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PubMed2003

[Mechanism of arsenic trioxide-induced apoptosis in hepatic blastoma cells HepG2].

OBJECTIVE: To investigate the mechanism of arsenic trioxide (As(2)O(3)) induced apoptosis in hepatic blastoma cells HepG2 and its effects on cell nuclear matrix related protein promyelocytic leukaemia (PML). METHODS: HepG2 cells were cultured in MEM medium and treated with different concentrations of As(2)O(3) for either 24 h or 96 h. In situ terminal deoxynucleotidyl transferase (TdT) labeling (TUNEL) and DNA ladder were applied to detect apoptosis. Confocal microscopy and western blot were performed to observe the expression of PML. RESULTS: TUNEL positive apoptotic cells and DNA ladder could be detected in As(2)O(3) treated groups. The expression of PML decreased in HepG2 cells with 2 micro mol/L As(2)O(3), and micropunctates characteristic of PML protein in HepG2 cell nuclei almost disappeared after the treatment of 5 micro mol/L As(2)O(3). CONCLUSION: As(2)O(3) induces HepG2 tumor cell apoptosis in a time- and concentration-dependent manner. As(2)O(3) may degradate the PML protein in HepG2 cell nuclei. The decreased expression of PML is closely correlated with apoptosis. Nuclear matrix associated protein PML could be the target of As(2)O(3) therapy.

Antineoplastic Agents↗
PubMed2005

Noninvasive testing for colorectal cancer: a review.

OBJECTIVES: Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States. Endoscopic screening is now in favor and its use is increasing, but overall participation rates are poor. A substantial percentage of the population will likely continue to resist endoscopic screening. As such, a noninvasive biomarker for the early detection of CRC remains a priority. Herein, we (i) review the currently available noninvasive screening markers for the early detection of CRC, (ii) discuss newer markers that have undergone preliminary testing, and (iii) introduce and explain potentially promising markers of the future. METHODS: The published literature on markers for early detection of CRC was identified using a MEDLINE/PubMed search with secondary review of cited publications. RESULTS: Noninvasive testing for CRC is most advanced in testing for stool fecal occult blood, globin, or DNA mutations. Study of abnormal mucins has also been explored. Research for serum-based markers is just beginning and includes serum proteomics, nuclear matrix proteins, and serum DNA testing. CONCLUSIONS: Serial guaiac-based fecal occult blood testing (FOBT) is simple, inexpensive, and proven effective at reducing mortality from CRC. Immunochemical fecal occult blood tests facilitate compliance and offer improved specificity, but at increased cost in comparison to FOBT. Fecal DNA testing may provide enhanced sensitivity for detection of CRC in comparison with FOBT, but its high cost limits its use for generalized screening. Rectal mucin testing requires additional evaluation to determine its sensitivity and specificity in comparison with guaiac-based FOBT. Serum tests, such as proteomics, nuclear matrix proteins, and serum DNA, are still in their infancy, but remain a hope for the future.

Biomarkers, Tumor↗
PubMed1987

Heat shock-induced changes in the structural stability of proteinaceous karyoskeletal elements in vitro and morphological effects in situ.

Karyoskeletal protein fractions prepared from Drosophila melanogaster embryos contain morphologically identifiable remnants of nuclear pore complexes and peripheral lamina as well as what appears to be an internal nuclear "matrix" (Fisher, P. A., M. Berrios, and G. Blobel, 1982, J. Cell Biol., 92: 674-686). Structural stability of these proteinaceous assemblies is dependent on thermal incubation in vitro (37 degrees C, 15 min) before subfractionation of nuclei. In the absence of such incubation, greater than 90% of the total karyoskeletal protein including major polypeptide components of internal "matrix," pore complexes, and the peripheral lamina, is solubilized by 1 M NaCl. In vivo heat shock induces karyoskeletal stabilization resembling that resulting from thermal incubation in vitro. Immunocytochemical studies have been used to establish the effects of heat shock on the organization and distribution of major karyoskeletal marker proteins in situ. Taken together, these results are consistent with the notion that in vivo, regulation of karyoskeletal plasticity (and perhaps form) may be a functionally significant component of the Drosophila heat shock response. They also have broad practical implications for studies pertaining to the structure and function of karyoskeletal protein (nuclear "matrix") fractions isolated from higher eukaryotic cells.

