The 5'-flanking cis-acting elements of the human ε-globin gene associates with the nuclear matrix and binds to the nuclear matrix proteins

The nucleax mains attachment regions(MARs) and the binding nuclear matrix proteins in the 5’-flalildng cisacting elements of the humanε-globin gene have been examined. Using in vitro DNA-matrix binding assay,it has been shown that the positive stage-specific regulatory element (ε-PREII, -446bp-419bp) upstream of this gene could specifically associate with the nuclear matrix from K562 cells, indicating thatε-PREII mad be an erythroidspecilic facultstive MAR. In gel mobility shift assay and Southwestern blotting assal an eothroid-specific nuclear matrix protein (ε-NMPk) in K562 cells has been revealed to bind to this positive regulatory element (E-PREII). Furthermore, we demonstrated that the silencer (-392hp -177bp) uP8tream of the humanε-globin gene could associate with the nuclear matrices from K562, HEL and Raji cells. In addition, the nucleax matrix proteins prepared from these three cell lines could also bind to this silencer, suggesting that this silencer element linght be a constitutive nuclear mains attachment region (constitutive MAR). Our results demonstrated that the nucleax madrid and nuclear mains proteins lxilght play an important role in the regulation of the human 5-globin gene expression.

The interaction between the human β-globin locus control region and nuclear matrix

Our previous study showed that hydroxyurea (Hu) could induce HEL cells to express humanβ-globin gene. However the molecular mechanisms by which the expression of β-globin gene is activated and regulated are poorly understood. Here we show that the binding patterns between the core DNA sequences (HS2 core sequence -10681- -10971 bp , HS3 core sequence -14991- -14716 bp and HS4 core sequence -18586- -18306 bp) of DNase I hypersensitive sites in the human β-globin LCR and nuclear matrix proteins isolated from Hu induced and uninduced HEL cells are quite different. Results demonstrated that nuclear matrix proteins might play important roles in regulating the expression of humanβ-like globin genes through their interaction with HSs (HS2,HS3 and HS4 core sequences) in the LCR. Moreover, the results obtained from the in vitro DNA-matrix binding assay showed that the core DNA sequences of DNase I hypersensitive sites (HS2, HS3 and HS4) were unable to bind to the nuclear matrix isolated from uninduced HEL cells; in addition, HS2 core DNA sequence was capable of binding to the nuclear matrix prepared from Hu-induced HEL cells, while both HS3 and HS4 core DNA sequences could not do so. Results indicated that the HS2 core DNA sequence may be a functional MAR (matrix attachment region). We suggest that the HS2 core DNA sequence binding to the nuclear matrix in Hu-induced HEL cells may open the structure of chromatin to make the LCR accessible to the promoter of β-globin gene and to promote its transcription.

Comparisons of voided urine cytology, nuclear matrix protein-22 and bladder tumor associated antigen tests for bladder cancer of geriatric male patients in Taiwan, China

Aim： To compare the results of bladder tumor associated antigen （BTA TRAK）, nuclear matrix protein 22 （NMP 22） and voided urine cytology （VUC） in detecting bladder cancer. Methods： A total of 135 elderly male and 50 healthy volunteers enrolled in this study were classified into three groups： （i） 93 patients with bladder cancer; （ii） 42 patients with urinary benign conditions; and （iii） 50 healthy volunteers. BTA TRAK and NMP 22 kits were used to detect bladder cancer. Voided urine cytology was used to compare the sensitivity and specificity of the screening tests. Results： The sensitivity and specificity of cytology, BTA TRAK and NMP 22 were 24% and 97%, 51% and 73%, 78% and 73%, respectively. The level of NMP 22 increased with tumor grading. The BTA TRAK kit has the lowest sensitivity among the screening tests. The NMP 22 with the best sensitivity can be an adjunct to cytology for evaluating bladder cancer. Conclusion： The NMP 22 test has a better correlation with the grading of the bladder cancer than BTA TRAK. As cytology units are typically not available in hospitals or in outpatient clinics, NMP 22 might be a promising tool for screening bladder cancer.

