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At least 865 records · Page 48Linked to original sources
PubMed1990

Evidence for DNA binding activity of numatrin (B23), a cell cycle-regulated nuclear matrix protein.

Stimulation of various cell types with growth factors is associated with a rapid induction in the synthesis of a nuclear matrix protein, termed 'numatrin' which was shown to be identical to the nucleolar protein B23. The abundance of numatrin was shown to be correlated with entry and progression through the S-phase. Thus, experiments were undertaken to examine whether numatrin also has DNA binding activity. Using whole nuclear extract, we showed that numatrin binds to both double-stranded (DS) DNA and to single-stranded (SS) DNA cellulose columns. Purified numatrin, which was extracted either under native conditions (in oligomeric form) or under urea conditions (in monomeric form), demonstrated significant binding to either [3H]DS-DNA or [3H]DS-DNA as shown by nitrocellulose filter binding assay. However, numatrin binding to DS-DNA was qualitatively and quantitatively different from its binding to SS-DNA. Thus, the binding of numatrin was several fold higher to DS-DNA as compared to SS-DNA. The binding to DS-DNA was reduced by 77% in the presence of 0.5 M NaCl, while the binding to SS-DNA was not affected under this condition. Furthermore, treatment of the native numatrin under conditions which caused monomerization of the protein resulted in a significant enhancement of numatrin binding to SS-DNA but did not affect the binding to DS-DNA. Following heparin-Sepharose chromatography purification (under native conditions), numatrin at picomole amounts showed significant binding to both DS-DNA and SS-DNA. Finally, numatrin was found to copurify with the complex of DNA polymerase alpha primase together with other proteins required for SV-40 in vitro replication activity. These results demonstrate that numatrin has DNA binding activity, and imply a possible role for numatrin/B23 in DNA-associated processes.

Blotting, Western↗
PubMed2002

Formation of nuclear matrix filaments by p27(BBP)/eIF6.

p27(BBP)/eIF6 is an evolutionarily conserved protein necessary for ribosome biogenesis which was cloned in mammals for its ability to bind the cytodomain of beta 4 integrin. In cultured cells, a conspicuous fraction of p27(BBP)/eIF6 is associated with the intermediate filaments/nuclear matrix (IF/NM) cytoskeleton. The mechanism of this association is not known. Here we show that in epidermis p27(BBP)/eIF6 is naturally associated with IF/NM. To analyze the intrinsic capability of p27(BBP)/eIF6 to generate cytoskeletal networks, the properties of the pure, recombinant, untagged protein were studied. Recombinant p27(BBP)/eIF6 binds beta 4 integrin. Upon dialysis against IF buffer, p27(BBP)/eIF6 forms polymers which, strikingly, have a morphology identical to NM filaments. Cross-linking experiments suggested that polymerization is favored by the formation of disulphide bridges. These data suggest that p27(BBP)/eIF6 is associated with the cytoskeleton, and contributes to formation of NM filaments. These findings help to settle the controversy on nuclear matrix.

Animals↗
PubMed1991

Localization of nuclear matrix proteins (NMPs) in multiple tissue types with NM-200.4 (an antibody strongly reactive with NMPs found in breast carcinoma).

Nuclear matrix proteins (NMP) are nonhistone proteins found in the nucleus of many eukaryotic cells. Furthermore certain NMPs are reported to be cell-type specific and expressed differentially by malignant cells. To study the specificity of NM-200.4 (an antibody reactive to NMPs extracted from cultured breast carcinoma cells of the T-47D line), cancers and benign tissues from multiple body sites were surveyed. All 17 breast carcinomas showed strong reactivity to tumor cell nuclei. Also nuclei from one of two lung carcinomas, a papillary thyroid carcinoma, an ovarian fibroma, and a lymphoma were strongly reactive. One leiomyosarcoma and a dermoid cyst were negative. Although 1 benign breast with duct hyperplasia showed moderate reactivity, only 1 of 10 benign breast biopsies without hyperplasia showed reactivity. Three of 4 skin biopsies, 2 liver biopsies, 6 of 9 kidney biopsies, and 5 of 10 gastrointestinal mucosal biopsies showed reactivity in benign nuclei. It is concluded that, although breast carcinoma nuclei showed the most consistent reactivity for NM-200.4, both benign and malignant nuclei from other body sites also show reactivity.

