthesis

Estrogen-stimulated in vitro RNA synthesis in rat uterine nuclear-myofibrilar fractions, isolated uterine nuclei, and nucleoli was selectively inhibited by NH₄Cl (400 μmoles) when added prior to the start of a 37 °C incubation. This inhibitory effect was not observed if the salt was added after the start (as early as 1 min) of a 37 °C incubation. The removal of NH₄Cl from reaction mixtures not yet incubated at 37 °C further reduced in vitro RNA synthesis in both control and hormone-treated nuclei. The data suggest that NH₄Cl (400 μmoles) added prior to, but not after, the start of a 37 °C incubation inhibited estrogen-stimulated rat uterine nucleolar RNA polymerase activity perhaps by removing a protein component(s) which is necessary for hormonal stimulation.     

Protein disulfide crosslinking stabilizes a polyoma large T antigen-host protein complex on the nuclear matrix

We have investigated the effects of intermolecular disulfide crosslinking and temperature-dependent insolubilization of nuclear proteins in vitro on the association of the polyoma large T antigen with the nuclear matrix in polyomavirus-infected mouse 3T6 cells. Nuclear matrices, prepared from polyomavirus-infected 3T6 cells by sequential extraction of isolated nuclei with 1% Triton X-100 (Triton wash), DNase I, and 2 M NaCl (high salt extract) at 4 degrees C, represented 18% of total nuclear protein. Incubation of nuclei with 1 mM sodium tetrathionate (NaTT) to induce disulfide crosslinks or at 37 degrees C to induce temperature-dependent insolubilization prior to extraction, transferred an additional 9-18% of the nuclear protein from the high salt extract to the nuclear matrix. This additional protein represented primarily an increased recovery of the same nuclear protein subset present in nuclear matrices prepared from untreated nuclei. Major constituents of chromatin including histones, hnRNP core proteins, and 98% of nuclear DNA were removed in the high salt extract following either incubation. Polyoma large T antigen was quantified in subcellular fractions by immunoblotting with rat anti-T ascites. Approximately 60-70% of the T antigen was retained in nuclei isolated in isotonic sucrose buffer at pH 7.2. Most (greater than 95%) of the T antigen retained in untreated nuclei was extracted by DNase-high salt treatment. Incubation at 37 degrees C or with NaTT transferred most (greater than 95%) of the T antigen to the nuclear matrix. T antigen solubilized from NaTT-treated matrices with 1% SDS sedimented on sucrose gradients as a large (50-S) complex. These complexes, isolated by immunoprecipitation with anti-T sera, were dissociated by reduction with 2-mercaptoethanol, and SDS-PAGE analysis revealed that T antigen was crosslinked in stoichiometric amounts to several host proteins: 150, 129, 72, and 70 kDa. These host proteins were not present in anti-T immunoprecipitates of solubilized nuclear matrices prepared from iodoacetamide-treated cells. Our results suggest that the majority of polyomavirus large T antigen in infected cells is localized to a specific subnuclear domain which is distinct from the bulk chromatin and is closely associated with the nuclear matrix.     

染色体端粒DNA与核骨架的结合关系（简报）

Nuclear matrix from HeLa cells was gently extracted with a high salt solution and treated with DNase I. DNA that remained associated with the nuclear matrix (N. M. DNA) and DNA fragments released into the supernatant (SN.DNA) were isolated respectively and dot hybridized to human telomere sequence (AGGGTT/TCCCAA)40 probe. As the time of DNase I treatment was extended, the amount of N. M. DNA decreased while the concentration of telomere sequence in N.M. DNA proportionally increased. These preliminary results suggest that the telomere sequence is tightly bound to nuclear matrix in HeLa cells.     