Altered profiles of nuclear matrix proteins during the differentiation of human gastric mucous adenocarcinoma MGc80-3 cells

AIM： To find and identify specific nuclear matrix proteins associated with proliferation and differentiation of carcinoma cells, which will be potential markers for cancer diagnosis and targets in cancer therapy. METHODS： Nuclear matrix proteins were selectively extracted from MGcS0-3 cells treated with or without hexamethylamine bisacetamide （HMBA）, and subjected to 2-D gel electrophoresis. The resulted protein patterns were analyzed by Melanie software. Spots of nuclear matrix proteins differentially expressed were excised and subjected to in situ digestion with trypsin. Peptide masses were obtained by matrix-assisted laser-desorption/ ionization time of flight mass spectrometry （MALDI-TOFMS） analysis and submitted for database searching using Mascot tool. RESULTS： The MGc80-3 cells were induced into differentiation by HMBA. There were 22 protein spots which changed remarkably in the nuclear matrix, from differentiation of MGcS0-3 cells compared to control. Eleven of which were identified. Seven proteinsactin, prohibitin, porin 31HL, heterogeneous nuclear dbonucleoprotein A2/B1, vimentin, ATP synthase, and heat shock protein 60 were downregulated, whereas three proteins - heat shock protein gp96, heat shock protein 90-beta, and valosin-containing protein were upregulated, and the oxygen-regulated protein was only found in the differentiated MGc80-3 cells. CONCLUSION： The induced differentiation of carcinoma cells is accompanied by the changes of nuclear matrix proteins. Further characterization of those proteins will show the mechanism of cellular proliferation and differentiation, as well as cancer differentiation.

Alreration of nuclear matrix-intermediate filament system and differential expression of nuclear matrix proteins during human hepatocarcinoma cell differentiation

AIM： To investigate the association between the configurational and compositional changes of nuclear matrix and the differentiation of carcinoma cells. METHODS： Cells cultured with or without 5 × 10^-3 mmol/L of hexamethylene bisacetamide （HMBA） on Nickel grids were treated by selective extraction and prepared for whole mount observation under electron microscopy. The samples were examined under transmission electron microscope. Nuclear matrix proteins were selectively extracted and subjected to subcellular proteomics study. The protein expression patterns were analyzed by PDQuest software. Spots of differentially expressed nuclear matrix proteins were excised and subjected to in situ digestion with trypsin. The peptides were analyzed by matrix-assisted laser- desorption/ionization time of flight mass spectrometry （MALDI-TOF-MS）. Data were submitted for database searching using Mascot tool （www.matrixscience.com）. RESULTS： The nuclear matrix （NM） and intermediate filament （IF） in SMMC-7721 hepatocarcinoma cells were found relatively sparse and arranged irregularly. The nuclear lamina was non-uniform, and two kinds of filaments were not tightly connected. After induction for differentiation by HMBA, the NM-IF filaments were concentrated and distributed uniformly. The heterogeneous population of filaments, including highly branched utrathin filaments could also be seen in the regular meshwork. The connection between the two kinds of filaments and the relatively thin, condensed and sharply demarcated lamina composed of intermediate- sized filaments was relatively fastened. Meanwhile, 21 NM proteins changed remarkably during SMMC-7721 cell differentiation. Four proteins, i.e. mutant Pystl, hypothetical protein, nucleophosminl, and LBP were downregulated, whereas four other proteins, eIF6, p44 subunit, 13-tubulin, and SIN3B were upregulated with the last one, SR2/ASF found only in the differentiated SMMC-7721 cells. CONCLUSION： The induced differentiation of SMMC-7721 cells by HMBA is accompanied by the configurational changes of nuclear matrix-intermediate filament （NM-IF） system and the compositional changes of nuclear matrix protein expression. These changes may be important morphological or functional indications of the cancer cell reversion.

Aberrant expression of nuclear matrix proteins during HMBA-induced differentiation of gastric cancer cells

AIM: To investigate the aberrant expression of nuclear matrix proteins in human gastric cancer cells before and after hexamethylene bisacetamide (HMBA) treatment.METHODS: Proteomics analysis of differential nuclear matrix proteins was performed by two dimensional electrophoresis polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.The expression levels of three nuclear matrix proteins were further confirmed by Western blotting and their locations in nuclear matrix filament were observed by quantum dots-based immunofluorescence.RESULTS: Proteomics analysis showed that 43 protein spots were significantly changed due to HMBA treatment.Fifteen proteins were identified in the HMBAinduced differentiation of gastric tumor cells.Eight proteins spots were down-regulated while seven were up-regulated.Among these proteins,prohibitin,nucleophosmin and hnRNP A2/B1 were significantly decreased in HMBA-treated human gastric cancer cells,and their locations in nuclear matrix were altered by HMBA.Our results proved the alteration of specific nuclear matrix proteins during the differentiation of human gastric cancer cells.And the aberrant expressions of nuclear matrix proteins were of significance in revealing the regulatory mechanism of tumor cell proliferation and differentiation.CONCLUSION: The aberrant expressions and intracellular redistributions of nuclear matrix proteins before and after HMBA treatment indicated that nuclear matrix proteins play a pivotal role in the differentiation of gastric cancer cells.

Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL-60 cells

The events of cell death and the expression of nuclear matrix protein (NMP) have been investigated in a promyelocytic leukemic cell line HL-60 induced with etoposide. By means of TUNEL assay, the nuclei displayed a characteristic morphology change, and the amount of apoptotic cells increased early and reached maximun about 39% after treatment with etoposide for 2 h. Nucleosomal DNA fragmentation was observed after treatment for 4 h. The morphological change of HL-60 cells, thus, occurred earlier than the appearance of DNA ladder. Total nuclear matrix proteins were analyzed by 2-dimensional gel electrophoresis. Differential expression of 59 nuclear matrix proteins was found in 4 h etoposide treated cells. Western blotting was then performed on three nuclear matrix acssociated proteins, PML, HSC70 and NuMA. The expression of the suppressor PML protein and heat shock protein HSC70 were significantly upregulated after etoposide treatment, while NuMA, a nuclear mitotic apparatus protein, was down regulated. These results demonstrate that significant biochemical alterations in nuclear matrix proteins take place during the apoptotic process.

Tropomyosin is localized in the nuclear matrix and chromosome scaffold of physarum polycephalum

The nuclei and chromosomes were isolated from plasmodia of Physarum polycephalum. The nuclear matrir and chromosome scaffold were obtained after the DNA and most of the proteins were extracted with DNase I and 2 M NaCl. SDS-PAGE analyses revealed that the nuclear matrir and chromosome scaffold contained a 37 kD polypeptide which is equivalent to tropomyosin in molecular weight. Immunofluorescence observations upon slide preparations labeled with anti-tropomyosin antibody showed that the nuclear matrix and chromosome scaffold emanated bright fluorescence, suggesting the presence of the antigen in them.Immunodotting results confirmed the presence of tropomyosin in the nuclear matrix and chromosome scaffold. Immunoelectron microscopic obserwtions further demonstrated that tropomyosin was dispersively distributed in the interphase nuclei and metaphase chromosomes.

CHANGES OF NUCLEAR MATRIX PROTEIN AND ITS RELATIONSHIP WITH c-erbB-2 IN HUMAN COLON ADENOCARCINOMA

Objective： Nuclear matrix protein is tissue, cell-type specific, and tumor-relative. It plays an important role in the regulation of intranuclear processes. Some researches also showed that a c-erbB-2 promoter-specific DNA-binding nuclear matrix protein is present only in malignant human breast tissues and induces mitogenesis and cell surface expression of the c-erbB-2 protein in resting NIH/3T3 cells. But it is not clear that how it in colon adenocarcinomas. Methods： Two-dimensional gel electrophoretic method was used for NMP identification and immunohistochemistry was used for c-erbB-2 detection in 12 cases of colon adenocarcinomas and matched adjacent normal colon tissues. Results： 5 different nuclear matrix proteins （named C1-C5） were identified in 12 colon adenocarcinoma specimens, but not in the matched adjacent normal colon tissues; 3 nuclear matrix proteins （named N1-N3） were identified in all 12 matched adjacent normal colon tissues, but not in colon adenocarcinoma specimens. A nuclear matrix protein （named N4） was detected in all of 9 moderated-well differentiated adenocarcinomas and all 12 matched adjacent normal colon tissues, but not in 3 poor-differentiated adenocarcinomas. All of the 10 colon adenocarcinomas which had the nuclear matrix protein C4 were c-erbB-2 expression positive. Conclusion： The data suggest that there are specific nuclear matrix proteins in colon adenocarcinomas and its subtypes, which maybe valuable to serve as markers of colon adenocarcinomas in future. Nuclear matrix protein C4 probably is a c-erbB-2 promotor-specific nuclear matrix protein in colon adenocarcinomas, and may induce the expression of c-erbB-2.

The detection of nuclear matrix in most primitive presentexisting eukaryote, Giardia lamblia

The nuclear matrix of diplomonad Giardia lamblia was detected for the first time with DGD embedmentsectioning- embedment free electron microscopy after a series of specific extractions. The result showed that archaezoa Giardia lamblia already possessed nuclear matrix within its two nuclei. The finest fibrils of the nuclear matrix of Giardia lamblia were measured to be about 11 to 13 nm in thickness. However, the nuclear lamina and nucleolus have never been observed. These results seem to suggest that nuclear matrix is an indispensable intranuclear structural component even in the primitive nucleus.