Animals↗
PubMed2004

Evaluation of nuclear matrix protein-22 as a clinical diagnostic marker for bladder cancer.

OBJECTIVES: To evaluate prospectively the prognostic power of urinary nuclear matrix protein-22 (NMP-22) for bladder cancer in Taiwanese screening and surveillance settings. METHODS: Single voided urine samples were obtained from 68 healthy individuals, 303 patients with benign urothelial diseases, and 28 patients with urogenital tumors. The NMP-22 levels in the urine samples were measured using enzyme-linked immunosorbent assay methods. RESULTS: The median NMP-22 level in healthy individuals and patients with benign and malignant disease was 5.9, 4.8, and 7.4 U/mL, respectively. The positive NMP-22 rate in healthy individuals and patients with benign and malignant disease was 4.4%, 17.2%, and 50%, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value was 50%, 82.8%, 21.2%, and 94.7%, respectively, using 7.5 U/mL as the cutoff value. CONCLUSIONS: Our data demonstrated that NMP-22 is not a good diagnostic tool for screening or follow-up surveillance of bladder cancer owing to its low sensitivity and positive predictive value. Nevertheless, it could be adopted as a tool to rule out the possibility or risk of developing bladder cancer because of its high negative predictive value in our study.

Adolescent↗
PubMed1995

Specificity and functional significance of DNA interaction with the nuclear matrix: new approaches to clarify the old questions.

In this chapter the specificity of chromosomal DNA partitioning into topological loops is discussed. Different experimental approaches used for the analysis of the above problem are critically reviewed. This discussion is followed by presentation of a novel approach for mapping the DNA loop anchorage sites that we have developed. This approach, based on the excision of the whole DNA loops by topoisomerase II-mediated DNA cleavage at matrix attachment sites, seems to constitute a unique tool for the analysis of topological organization of chromosomal DNA in living cells. We also discuss experimental results indicating that the DNA-loop anchorage sites form "weak points" in chromosomes that are preferentially sensitive to cleavage with both endogenous and exogenous nucleases. In connection with this discussion, rationales for the supposition that DNA loops constitute basic units of eukaryotic genome organization and evolution are considered. The chapter concludes by suggesting a new model of spatial organization of eukaryotic genome within the cell nucleus that resolves apparent contradictions between different data on the specificity of DNA interaction with the nuclear matrix.

Animals↗
PubMed2001

Hairless is translocated to the nucleus via a novel bipartite nuclear localization signal and is associated with the nuclear matrix.

Hair follicle cycling is an exquisitely regulated and dynamic process consisting of phases of growth, regression and quiescence. The transitions between the phases are governed by a growing number of regulatory proteins, including transcription factors. The hairless (hr) gene encodes a putative transcription factor that is highly expressed in the skin, where it appears to be an essential regulator during the regression of the catagen hair follicle. In hairless mice, as well as humans with congenital atrichia, the absence of hr gene function initiates a premature and abnormal catagen due to a dysregulation of apoptosis and cell adhesion, and defects in the signaling required for hair follicle remodeling. Here, we report structure-function studies of the hairless gene product, in which we identify a novel bipartite nuclear localization signal (NLS) of the form KRA(X13) PKR. Deletion analysis of the mouse hr gene mapped the NLS to amino acid residues 409-427. Indirect immunofluorescence microscopy of cells transiently transfected with hairless-green fluorescent fusion proteins demonstrated that these amino acid residues are necessary and sufficient for nuclear localization. Furthermore, nuclear fractionation analysis revealed that the hr protein is associated with components of the nuclear matrix.

3T3 Cells↗
PubMed2000

Structural association of Bax with nuclear matrix and cytomatrix revealed by embedment-free immunogold electron microscopy.

Bax is a cellular protein functioning as a promoter of apoptosis. It is ultrastructurally associated with mitochondrial membranes, where it participates in permeability transition pore formation. By employing embedment-free electron microscopy (EFEM), we present evidence that Bax is also associated with the nuclear matrix and cytomatrix of cultured human tumour cells (COLO 205, PA-1, U-373 MG). Extracted cellular scaffolds were probed with anti-Bax antibody using the immunogold electron microscopy technique. Bax immunoreactivity was found on 10-15 nm intermediate filaments of karyo- and cytoskeleton, stretched between the nucleus, nuclear lamina and cell periphery. Bax immunoreactivity was preferentially localized to certain areas of filaments (spot-like). The target molecules for Bax binding in the cellular matrix and their physiological significance remain to be established.

