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PubMed1983

Alkylating agent interactions with the nuclear matrix.

The interrelationship of DNA to the nuclear matrix is integral to the organization of chromatin within the nucleus and to the DNA replication process. The influence of nitrosourea and nitrogen mustard interactions with the nuclear matrix were studied in log phase HeLa cells. Alkylation of the nuclear matrix by chlorozotocin (CLZ) or 1-(2-chloroethyl-3-cyclohexyl)-1-nitrosourea (CCNU) was 1.58 and 1.27 pmoles drug/micrograms protein, respectively, whereas carbamoylation by CCNU was 32.5 pmoles/micrograms. These constituted approximately 30% of the total (nuclear) drug modifications. The structural matricin fibrillar components of the matrix were alkylated and carbamoylated twice as much as the ribonuclear protein elements (RNP). However, when alkylations are measured per microgram of protein, the ratio of covalently bound drug to RNP:matricin was 1.2 for both CLZ and CCNU. The RNP:matricin carbamoylation ratio for CCNU was 0.9. The importance of DNA and matrix protein alkylations to the process of reassociation was studied. Under control conditions, in vitro, approximately 80% of the DNA was associated with the matrix at a protein:DNA ratio (micrograms for micrograms) of 50:1. Direct alkylation or carbamoylation of the matrix proteins did not affect these DNA-protein interactions. However, using in vitro alkylated DNA (1 alkylation/10(2) base pairs), there was a 60% reduction of the alkylated nucleic acid bound to the matrix at the same protein: DNA ratio. The reduced binding of DNA to matrix may be a function of interference with the DNA recognition sites by alkylation of specific bases. The interference of DNA-matrix association by DNA alkylation may contribute to the cytotoxic activity of these antineoplastic agents.

Alkylating Agents↗
PubMedDate not supplied

Bcl-2 and Bax proteins are nuclear matrix associated proteins.

Bcl-2 and Bax proteins are implicated in the regulation of apoptosis. Nuclear matrix has been demonstrated to be associated with a vast array of functional and regulatory properties of cells. NuMA is one member of a class of nuclear matrix proteins that resides in both the nucleus and mitotic apparatus. The nuclear lamins appear to form a thin fibrous structure immediately underlying the inner nuclear membrane of eukaryotic cell nuclei. The association of bcl-2 and Bax protein with nuclear matrix in glioblastoma cell line U343 was studied by confocal microscopy and Western blotting. Confocal microscopic images display that bcl-2 was localized at the peripheral of the nuclear matrix and Bax protein was located in the nuclear matrix. Western blotting detected a 26 kDa bcl-2 band and a specific band of Bax at around 66 kDa in nuclear matrix proteins. Our results suggest that bcl-2 and Bax proteins are nuclear matrix associated proteins.

Antigens, Nuclear↗
PubMed1998

[Two proteins from the nuclear matrix bind osteopontin gene promoter].

DNA transcription and replication are organized by the nuclear matrix. It has been recently shown that the osteocalcin gene promoter binds specifically two proteins, NMP-1 and NMP-2 (nuclear matrix proteins 1 and 2), from cell line ROS 17/2.8 (Bidwell et al., 1993). Osteocalcin in one of the proteins taking part in extracellular matrix mineralization during osteogenic differentiation. Osteopontin gene is also active at the late stages of osteogenesis, so we supposed that its promoter could be bound to the nuclear matrix as well. Among the nuclear matrix proteins of ROS 17/2.8 we have found a protein factor which binds the promoter region of the osteopontin gene specifically with the help of gel shift assay. Two proteins with isoelectric points of pH > 8.0 and mol. masses of 38 and 43 kDa, respectively, have been purified by affinity chromatography with the promoter region of osteopontin attached to cellulose. The mol. masses of the revealed proteins are similar to those of already characterized NMP-2 (38 kDa) and one of subunits of heterodimer NMP-1 (43 kDa). Computer analysis shows that the binding sites for these proteins exist in the osteopontin gene promoter region. So, it is very likely that we dealt with the same proteins. A further investigation could lead to characterization of a group of nuclear matrix attached osteogenic-specific transcriptional factors.

