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PubMed1992

Construction of the nuclear matrix at the transition from maternal to zygotic control of development in the mouse: an immunocytochemical study.

The nuclear matrix is thought to be responsible for DNA organization, DNA replication, RNA synthesis, and RNA processing. We have looked for the presence of nuclear matrix antigens during early mouse embryogenesis. Antibodies to peripheral and interior antigens (P1, Pl1, Pl2, and lamin B) were used to immunolocalize nuclear matrix antigens in germinal vesicle oocytes, metaphase II oocytes, zygotes, two-cell-stage embryos, and eight-cell stage embryos. All antibodies reacted with the nuclei of germinal vesicle oocytes, and two- and eight-cell-stage embryos; however, only P1 and lamin B were present at the pronuclear stage. In eggs collected at the pronuclear stage and cultured to the late two-cell stage in the presence of alpha-amanitin, the matrix morphology was altered for Pl1 and Pl2. alpha-Amanitin had no affect on the distribution of P1 or lamin B antigens. If alpha-amanitin was added 2 hr after cleavage to the two-cell stage, the normal staining pattern of Pl2 was retained. These results suggest that the presence of specific components of an internal matrix is correlated with normal genomic activity.

Animals↗
PubMed1993

Immunological characterization of lamins in the nuclear matrix of onion cells.

We have used polyclonal and monoclonal antibodies against different lamins from vertebrates, and the IFA antibody recognizing all kinds of intermediate filament proteins, to investigate the lamins of the nuclear matrix of Allium cepa meristematic root cells. All the antibodies react in the onion nuclear matrix with bands in the range of 60-65 kDa, which are enriched in the nuclear matrix after urea extraction, and do not crossreact with other antibodies recognizing intermediate filaments in plants (AFB, anti-vimentin and MAC 322), ruling out crossreaction with contaminating intermediate filaments of cytoplasmic bundles. In 2-D blots the chicken anti-lamin serum reacts with one spot at 65 kDa and pI 6.8 and the anti B-type lamin antibodies with another one at 64 kDa and pI 5.75. Both crossreact with IFA. The lamin is localized at the nuclear periphery and the lamina by indirect immunofluorescence. Immunogold labelling of nuclear matrix sections reveals that the protein is not only associated with the lamina, but also with the internal matrix. Taken together these results reveal that higher plants, which do not possess an organized network of cytoplasmic intermediate filaments, nevertheless present a well-organized lamina containing lamins in which at least one of them is immunologically related to vertebrate lamin B. Our data confirm that lamins are very old members of the intermediate filament proteins that have been better conserved in plants during evolution than their cytoplasmic counterparts.

Allium↗
PubMed1985

Nuclear matrix modifications at different stages of infection by herpes simplex virus type 1.

In BHK-21 cells infected with herpes simplex virus type 1 many virus-induced proteins were found attached to the nuclear matrix. To understand the role of this cell fraction during virogenesis, matrix-associated proteins were analysed at different stages of infection. All the immediate-early protein species were bound to the nuclear matrix and their association with this structure was stable. During the first few hours of infection, the pattern of virus-induced proteins attached to the nuclear matrix remained identical, indicating that polypeptides from the early group are not associated with this cell fraction. Among the late proteins, which are generally structural proteins, 60% of the nuclear proteins were tightly bound to the nuclear matrix. This suggests that the nuclear matrix is involved in at least two different events during virogenesis, regulation of viral infection and assembly of viral capsids.

Animals↗
PubMed1996

Bladder cancer-associated nuclear matrix proteins.

The early diagnosis of bladder cancer is central to the effective treatment of the disease. Presently, there are no methods available to easily and specifically identify the presence of bladder cancer cells. The prevailing method for the detection of bladder cancer is the identification of bladder cancer cells by morphological examination of exfoliated cells or biopsy material by a pathologist. A hallmark of the malignant or transformed phenotype is an abnormal nuclear shape, the presence of multiple nucleoli, and altered patterns of chromatin organization. Nuclear structural alterations are so prevalent in cancer cells that they are commonly used as markers of transformation for many types of cancer. Nuclear shape is determined by the nuclear matrix, the dynamic skeleton of the nucleus. The nuclear matrix is the structural component of the nucleus that determines nuclear morphology, organizes the DNA in a three-dimensional fashion that is tissue specific, and has a central role in the regulation of a number of nuclear processes, including the regulation of DNA replication and gene expression. Previous investigations into prostate and breast cancer have revealed that nuclear matrix protein (NMP) composition undergoes alterations with transformation and that the nuclear matrix can serve as a marker for the malignant phenotype. In this study, we have identified NMPs with which it is possible to differentiate human bladder tumors from normal bladder epithelial cells. We examined the NMP composition of 17 matched tumor and normal samples from patients undergoing surgery for bladder cancer. We have identified six proteins present in all tumor samples that are not present in the corresponding normal samples and three proteins that are unique to the normal bladder tissues in comparison with the tumor samples. Five of the six bladder cancer-associated proteins were also identified in three human bladder cancer cells lines examined (253j, UMUC-2, and T24). Therefore, we have demonstrated that nuclear matrix composition is able to differentiate bladder cancer from normal bladder tissue and may provide useful tools for early detection and recurrence of the disease. Importantly, these markers may provide valuable tools for cytopathological screening for bladder carcinoma.