Animals↗
PubMed2005

Bladder cancer screening and monitoring of 4,4'-methylenebis(2-chloroaniline) exposure among workers in Taiwan.

OBJECTIVES: Transitional cell carcinoma of the urinary bladder is associated with occupational exposure to 4,4'-methylenebis(2-chloroaniline) (MBOCA). A program to monitor MBOCA levels in the work environment and to screen for bladder cancer was performed at four MBOCA manufacturing factories. METHODS: The U.S. Occupational Safety and Health Administration analytic method No. 24 was adopted in this study to measure air MBOCA concentrations. A total of 70 MBOCA-exposed workers and another 92 nonexposed workers were recruited for screening. Urine occult blood tests, urine cytology, tests for the urine tumor marker nuclear matrix protein, and abdominal ultrasonography were performed in all patients. Intravenous urography and cystoscopy were used to confirm the presence of bladder cancer. RESULTS: The air concentration of MBOCA was greatest in the purification area (0.23 to 0.41 mg/m3), followed by the washing area (less than 0.02 to 0.08 mg/m3) and neutralization area (less than 0.05 to 0.06 mg/m3). This study identified a current worker with proved bladder cancer. In addition, we also identified 1 worker with suspected malignant cells on urine cytology and 1 worker with atypical cytology combined with gross hematuria. Although the prevalence of atypical urinary cells and the nuclear matrix protein 22 tumor marker was not significantly different between the MBOCA-exposed workers and nonexposed workers as a whole or when grouped by sex, the prevalence of positive occult blood was marginally significantly (P = 0.055) greater in male exposed workers (18%) than in male nonexposed workers (7%). CONCLUSIONS: The findings of this study support the conclusions from other studies that MBOCA is potentially carcinogenic to humans. Control measures are needed to prevent overexposure from inhalation and skin absorption.

Adult↗
PubMed2000

The RING finger protein SNURF modulates nuclear trafficking of the androgen receptor.

The androgen receptor (AR) is a transcription factor that mediates androgen action. We have used the green fluorescent protein (GFP) technique to investigate dynamics of nuclear trafficking of human AR in living cells. In the absence of ligand, the GFP-AR fusion protein is distributed between cytoplasm and nuclei. Androgen exposure leads to a rapid and complete import of GFP-AR to nuclei of CV-1 cells (>=90% nuclear in 30 minutes), whereas a pure antiandrogen, Casodex, elicits a slower (<40% nuclear in 30 minutes) and incomplete transfer. Unliganded ARs with mutations in the basic amino acids of the bipartite nuclear localization signal (NLS) within the second zinc finger and the hinge region are predominantly cytoplasmic and their androgen-dependent nuclear import is severely compromised ((3/4)20% nuclear in 30 minutes). Interestingly, substitutions of the Leu residues flanking the bipartite NLS lead to inefficient nuclear transfer in response to androgen ((3/4)20% nuclear in 30 minutes). The ligand-binding domain of AR, which represses bipartite NLS activity, contains an agonist-specific NLS. The small nuclear RING finger protein SNURF, which interacts with AR through a region overlapping with the bipartite NLS, facilitates AR import to nuclei and retards its export on hormone withdrawal. More AR is associated with the nuclear matrix in the presence than absence of coexpressed SNURF. We suggest that the SNURF-mediated tethering of AR in nuclei represents a novel mechanism for activating steroid receptor functions.

Amino Acid Sequence↗
PubMed1986

Replication origins are attached to the nuclear skeleton.

DNA fragments containing replication origins (oriDNA) were isolated from a chicken erythroblast cell line by a modified procedure of Zannis-Hadjopoulos et al. and studied in the renaturation reaction driven by either total or nuclear matrix DNA (nmDNA) from the same cells or from mature erythrocytes. We found that the unique sequences of nmDNA from erythroblasts (5 kb long) represented a specific subset of sequences constituting about a quarter of total DNA unique sequences, while the erythrocyte nmDNA 5 kb fragments constitute only about one tenth of total unique DNA and all are recovered among erythroblast nmDNA. Virtually all oriDNA sequences are present in the fraction of erythrocyte nmDNA. Thereafter, the putative positions of replication origins within the alpha-globine gene domain have been mapped by hybridization experiments. They were found to coincide with the previously established positions of permanent sites of DNA attachment to the nuclear matrix.

