人成骨肉瘤MG-63细胞在HMBA诱导分化后核基质蛋白表达的变化

HMBA诱导处理人成骨肉瘤MG-63细胞分化后,选择性抽提核基质蛋白,经双向凝胶电泳技术分析．共有12个蛋白点表达发生变化,经肽指纹图谱分析鉴定了9个蛋白质,其中,MHCⅡ类抗原、IFN刺激的基因因子3α蛋白、DKFZp434M2221．1蛋白、8-羟基-鸟嘌呤糖基化酶同功酶oggl和波形蛋白表达上调,hnRNP A2/B1和肌动蛋白表达下调,60S核糖体蛋白L21和ST2蛋白仅在分化的MG-63细胞中表达。研究结果表明肿瘤细胞增殖分化过程中伴随核基质蛋白表达的变化,并为深入揭示核基质蛋白与细胞癌变和逆转的关系以及阐明细胞增殖分化的基因表达调控原理提供了依据。     

Differential expression of nuclear matrix proteins during the differentiation of human neuroblastoma SK‐N‐SH cells induced by retinoic acid

To investigate the alteration of nuclear matrix proteins (NMPs) during the differentiation of neuroblastoma SK‐N‐SH cells induced by retinoic acid (RA), differentiation markers were detected by immunocytochemistry and NMPs were selectively extracted and subjected to two‐dimensional gel electrophoresis analysis. Immunocytochemical observation demonstrated that the expression of neuronal markers was up‐regulated in SK‐N‐SH cells following RA treatment. Meanwhile, 52 NMPs (41 of which were identified) changed significantly during SK‐N‐SH differentiation; four of these NMPs were further confirmed by immunoblotting. This study suggests that the differentiation of neuroblastoma cells was accompanied by the altered expression of neuronal markers and NMPs. The presence of some differentially expressed NMPs was related to the proliferation and differentiation of neuroblastomas. Our results may help to reveal the relationship between NMPs and neuroblastoma carcinogenesis and reversion, as well as elucidate the regulatory principals driving neural cell proliferation and differentiation. J. Cell. Biochem. 106: 849–857, 2009. © 2009 Wiley‐Liss, Inc.     

Localization of nucleophosmin in nuclear matrix and changes in its expression during the differentiation of human neuroblastoma induced by retinoic acid

In this article, we selectively extracted the nuclear matrix and intermediate filament system of human neuroblastoma SK‐N‐SH cells pre‐ and post‐treated with retinoic acid (RA). The distribution of nucleophosmin (NPM) in the nuclear matrix and its colocalization with several products of related genes were investigated. Results from two‐dimensional gel electrophoresis and MALDI‐TOF showed that NPM was a component of the nuclear matrix and its expression in SK‐N‐SH cells post‐treated with RA was down‐regulated. Immunofluorescent microscopy observations further showed that NPM was localized in the nuclear matrix of SK‐N‐SH cells, and its expression level and distribution were altered after treatment with RA. The colocalization of NPM with c‐myc, c‐fos, p53, and Rb in SK‐N‐SH cells was observed under a laser scanning confocal microscope, but the colocalization region was changed by RA. Our results prove that NPM is a nuclear matrix protein, which is localized in nuclear matrix fibers. The colocalization of NPM with its related genes and oncogenes affect the differentiation of SK‐N‐SH cells. The expression of NPM and its distribution in the process of cell differentiation deserve more intensive investigation. J. Cell. Biochem. 111: 67–74, 2010. © 2010 Wiley‐Liss, Inc.     

Nuclear matrix protein,prohibitin, was down‐regulated and translocated from nucleus to cytoplasm during the differentiation of osteosarcoma MG‐63 cells induced by ginsenoside Rg1, cinnamic acid,and tanshinone IIA (RCT)