Cytoplasm↗
PubMed1993

A correlation between DNA-nuclear matrix binding and relative radiosensitivity in two human squamous cell carcinoma cell lines.

Three aspects of DNA topology were examined in two human squamous cell carcinoma lines of differing radiosensitivity (SQ-9G, D0 = 1.46 Gy; and SQ-20B, D0 = 2.36 Gy). High-salt-extracted nuclei (nucleoids) were taken from gamma-irradiated cells, stained with ethidium bromide and examined by flow cytometry. After 5 Gy, nucleoids from SQ-9G cells became 30% less efficient at adopting positive DNA supercoils than were unirradiated controls. In contrast, only a 4% difference was found with the radioresistant SQ-20B line. Both lines produced positive supercoils more efficiently after irradiation if first exposed to the topoisomerase II inhibitor VP16. Ethidium bromide titration of nucleoids was consistent with each containing similar numbers and sizes of DNA loops. In each line approximately 30-35% of DNA was accessible to trioxsalen, as shown by inter-strand crosslinking after UV photo-activation. Exhaustive digestion of nuclear DNA by DNase I removed more DNA from the radiosensitive than from the radioresistant cell line (12% vs 28% remaining). This difference was thought to be due to the increased accessibility of SQ-9G DNA in vitro. We suggest that a looser association of SQ-9G DNA with the nuclear matrix both promotes DNase I digestion and affects the ability of SQ-9G nucleoids to maintain positive DNA supercoils after irradiation. These data implicate the DNA matrix attachment region in the expression of radiation sensitivity in the cell lines studied.

Carcinoma, Squamous Cell↗
PubMed2001

Urinary levels of nuclear matrix protein 22 in patients with urinary diversion.

OBJECTIVES: To evaluate the influence of various types of urinary diversion on urinary nuclear matrix protein 22 (U-NMP22) levels. METHODS: U-NMP22 values were determined for 38 urinary diversion patients without upper urinary tract cancer. The patients were divided into three groups: tubeless cutaneous ureterostomy (n = 12), ileal conduit (n = 15), and continent urinary reservoir (n = 11). The mean values and false-positive rates of U-NMP22 (cutoff value 12 U/mL) were compared among the three groups. RESULTS: The mean +/- standard error U-NMP22 value for the ureterostomy, ileal conduit, and reservoir groups was 20.1 +/- 5.8 U/mL, 335.6 +/- 63.5 U/mL, and 671.8 +/- 220.4 U/mL, respectively (P = 0.0030). The false-positive rate of U-NMP22 for the ureterostomy, ileal conduit, and reservoir groups was 41.7%, 100%, and 100%, respectively. In the ureterostomy group, the exclusion of patients with pyuria improved the false-positive rate of U-NMP22 from 41.7% to 12.5%. CONCLUSIONS: U-NMP22 levels may be useful in the diagnosis of upper urinary tract cancer in patients with a tubeless cutaneous ureterostomy. However, in patients with urinary diversion using a bowel segment, U-NMP22 has no diagnostic value because of the high U-NMP22 levels.

Aged↗
PubMed1988

Cell cycle dependence of thyroid hormone nuclear receptors in cultured GC cells: relationship to nuclear matrix.

The DNA synthesis (S) phase of cultured GC cells, a clonal rat pituitary tumor cell line, is characterized by increases in nuclear receptors for T3 and glucocorticoid (G) hormones. However, this increased receptor abundance appears functionally dissociated from the GH gene since GH messenger RNA (mRNA) synthesis is decreased in S-phase cells. We have now examined a putative structural correlate of this dissociation by measuring the abundance of T3 and G receptors and the GH gene in the nuclear matrix (NM)/scaffold fraction. NM of control and S-phase cultures both contained 30-50% of G receptors. Thirty to 50% of T3 receptors were also localized to NM of asynchronous cultures, but T3 receptor abundance (femtomoles per 100 micrograms of protein) was significantly decreased in NM of S-phase cultures: Exp 1 (control, 140 +/- 6.2; S-phase, 56.5 +/- 0.8; Exp 2 (control, 170 +/- 12; S-phase, 105 +/- 2.4). Nuclear scaffolds were digested with restriction enzymes to solubilize DNA loop structures and probed with GH complementary DNA (cDNA) to examine the distribution of transcribed regions of the GH gene. EcoR1 digestion resulted in an 11 kilobase fragment including the reported regulatory sequences for T3 receptors. No specific differences in GH gene localization to nuclear scaffold were observed between asynchronous and S-phase cultures or in different hormonal states. Thus, GH gene localization to nuclear scaffolds was not correlated with changes in transcription induced by T3 and G hormones or position in the cell cycle. These studies suggest that decreased concentration of T3 receptors in the NM fraction of S-phase cultures may be a structural correlate for cell cycle regulation of T3 receptor function.