Animals↗
PubMed1994

Induction of DNA damage in gamma-irradiated nuclei stripped of nuclear protein classes: differential modulation of double-strand break and DNA-protein crosslink formation.

The influence of chromatin proteins on the induction of DNA double-strand breaks (dsb) and DNA-protein crosslinks (dpc) by gamma-radiation was investigated. Low molecular weight non-histone proteins and classes of histones were extracted with increasing concentrations of NaCl, whereas nuclear matrix proteins were not extractable even by 2.0 M NaCl. The yield of dsb increased with progressive removal of proteins from chromatin. Whilst removal of low molecular weight non-histone proteins and histone H1 resulted in small increases in the production of dsb, removal of histones H2A/H2B, all histones, or all proteins led to 18.4, 46.4 and 55.5-fold increases in the yield of dsb, respectively, relative to irradiated cells. Therefore, both histones and non-histone proteins contribute to the radioprotection of DNA, core histones being the major radio-protectors. In contrast, depletion of chromatin proteins caused little or no effect on the induction of dpc until the chromatin was extracted with > or = 1.4 M NaCl. However, our studies indicated no direct, quantitative correlation between the removal of histones and the induction of dpc. The data support our previous conclusion that nuclear matrix protein rather than the majority of the histones are the predominant substrates for dpc production, although the involvement of a subset of tightly bound histones (H3 and H4) has not been excluded. This finding demonstrates that chromatin proteins can differentially modify the yield of two types of radiation-induced DNA lesions.

Animals↗
PubMed1997

Cell specific nuclear antigens of boar spermatozoa.

Demembranated boar sperm heads were differentially extracted at conditions involving high salt-urea, proteolysis and DNase I cleavage that mimic the conditions promoting the in vivo decondensation of the fertilizing sperm nucleus in the egg ooplasm. The sperm-unique subset of proteins was studied which remained bound in the residual salt-resistant nuclear structure operationally defined as sperm nuclear matrix. By means of polyvalent antisera the immune specificity of the sperm nucleoprotein complex was estimated using ELISA and microcomplement fixation test as compared to somatic type dehistonized chromatin of boar liver. To define immunologically specific sperm DNA-associated proteins hybridomas were generated by fusing lymphocytes immunized with boar sperm protein/DNA complex. Monoclonal antibodies were selected (Mab 1A8, 1B3, 2B5, 2H5 and 3A4) which identified protein moieties in the sperm DNA-tight binding proteins complex resistant to cleavage with DNase I and sensitive upon digestion with high concentration of proteases. No appreciable reactivity was recorded of the antibodies to somatic chromatin and no significant binding to ssDNA. A polypeptide in the residual sperm nuclear structure of apparent Mr 27 kDa was recognized by Mab 3A4 as detected by Western blotting. The enhanced reactivity to the DNase I digested sperm nuclear fraction (except for Mab 2H5) suggests that DNA protected from nuclease digestion by a protein might be essential for immune reactivity and full antigenic integrity as well as the dependence of the cognate proteins on the binding to DNA for antigenicity and immune specificity. The functioning of the identified putative sperm specific proteins is anticipated in the structural rearrangement of chromatin in the zygote.

Animals↗
PubMed1998

Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus.

What determines the nuclear organization within a cell and whether this organization itself can impose cellular function within a tissue remains unknown. To explore the relationship between nuclear organization and tissue architecture and function, we used a model of human mammary epithelial cell acinar morphogenesis. When cultured within a reconstituted basement membrane (rBM), HMT-3522 cells form polarized and growth-arrested tissue-like acini with a central lumen and deposit an endogenous BM. We show that rBM-induced morphogenesis is accompanied by relocalization of the nuclear matrix proteins NuMA, splicing factor SRm160, and cell cycle regulator Rb. These proteins had distinct distribution patterns specific for proliferation, growth arrest, and acini formation, whereas the distribution of the nuclear lamina protein, lamin B, remained unchanged. NuMA relocalized to foci, which coalesced into larger assemblies as morphogenesis progressed. Perturbation of histone acetylation in the acini by trichostatin A treatment altered chromatin structure, disrupted NuMA foci, and induced cell proliferation. Moreover, treatment of transiently permeabilized acini with a NuMA antibody led to the disruption of NuMA foci, alteration of histone acetylation, activation of metalloproteases, and breakdown of the endogenous BM. These results experimentally demonstrate a dynamic interaction between the extracellular matrix, nuclear organization, and tissue phenotype. They further show that rather than passively reflecting changes in gene expression, nuclear organization itself can modulate the cellular and tissue phenotype.

