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PubMed1999

Interrelationships of transcriptional machinery with nuclear architecture.

There is increasing evidence for functional linkages between nuclear architecture and the regulation of gene expression. The nuclear matrix provides a paradigm for involvement of nuclear morphology in assembly of the biochemical machinery for physiological control of transcription. Mechanisms are being experimentally defined that direct regulatory factors to subnuclear sites that support gene expression. Consequently, we are gaining insight into the rules that govern transcriptional control within the three-dimensional context of nuclear organization.

Animals↗
PubMed1998

The new paradigm: integrating genomic function and nuclear architecture.

A new view of the cell nucleus is emerging based on the functional dynamics of nuclear architecture. The striking structural preservation of a variety of genomic processes on the nuclear matrix provides an important approach for correlating nuclear form and function. In situ labeling coupled with three-dimensional microscopy and computer imaging techniques shows that DNA replication and transcription sites are organized into higher-order units, or "zones," in the cell nucleus. The dynamic interplay and "re-zoning" of replication and transcription regions during the cell cycle may form the structural basis for the elaborate global coordination of replicational and transcriptional programs in the mammalian cell.

Animals↗
PubMed1992

DNA polymerase-delta from the silk glands of Bombyx mori.

The silk gland of Bombyx mori is a terminally differentiated tissue in which DNA replication continues without cell or nuclear division during larval development. DNA polymerase-delta activity increases in the posterior and middle silk glands during the development period, reaching maximal levels in the middle of the fifth instar larvae. The enzyme has been purified to homogeneity by a series of column chromatographic and affinity purification steps. It is a multimer comprising of three heterogeneous subunits, M(r) 170,000, 70,000, and 42,000. An auxiliary protein from B. mori silk glands, analogous to the proliferating cell nuclear antigen, enhances the processivity of the enzyme and stimulates catalytic activity by 3-fold. This auxiliary protein has also been purified to homogeneity. It is a dimer comprised of a single type M(r) 40,000 subunit. Polymerase-delta possesses an intrinsic 3'----5' exonuclease activity which participates in proofreading by mismatch repair during DNA synthesis and is devoid of any primase activity. DNA polymerase-delta activity could be further distinguished from polymerase-alpha from the same tissue based on its sensitivity to various inhibitors and polyclonal antibodies to the individual enzymes. Like DNA polymerase-alpha, polymerase-delta is also tightly associated with the nuclear matrix. The polymerase alpha-primase complex could be readily separated from polymerase-delta (exonuclease) in the purification protocol adopted. DNA polymerase-delta from B. mori silk glands resembles the mammalian delta-polymerases. Considering that both DNA polymerase-delta and -alpha are present in nearly equal amounts in this highly replicative tissue and their close association with the nuclear matrix, the involvement of both the enzymes in the chromosomal endoreplication process in B. mori is strongly implicated.

Animals↗
PubMed1998

Nuclear structure--skeletal gene expression interrelationships.

Using the osteocalcin gene as a paradigm for bone tissue-specific transcription, evidence is presented for functional linkage of nuclear architecture with developmental and steroid hormone-responsive control. The involvement of chromatin structure, nucleosome organization and the nuclear matrix is evaluated within the context of integrating regulatory signals that activate and/or suppress transcription of the osteocalcin gene. Mechanisms are evaluated which direct the bone and hematopoietic-specific acute myelogenous leukemia/core binding factor (AML/CBF) transcription factors to nuclear matrix-associated subnuclear domains that are competent to support expression.

Animals↗
PubMed1985

Eukaryotic type I topoisomerase is enriched in the nucleolus and catalytically active on ribosomal DNA.

The distribution of eukaryotic DNA topoisomerase I in the cell has been analyzed at four levels: (i) at the level of the nuclear matrix; (ii) at the cytological level by immunofluorescence of whole cells; (iii) at the electron microscopic level using the protein A/colloidal gold technique; and (iv) at the level of DNA to identify in situ the sequence upon which topoisomerase I is catalytically active. Although topoisomerase I is clearly distributed non-randomly in the nucleus, the unique distribution of the enzyme is not related to the nuclear matrix. The data support the conclusion that topoisomerase I is heavily concentrated in the nucleolus of the cell; furthermore, particular regions within the nucleolus are depleted of topoisomerase. A technique has been developed which allows isolation and analysis of the cellular DNA sequences covalently attached to topoisomerase. Ribosomal DNA sequences are at least 20-fold enriched in topoisomerase/DNA complexes isolated directly from a chromosomal setting, relative to total DNA. This is the first direct evidence that topoisomerase I is catalytically active on ribosomal DNA in vivo.