NUCLEAR MATRIX PROTEIN IN LEUKEMIA CELLS

Objective: To compare the composition of nuclear matrix proteins (NMP) between leukemia cells and normal bone marrow cells. Methods: NMP was isolated by high-salt extraction and identified in acute and chronic myelogenous leukemia cells as well as in the blast phase of chronic leukemia. On SDS-PAGE, NMPs with molecular myelogenous ferment from what were seen in normal bone marrow cells were present in both acute and chronic myelogenous leukemia. Conclusion: Marked changes of NMP, not only in contents but also in compositions, exist in leukemic cells compared with normal bone marrow cells. NMP may serve as a target of chemotherapeutic drug against leukemia.

Association of DNA with nuclear matrix in in vitro assembled nuclei induced by rDNA from Tetrahymena shanghaiensis in Xenopus egg extracts

The nuclei assembled from exogenous DNA or chromatin in egg extracts resemble their in vivo counterparts in many aspects. However, the distribution pattern of DNA in these nuclei remains unknown. We introduced rDNA from the macronuclei of Tetrabymena into Xenopus cellfree extracts to examine the association of specific DNA sequences with nuclear matrix (NM) in the nuclei assembled in vitro. Our previous works showed the 5’NTS (nontranscription sequences) of the rDNA specifically bind to the NM system in the macronuclei. We show now the rDNA could induce chromatin assembly and nuclear formation in Xenopus cell-free system. When we extracted the NM system and compared the binding affinity of different regions of rDNA with the NM system, we found that the 5’NTS still hold their binding affinity with insoluble structure of the assembled nuclei in the extracts of Xenopus eggs.

On the history of nuclear matrix manifestation

The nonchromatin proteinous residue of the cell nucleus was revealed in our laboratory as early aJs in 1948and then identified by light and electron microscoPy as residual nucleoli. intranuclear network and nuclear envelope before 1960. This structure termed afterwards as "nuclear residue", "nuclear skeleton", "nuclear cage" 3 "nuclearcarcass" etc., was much later (in 1974) isolated, studied,and entitled as "nuclear matrix" by Berezney and Coffey,to whom the discovery of this residual structure is of ten wronly ascribed. The real history of nuclear matrix manifestation is reported in this paper.

Nuclear lamina-like filaments and nuclear matrix in Allium cepa as revealed by scanning electron microscopy

In this study, freeze - fractured specimens of Allium cepa root tip meristems were examined under the scanning electron microscope (SEM). This technique permitted the visualization of the outer membrane of the nuclear envelope with nuclear pore complexes and polyribosomes. Some of the cell nuclei prepared with this procedure had fissures of various widths on their nuclear envelopes through which the nuclear lamina-like filaments (LLF) underneath the nucleoplasmic side of the envelopes were clearly visible. The diameters of these filaments varied between 25 and 125 nm. Many of the LLFs showed granular thickenings at places, and were attached to the inner surface of nuclear envelope in some regions. Similar LLFs were also seen at the peripheries of the freeze -fractured faces of nuclei. Meanwhile,the spatial relation between the nuclear matrix filaments (NMF) and other nuclear structures (nucleoli, chromatin and peripheral lamina - like filaments) was revealed in these fractured preparations. In addition, the methods and techniques in studying the nuclear lamina morphology and the roles played by NMFs in activities of various nuclear structures were discussed in brief.

Phosphorylation and biosynthesis of high molecular weight proteins of tumor nuclear matrix

Our previous studies showed a predominance of high molecular weight protein group in tumor nuclear matrices. Contrary to normal cells, proteins of this group are preferentially phosphorylated. Phosphoproteins of hepatoma nuclear matrix are selectively subjected to rapid proteolysis. By alkali treatment and a monoclonal antibody against phosphotyrosyl residue the presence of two high molecular weight bands of phosphotyrosyl-containing proteins was detected in nuclear matrices of tumor but not of normal liver cells. High molecular weight protein group of tumor nuclear matrices revealed also a rapid turnover and preferential incorporation of labeled amino acids selectively inhibited by chloramphenicol.

STUDY ON NUCLEAR MATRIX PROTEINS FROM HUMAN BREAST CARCINOMA

Objective To investigate the marker protein of human breast carcinoma from nuclear matrix proteins (NMPs). Methods NMPs were injected subcutaneously into rabbit to get antiserum, which was used to detect the NMPs specificity for breast carcinoma. Results There was an apparent positive band (100 kD) in the NMPs of breast carcinoma, which did not exist in normal breast and other tumors that were detected.Conclusion One or one group of 100 kD NMPs were found to be related to human breast carcinoma, which may be involved in the carcinogenesis and development of human breast carcinoma and valuable for breast carcinoma diagnosis.

