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PubMed2003

A possible role of cholesterol-sphingomyelin/phosphatidylcholine in nuclear matrix during rat liver regeneration.

BACKGROUND/AIMS: Phospholipids and cholesterol in chromatin have been previously demonstrated. The lipid fraction changes during cell proliferation in relation to activation of enzymes of phospholipid metabolism. The aim of the present work is to clarify if chromatin lipids may derive or not from nuclear matrix and if they have different roles. METHODS: The subnuclear fractions were isolated from rat hepatocyte nuclei and the lipid fraction was extracted and analysed by chromatography in normal and regenerating liver. The phosphatidylcholine-sphingomyelin metabolism enzymes activity was assayed, by using radioactive substrates. RESULTS: In nuclear matrix, cholesterol and sphingomyelin are respectively five and three times higher than those present in chromatin; the amount of phosphatidylcholine, which it is enriched in saturated fatty acids, is lower, thus indicating a less fluid structure. The lower content in phosphatidylcholine may be justified by the phosphatidylcholine-dependent phospholipase C activity, which increases during liver regeneration, reaching a peak at the beginning of S-phase, when also cholesterol and sphingomyelin increase. CONCLUSIONS: The nuclear matrix lipids are independent from chromatin lipids; the ratio cholesterol-sphingomyelin/phosphatidylcholine is higher and, as a consequence, nuclear matrix is less fluid in relation to DNA synthesis, suggesting a specific role of nuclear matrix as a structure involved in DNA duplication.

Animals↗
PubMed2006

[Changes of nuclear matrix proteins during differentiation of human osteosarcoma MG-63 cells induced by HMBA].

Human osteosarcoma MG-63 cells were induced into differentiation by hexamethylene bisacetamide (HMBA). Its nuclear matrix proteins were selectively extracted, and subjected to two dimensional gel electrophoresis analysis. The resulted protein patterns were analyzed by Melanie software. There were 12 spots changed remarkably during the differentiation induced by HMBA, nine of which were identified. The up-regulated proteins were identified as MHC class II antigen, interferon-stimulated gene factor 3d, hypothetical protein DKFZp434M2221.1,8-hydroxy-guanine glycosylase homolog ogg1, and vimentin. The down-regulated ones were hnRNP A2/B1 and actin; and two newly expressed proteins under induction were 60S ribosomal protein L21 and ST2 protein. This study suggests that the induced differentiation of carcinoma cells is accompanied with changes of nuclear matrix proteins, and confirms the presence of some specific nuclear matrix proteins associated with carcinoma cell growth and differentiation. The changed nuclear matrix proteins are potential markers for cancer diagnosis or targets for cancer therapy.

Acetamides↗
PubMed1998

Mechanism of protein kinase CK2 association with nuclear matrix: role of disulfide bond formation.

Nuclear matrix (NM) appears to be an intranuclear locale for significant and dynamic association of the ubiquitous multifunctional messenger-independent serine/threonine protein kinase CK2 that has been implicated in growth control [Tawfic et al. (1996): J Cell Biochem 61:165-171]. We have examined the nature of the association of CK2 with the NM. Nuclei prepared in the presence of a sulfhydryl-blocking reagent such as iodoacetamide demonstrate a reduction in the amount of CK2 associated with the NM to less than 5% of the control. On the other hand, when nuclei are treated with the sulfhydryl crosslinking reagent sodium tetrathionate, NM-associated CK2 increases severalfold. Treatment of nuclei with sodium tetrathionate followed by 2-mercaptoethanol blocks this increase. Nuclei isolated from rat liver and prostate behaved similarly, suggesting an identical mode of association of CK2 with the NM regardless of the organ. These results indicate a role of sulfhydryl interactions such that NM anchoring of CK2 occurs via its beta subunit, which contains several vicinal cysteine residues. Further, various sulfhydryl-blocking reagents inhibited CK2 activity in a concentration-dependent manner, and the inhibitory effect was reversed by agents such as dithiothreitol, implying that cysteine residues in the CK2 play a role in its catalytic activity.

Animals↗
PubMed2002

Dynamics of replication foci and nuclear matrix during S phase in Allium cepa L. cells.