Antigens, Nuclear↗
PubMed1987

Alterations in the nuclear matrix protein mass correlate with heat-induced inhibition of DNA single-strand-break repair.

The total protein mass co-isolating with the nuclear matrix or nucleoid from Chinese hamster ovary (CHO) cells was observed to increase in heated cells as a function of increasing exposure temperature between 43 degrees C and 45 degrees C or of exposure time at any temperature. The sedimentation distance of the CHO cell nucleoid in sucrose gradients increased with increasing exposure time at 45 degrees C. Both these nuclear alterations correlated in a log-linear manner with heat-induced inhibition of DNA strand break repair. A two-fold threshold increase in nuclear matrix protein mass preceded any substantial inhibition of repair of DNA single-strand breaks. When preheated cells (45 degrees C for 15 min) were incubated at 37 degrees C the nuclear matrix protein mass and nucleoid sedimentation recovered with a half-time of about 5 h, while DNA single-strand-break repair recovered with a half-time of about 2 h. When preheated cells were placed at 41 degrees C (step-down heating; SDH) a further increase was observed in the nuclear matrix protein mass and the half-time of DNA strand break repair, while nucleoid sedimentation recovered toward control values. These results implicate alterations in the protein mass of the nuclear matrix in heat-induced inhibition of repair of DNA single-strand breaks.

Animals↗
PubMed1990

A nuclear matrix protein binds very tightly to DNA in the avian beta-globin gene enhancer.

Current evidence suggests that DNA is covalently attached to proteins in the nuclear matrix of eukaryotic cells and that specific DNA sequences are tightly associated with the nuclear matrix. However, it has not been documented that specific DNA sequences can become covalently attached to nuclear matrix protein. We have examined the binding of cloned DNA sequences that contain the avian beta-globin gene enhancer, a region previously shown to be matrix associated in erythroid cells in vivo, with nuclear matrices from several avian tissue sources to determine if covalent DNA-protein bonds are formed. Our results indicate that sequence-specific DNA-protein complexes that are resistant to denaturation by SDS, boiling, and phenol and disulfide reduction are formed. Excess protein, capable of forming very tight bonds with DNA that contains the beta-globin gene enhancer, is present in cells in which matrix attachment of this DNA sequence is not detected in vivo. Evidence is presented that suggests that the protein to which DNA forms very tight bonds is not topoisomerase II. These results are discussed in relation to current models of the nuclear matrix and the utility of in vitro assays of matrix attachment regions using cloned DNA.

Animals↗
PubMed1978

[Replication and reassociation kinetics of nuclear matrix DNA from regenerating rat liver].

Fraction 1, containing 65--70% of nuclear DNA (nDNA) was extracted from isolated rat liver nuclei. Then nuclear matrix fraction, containing 30--35% of nDNA was extracted with 1.2 M NaCl. About 1% of nDNA war discovered in residual matrix. Specific activity of residual matrix labelled DNA within 1 and 3 min. after the injection of 14C-orotic acid into the liver portal vein (24 hours after hepatectomy) was respectively in 70 and 50 times higher, and that of matrix DNA--in 45 and 20 times as high as in fraction 1 DNA. Thus, replication begins from nuclear matrix DNA. The latter is enriched with unique sequences by 10%, as compared with total nDNA, and its reassociation kinetics does not change at different stages of the cell cycle (0 and 24 hours after partial hepatectomy). It is suggested, that DNA does not migrate with respect to nuclear matrix under replication.

Animals↗
PubMed1987

Enhanced processivity of nuclear matrix bound DNA polymerase alpha from regenerating rat liver.