Cell Nucleus↗
PubMed1984

Increased levels of nuclear androgen receptors in hyperplastic prostate of aging men.

Several publications indicate the dihydrotestosterone content of hyperplastic prostatic tissue from man and dog is increased. There is limited information concerning an abnormality in the androgen receptor content in this disorder. In men, we determined the androgen receptor concentration in cytosol, nuclei, the nuclear matrix, and nuclease solubilized salt extract fractions from benign prostatic hyperplasia (BPH) and normal prostate. Nine BPH cases (age 61 +/- 7, mean +/- SD) and seven controls (age 29 +/- 10.6) were compared. Prostates were obtained during autopsy performed within 12 h after death and frozen at -70 degrees C until analysis. Four grams of tissue were homogenized and centrifuged for separation of nuclei and cytosol. Androgen receptors were estimated with an improved assay procedure. The androgen receptor content per mg of DNA in whole nuclei and in the salt extract fraction (P less than 0.01 and less than 0.05 respectively) was higher in BPH cases than in controls. Cytosol did not show a significant difference. For both groups, receptors were undetectable in the nuclear matrix. We speculate that increased androgen receptor bound to chromatin of hyperplastic tissue may be causally related to the development of BPH in aging man.

Adult↗
PubMed1992

A coiled-coil related protein specific for synapsed regions of meiotic prophase chromosomes.

Synaptonemal complexes (SCs) are structures that are formed between homologous chromosomes during meiotic prophase. They are probably involved in chromosome pairing and recombination. Using a monoclonal anti-SC antibody we isolated cDNAs encoding a major component of SCs which is localized specifically in synapsed segments of meiotic prophase chromosomes. The protein predicted from the nucleotide sequence of a full-length cDNA, named SCP1, consists of 946 amino acid residues and has a molecular weight of 111 kDa. It shares several features with nuclear lamins and some recently identified nuclear matrix proteins. The major part of SCP1 consists of long stretches capable of forming amphipathic alpha-helices. This region shows amino acid sequence similarity to the coiled-coil region of myosin heavy chain. A leucine zipper is included in this region. The carboxy-terminus has two small basic domains and several S/T-P-X-X motifs, which are characteristic of DNA-binding proteins. One of these motifs is a potential target site for p34cdc2 protein kinase. The amino-terminus is acidic and relatively proline-rich, but does not contain the S/T-P-X-X motif. The transcription of the gene encoding SCP1 is restricted to zygotene-diplotene spermatocytes. A polyclonal antiserum raised against the fusion protein of one of the cDNA clones recognizes a single protein on Western blots of isolated SCs, with an electrophoretic mobility identical to that of the antigen recognized by the original monoclonal antibody (mAb), IX5B2. From a detailed comparison of the immunogold labelling of rat SCs by mAb IX5B2 and the polyclonal anti-fusion protein antiserum respectively, we tentatively infer that the carboxy-terminus of SCP1 is orientated towards the lateral elements and that the other domains of the protein extend towards the central region between the lateral elements. We conclude that SCP1 is the major component of the transverse filaments of SCs, and speculate that it has evolved by specialization of a nuclear matrix protein.

Amino Acid Sequence↗
PubMed1999

The A-kinase-anchoring protein AKAP95 is a multivalent protein with a key role in chromatin condensation at mitosis.