Animals↗
PubMed1986

Correspondence of two nuclear networks observed in situ with the nuclear matrix.

In resting lymphocytes, three well defined networks are observed and attempts were made to superimpose them upon the networks described in isolated nuclear matrices. These three nuclear structures are seen after DNase and RNase treatment. They are digested by pepsin but their sensitivity to this enzyme is different. The more resistant network corresponds to the outer lamina of the isolated nuclear matrix. The second network is located in the inter-chromatin area. It is more sensitive to pepsin than the lamina and this sensitivity is increased ten-fold when digestion with pepsin is preceded by RNase digestion. This network corresponds to the internal network of isolated nuclear matrices. The third network is located in the intrachromatin area and is the most sensitive to pepsin action.

3,3'-Diaminobenzidine↗
PubMed1982

Nonrandom distribution of repeated DNA sequences with respect to supercoiled loops and the nuclear matrix.

The DNA in a eukaryotic nucleus is arranged into a series of supercoiled loops that are anchored at their bases to the nuclear matrix. We have analyzed the DNA sequences that are closest to the matrix attachment points for their relative content of specific repeated sequences. Sequences were enriched (mouse satellite, human Alu family) or depleted (mouse EcoRI repeat, monkey alpha component), depending on the specific sequence and species examined. These results can be understood in terms of a nonrandom arrangement of DNA sequences with respect to nuclear DNA loops.

Animals↗
PubMed1998

PTH-responsive osteoblast nuclear matrix architectural transcription factor binds to the rat type I collagen promoter.

In connective tissue, cell structure contributes to type I collagen expression. Differences in osteoblast microarchitecture may account for the two distinct cis elements regulating basal expression, in vivo and in vitro, of the rat type I collagen alpha1(I) polypeptide chain (COL1A1). The COL1A1 promoter conformation may be the penultimate culmination of osteoblast structure. Architectural transcription factors bind to the minor groove of AT-rich DNA and bend it, altering interactions between other trans-acting proteins. Similarly, nuclear matrix (NM) proteins bind to the minor groove of AT-rich matrix-attachment regions, regulating transcription by altering DNA structure. We propose that osteoblast NM architectural transcription factors link cell structure to promoter geometry and COL1A1 transcription. Our objective was to identify potential osteoblast NM architectural transcription factors near the in vitro and in vivo regulatory regions of the rat COL1A1 promoter. Nuclear protein-promoter interactions were analyzed by gel shift analysis and related techniques. NM extracts were derived from rat osteosarcoma cells and from rat bone. The NM protein, NMP4, and a soluble nuclear protein, NP, both bound to two homologous poly(dT) elements within the COL1A1 in vitro regulatory region and proximal to the in vivo regulatory element. These proteins bound within the minor groove and bent the DNA. Parathyroid hormone increased NP/NMP4 binding to both poly(dT) elements and decreased COL1A1 mRNA in the osteosarcoma cells. NP/NMP4-COL1A1 promoter interactions may represent a molecular pathway by which osteoblast structure is coupled to COL1A1 expression.

Animals↗
PubMed1999

Nuclear matrix protein levels in burn patients with multiple organ dysfunction syndrome.

In this study, we investigated multiple organ dysfunction syndrome (MODS) and nuclear matrix protein (NMP), which is an indicator for apoptosis, in burn patients. The subjects were 17 patients with a total burn surface area (TBSA) of 26% or more. Eight of them had MODS, and nine did not. Seven patients died, and 10 survived. The NMP value in the group with MODS (610 +/- 318 units/ml) was significantly higher than in the group without MODS (82 +/- 31 units/ml). The NMP values of the patients who died (645 +/- 326 units/ml) were significantly higher than in the group who survived (111 +/- 95 units/ml), and they rose significantly as the number of impaired organs increased. Our findings suggested that the NMP level in burn cases associated with MODS was higher when apoptosis developed and larger numbers of organs were impaired.