Translocation of DNA during in vitro DNA synthesis on nuclear matrix bound replicational assemblies from regenerating rat liver was determined by measuring the processivity (average number of nucleotides added following one productive binding event of the polymerase to the DNA template) of nuclear matrix bound DNA polymerase alpha with poly(dT).oligo(A)10 as template primer. The matrix-bound polymerase had an average processivity (28.4 nucleotides) that was severalfold higher than the bulk nuclear DNA polymerase alpha activity extracted during nuclear matrix preparation (8.9 nucleotides). ATP at 1 mM markedly enhanced the activity and processivity of the matrix-bound polymerase but not the corresponding salt-soluble enzyme. The majority of the ATP-dependent activity and processivity enhancement was completed by 100 microM ATP and included products ranging up to full template length (1000-1200 nucleotides). Average processivity of the net ATP-stimulated polymerase activity exceeded 80 nucleotides with virtually all the DNA products greater than 50 nucleotides. Release of nuclear matrix bound DNA polymerase alpha by sonication resulted in a loss of ATP stimulation of activity and a corresponding decrease in processivity to a level similar to that of the salt-soluble polymerase (6.8 nucleotides). All nucleoside di- and triphosphates were as effective as ATP. Stimulation of both activity and processivity by the nonhydrolyzable ATP analogues adenosine 5'-O-(3-thiotriphosphate), 5'-adenylyl imidodiphosphate, and adenosine 5'-O-(1-thiotriphosphate) further suggested that the hydrolysis of ATP is not required for enhancement to occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

Multiple cis-acting sequences implicate function diversity in nuclear matrix attachment regions of bovine mammary gland.

Chromosomal DNA in higher eukaryotes is spatially organized into loops by periodic attachment to the nuclear matrix at its base via a specific matrix attachment region (MAR). In order to study the nature of DNA sequences that affixed the loops to the nuclear matrix, we have cloned the MAR DNA from bovine lactating mammary tissues. In vitro binding assay showed that the cloned fragments could be co-complexed with nuclear matrix proteins to form insoluble complex easily removed by centrifugation. Sequences of the two chosen MAR loci are composed of TG-, CA- and GA- blocks, as well as the ATTA motifs. Both the MAR loci show numerous replication/transcription factor binding sites, enhancer motifs, several perfect or imperfect inverted repeats, and sequences sharing the common features of the potential DNA bending core sequence. The possibility that a combination of different elements in the same DNA sequence may function as either positive or negative regulatory elements in controlling a variety of cellular and developmental processes is discussed.

Animals↗
PubMed1990

Replication forks are associated with the nuclear matrix.

It has been proposed that DNA in eukaryotic cells is synthesized via replication complexes that are fixed to a proteinaceous nuclear matrix. This model has not been universally accepted because the matrix and its associated DNA are usually prepared under hypertonic conditions that could facilitate non-specific aggregation of macromolecules. We therefore investigated whether different ionic conditions can significantly affect the association of nascent DNA with the nuclear matrix in cultured mammalian cells. Matrices were prepared either by a high salt method or by hypotonic or isotonic LIS extraction. Chromosomal DNA was subsequently removed by digestion with either DNAse I or EcoRI. With all methods of preparation, we found that newly synthesized DNA preferentially partitioned with the nuclear matrix. Furthermore, when the matrix-attached DNA fraction was analyzed by two-dimensional gel electrophoresis, we found that it was markedly enriched for replication forks. We therefore conclude that attachment of DNA to the matrix in the vicinity of replication forks is not induced by conditions of high ionic strength, and that replication may, indeed, occur on or near the skeletal framework provided by the nuclear matrix. From a practical standpoint, our findings suggest a strategy for greatly increasing the sensitivity of two important new gel electrophoretic methods for the direct mapping of replication fork movement through defined chromosomal domains in mammalian cells.

Cells, Cultured↗
PubMed1987

Binding of the DNA polymerase alpha-DNA primase complex to the nuclear matrix in HeLa cells.