Ginsenoside Rg1, cinnamic acid, and tanshinone IIA (RCT) are effective anticancer and antioxidant constituents of traditional Chinese herbal medicines of Ginseng, Xuanseng, and Danseng. The molecular mechanisms of anticancer effects of those constituents and their targets are unknown. Prohibitin, an inner membrane‐bound chaperone in mitochondrion involved in the regulation of cell growth, proliferation, differentiation, aging, and apoptosis, was chosen as a candidate molecular target because of its frequent up‐regulation in various cancer cells. We demonstrated that prohibitin existed in the filaments of the nuclear matrix of the MG‐63 cell and its expression was down‐regulated by the treatment of RCT using proteomic methodologies and Western blot analysis. Immunogold electro‐microscopy also found that prohibitin was localized on nuclear matrix intermediate filaments (NM‐IF) that had undergone restorational changes after RCT treatment. Prohibitin may function as a molecular chaperone that might interact with multiple oncogenes and tumor suppressor genes. We found that oncogenes c‐myc and c‐fos and tumor suppressor genes P53 and Rb were regulated by RCT as well and that these gene products co‐localized with prohibitin. Our study identified prohibitin as a molecular target of the effective anticancer constituents of Ginseng, Xuanseng, and Danseng that down‐regulated prohibitin in nuclear matrix, changed prohibtin trafficking from nucleolus to cytoplasm, and regulated several oncogenes and tumor suppressor genes. Prohibitin downregulation and cellular trafficking from nucleolus to cytoplasm indicated RCT protective roles in cancer prevention and treatment. J. Cell. Biochem. 108: 926–934, 2009. © 2009 Wiley‐Liss, Inc.     

人参皂苷Rg1组合诱导人成骨肉瘤MG-63细胞分化过程中Prohibitin的表达与定位变化

选择性抽提经人参皂苷Rg1组合（RCT）诱导处理前后的人成骨肉瘤MG-63细胞核基质,对prohibitin在核基质中的存在、分布及其与相关基因产物在RCT处理前后MG-63细胞中的共定位关系进行观察研究.蛋白质组学分析结果显示,prohibitin存在于人成骨肉瘤MG-63细胞核基质蛋白组分中,并在RCT处理后细胞核基质中表达下调;蛋白质印迹杂交确证了prohibitin在MG-63细胞核基质中的存在及其在RCT处理后下调变化;免疫荧光显微镜观察进一步证实prohibitin定位在核基质上,经RCT处理后出现分布位置与表达水平变化;激光共聚焦显微镜观察可见prohibitin与c-Fos、c-Myc、p53和Rb基因产物均存在共定位关系,并在RCT处理后共定位分布区域出现变化.本研究证实了prohibitin是一种新发现的核基质蛋白,其在核基质上的定位与表达在RCT诱导分化前后发生显著变化,并与相关癌基因、抑癌基因产物存在共定位关系.实验表明RCT处理引起的prohibitin的变化与人成骨肉瘤MG-63细胞的诱导分化与调控具有密切关系,为深入揭示RCT等中药有效成分诱导肿瘤细胞分化的机理提供了重要科学依据和深入探索的新方向.     

The localization of hnRNP A2/B1 in nuclear matrix and the aberrant expression during the RA-induced differentiation of human neuroblastoma SK-N-SH cells

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is involved in the synthesis of RNA. Its expression is up-regulated in many tumor cell lines. In this study, we investigated the distribution of hnRNP A2/B1 in the nuclear matrix, including its co-localization with expression products of related genes. Results from 2-DE PAGE and MS showed that hnRNP A2/B1 is involved with components of nuclear matrix proteins of SK-N-SH cells, and that its expression level is down-regulated after retinoic acid (RA) treatment. Protein immunoblotting results further confirm the existence of hnRNP A2/B1 in the nuclear matrix, as well as its down-regulation after RA treatment. Immunofluorescence microscopy observation showed that hnRNP A2/B1 localized in nuclear matrix of SK-N-SH cells and its distribution regions were altered after RA treatment. Laser scanning confocal microscopy observation showed that hnRNP A2/B1 co-localized with c-Myc, c-Fos, P53, and Rb in SK-N-SH cells. The co-localized region was altered as a result of RA treatment. Our data proved that hnRNP A2/B1 is a nuclear matrix protein and can be up-regulated in human neuroblastoma. The expression and distribution of hnRNP A2/B1 can affect the differentiation of SK-N-SH cells, as well as its co-localization with related oncogenes and tumor suppressor genes.     