Human papillomavirus 16 E6 is associated with the nuclear matrix of esophageal carcinoma cells

AIM: To explore the etiologic role of HPV infection in esophageal carcinoma, and the association of HPV-16 E6with the nuclear metrix of carcinoma cells.METHODS: Two esophageal carcinoma cell lines, EC/CUHK1 and EC/CUHK2, were tested for HPV-16 E6subgenetic fragment by polymerase chain reaction amplification of virus DNA associated nuclear matrix. RT-PCR and immunocytochemistry were also used to visualizethe expression of E6 subgene in the cells.RESULTS: The HPV-16 E6 subgenetic fragment wes found to be present in nuclear metrix-associeted DNA, E6oncoprotein localized in the nucleus where it is tightly associated with nuclear matrix after sequential extraction in EC/CUHK2 cells. It was not detected, however, in EC/CUHK1 cells.CONCLUSION: The interaction between HPV-16 E6 and nuclear matrix may contribute to the virus induced carcinogenesis in esophageal carcinoma.

Identification of the nuclear matrix and chromosome scaffold in dinoflagellate Crypthecodinium cohnii^1

Dinoflagellate is one of the primitive eukaryotes, whose nucleus may represent one of the transition stages from prokaryotic nucleoid to typical eukaryotic nucleus. Using selective extraction together with embeddment - free section and whole mount electron microscopy, a delicate nuclear matrix filament network was shown, for the first time, in dinoflagellate Crypthecodinium cohnii nucleus. Chromosome residues are connected with nuclear matrix filaments to form a complete network spreading over the nucleus. Moreover, we demonstrated that the dinoflagellate chromosome retains a protein scaffold after the depletion of DNA and soluble proteins. This scaffold preserves the characteristic morphology of the chromosome. Two dimensional elec-trophoreses indicated that the nuclear matrix and chromosome scaffold are mainly composed of acidic proteins. Our results demonstrated that a framework similar to the nuclear matrix and chromosome scaffold in mammalian cells appears in this primitive eukaryote,suggesting that these structures may have been originated from the early stages of eukaryote evolution.

Changes of Nuclear Matrix Proteins Following the Differentiation of Human Osteosarcoma MG-63 Cells

Human osteosarcoma MG-63 cells were induced into differentiation by 5 mmol/L hexamethylene bisacetamide （HMBA）. Their nuclear matrix proteins （NMPs） were selectively extracted and subjected to two-dimensional gel electrophoresis analysis. The results of protein patterns were analyzed by Melanie software. The spots of differentially expressed NMPs were excised and subjected to in situ digestion with trypsin. The maps of peptide mass fingerprinting were obtained by MALDI-TOF-MS analysis, and were submitted for NCBI database searches by Mascot tool. There were twelve spots changed remarkably during the differentiation induced by HMBA, nine of which were identified. The roles of the regulated proteins during the MG-63 differentiation were analyzed. This study suggests that the induced differentiation of cancer cells is accompanied by the changes of NMPs, and confirms the presence of some specific NMPs related to the cancer cell proliferation and differentiation. The changed NMPs are potential markers for cancer diagnosis or targets for cancer therapy.

Nuclear matrix associated protein PML: an arsenic trioxide apoptosis therapeutic target protein in HepG2 cells

Objective To investigate arsenic trioxide (As 2O 3)-induced apoptosis and the effects on cell nuclear matrix related protein promyelocytic leukaemia (PML). Methods HepG2 cells were cultured in MEM medium and treated with 0.5, 2, 5 and 10 μmol/L As 2O 3 for either 24 h or 96 h at each concentration. In situ terminal deoxynucleotidyl transferase (TdT) labeling (TUNEL) and DNA ladders were used to detect apoptosis. Confocal microscopy and Western blotting were used to observe the expression of PML. Results The growth rates of HepG2 cells were slower in the As 2O 3 treated than the untreated control group. DNA ladder and TUNEL positive apoptotic cells could be detected in As 2O 3 treated groups. The expression of PML decreased in HepG2 cells with 2 μmol/L As 2O 3 treatment. Confocal images demonstrated that the expression of PML protein in HepG2 cell nuclei decreased after treatment with 2 μmol/L As 2O 3, and micropunctates characteristic of PML protein in HepG2 cell nuclei disappeared after treatment with 5 μmol/L As 2O 3.Conclusions Our results show that arsenic trioxide can significantly inhibit the growth of HepG2 cells in vitro. As 2O 3 induces apoptosis in HepG2 tumor cells in a time and concentration dependent manner. As 2O 3 may degrade the PML protein in HepG2 cell nuclei. The decreased expression of PML in As 2O 3 treated tumor cells is most likely to be caused by apoptosis. Nuclear matrix associated protein PML could be the target of As 2O 3 therapy.