Animals↗
PubMed1988

In situ preparation of the nuclear matrix of Physarum polycephalum: ultrastructural and biochemical analysis of different matrix isolation procedures.

A novel method for in situ preparation of nuclear matrix from whole plasmodia of Physarum polycephalum without isolation of nuclei is presented. Plasmodia are encapsulated in agarose beads and after solubilization of the cytoplasm the nuclear matrix is prepared. With this quick and easy technique nuclear matrix can be reproducibly prepared with perfect recovery. We compared the ultrastructural and biochemical properties of the matrix after three different matrix isolation procedures: preparation with high salt, ammonium sulphate and lithium diiodosalicylic acid. The results show that the ultrastructure and protein composition of the three types of matrix are very similar or even identical. We conclude that many of the conflicting results on nuclear matrix in the literature are due to perturbations of nuclear integrity during the isolation of nuclei. For this reason the new in situ method is an important approach in the standardization of nuclear matrix isolation.

Cell Nucleus↗
PubMed2003

Nuclear matrix bound fibroblast growth factor receptor is associated with splicing factor rich and transcriptionally active nuclear speckles.

We have used confocal microscopy combined with computer image analysis to evaluate the functional significance of a constitutively expressed form of the receptor tyrosine kinase FGFR1 (fibroblast growth factor receptor 1) in the nucleus of rapidly proliferating serum stimulated TE 671 cells, a medullobastoma human cell line. Our results demonstrate a limited number of large sites and numerous smaller sites of FGFR1 in the nuclear interior. The larger sites showed virtually complete colocalization (>90%) with splicing factor rich nuclear speckles while the smaller sites showed very limited overlap (<20%). Similar results were found for several other proliferating cell lines grown in culture. An in situ transcription assay was used to determine colocalization with transcription sites by incorporating 5-bromouridine triphosphate (BrUTP) followed by dual staining for BrUTP and FGFR1. These results combined with those from using an antibody against the large subunit of RNA polymerase II suggest a significant degree of colocalization (26-38%) over both the large and small sites. No colocalization was detected with sites of DNA replication. The spatial arrangements of FGFR1 sites and colocalization with nuclear speckles were maintained following extraction for nuclear matrix. Moreover, immunoblots indicated a significant enrichment of FGFR1 in the nuclear matrix fraction. Our findings suggest an involvement of a nuclear matrix bound FGFR1 in transcriptional and RNA processing events in the cell nucleus. We further propose that nuclear speckles, aside from a role in transcriptional/RNA processing events, may serve as fundamental regulatory factories for the integration of diverse signaling and regulatory factors that impact transcription and cellular regulation.

Animals↗
PubMedDate not supplied

Gel electrophoretic analysis of nuclear matrix fractions isolated from different human cell lines.

The nuclear matrix is operationally defined as the structure that remains after nuclei are extracted with nonionic detergent and with high salt and are digested with nucleases. Thus the nuclear matrix protein composition is critically dependent on the isolation conditions. We have compared nuclear matrices isolated from human cell lines by two different methods. First, isolated nuclei were extracted as above to obtain a matrix fraction. This method showed a substantial contamination by cytoplasmic intermediate filaments but immunization of mice resulted in antibodies recognizing nuclei and the mitotic spindle apparatus. Second, a nuclear matrix fraction was made by extracting whole cells as above and dissolving the residue in urea and dialysing against an assembly buffer to precipitate intermediate filament proteins (Fey, E. G. and Penman, S., Proc. Natl. Acad. Sci. USA 1988, 85, 121-125). Such fractions showed complex protein patterns in silver-stained two-dimensional gels for four cell lines: HeLa, MCF-7, SW13 and the U333CG/343MG glioma line. While some proteins in the nuclear matrix fraction were common to all cell lines, others appeared cell-line specific. Two-dimensional gels and the immunoresponse in mice again showed contamination of these preparations with cytoplasmic proteins. These results clearly show the difficulties associated with protein chemical analysis of nuclear matrices: the preparations have substantial cytoplasmic contamination, the polypeptide composition is extremely complex and the yield of individual polypeptides is low. Thus, without further experiments one cannot say which proteins are true nuclear matrix components.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex Neoplasms↗
PubMed1986

Association of RNA with the cytoskeleton and the nuclear matrix.