Tissue matrix protein expression in human osteoblasts,osteosarcoma tumors,and osteosarcoma cell lines

Treatment for osteosarcoma is problematic because there are no prognostic markers. Diagnosis is primarily limited to cytologic grading. Oncogenesis alters cell structure therefore osteoblast tissue matrix proteins (extracellular matrix, cytoskeletal, intermediate filament, and nuclear matrix proteins), components of the cell substructure, are candidates for osteosarcoma markers. Structural proteins of the extracellular matrix, e.g. the collagens, are useful for diagnosis but not for tumors that produce little osteoid. To identify principal cellular tissue matrix proteins that distinguish normal from transformed human osteoblasts, their expression in normal osteoblasts, two osteosarcoma cell lines, and three primary osteosarcoma tumors were compared. The tumors were graded as (i) intermediate, (ii) high, and (iii) high grade recurrent. The 1-D SDS/PAGE profiles of the major components of the nuclear matrix and intermediate filament fractions from normal osteoblasts did not vary with biopsy site, age, or sex of patients. These profiles included known cytoskeletal proteins and OB250, a ∼250 kD protein(s) observed in the intermediate filament fraction. A loss of protein bands, including OB250, was observed in the osteosarcoma cell lines and tumors. The intermediate and high grade tumors exhibited nearly identical protein profiles including potential tumor-specific proteins and collagen, consistent with the presence of intracellular collagen fibers in osteosarcoma. A microsequence was obtained for OT25, a novel low molecular weight protein observed in osteosarcoma cell lines. Fibrinogen γ-chain, a protein that mediates cell adhesion was recovered from the high grade recurrent tumor. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ad-IL-24对MG-63人骨肉瘤细胞抑癌效应的体内外实验

本研究旨在分析腺病毒携带的IL-24基因在体内外对人骨肉瘤细胞生长抑制效应及其分子机制。将Ad-IL-24重组腺病毒感染MG-63细胞,用荧光显微镜、RT-PCR法检测IL-24在MG-63细胞中的转录和表达;MTT法、流式细胞技术和Hoechst染色法检测IL-24基因的表达对MG-63细胞的生长抑制和凋亡效应;半定量RT-PCR法检测IL-24基因的表达对MG-63细胞中的bcl-2、bax、caspase-3相关基因表达的影响。用Ad-IL-24重组腺病毒在MG-63骨肉瘤荷瘤裸鼠的瘤体内进行注射治疗,观察肿瘤生长变化,15d后处死裸鼠,摘除瘤体,称瘤重。并通过免疫组化法检测Bcl-2、Bax、Caspase-3等与细胞凋亡相关因子的表达。结果表明Ad-IL-24重组腺病毒感染MG-63细胞后,能明显抑制MG-63细胞增殖,并能通过上调细胞中bax、caspase-3和下调bcl-2基因表达,诱导细胞凋亡,呈现出典型细胞凋亡形态学变化。Ad-IL-24重组腺病毒瘤内注射MG-63裸鼠荷瘤骨肉瘤移植瘤后,能显著抑制肿瘤生长,瘤重的抑制率可达52%,免疫组化结果显示Ad-IL-24重组腺病毒能明显上调与细胞...     

Interaction of MAR-sequences with nuclear matrix proteins.

The recent discovery of DNA sequences responsible for the specific attachment of chromosomal DNA to the nuclear skeleton (MARs/SARs) was an important step towards our understanding of the functional and structural organization of eukaryotic chromatin [Mirkovitch et al.: Cell 44:273-282, 1984; Cockerill and Garrard: Cell 44:273-282, 1986]. A most important question, however, remains the nature of the matrix proteins involved in the specific binding of the MARs. It has been shown that topoisomerase II and histone H1 were capable of a specific interaction with SARs by the formation of precipitable complexes [Adachi et al.: EMBO J8:3997-4006, 1989; Izaurralde et al.: J Mol Biol 210:573-585, 1989]. Here, applying a different approach, we were able to "visualize" some of the skeletal proteins recognizing and specifically binding MAR-sequences. It is shown that the major matrix proteins are practically the same in both salt- and LIS-extracted matrices. However, the relative MAR-binding activity of the individual protein components may be different, depending on the method of matrix preparation. The immunological approach applied here allowed us to identify some of the individual MAR-binding matrix proteins. Histone H1 and nuclear actin are shown to be not only important components of the matrix, but to be involved in a highly efficient interaction with MAR-sequences as well. Evidence is presented that proteins recognized by the anti-HMG antibodies also participate in MAR-interactions.     