It is well-known that there are multiple forms of DNA polymerase alpha. In order to determine which form(s) is (are) tightly bound, the activities were dissociated from DNA-poor nuclear matrices, with octyl beta-D-glucoside. Sucrose gradient sedimentation analysis revealed three bands with s values of 7.5, 10.5, and 13. The 7.5S form was free of DNA primase and represented only 10% of the total DNA polymerase alpha bound to the nuclear matrix. The 13S and the 10.5S forms each contained DNA primase activity. The 10.5S form comprised 85% of the DNA polymerase alpha activity and 95% of the DNA primase activity, dissociated from the nuclear matrix. Neither temperature of nuclease digestion nor various salt treatments of nuclei had significant effects on the proportions of DNA polymerase alpha and DNA primase activities bound to, or subsequently dissociated from, nuclear matrices. In a comparison of primase activity bound to the nuclear matrix, dissociated from the nuclear matrix, and in the soluble fraction, it was found that the bound activity had a lower ATP dependence, had less KCl inhibition, and was less sensitive to heat, compared to the dissociated and soluble activities. No differences in Mg2+ or pH dependence were noted. The amounts of DNA polymerase alpha and DNA primase activities bound to the nuclear matrix varied over the cell cycle of synchronized cells. Over the S phase, there were two peaks of matrix-bound DNA primase and two peaks of subsequently dissociated DNA polymerase alpha-DNA primase complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Cycle↗
PubMed2002

DNA damage-dependent interaction of the nuclear matrix protein C1D with Translin-associated factor X (TRAX).

The nuclear matrix protein C1D is an activator of the DNA-dependent protein kinase (DNA-PK), which is essential for the repair of DNA double-strand breaks (DSBs) and V(D)J recombination. C1D is phosphorylated very efficiently by DNA-PK, and its mRNA and protein levels are induced upon gamma-irradiation, suggesting that C1D may play a role in repair of DSBs in vivo. In an attempt to identify the biological function of C1D, we have employed the yeast two-hybrid system and found that C1D interacts specifically with Translin-associated factor X, TRAX. Although the biological function of TRAX remains unknown, its bipartite nuclear targeting sequences suggest a role for TRAX in the movement of associated proteins, including Translin, into the nucleus. We show that C1D and TRAX interact specifically in both yeast and mammalian cells. Interestingly, however, interaction of these two proteins in mammalian cells only occur following gamma-irradiation, raising the possibility of involvement of TRAX in DNA double-strand break repair and providing evidence for biological functions of the nuclear matrix protein C1D and TRAX. Moreover, we show, using fluorescently tagged proteins, that the relative expression levels of TRAX and Translin affect their subcellular localization. These results suggest that one role for C1D may be to regulate TRAX/Translin complex formation.

Animals↗
PubMed1990

Increase of 63 kDa protein kinase in the nuclear matrix of HL-60 cells during differentiation by retinoic acid.

The changes of protein kinases (PKs) in the nuclear matrix of HL-60 cells during the differentiation by retinoic acid (RA) were examined by in situ assay after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the nuclear matrix of HL-60, at least seven species of PKs (83, 63, 58, 46, 42, 38, and 31 kDa) were always detected. Among these PKs, 63 kDa PK was increased dramatically during differentiation by RA. Two-dimensional electrophoresis revealed that two species of 63 kDa PKs (pI 5.0 and 5.4) were presented in the nuclear matrix and increased similarly during the differentiation. This increase of 63 kDa PKs may be related to the lobulation of the nucleus and the nuclear matrix accompanying the differentiation by RA.

Cell Nucleus↗
PubMed2000

The nuclear matrix as a target for viral and cellular oncogenes.

As the key integrator of nuclear structure and function, the nuclear matrix is likely to be an important target for structural and functional alterations during the process of neoplastic transformation. Here I summarize and discuss data demonstrating that the major transforming protein of the small DNA tumor virus simian virus 40 (SV40), the SV40 large tumor antigen (large T), specifically targets the chromatin and the nuclear matrix during viral transformation. I then turn to recent evidence endorsing the concept that mutant p53--the most commonly expressed oncogene in human cancer--might exert its oncogenic activities by specifically interacting with the nuclear matrix. The data suggest that SV40 large T and mutant p53 might be members of a new family of oncogenes that exert their oncogenic functions by directly modulating nuclear structure and function.

Animals↗
PubMed1993

Adenovirus precursor to terminal protein interacts with the nuclear matrix in vivo and in vitro.