The sequential organisation of replication foci during S phase in onion ( Allium cepa) and their relationship to the nuclear matrix were investigated. To discern their structural features and temporal firing sequence, immunodetection of 5-bromo-2'-deoxyuridine (BrdU) was carried out after in vivo feeding in synchronised cells released from a 14-h-long hydroxyurea block. Replication foci consisted of small replication granules, called replisomes, which clustered together. Analysis of synchronous binucleate cells that maintained in their two nuclei the specular symmetry of distribution of sister chromosomes in anaphase, showed that replication starts in small replication foci at the telomeric pole (pattern I), though the telomeres themselves formed large foci that were late-replicating. The rDNA replication foci (pattern II) also become replicated in early S phase. Replication of large foci, including the heterochromatin (IV), occurred in late S phase and finished at the centromeric nuclear pole (pattern V). Labelling of proliferating cell nuclear antigen (PCNA) in nuclear matrices, prepared from S-phase nuclei after extensive DNase digestion, demonstrated that replication foci were always stably anchored to the nuclear matrix. Thus, association with the nucleoskeleton is not exclusively mediated by the replicating or nascent DNA. The overlapping of patterns I, II and III in the nuclear matrix, in contrast to the results of BrdU localisation in nuclei, suggests that PCNA becomes associated with the nuclear matrix before the replication foci are operative, and remains bound during replication.

Bromodeoxyuridine↗
PubMed1996

The utilization of nuclear matrix proteins for cancer diagnosis.

There is a great need for improved biomarkers in the areas of cancer diagnosis and treatment. Cancer-specific nuclear matrix proteins may provide clinicians with improved biomarkers for earlier diagnosis as well as improved therapies. The nuclear matrix is the RNA-protein skeleton of the nucleus that has structural and functional roles within the cell. Nuclear matrix proteins of a variety of cell lines and tissues, both normal and cancerous, have now been examined and are beginning to be characterized. After comparison of tumor and normal tissues as well as distinct tissue-specific and cancer-specific differences. It is these proteins differences that provide possible biomarkers that may allow for earlier detection of cancer and thus potentially increase the chance of survival.

Animals↗
PubMed2000

Atrophin-1, the dentato-rubral and pallido-luysian atrophy gene product, interacts with ETO/MTG8 in the nuclear matrix and represses transcription.

Dentato-rubral and pallido-luysian atrophy (DRPLA) is one of the family of neurodegenerative diseases caused by expansion of a polyglutamine tract. The drpla gene product, atrophin-1, is widely expressed, has no known function or activity, and is found in both the nuclear and cytoplasmic compartments of neurons. Truncated fragments of atrophin-1 accumulate in neuronal nuclei in a transgenic mouse model of DRPLA, and may underlie the disease phenotype. Using the yeast two-hybrid system, we identified ETO/MTG8, a component of nuclear receptor corepressor complexes, as an atrophin-1-interacting protein. When cotransfected into Neuro-2a cells, atrophin-1 and ETO/MTG8 colocalize in discrete nuclear structures that contain endogenous mSin3A and histone deacetylases. These structures are sodium dodecyl sulfate-soluble and associated with the nuclear matrix. Cotransfection of ETO/MTG8 with atrophin-1 recruits atrophin-1 to the nuclear matrix, while atrophin-1 and ETO/MTG8 cofractionate in nuclear matrix preparations from brains of DRPLA transgenic mice. Furthermore, in a cell transfection-based assay, atrophin-1 represses transcription. Together, these results suggest that atrophin-1 associates with nuclear receptor corepressor complexes and is involved in transcriptional regulation. Emerging links between disease-associated polyglutamine proteins, nuclear receptors, translocation-leukemia proteins, and the nuclear matrix may have important repercussions for the pathobiology of this family of neurodegenerative disorders.

Animals↗
PubMed1994

The effect of in vitro heating on the distribution of nuclear matrix polypeptides in HeLa cells.

The in situ nuclear matrix was obtained from HeLa cells. After permeabilization with nonionic detergent, the resulting structures were incubated for 1 h at 37 degrees C to determine whether or not such an incubation might result in the redistribution of nuclear polypeptides which resisted extraction with buffers of high-ionic strength (1.6 M NaCl or 0.25 M (NH4)2SO4 as well as DNase I digestion. Using indirect immunofluorescence experiments and monoclonal antibodies we show that heating to 37 degrees C changes the distribution of a 160 kDa protein previously shown to be a component of the inner matrix network. On the other hand, a 125 kDa polypeptide was not affected at all by the incubation. Our results clearly indicate that the inclusion of a 37 degrees C incubation (for example during digestion with DNase I) in the protocol to obtain the in situ nuclear matrix can result in the formation of in vitro artifacts.

Blotting, Western↗
PubMedDate not supplied

[Characteristics of DNA sequences in the sites of permanent attachment to the nuclear matrix located in the vicinity of replication initiation site].