Protein kinase A (PKA) and the nuclear A-kinase-anchoring protein AKAP95 have previously been shown to localize in separate compartments in interphase but associate at mitosis. We demonstrate here a role for the mitotic AKAP95-PKA complex. In HeLa cells, AKAP95 is associated with the nuclear matrix in interphase and redistributes mostly into a chromatin fraction at mitosis. In a cytosolic extract derived from mitotic cells, AKAP95 recruits the RIIalpha regulatory subunit of PKA onto chromatin. Intranuclear immunoblocking of AKAP95 inhibits chromosome condensation at mitosis and in mitotic extract in a PKA-independent manner. Immunodepletion of AKAP95 from the extract or immunoblocking of AKAP95 at metaphase induces premature chromatin decondensation. Condensation is restored in vitro by a recombinant AKAP95 fragment comprising the 306-carboxy-terminal amino acids of the protein. Maintenance of condensed chromatin requires PKA binding to chromatin-associated AKAP95 and cAMP signaling through PKA. Chromatin-associated AKAP95 interacts with Eg7, the human homologue of Xenopus pEg7, a component of the 13S condensin complex. Moreover, immunoblocking nuclear AKAP95 inhibits the recruitment of Eg7 to chromatin in vitro. We propose that AKAP95 is a multivalent molecule that in addition to anchoring a cAMP/PKA-signaling complex onto chromosomes, plays a role in regulating chromosome structure at mitosis.

Antibodies↗
PubMed2001

The 5' boundary of the human apolipoprotein B chromatin domain in intestinal cells.

The 5' boundary of the chromosomal domain of the human apolipoprotein B (apoB) gene in intestinal cells has been localized and characterized. It is composed of two kinds of boundary elements; the first, functional boundary is an insulator activity exhibited by a 1.8 kb DNA fragment located between -58 and -56 kb upstream of the human apoB promoter. In this region, an enhancer-blocking activity has been mapped to a CTCF binding site that is located upstream of two apoB intestinal enhancers (IEs), the 315 IE and the 485 IE. The CTCF site represents a boundary between two types of chromatin structure: an open, DNaseI-sensitive region 3' of the CTCF site containing the intestinal regulatory elements and a closed, DNaseI-resistant region 5' of the CTCF site. The 1.8 kb fragment harboring the CTCF site also insulated mini-white transgenes against position effects in Drosophila melanogaster. The second, structural boundary is represented by a nuclear matrix attachment region (MAR), situated about 3 kb 5' of the CTCF site. This MAR may represent the 5' anchorage site for a chromosomal loop that functions to bring the intestinal regulatory elements closer to the apoB promoter.

5' Untranslated Regions↗
PubMed1997

A far upstream cis-element is required for Wilms' tumor-1 (WT1) gene expression in renal cell culture.

To identify novel cis-regulatory elements responsible for the tissue-restricted expression pattern of the Wilms' tumor-1 (WT1) gene, we mapped a total of 11 DNase I-hypersensitive sites in the 5'-flanking region and first intron of the human gene, six of which were specific for WT1 expressing cell lines. A 1.4-kilobase (kb) fragment from the mouse wt1 5'-flanking region contained cross-hybridizing sequence with significant homology to a region of DNase I hypersensitivity in the human WT1 gene which bound to nuclear matrix in human fetal kidney 293 cells. None of the DNase I-hypersensitive sites/matrix attachment regions, either alone or in combination, were sufficient for tissue-specific WT1 expression in transient and stably transfected cell lines. However, stable transfection of an approximately 620-kb yeast artificial chromosome (YAC) that carried the entire mouse wt1 locus into 293 cells resulted in wt1 (trans)gene expression at a level of approximately 30% of the endogenous human gene. Deletion of the 1.4-kb cross-hybridizing mouse fragment, located approximately 15 kb upstream of the transcription start site, caused complete loss of wt1 gene expression in the YAC-transfected 293 cells. In summary, we have identified a far upstream element that contains a region of DNase I hypersensitivity and that binds to nuclear matrix. This element includes phylogenetically conserved sequence and is required, although not sufficient, for mouse wt1 gene expression in human fetal kidney cells in culture.

Animals↗
PubMed1996

Scaffold/matrix-attached regions: topological switches with multiple regulatory functions.