Adult↗
PubMed1996

Contrasting effects of the SATB1 core nuclear matrix attachment region and flanking sequences of the keratin 18 gene in transgenic mice.

The 2.3 kb and 3.5 kb of DNA that flank the human keratin 18 (K18) gene and synthetic nuclear matrix attachment regions (MAR) composed of the binding sites for the SATB1 nuclear protein were fused to a reporter gene that utilizes the mouse metallothionein promoter and the human growth hormone gene (MThGH). Transgenic mice were generated from both constructions and the control MThGH gene to test K18 and SATB1 MAR sequences for the ability to insulate the reporter gene from integration site-specific position effects. The MThGH control gene was variably expressed in brain, heart, intestine, kidney, liver, and testes, confirming previous studies. In contrast, the MThGH gene insulated by the K18 flanking sequences was expressed in the same tissues of four independent transgenic animals at levels correlated with the copy number except for intestine. The average level of expression on a per gene basis of the K18 insulated gene was from 9- to 49-fold higher than the control. The MThGH gene linked to the SATB1 MAR sequences was completely repressed in the brains and kidneys of all six transgenic mice. However, expression was nearly as efficient in testes as the K18-insulated gene. Both the SATB1 MAR and the K18 flanking sequences confer position-independent transcriptional status on the reporter gene in some or many tissues. However, the effects are stimulatory for the K18 elements and generally suppressive for the SATB1 MAR elements.

Animals↗
PubMed2001

[Clinical evaluation of urinary nuclear matrix protein 22 as a marker for bladder cancer].

The purpose of this study is to evaluate the clinical usefulness of urinary nuclear matrix protein 22 (NMP22) as a marker for bladder cancer. We examined the positive rates of NMP22 test, urinary cytology and bladder tumor antigen (BTA) test, and compared the positive rate of NMP22 test with that in urinary cytology and BTA test. Urine samples were obtained from 50 patients with histologically confirmed bladder cancer before the treatment. The samples were examined by NMP22 test, urinary cytology and BTA test. In 50 patients with bladder cancer, the overall positive rate was 40% for NMP22 test, 40% for urinary cytology, and 16% for BTA test. A combination of NMP22 test and urinary cytology showed a significantly higher positive rate (54%) as compared to NMP22 test or urinary cytology alone. When NMP22 test and urinary cytology were compared for tumor size, number, shape, stage and grade, NMP22 test showed a significant higher positive rate than urinary cytology in grade 1 bladder cancer. In conclusion, although NMP22 test and urinary cytology gave a similar positive rate, a combination of NMP22 test and urinary cytology is more useful than the NMP22 test or urinary cytology alone for monitoring of bladder cancer.

Adult↗
PubMed2004

Colon cancer specific nuclear matrix protein alterations in human colonic adenomatous polyps.

Most colon cancers arise within preexisting adenomatous polyps or adenomas. The slow evolution from the non-invasive premalignant lesion, the adenomatous polyp, to invasive cancer supports a strategy of early detection. Recently, we identified unique nuclear matrix proteins (NMPs) specific for colon cancer (CC2, CC3, CC4, CC5). Most of the NMPs identified are common to all cell types, but several identified NMPs are tissue and cell line specific. The objective of this study is to describe and characterize the NMP profile of premalignant adenomatous colon polyps. Specifically when in the adenoma-carcinoma sequence four specific colon cancer NMPs, previously described, appear. Using two-dimensional (2-D) gel analysis 20 colon polyps (one juvenile polyp, six tubular adenoma (TA), seven tubulovillous adenoma (TVA), six TVA with focal high-grade dysplasia (HGD), were analyzed for the presence of four (CC2, CC3, CC4, CC5) specific NMPs. CC2 was not seen in any of the premalignant polyps. CC5 was present in only two premalignant TVA with HGD and in one TA. CC3 and CC4 were present in most adenomas. None of the NMPs were seen in the juvenile polyp, which is not considered to be a precursor of colon cancer. CC2 and CC5 are NMPs expressed at the junction of an advanced adenoma and invasive colorectal cancer. CC3 and CC4 are expressed earlier in the evolution of adenomatous polyps. Development of an assay to these proteins may serve as a new method for early detection of colorectal cancer.

Adenomatous Polyps↗