The permanent DNA attachment sites to the nuclear matrix in the domain of chicken alpha-globin genes originally found in erythrocyte nuclei are shown to exist in sperm and cultured fibroblast cells too. Short fragments of permanently attached to the nuclear matrix DNA have been cloned and sequenced. A primary structure of a 1.7 k.b. fragment from 5'-region of chicken alpha-globin gene domain containing both replication origin and permanent attachment site has been determined. A region possessing homologies with papovaviral replication origins and putative mammalian ARS elements has been found on the 1.7 k.b. fragment. A region containing short internal repeats and GC-rich motifs has also been found. Similar motifs were observed in several of the cloned short fragments of DNA permanently attached to the nuclear matrix.

Animals↗
PubMedDate not supplied

Microwave fixation of nuclear matrix in tumor cells.

Microwave irradiation provides good fixation of human and animal tissues for light and electron microscopy. In this study, microwave irradiation was used for the fixation of cytoplasmic and nuclear matrix in tumor cells. The nuclear matrix appears well preserved and exhibits a network formed by thick and thin filaments. Hence microwave fixation can be used as a quick and effective method for the study of the morphology of nuclear matrix.

Animals↗
PubMed1994

Histone acetyltransferase is associated with the nuclear matrix.

Only a small fraction of the adult chicken erythrocyte histones is involved in dynamic acetylation. We have reported previously that the rapidly acetylated and deacetylated H4 histones are primarily associated with the transcriptionally active DNA-enriched chromatin fragments that remain attached to the residual nuclear material following micrococcal nuclease digestion and chromatin solubilization. Furthermore, this nuclear fraction contained most of the histone deacetylase activity. In this study we show that the bulk of the nuclear histone acetyltransferase activity is located with the insoluble residual nuclear material. We demonstrate that in vitro the enzymes associated with the residual nuclear material catalyze reversible acetylation when the endogenous histones of the nuclear skeleton-bound chromatin fragments are used as substrate. Nuclear matrices isolated from adult chicken immature erythrocyte and trout liver nuclei had 60-76% of the nuclear histone acetyltransferase activity. Procedures that solubilized the internal nuclear matrix also resulted in the release of the enzyme from the nuclear matrix. Together, our observations suggest that histone acetyltransferase and deacetylase are associated with the internal nuclear matrix, and one of the functions of these enzymes may be to mediate a dynamic attachment between transcriptionally active chromatin and the nuclear matrix.

Acetates↗
PubMed1993

ors12, a mammalian autonomously replicating DNA sequence, associates with the nuclear matrix in a cell cycle-dependent manner.

Origin enriched sequence ors8 and ors12, have been isolated previously by extrusion of nascent CV-1 cell DNA from replication bubbles at the onset of S-phase. Both have been shown to direct autonomous DNA replication in vivo and in vitro. Here, we have examined the association of genomic ors8 and ors12 with the nuclear matrix in asynchronous and synchronized CV-1 cells. In asynchronously growing cells, ors8 was found to be randomly distributed, while ors12 was found to be enriched on the nuclear matrix. Using an in vitro binding assay, we determined that ors12 contains two attachment sites, each located in AT-rich domains. Surprisingly, in early and mid-S-phase cells, ors12 homologous sequences were recovered mainly from the DNA loops, while in late-S the majority had shifted to positions on the nuclear matrix. In contrast, the distribution of ors8 over the matrix and loop DNA fractions did not change during the cell cycle. By bromodeoxyuridine substitution of replicating DNA, followed by immunoprecipitation with anti-bromodeoxyuridine antibodies and PCR amplification, we demonstrated that ors12 replicates almost exclusively on the matrix in early and mid-S-phase; replicating ors8 was also found to be enriched on the matrix in early S-phase. Chase experiments showed that the ors12 sequences labelled with bromodeoxyuridine in the first 2 hours of S-phase remain attached to the nuclear matrix, resulting in an accumulation of ors12 on the nuclear matrix at the end of the S period.

Animals↗
PubMed2005

[Proteome analysis of nuclear matrix proteins during arsenic trioxide induced apoptosis in K562 cells].