Heteronuclear RNA (hnRNA) is preferentially associated (76%) with the nuclear matrix in mammalian cells. Active mRNA, in the form of polyribosomes, is associated (greater than 97%) with the cytoskeletal framework. In this report, we present evidence that the association of both hnRNA and mRNA with structural networks of the cell may be essential features of gene expression. To study the association of polyribosomes with the cytoskeletal framework, cytochalasin D was used to release mRNA from the cytoskeletal framework. Protein synthesis was inhibited by cytochalasin D in direct proportion to the release of mRNA. The released mRNA is unaltered in its translatability as measured in vitro but is no longer translated in the cytochalasin-treated HeLa cells. The residual protein synthesis occurs on polyribosomes that are reduced in amount but display a normal sedimentation distribution. The results support the hypothesis that mRNA binding to the cytoskeletal framework is necessary, though not sufficient, for translation. Further fractionation of the cytoskeletal framework separates nuclear constituents into three distinct protein fractions. Chromatin proteins and 94% of the DNA are released by 0.25 M-ammonium sulphate after inter-nucleosomal DNA is cut with DNase I. The resulting structure retains 76% of the hnRNA in the form of ribonucleoprotein and is designated the RNP-containing nuclear matrix. The proteins of hnRNP complex are those associated with the nucleus only if RNA is intact. These proteins and 97% of the hnRNA are released after brief digestion with RNase A. Visualizing the nuclear matrix using resinless sections shows that nuclear RNA plays an important role in the organization of the nuclear matrix. Electron micrographs of resinless sections show the interior of the matrix to be a three-dimensional network of thick filaments bounded by the nuclear lamina. The filaments are densely covered with 20-30 nm electron-dense particles, which may contain the hnRNA. The RNP-depleted matrix is disordered and the interior fibres aggregated. These results suggest that hnRNA is involved in the spatial organization of the interior of the nuclear matrix.

Animals↗
PubMed2000

The protein composition of the hepatocyte nuclear matrix is differentiation-stage specific.

The protein composition of hepatocyte nuclear matrices was examined in rats from the 16th day of gestation to 75 days after birth (adult). An overall increase in size of the nuclear matrix was accompanied by quantitative and qualitative changes in its protein content. Quantitative changes of the major proteins of the peripheral lamina surrounding the isolated nuclear matrix were detected. By Western analysis we established that in pre- and postnatal nuclear matrices the relative concentrations of lamin C were greater than lamin A. After birth, the relative concentrations of both lamins progressively increased. In the adult nuclear matrix, the concentration of lamin A was greater than lamin C. In contrast, the relative concentrations of lamin B remained unchanged throughout development and growth. The relative concentrations of two nuclear matrix-associated regulatory proteins studied changed with development and growth: transcription factor C/EBPalpha isoforms, which were detected during the gestation period, increased notably after the first postnatal day, attaining a maximum at the adult stage; the high concentrations of the proliferating cell nuclear antigen (PCNA) perceptibly decreased after the 21st prenatal day. Changes in the composition of the nuclear matrix protein suggest that this structure coordinates nuclear functioning during cell differentiation.

Animals↗
PubMed1996

Nuclear matrix as an anchor for protein kinase CK2 nuclear signalling.

Nuclear matrix (NM) is not only the structural basis for nuclear shape but also is intimately involved in nuclear functional activities. Among the modulatory factors that may affect these diverse activities are the signals that may influence the state or composition of the NM proteins. One such mechanism for altering the functional activity of at least some NM proteins may be the extent of their phosphorylation. Protein kinase CK2 appears to associate with NM and to phosphorylate a number of NM-associated proteins. Chromatin- and NM-associated CK2 is rapidly modulated by mitogenic signals. We propose that NM serves as a physiological anchor for nuclear signalling of protein kinase CK2 which may influence functions of NM such as transcription of active genes and growth.