Animals↗
PubMed2003

NUFIP1 (nuclear FMRP interacting protein 1) is a nucleocytoplasmic shuttling protein associated with active synaptoneurosomes.

Fragile X syndrome, the most common cause of inherited mental retardation, is caused by the absence of FMRP (Fragile X Mental Retardation Protein). FMRP is an RNA binding protein reported to be involved in translational control, notably at postsynaptic sites of protein synthesis as a part of a multiprotein/mRNA complex. One of the FMRP interactors, NUFIP1, is an RNA binding protein with an expression profile matching that of FMRP. We now show that in the nucleus NUFIP1 is localized in the nuclear matrix in RNA-containing structures lying in the proximity of, but not overlapping with, sites of nascent RNA. NUFIP1 is also present in the cytoplasm, where it is associated with ribosomes, similarly to FMRP. In neurons NUFIP1 can be detected in functional synaptoneurosomes, colocalizing with ribosomes. Consistent with its subcellular localization in both nucleus and cytoplasm, we show that NUFIP1 contains a functional CRM1-dependent nuclear export signal and is able to shuttle between these two cellular compartments. These findings suggest the involvement of NUFIP1 in the export and localization of mRNA and, in association with FMRP, in the regulation of local protein synthesis near synapses.

Active Transport, Cell Nucleus↗
PubMed1991

A novel protein related to cell cycle-dependent alterations of chromatin structure.

We have detected a novel nuclear antigen, AF-2, which appears to be involved in cell cycle-dependent alterations of chromatin structure. Specific monoclonal antibodies detect the antigen spread over the whole cell during mitosis and in islet-like structures in the nuclei of a subpopulation of cells in interphase. Upon nucleolytic digestion of fixed cells, the antigen becomes available to the antibodies in all cells, indicating that AF-2 antigen is present during the whole cell cycle but differentially accessible. Digestion with the single strand specific S1 nuclease reveals that the alteration of chromatin structure induced by the introduction of nicks into the DNA rather than the digestion of DNA bound to the immunogenic epitope accounts for the change in accessibility of AF-2 antigen in interphase nuclei. The epitope recognized by the antibody in human cells is present in two polypeptides of 65 and 36 kDa, respectively, which are tightly bound to chromatin and cross-linkable to the nuclear matrix. The proteins also occur in the midbody during cytokinesis. The immunogenic epitope is conserved between man and fission yeast.

Antibodies, Monoclonal↗
PubMedDate not supplied

[Regulatory potential of S/MAR elements in transient expression].

S/MARs (scaffold/matrix attachment regions) are the DNA regions that are involved in the interaction with the nuclear matrix and are identified by in vitro methods. According to the available information, S/MARs possess an insulating activity, i.e., the ability to block the interaction between the enhancer and promoter in vivo, and are, probably, intact insulators or their fragments. Nevertheless, there is still no direct proof for this correspondence. To obtain additional information on the insulator activity of S/MARs, we selected five DNA fragments of different lengths and affinities for the nuclear matrix from the previously constructed library of S/MARs and tested their ability to serve as insulators. Two of five elements exhibited an insulator (enhancer-blocking) activity upon the transient transfection of CHO cells. None of the S/MARs displayed either promoter or enhancer/silencer activities in these cells.

Animals↗
PubMed2000

DNA-protein cross-linking in nuclei of immature and mature chicken erythrocytes.