OBJECTIVE: To investigate arsenic trioxide (As(2)O(3))-target interactions at the level of nuclear matrix (NM) in chronic myelogenous leukemia cell line K562 by proteomics. METHODS: DNA fragmentation analysis was used for As(2)O(3) induced apoptosis of K562 cells. The nuclear matrix proteins were analyzed by high-resolution two-dimensional gel electrophoresis and computer-assisted image analysis. RESULTS: While more than 200 protein spots were shared among the nuclear matrices, about 18 distinct spots were found characteristic of As(2)O(3) treated cells. Onset of mass mange apoptosis, and the profiling of nuclear matrix proteins had been alternated and it was a more sensitive indicator than nucleosomal DNA fragmentation against As(2)O(3) treatment. CONCLUSION: As(2)O(3) induced apoptosis in K562 cells in a dose-time-dependent manner. As(2)O(3) might be clinically useful in treatment of chronic myelogenous leukemia and the changes of nuclear matrix proteins in the treated cells can be used as a useful indicator for the treatment.

Antineoplastic Agents↗
PubMed1993

Copper ion-mediated sensitization of nuclear matrix attachment sites to ionizing radiation.

Exposure of mammalian cells to ionizing radiation induces nuclear matrix proteins and their attached transcribing DNA sequences to form cross-links. To characterize the cellular and matrix components necessary for DNA-protein crosslink (DPC) formation, DPC yields have been examined in isolated nuclear matrices and in the intermediate steps during cell fractionation. It was found that, in both unirradiated and irradiated cells, all components of DPC are retained in isolated nuclei, and the formed DPC are retained as well during the cell fractionation procedure resulting in nuclear matrices. In contrast, nuclear matrices isolated from unirradiated cells are deficient in the ability to form DPC upon irradiation, indicating that elements necessary for DPC production have been disrupted or removed during the isolation procedure. When isolated nuclei were irradiated, the yield of radiation-induced DPC was about 2-fold higher than that for intact cells, presumably due to the removal of soluble cellular scavengers during the isolation procedure. Treatment of nuclei with Cu2+ to stabilize nuclear structural organization during the preparation of the nuclear matrix caused additional DNA, especially the matrix-associated newly replicated DNA, to become bound to protein. Such treatment also enhanced radiation-induced DPC production which was sensitive to OH radical scavengers. Moreover, radiation-induced DPC production in Cu(2+)-treated nuclei was more sensitive to EDTA and catalase than in untreated nuclei. It is therefore proposed that excess DPC induction in Cu(2+)-treated nuclei occurs preferentially at the sites of Cu2+ binding to chromatin where hydroxyl radicals are produced repeatedly through the Fenton reaction.

Animals↗
PubMed1993

Multitude of inverted repeats characterizes a class of anchorage sites of chromatin loops to the nuclear matrix.

In order to understand the nature of DNA sequences that organize chromatin into domains or loops, we have cloned the nuclear matrix DNA (1.7% of the total DNA) from human myelogenous leukemia cells in culture. Nuclear matrix is formed by interactions between specific stretches of DNA of about 0.1 to 5.0 kb with protein transcription factors, nuclear enzymes, and structural proteins. Nuclear matrix is believed to be the exclusive nuclear microenvironment in which initiation of DNA replication, transcription, and repair take place. The matrix attachment regions (MARs) of DNA have transcriptional enhancer activity, harbor the origins of replication of the human genome, and define the borders between neighboring chromatin loops. In this study we report the sequence of the human MAR fragment 19.2 of a size of 542 bp. Hum. MAR 19.2 is composed of TG-, CA-, CT-, and GA-rich blocks and shows 8 perfect and imperfect inverted repeats. Thus, we have identified a novel class of MARs with sequence characteristics divergent from the AT-rich class of MARs. The inverted repeats of the 19.2 sequence might be stabilized into their cruciform configuration by torsional strain and by specific transcription/replication protein factors. This MAR might function in the initiation of replication of the flanking chromatin domain and in the regulation of the transcriptional activity of the gene(s) that reside in this domain.

Base Sequence↗
PubMed1986

Cell cycle-dependent expression of nuclear matrix proteins of Ehrlich ascites cells studied by in vitro translation.

A combination of methods was used to study the cell cycle-dependent expression of nuclear matrix proteins of Ehrlich ascites cells: Separation of asynchronous cells growing in vivo into fractions of G1-, S- and G2- phase cells by centrifugal elutriation with less than 10% cross-contamination. Isolation of poly(A+) RNA populations from total cytoplasmic RNA by affinity chromatography on messenger affinity paper (mAP). In vitro translation of poly(A+) RNA from asynchronous and phase synchronous cells. Immunoprecipitation of in vitro synthesized nuclear matrix proteins by a monoclonal antibody with anti-lamin specificity (PKB8) and by a polyspecific anti-nuclear matrix serum (AMS5) followed by analysis of immunoprecipitated materials on SDS-polyacrylamide gels. The results indicate that mRNAs for nuclear matrix-associated proteins including the lamins B and C are either exclusively or at least predominantly present in the cytoplasm of cells in S phase suggesting a high rate of in vivo synthesis of these proteins during S phase. This is consistent with an anticipated biological function of the nuclear matrix which is considered to organize parental and newly synthesized DNA in higher order structures.