Animals↗
PubMed1992

Partial characterization of nuclear matrix attachment regions from human fibroblast DNA using Alu-polymerase chain reaction.

The proteinaceous nuclear matrix of mammalian cell nuclei has been suggested to be involved in the regulation of chromatin structure, DNA replication, and gene expression. Interaction between cellular DNA and the nuclear matrix is mediated by putative DNA binding sequences, matrix attachment regions (MARs), which may become altered during early events in cellular transformation. Among the cellular changes occurring during the development of neoplasia, all of which may potentially involve the nuclear matrix, are alterations in nuclear structure, loss of control of DNA replication, and significant modifications of cellular gene expression. Therefore, a better understanding of the interaction between DNA and the nuclear matrix is needed. Isolated matrix associated DNA from pulse labeled SV40 transformed human fibroblasts was shown to be enriched in newly replicated DNA, confirming the association of DNA replication with the nuclear matrix as observed by others. Subgenomic fractions of matrix associated DNA enriched in putative MARs sites were prepared from quiescent and logarithmically growing normal human fibroblasts and SV40 transformed human fibroblasts. These fractions of DNA were analyzed by Alu-polymerase chain reaction and agarose gel electrophoresis, revealing complex and unique patterns of DNA products for each cell type investigated. A number of prominent DNA fragments with similar molecular size were found to be present in the amplified DNA products of each DNA source, suggesting that these DNA fragments may represent common DNA sequences which contain MARs sites or which are associated with MARs sites. The application of Alu-polymerase chain reaction to the molecular analysis of nuclear matrix associated DNA may facilitate the isolation and characterization of potentially new human MARs sequences.

DNA↗
PubMed1990

Characterization of SV40 T antigen associated with the nuclear matrix.

Simian virus 40 (SV40) T antigen associated with the nuclear matrix of SV40-infected TC7 cells has been characterized. Pulse-chase studies on the turnover of T antigen in the different subcellular fractions show that T antigen turns over most rapidly in its association with the purified SV40 nucleoprotein complexes (NPCs) and undergoes a slower rate of turnover in its association with the nuclear matrix. In contrast, turnover of SV40 T antigen in its association with the other subcellular fractions is not detected during the same period of time. Tryptic peptide maps establish that NPC-associated T antigen and nuclear matrix-associated T antigen are chemically related, in that they have two additional methionine-containing peptides that are not found in the majority of T antigen molecules. The association of T antigen with the nuclear matrix is independent of SV40 DNA replication since T antigen is still present in the nuclear matrix after a 1-hr shift-up of tsA58-infected cells to the nonpermissive temperature. In addition, T antigen is associated with the nuclear matrices of both C6 and Cos7 transformed cells, indicating that the association of T antigen with the nuclear matrix is independent of its ability to initiate and support SV40 DNA replication.

Animals↗
PubMed2000

Half a century of "the nuclear matrix".

A cell fraction that would today be termed "the nuclear matrix" was first described and patented in 1948 by Russian investigators. In 1974 this fraction was rediscovered and promoted as a fundamental organizing principle of eukaryotic gene expression. Yet, convincing evidence for this functional role of the nuclear matrix has been elusive and has recently been further challenged. What do we really know about the nonchromatin elements (if any) of internal nuclear structure? Are there objective reasons (as opposed to thinly veiled disdain) to question experiments that use harsh nuclear extraction steps and precipitation-prone conditions? Are the known biophysical properties of the nucleoplasm in vivo consistent with the existence of an extensive network of anastomosing filaments coursing dendritically throughout the interchromatin space? To what extent may the genome itself contribute information for its own quarternary structure in the interphase nucleus? These questions and recent work that bears on the mystique of the nuclear matrix are addressed in this essay. The degree to which gene expression literally depends on nonchromatin nuclear structure as a facilitating organizational format remains an intriguing but unsolved issue in eukaryotic cell biology, and considerable skepticism continues to surround the nuclear matrix fraction as an accurate representation of the in vivo situation.

Animals↗
PubMed1983

Herpes simplex virus and protein transport are associated with the cytoskeletal framework and the nuclear matrix in infected BSC-1 cells.