DNA-protein cross-linkages were formed in isolated nuclei from immature and mature chicken erythrocytes by reaction with cis-diammine dichloroplatinum. On the basis of electrophoretic behaviour, the most abundant proteins involved in the cross-linking appeared to be present also in preparations of nuclear matrix. The maturation of the erythrocyte, which is accompanied by transcriptional inactivation, leads to a decrease in the amount of DNA-interacting proteins, to a loss of proteins capable of a specific recognition of DNA sequences and, unexpectedly, to the appearence of some new DNA-protein interactions. At least three cross-linked proteins were found predominantly or exclusively in nuclei of immature cells, and three others in those of mature ones. The three DNA-bound proteins, typical of mature erythrocytes, were not found among the components of a high-salt preparation of nuclear matrix. The results obtained suggest that, in addition to the well-known histone H5 and MENT protein, these newly identified DNA-bound proteins contribute to the formation of the condensed, inactive chromatin characteristic of mature erythrocyte.

Animals↗
PubMed1988

Lectin-binding proteins in nuclear preparations from rat liver and malignant tumors.

Lectin binding [concanavalin A, biotinylated ricinus communis agglutinin, and biotinylated succinylated wheat germ agglutinin (B-SWGA)] was used to detect the glycosylated proteins associated with a residual protein fraction [insoluble in 4% sodium dodecyl sulfate and termed the nuclear residual fraction (NRF)] or with nuclear matrix preparations from normal rat liver, azo dye (3'-MeDAB)-induced rat hepatoma, and Walker 256 transplantable carcinosarcoma. One- and two-dimensional gel electrophoresis were used with lectins, polyclonal antisera, and monoclonal antibody binding to characterize some of the glycoconjugates. Two polypeptide bands with approximate molecular weights of 95,000 and 55,000, shown previously to be present only in the induced tumor cells and the Walker 256 tumor, were reactive with lectins. In addition, a Mr 62,000 protein reacted only with B-SWGA in the nuclear matrix fractions from normal rat liver and the induced hepatoma. A polypeptide band (approximate molecular weight, 213,000) in the Walker 256 NRF reacted with concanavalin A and biotinylated ricinus communis agglutinin. One polypeptide band (approximate molecular weight, 182,000) reacted with concanavalin A in all three tissues, with biotinylated ricinus communis agglutinin and B-SWGA in the Walker NRF, and with B-SWGA in the hepatoma NRF. Another polypeptide band (approximate molecular weight, 138,000), reactive with all three lectins, was present in all three tissues. Our findings are consistent with previous reports of lectin binding proteins in the eukaryotic cell nucleus and indicate that certain glycoproteins isolated in nuclear preparations are found specifically in 3'-MeDAB-induced hepatoma and Walker 256 transplantable carcinosarcoma.

Animals↗
PubMedDate not supplied

Induction of apoptosis in breast cancer cells in response to vitamin D and antiestrogens.

1,25-Dihydroxycholecalciferol D3 (1,25(OH)2D3), the active metabolite of vitamin D, is a potent inhibitor of breast cancer cell growth both in vivo and in vitro. We have shown that MCF-7 cells treated with 100 nM 1,25(OH)2D3 exhibit characteristic apoptotic morphology (pyknotic nuclei, chromatin and cytoplasmic condensation, nuclear matrix protein reorganization) within 48 h. In the experiments reported here, we examined the interactions between 1,25(OH)2D3 and the antiestrogen 4-hydroxytamoxifen (TAM), which also induces apoptosis in MCF-7 cells. Our data suggest that TAM significantly potentiates the reduction in cell number induced by 1,25(OH)2D3 alone. Combined treatment with 1,25(OH)2D3 and TAM enhances the degree of apoptosis assessed using morphological markers that identify chromatin and nuclear matrix protein condensation. We have selected a subclone of MCF-7 cells resistant to 1,25(OH)2D3 (MCF-7D3Res). These cells express the vitamin D receptor and exhibit doubling times comparable to the parental MCF-7 cells, even when grown in 100 mM 1,25(OH)2D3. Treatment of both parental and resistant MCF-7 cells with TAM induces apoptosis and clusterin. These data emphasize that apoptosis can be induced in MCF-7 cells either by activation of vitamin-D-mediated signalling or disruption of estrogen-dependent signalling.

Apoptosis↗
PubMed1998

Radioprotection of cellular chromatin by the polyamines spermine and putrescine: preferential action against formation of DNA-protein crosslinks.