The adenovirus precursor to the terminal protein (pTP), expressed in a vaccinia virus expression system or in native adenovirus, was assayed for its ability to interact with the nuclear matrix. Biochemical function was measured by determining the relative amount of pTP protein or of adenovirus DNA that remained associated with the nuclear matrix after extensive washing. pTP was retained on the matrix whereas beta-galactosidase was not, as assayed by quantitative immunoblot analysis. Nuclear matrix isolated from adenovirus-infected HeLa cells retained bound adenovirus DNA even when washed with 1 M guanidine hydrochloride; this interaction could be inhibited by added purified pTP protein. Analogous experiments with matrix isolated from HeLa cells infected with a recombinant vaccinia virus that expressed pTP showed a similar retention of pTP protein; this association could also be inhibited by added pTP protein. Binding of pTP to nuclear matrix isolated from uninfected cells was saturable, with an apparent Kd of 250 nM and an estimated 2.8 x 10(6) sites for pTP binding per cell nucleus. The association of pTP with matrix is postulated to help direct adenovirus replication complexes to the appropriate locale within the nucleus.

Adenoviridae↗
PubMed2005

Caspase-3 is required in the apoptotic disintegration of the nuclear matrix.

Apoptotic breakdown of cellular structures is largely mediated by caspases. One target of degradation is a proteinaceous framework of the nucleus termed the nuclear matrix. We compared the apoptotic changes of the nuclear matrix in staurosporine-treated caspase-3-deficient MCF-7 cells transfected with intact CASP-3 gene (MCF-7c3) or an empty vector (MCF-7v) as a control. Nuclear Mitotic Apparatus protein (NuMA), lamin A/C and lamin B were used as markers for internal nuclear matrix and peripheral nuclear lamina, respectively. In both cell lines, staurosporine induced rapid cytoplasmic shrinkage and partial chromatin condensation. MCF-7c3 cells formed apoptotic bodies, whereas MCF-7v cells did not. NuMA and lamins were actively cleaved in MCF-7c3 cells following caspase-3 activation, but only minimal or no cleavage was detected in MCF-7v cells. Interestingly, lamin B but not lamin A/C was relocated into cytoplasmic granules in apoptotic MCF-7v cells. Pancaspase inhibitor, z-VAD-fmk, prevented the apoptotic changes, while caspase-3 inhibitor, z-DEVD-fmk, induced lamin B granules in both cell lines. These results show that caspase-3 is involved in the cleavage of NuMA and lamins either directly or by activating other proteases. This may be essential for disintegration of the nuclear structure during apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed2003

c-Abl tyrosine kinase selectively regulates p73 nuclear matrix association.

p73 is a structural and functional homologue of the p53 tumor-suppressor protein. Like p53, p73 is activated in response to DNA-damaging insults to induce cell cycle arrest or apoptosis. Under these conditions p73 is tyrosine-phosphorylated by c-Abl, a prerequisite modification for p73 to elicit cell death in fibroblasts. In this study we report that in response to ionizing radiation, p73 undergoes nuclear redistribution and becomes associated with the nuclear matrix. This association is c-Abl-dependent because it was not observed in cells that are defective in c-Abl kinase activation. Moreover, STI-571, a specific c-Abl kinase inhibitor, is sufficient to block significantly p73 alpha nuclear matrix association. The observed c-Abl dependence of nuclear matrix association was recapitulated in the heterologous baculovirus system. Under these conditions p73 alpha but not p53 is specifically tyrosine-phosphorylated by c-Abl. Moreover, the phosphorylated p73 alpha is predominantly found in association with the nuclear matrix. Thus, in response to ionizing radiation p73 is modified in a c-Abl-dependent manner and undergoes nuclear redistribution and translocates to associate with the nuclear matrix. Our data describe a novel mechanism of p73 regulation.

Animals↗
PubMed1996

Utilization of nuclear matrix proteins for cancer diagnosis.

Circulating tumor markers have been used increasingly in recent years as clinical tools for cancer diagnosis and management. This review presents a brief discussion of currently available tumor-associated antigens. Included is an overview of different functional classes of circulating markers and their clinical applications. The limitations of some traditional tumor markers presently in widespread use are discussed in the context of the properties exhibited by an ideal tumor marker. The nuclear matrix provides structural support for the nucleus and plays a dynamic role in the spatial organization of the genome and in the control of DNA replication and transcription. The recovery of increased amounts of specific nuclear matrix proteins in several different cancers has led to the further study of some of these proteins as a new class of tumor markers. Progress on the use of a nuclear matrix protein known as NuMA as a marker for bladder cancer is presented, including results of a recently completed multisite clinical trial. Additional studies on the potential utility of nuclear matrix proteins as markers for prostate cancer are also presented. Nuclear matrix proteins could provide for the development of assays with increased efficacy for the diagnosis and treatment of cancer.

Antibodies, Monoclonal↗