Triton cytoskeletons and nuclear matrices were prepared from herpes simplex virus (HSV)-infected cells by a sequential fractionation scheme. Electron microscopic studies revealed the association of mature HSV with the filamentous network of the nuclear matrix. Indirect immunofluorescence assays with monoclonal antibodies revealed that ICP5, the major capsid protein, accumulated on the nuclear matrix while ICP8, the major viral DNA binding protein, accumulates in the chromatin fraction that can be separated from the nuclear matrix by extraction with DNase and salt. Pulse-chase experiments confirmed the kinetics studies of D. M. Knipe and A. E. Spang (J. Virol. 43, 314-324, 1982) and showed that ICP5 is transported after a lag from the cytoplasmic framework to the nuclear matrix, while ICP8 is transported faster to the chromatin fraction.

Animals↗
PubMed1996

ATP-dependent release of glucocorticoid receptors from the nuclear matrix.

Glucocorticoid receptors (GRs) have the capacity to shuttle between the nuclear and cytoplasmic compartments, sharing that trait with other steroid receptors and unrelated nuclear proteins of diverse function. Although nuclear import of steroid receptors, like that of nearly all other karyophilic proteins examined to date, requires ATP, there appear to be different energetic requirements for export of proteins, including steroid receptors, from nuclei. In an attempt to reveal which steps, if any, in the nuclear export pathway utilized by steroid receptors require ATP, we have used indirect immunofluorescence to visualize GRs within cells subjected to a reversible ATP depletion. Under conditions which lead to >95% depletion of cellular ATP levels within 90 min, GRs remain localized within nuclei and do not efflux into the cytoplasm. Under analogous conditions of ATP depletion, transfected progesterone receptors are also retained within nuclei. Importantly, GRs which accumulate within nuclei of ATP-depleted cells are distinguished from nuclear receptors in metabolically active cells by their resistance to in situ extraction with a hypotonic, detergent-containing buffer. GRs in ATP-depleted cells are not permanently trapped in this nuclear compartment, as nuclear receptors rapidly regain their capacity to be extracted upon restoration of cellular ATP, even in the absence of de novo protein synthesis. More extensive extraction of cells with high salt and detergent, coupled with DNase I digestion, established that a significant fraction of GRs in ATP-depleted cells are associated with an RNA-containing nuclear matrix. Quantitative Western blot (immunoblot) analysis confirmed the dramatic increase in GR binding to the nuclear matrix of ATP-depleted cells, while confocal microscopy revealed that GRs are bound to the matrix throughout all planes of the nucleus. ATP depletion does not lead to wholesale collapse of nuclear proteins onto the matrix, as the interaction of a subpopulation of simian virus 40 large tumor antigen with the nuclear matrix is not quantitatively altered in ATP-depleted Cos-1 cells. Nuclear GRs which are not bound to the nuclear matrix of metabolically active cells (i.e., a DNA-binding domain deletion mutant and a beta-galactosidase chimera possessing the GR nuclear localization signal sequence) are not recruited to the matrix upon depletion of cellular ATP. Thus, it appears that ATP depletion does not expose the GR to nuclear matrix interactions which are not normally encountered in cells but merely alters the dynamics of such interactions. The dynamic association of steroid receptors with the nuclear matrix may provide a mechanism which is utilized by these regulable transcription factors to facilitate their efficient scanning of the genome.

Adenosine Triphosphate↗
PubMed1983

The association of transcriptionally active genes with the nuclear matrix of the chicken oviduct.

Eucaryotic DNA is organized into a series of supercoiled loops that are anchored to the nuclear matrix. When these DNA loops are cleaved by endonucleases, the DNA sequences which remain associated with the nuclear matrix can be recovered and analyzed for their content of specific genes. Using restriction endonucleases to cleave the loops, we demonstrate that ovalbumin and conalbumin gene sequences are preferentially associated with the nuclear matrix of hen oviduct cells but not with the nuclear matrix of hen brain cells. Furthermore, we determined that several regions of the ovalbumin gene were independently attached to the nuclear matrix of hen oviduct cells. This included sequences located 3.8 kb downstream from the 3' end of the ovalbumin gene transcription unit. To determine whether the nuclear matrix association of the ovalbumin gene was regulated by hormones, we examined the oviduct cells of chicks that underwent primary estrogen stimulation, estrogen withdrawal and secondary estrogen stimulation. Ovalbumin gene sequences selectively dissociated from the chick oviduct nuclear matrix during estrogen withdrawal and reassociated with the nuclear matrix following restimulation.