The nuclear matrix or scaffold that can be prepared and investigated in vitro has an affinity for distinct topological forms of DNA and for sequences that permit the induction of such a topology by their association with a corresponding set of proteins (so-called matrix- or scaffold-attached regions, S/MARS). As a consequence, S/MARS are regarded as topological sinks that can be regulated according to activity-related requirements. Here we develop a switching model to understand the biological functions of S/MARS, which are able to augment transcriptional processes and form barriers between independently regulated domains. The current literature is screened for examples supporting such a mechanism and novel approaches are suggested for their further elucidation.

Animals↗
PubMed1994

ScII: an abundant chromosome scaffold protein is a member of a family of putative ATPases with an unusual predicted tertiary structure.

Here, we describe the cloning and characterization of ScII, the second most abundant protein after topoisomerase II, of the chromosome scaffold fraction to be identified. ScII is structurally related to a protein, Smc1p, previously found to be required for accurate chromosome segregation in Saccharomyces cerevisiae. ScII and the other members of the emerging family of SMC1-like proteins are likely to be novel ATPases, with NTP-binding A and B sites separated by two lengthy regions predicted to form an alpha-helical coiled-coil. Analysis of the ScII B site predicted that ScII might use ATP by a mechanism similar to the bacterial recN DNA repair and recombination enzyme. ScII is a mitosis-specific scaffold protein that colocalizes with topoisomerase II in mitotic chromosomes. However, ScII appears not to be associated with the interphase nuclear matrix. ScII might thus play a role in mitotic processes such as chromosome condensation or sister chromatid disjunction, both of which have been previously shown to involve topoisomerase II.

Adenosine Triphosphatases↗
PubMed1990

Nuclear localization of Semliki Forest virus-specific nonstructural protein nsP2.

About 50% of Semliki Forest virus-specific nonstructural protein nsP2 is associated with the nuclear fraction in virus-infected BHK cells. Transport into the nucleus must be specific, since only trace amounts of nsP3 and nsP4 and about 13% of nsP1, all derived from the same polyprotein, were found in the nucleus. Subfractionation of [35S]methionine-labeled Semliki Forest virus-infected cells showed that 80 to 90% of the nuclear nsP2 was associated with the nuclear matrix. Indirect immunofluorescence, with anti-nsP2 antiserum, showed the most intensive staining of structures which by Nomarski optics appeared to be nucleoli. In the presence of 1 to 5 micrograms of dactinomycin per ml the nuclei were stained evenly and no nucleoli could be found. Transport of nsP2 into the nucleus occurred early in infection and was fairly rapid. A cDNA encoding the complete nsP2 was isolated by the polymerase chain reaction technique and ligated into a simian virus 40 expression vector derivative. When BHK cells were transfected with this pSV-NS2 vector by the lipofection procedure, nsP2 was expressed in about 1 to 5% of the cells, as shown by indirect immunofluorescence. In positively transfected cells the immunofluorescence stain was most intensive in the nucleoli. Thus, Semliki Forest virus-specific nsP2 must have information which directs it into the nuclear matrix and, more specifically, into the nucleoli.

Animals↗
PubMed1991

Interaction between ionizing radiation and supercoiled DNA within human tumor cells.

We have analyzed DNA supercoiling within histone-free nuclei (nucleoids) using four human squamous cell carcinoma cell lines that express varying degrees of radiosensitivity. The entire DNA, arranged as negative supercoiled loops attached to the nuclear matrix, was extracted from single cells, stained with ethidium bromide, and passed through a flow cytometer recording both light scatter and red (DNA) fluorescence. Supercoiled loops of DNA from all cells were unwound with a low concentration of ethidium bromide, as seen by increased light scatter. Nucleoids from radiosensitive but not radioresistant cells resisted the transition from zero to positive supercoiling at higher concentrations of ethidium bromide. The profile of red DNA fluorescence from ethidium bromide-stained nucleoids showed that the radiosensitive cells expressed a greater variation in the total amount of ethidium bromide bound. After 12 Gy of gamma-radiation, radiosensitive cell lines produced nucleoids that contained a greater proportion of relaxed supercoiled DNA, making them larger than those from radioresistant cell lines. We suggest these observations are secondary effects resulting from an altered affinity between supercoiled looped DNA and the nuclear matrix. Combined with radiation damage, these structural alterations may lead to a more complex type of damage to repair within the radiosensitive cell lines.

Carcinoma, Squamous Cell↗