Spermine is an efficient radioprotector of plasmid or viral DNA and of viral minichromosomes by a mechanism involving radical scavenging and the induction of compaction and aggregation of DNA. Based on radioprotection of SV40 minichromosomes at a lower spermine concentration than needed for SV40 DNA, Newton et al. (Radiat. Res. 145, 776-780, 1996) proposed that the differential concentration dependence could account for the greater radiosensitivity of open regions of cellular chromatin compared to bulk inactive chromatin at physiological levels of spermine. However, we recently reported that, whereas the effects of spermine on the formation of DNA double-strand breaks (DSBs) in dehistonized V79 cell DNA (nucleoids) were consistent with spermine-induced DNA compaction, spermine provided no radioprotection of native chromatin and only modest radioprotection of histone H1-depleted chromatin (Chiu and Oleinick, Radiat. Res. 148, 188-192, 1997). To further characterize the radioprotection of cellular chromatin by spermine, radiation-induced DNA-protein crosslinks (DPCs) were investigated, because of evidence that these lesions occur preferentially at or near the sites of anchorage of chromosomes to the nuclear matrix. In contrast to the relatively inefficient radioprotection of V79 cell chromatin against the formation of DSBs, low concentrations (<0.1 mM) of spermine or putrescine provided partial radioprotection against the formation of DPCs in both native and H1-depleted chromatin. Whereas all DPCs generated by the irradiation of chromatin, above the level generated in intact cells, could be blocked by 5 mM spermine, less than half could be blocked by 5 mM putrescine. The difference in efficiency of radioprotection of native chromatin by the two polyamines can be accounted for by assuming that the binding of spermine is 10 times as efficient as the binding of putrescine. The results suggest that (a) both spermine and putrescine bind preferentially and with high affinity at matrix-associated sites of formation of DPCs, disrupting the associations between DNA and protein that are essential for formation of DPCs and/or scavenging hydroxyl radicals at these sites; (b) a smaller fraction of the sites are susceptible to putrescine than to spermine; and (c) endogenous spermine is a major radioprotector of cells against the formation of DPCs, either because of specific features of the lesion or because of the site of lesion formation at the nuclear matrix.

Animals↗
PubMed1996

Inhibition of prostate cancer growth by 9-aminocamptothecin and estramustine.

OBJECTIVES: Hormone-refractory prostate cancer continues to be associated with a very poor prognosis. Agents that interact with the nuclear matrix have been demonstrated to have activity against hormone-refractory prostate cancer. It was the aim of this study to assess the activity of estramustine, an estradiol-nitrogen mustard conjugate, and 9-aminocamptothecin (9-AC), a topoisomerase I inhibitor, in a preclinical model of hormone-refractory prostate cancer. METHODS: We used the Dunning rat prostatic adenocarcinoma model to demonstrate that the combination of estramustine and 9-AC interacts at the level of the nuclear matrix to inhibit the growth of prostate cancer cells. RESULTS: We demonstrate that the combination of these two agents at pharmacologically achievable doses are cytotoxic to rat and human prostate cancer cells in vitro and in vivo in the rat. CONCLUSIONS: The combination of the two drugs was significantly more cytotoxic than either drug alone. We have instituted a Phase II clinical trial in patients with hormone-refractory prostate cancer using 9-AC based on these preclinical findings.

Adenocarcinoma↗
PubMed1999

Neuronal differentiation of NT2/D1 teratocarcinoma cells is accompanied by a loss of lamin A/C expression and an increase in lamin B1 expression.

Nuclear lamins are prominent elements of the nuclear matrix and are expressed in cell type-specific and differentiation state-specific patterns. A few observations have indicated that nervous tissue may display unusual patterns of lamin expression, in that some neurons appear to lack A-type lamins, which are generally prominently expressed in terminally differentiated, postmitotic cells. To investigate lamin expression patterns during the differentiation of a teratocarcinoma cell line into neurons, NT2/D1 cells were induced to differentiate with retinoic acid treatment. Lamin expression and organization during differentiation in vitro were examined by quantitative immunofluorescence and immunoblotting methods. Undifferentiated NT2/D1 cells were all strongly labeled with an anti-lamin B1 antibody, but displayed marked variation in A/C lamin immunoreactivity. After differentiation, neuronal nuclear envelopes were significantly more strongly labeled by anti-lamin B1 antibody than those of undifferentiated cells, but completely lacked A/C lamin immunoreactivity. In contrast, nonneuronal cells displayed a slight reduction in B1 lamin immunoreactivity, along with a distinct increase in A/C lamin levels. The loss of lamin A/C expression in NT2/D1 neurons is contrary to the pattern normally observed in most somatic cell types during early development and indicates that the nuclear matrix of some neurons, along with certain neuroendocrine and hematopoietic cells, is uniquely specialized in this regard.