The effect of sodium tetrathionate stabilization on the distribution of three nuclear matrix proteins in human K562 erythroleukemia cells

Using both conventional fluorescence and confocal laser scanning microscopy we have investigated wheter or not stabilization of isolated human erythroleukemic nuclei with sodium tetrathionate can maintain in the nuclear matrix the same spatial distribution of three polypeptides (M_r 160 kDa and 125 kDa, previously shown to be components of the internal nuclear matrix plus the 180-kDa nucleolar isoform of DNA topoisomerase II) as seen in permeabilized cells. The incubation of isolated nuclei in the presence of 2 mM sodium tetrathionate was performed at 0° C or 37° C. The matrix fraction retained 20–40% of nuclear protein, depending on the temperature at which the chemical stabilization was executed. Western blot analysis revealed that the proteins studied were completely retained in the high-salt resistant matrix. Indirect immunofluorescence experiments showed that the distribution of the three antigens in the final matrix closely resembled that detected in permeabilized cells, particularly when the stabilization was performed at 37° C. This conclusion was also strengthened by analysis of cells, isolated nuclei and the nuclear matrix by means of confocal laser scanning microscopy. We conclude that sodium tetrathionate stabilization of isolated nuclei does not alter the spatial distribution of some nuclear matrix proteins.     

C-value paradox in angiosperm plant species. I. Sensitivity to DNase I in species with different 2C DNA content.

The experimental conditions for DNAase I digestion in situ for plant nuclei have been presented. Cytophotometric measurements of DNA loss performed on Feulgen-stained nuclei of three species differing in 2C DNA, heterochromatin and condensed euchromatin contents have shown that the lower 2C DNA amount the higher is DNase I sensitivity. Heterochromatin and some fractions of euchromatin are DNase I resistant. Microdensitometric measurements along M chromosome in Vicia faba have demonstrated the sites hypersensitive to DNase I.     

Electrochemical assay for deoxyribonuclease I activity

A thiolated oligonucleotide having three ferrocenes was immobilized on a gold electrode through the sulfur-gold linkage. This electrode showed a current response based on the redox reaction of the ferrocene moieties and this response was decreased after treatment with deoxyribonuclease I (DNase I), suggesting the disappearance of the ferrocene moieties on the electrode by the DNase I digestion. A linear correlation between i₀ and i, which are current peaks before and after DNase I treatment, respectively, was observed and this slope was decreased with increase in the amount of DNase I. No current decrease was observed in the presence of EDTA or RNase A instead of DNase I. These results suggested that the current decrease responded specifically to the amount of DNase I and this electrode could be used for an electrochemical DNase I assay. Under the optimum conditions of DNase I digestion at 37 °C for 30 min, a quantitative analysis could be achieved in the range of 10⁻⁴-10⁻² units/μl of DNase I.     

Metal-ion effects on proteolysis and stability in secondary lysosomes of mouse kidney

In previous studies, in vitro digestion of [^{1 2 5}I] ribonuclease by lysosomes of mouse kidney was limited because breakdown, which was rapid at first slowed markedly so that most of the labeled protein escaped degradation. We now describe incubation conditions which allow digestion to proceed until approximately 70% of the exogenous protein label is released in acid-soluble from, after 30–45 min at 37°C. Such activity is seen with either the addition of EDTA or incubation of concentrated cell particle suspensions. EDTA is effective in low concentrations and shows the same stimulation of digestion over a range of approximately 10⁻⁶−10⁻³ M. Other chelating agents have similar effects; dipyridyl and hydroxquinoline are as effective as EDTA, o-phenanthroline and diethyldithiocarbamate are slightly less effective. When the incubation medium had been treated with a chelating resin, Chelex 100, dilute suspensions of lysosomes were as active as those in EDTA. These results lead to the conclusion that metal ions, present as contaminants in very small concentraions, inhibit the activity of mouse kidney lysosomes.The effect of the metal ions is to diminish lysosomal stability, leading to release of intact labeled ribonuclease in non-sedimentable form. Interaction between lysosomes and metal, leading to inhibition of digestion upon heating occurs at low temperature, but breakdown requires incubation at 37°C and may be autolytic. In contrast to chelators, mercaptoethanol is without marked effect on stability; the stimulation in digestion rate caused by this agent is due either to a direct effect on the lysosomal enzymes or to a non-destructive influence on the lysosomal structure.