Animals↗
PubMed2000

Noncovalent binding of poly(ADP-ribose) to nuclear matrix proteins: developmental changes and tissue specificity.

Poly(ADP-ribose) is a nuclear polynucleotide involved in the regulation of chromatin functions via covalent and/or noncovalent modification of nuclear proteins. Using a binding assay on protein blots, we searched for poly(ADP-ribose) binding proteins in nuclear matrices from testes of differently aged rats as well as from various adult rat tissues (brain, liver, spleen). We found that nuclear matrix proteins represent a significant subset of the nuclear proteins that can establish noncovalent interactions with poly(ADP-ribose). The profiles of poly(ADP-ribose) binding nuclear matrix proteins appeared to be tissue-specific and changed during postnatal development in the testis. The isolation and analysis of endogenous poly-(ADP-ribose) from rat testes showed that the ADP-ribose polymers that bind nuclear matrix proteins in vitro are also present under physiologic conditions in vivo. These results further substantiate the possibility that poly(ADP-ribose) may affect chromatin functions through noncovalent interaction with specific protein targets, including nuclear matrix components.

Animals↗
PubMed1999

Nuclear matrix proteins and their potential applications to diagnostic pathology.

The nuclear matrix is the nonchromatin scaffolding of the cell nucleus that confers nuclear shape, organizes the nuclear chromatin, and regulates many important intranuclear biochemical events. Although our understanding of the nuclear matrix and its proteins is still evolving, it is clear that nuclear matrix proteins (NMPs) hold considerable promise as diagnostic tools for pathologists. Early evidence suggests that NMPs may be useful biomarkers of neoplastic disease in serum, body fluids, and tissues. NMPs are also potential candidates for use as tumor prognostic factors and targets of anticancer drugs. Moreover, NMPs may hold the key to understanding important cellular events, such as neoplastic transformation, steroid hormone binding, and apoptosis. Despite impressive gains made by cellular biologists and biochemists toward understanding the structure and function of the nuclear matrix, many of the potential applications of NMPs to diagnostic pathology are largely unexplored. Thus, NMPs should prove an exciting and fruitful area of investigation for experimental and clinical pathologists who are interested in developing diagnostic tests for detecting, quantitating, and characterizing these proteins in human tissues and body fluids and translating these applications into the clinical pathology laboratory.

Antigens, Nuclear↗
PubMed2006

Unichrom, a novel nuclear matrix protein, binds to the Ars insulator and canonical MARs.

Eukaryotic genomic DNA is organized into loop structures by attachments to the nuclear matrix. These attachments to the nuclear matrix have been supposed to form the boundaries of chromosomal DNA. Insulators or boundary elements are defined by two characteristics: they interrupt promoter-enhancer communications when inserted between them, and they suppress the silencing of transgenes stably integrated into inactive chromosomal domains. We recently identified an insulator element in the upstream region of the sea urchin arylsulfatase (HpArs) gene that shows both enhancer blocking and suppression of position effects. Here, we report that Unichrom, originally identified by its G-stretch DNA binding capability, is a nuclear matrix protein that binds to the Ars insulator and canonical nuclear matrix attachment regions (MARs). We also show that Unichrom recognizes the minor groove of the AT-rich region within the Ars insulator, which may have a base-unpairing property, as well as the G-stretch DNA. Furthermore, Unichrom selectively interacts with poly(dG).poly(dC), poly(dA).poly(dT) and poly(dAT).poly(dAT), but not with poly(dGC).poly(dGC). Unichrom also shows high affinity for single-stranded G- and C-stretches. We discuss the DNA binding motif of Unichrom and the function of Unichrom in the nuclear matrix.

Animals↗