Cell Differentiation↗
PubMed1988

Intranuclear distribution of SV40 large T-antigen and transformation-related protein p53 in abortively infected cells.

The intranuclear localization of SV40 T-antigen (T-Ag) and the cellular protein p53 was studied in SV40 abortively infected baby mouse kidney cells using two complementary methods of ultrastructural immunocytochemistry in combination with preferential staining of nuclear RNP components and electron microscope autoradiography. Both proteins were revealed in association with peri- and interchromatin RNP fibrils containing the newly synthesized hnRNA. In addition, T-Ag and p53 remained bound, at least in part, to the residual internal nuclear matrix following nuclease and salt extractions of infected cells. The localization of T-Ag was different in SV40 lytically infected monkey kidney cells since, in addition to hnRNP fibrils, the viral protein was also associated with cellular chromatin. However, when lytic infection was performed in conditions of blocked viral DNA replication, T-Ag was no longer associated with the cellular chromatin but remained bound to the hnRNP fibrils. We conclude that the transforming and lytic functions of T-Ag can be distinguished by different subnuclear distributions. The significance of the association of T-Ag and p53 with hnRNP fibrils and the internal nuclear matrix is discussed in relation to the role of these structures in the control of cellular mRNA biogenesis.

Antibodies, Monoclonal↗
PubMed2003

Extracellular matrix and nuclear abnormalities in skeletal muscle of a patient with Walker-Warburg syndrome caused by POMT1 mutation.

Walker-Warburg syndrome (WWS) is an autosomal recessive disorder characterized by congenital muscular dystrophy, structural eye abnormalities and severe brain malformations. We performed an immunohistochemical and electron microscopy study of a muscle biopsy from a patient affected by WWS carrying a homozygous frameshift mutation in O-mannosyltransferase 1 gene (POMT1). alpha-Dystroglycan glycosylated epitope was not detected in muscle fibers and intramuscular peripheral nerves. Laminin alpha2 chain and perlecan were reduced in muscle fibers and well preserved in intramuscular peripheral nerves. The basal lamina in several muscle fibers showed discontinuities and detachment from the plasmalemma. Most nuclei, including myonuclei and satellite cell nuclei, showed detachment or complete absence of peripheral heterochromatin from the nuclear envelope. Apoptotic changes were detected in 3% of muscle fibers. The particular combination of basal lamina and nuclear changes may suggest that a complex pathogenetic mechanism, affecting several subcellular compartments, underlies the degenerative process in WWS muscle.

Antigens, CD↗
PubMed2000

Spectrin-like proteins in plant nuclei.

We analysed the presence and localization of spectrin-like proteins in nuclei of various plant tissues, using several anti-erythrocyte spectrin antibodies on isolated pea nuclei and nuclei in cells. Western blots of extracted purified pea nuclei show a cross-reactive pair of bands at 220-240 kDa, typical for human erythrocyte spectrin, and a prominent 60 kDa band. Immunolocalization by means of confocal laser scanning microscopy reveals spectrin-like proteins in distinct spots equally distributed in the nucleoplasm and over the nuclear periphery, independent of the origin of the anti-spectrin antibodies used. In some nuclei tracks of spectrin-like proteins are also observed. No signal is present in nucleoli. The amount and intensity of signal increases when nuclei were extracted, successively, with detergents, DNase I and RNase A, and high salt, indicating that the spectrin-like protein is associated with the nuclear matrix. The labelling is similar in nuclei of various plant tissues. These data are the first that show the presence and localization of spectrin-like epitopes in plant nuclei, where they may stabilize specific interchromatin domains.

Cell Fractionation↗