Animals↗
PubMed1993

DNA and proteins of the nuclear matrix are the main targets of benzo[a]pyrene's action in rat hepatocytes.

The binding of [14C]benzo[a]pyrene (B[a]P) to DNA and proteins in total nuclei and subnuclear fractions of cultured rat hepatocytes was compared. The main targets of B[a]P were non-histone high molecular weight proteins of the nuclear matrix and DNA sequences attached to this structure. Following 24 h exposure to B[a]P the amounts of adducts in the nuclear matrix DNA and proteins were twice as high as in total nuclei. After withdrawal of the carcinogen containing medium the level of B[a]P-induced adducts gradually decreased but always remained the highest in the nuclear matrix proteins. Removal of adducts from the nuclear matrix DNA was more efficient than from the other DNA fractions, and 72 h after exposure to the carcinogen the level of DNA adducts in this fraction was similar to that in total nuclei.

Animals↗
PubMed2002

The interaction between the human beta-globin locus control region and nuclear matrix.

Our previous study showed that hydroxyurea (Hu) could induce HEL cells to express human beta-globin gene. However the molecular mechanisms by which the expression of beta-globin gene is activated and regulated are poorly understood. Here we show that the binding patterns between the core DNA sequences (HS2 core sequence -10681 approximately -10971 bp, HS3 core sequence -14991 approximately -14716 bp and HS4 core sequence -18586 approximately -18306 bp) of DNase I hypersensitive sites in the human beta-globin LCR and nuclear matrix proteins isolated from Hu induced and uninduced HEL cells are quite different. Results demonstrated that nuclear matrix proteins might play important roles in regulating the expression of human beta-like globin genes through their interaction with HSs (HS2, HS3 and HS4 core sequences) in the LCR. Moreover, the results obtained from the in vitro DNA-matrix binding assay showed that the core DNA sequences of DNase I hypersensitive sites (HS2, HS3 and HS4) were unable to bind to the nuclear matrix isolated from uninduced HEL cells; in addition, HS2 core DNA sequence was capable of binding to the nuclear matrix prepared from Hu-induced HEL cells, while both HS3 and HS4 core DNA sequences could not do so. Results indicated that the HS2 core DNA sequence may be a functional MAR (matrix attachment region). We suggest that the HS2 core DNA sequence binding to the nuclear matrix in Hu-induced HEL cells may open the structure of chromatin to make the LCR accessible to the promoter of beta-globin gene and to promote its transcription.

Binding Sites↗
PubMed1990

Decreased nuclear matrix DNA topoisomerase II in human leukemia cells resistant to VM-26 and m-AMSA.

CEM leukemia cells selected for resistance to VM-26 (CEM/VM-1) are cross-resistant to various other DNA topoisomerase II inhibitors but not to Vinca alkaloids. Since DNA topoisomerase II is a major protein of the nuclear matrix, we asked if alterations in nuclear matrix topoisomerase II might be important in this form of multidrug resistance. Pretreatment of drug-sensitive CEM cells for 2 h with either 5 microM VM-26 or 3 microM m-AMSA reduced the specific activity of newly replicated DNA on the nuclear matrix by 75 and 50%, respectively, relative to that of the bulk DNA. However, neither VM-26 nor m-AMSA affected the relative specific activity of nascent DNA isolated from the nuclear matrices of drug-resistant CEM/VM-1 cells. The decatenating and unknotting activities of DNA topoisomerase II were 6- and 7-fold lower, respectively, in the nuclear matrix preparations from the CEM/VM-1 cells compared to parental CEM cells. Western blot analysis revealed that the amount of immunoreactive topoisomerase II in the nuclear matrices of the CEM/VM-1 cells was decreased 3.2-fold relative to that in CEM cells, but there was no significant difference in the amount of enzyme present in the nonmatrix (1.5 M salt soluble) fractions of nuclei from these cell lines. Increasing the NaCl concentration used in the matrix isolation procedure from 0.2 to 1.8 M resulted in a progressive decrease in the specific activity of topoisomerase II in matrices of CEM/VM-1 but not CEM cells, which suggested that the association of the enzyme with the matrix is altered in the resistant cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amsacrine↗