Nonchromatin nuclear proteins of mammalian lens epithelial cells

The nuclear matrix (NM) proteins of six tissue cultured lens epithelial cell lines and one embryonic rabbit epidermal cell line were analyzed to determine possible tissue and species specificity of these proteins. The NM proteins were isolated by the modified Penman technique. The tissue cultured cells were pulsed with [³⁵S] methionine and nuclear matrix proteins were fractionated by two-dimensional (2-D) gel electrophoresis. The 2-D gels were dried and autoradiographed. The relative abundance of spot patterns of nuclear matrix proteins of different cells were compared. The data from these experiments revealed that all the examined cell lines have distinct spot patterns, however, all of NM profile showed a spot pattern in the 45 kDa region with acidic pH. Some of these spots cross-reacted with anti-vimentin antibodies, whereas a prominent protein spot in this region did not cross react with either vimentin or actin antibodies. The observed variations in the NM protein patterns of lens epithelial cells may reflect tissue and species specificity and also a role in the regulatory properties of these nuclear proteins in the eye tissue development. J. Cell. Biochem. 64:644–650. © 1997 Wiley-Liss, Inc.     

Similarity between nuclear matrix proteins of various cells revealed by an improved isolation method

Comparative analysis of nuclear matrix proteins by two-dimensional electrophoresis may be greatly impaired by copurifying cytoskeletal proteins. The present data show that the bulk of adhering cytofilaments may mechanically be removed by shearing of nuclei pretreated with vanadyl ribonucleoside complexes. Potential mechanisms of action not based on ribonuclease inhibition are discussed. To individually preserve the integrity of nuclear structures, we developed protocols for the preparation of nuclear matrices from three categories of cells, namely leukocytes, cultured cells, and tissue cells. As exemplified with material from human lymphocytes, cultured amniotic cells, and liver tissue cells, the resulting patterns of nuclear matrix proteins appeared quite similar. Approximately 300 spots were shared among the cell types. Forty-nine of these were identified, 21 comprising heterogeneous nuclear ribonucleoproteins. Heterogeneous nuclear ribonucleoproteins L and nuclear lamin B2 isoforms were identified by amino acid sequencing and mass spectrometry. However, individually expressed proteins, such as the proliferating cell nuclear antigen, also pertained following application of the protocols. Thus, enhanced resolution and comparability of proteins improve systematic analyses of nuclear matrix proteins from various cellular sources. J. Cell. Biochem. 71:363–374, 1998. © 1998 Wiley-Liss, Inc.     

Dynamic continuity of nuclear and mitotic matrix proteins in the cell cycle

The eukaryotic cell nucleus is a membrane-enclosed compartment containing the genome and associated molecules supported by a highly insoluble filamentous network known as the nucleoskeleton or nuclear matrix. The nuclear matrix is believed to play roles in maintaining nuclear architecture and organizing nuclear metabolism. Recently, advances in microscopic techniques and the availability of new molecular probes have made it possible to localize functional domains within the nuclear matrix and demonstrate dynamic interactions between both soluble and insoluble components involved in the control of multiple nuclear transactions. Like the cytoplasm and its skeleton, the nucleoplasm is highly structured and very crowded with an equally complex skeletal framework. In fact, there is growing evidence that the two skeletal systems are functionally contiguous, providing a dynamic cellular matrix connecting the cell surface with the genome. If we impose cell cycle dynamics upon this skeletal organization, it is obvious that the genome and associated nuclear matrix must undergo a major structural transition during mitosis, being disassembled and/or reorganized in late G2 and reassembled again in daughter nuclei. However, recent evidence from our laboratory and elsewhere suggests that much of the nuclear matrix is used to form the mitotic apparatus (MA). Indeed, both facultative and constitutive matrix-associated proteins such as NuMA, CENP-B, CENP-F, and the retinoblastoma protein (Rb) associate within and around the MA. During mitosis, the nuclear matrix proteins may either become inert “passengers” or assume critical functions in partitioning the genome into newly formed G1 nuclei. Therefore, we support the view that the nuclear matrix exists as a dynamic architectural continuum, embracing the genome and maintaining cellular regulation throughout the cell cycle. © 1996 Wiley-Liss, Inc.     

Influence of substrate composition on human embryonic stem cell differentiation and extracellular matrix production in embryoid bodies

Stem cells reside in specialized niches in vivo. Specific factors, including the extracellular matrix (ECM), in these niches are directly responsible for maintaining the stem cell population. During development, components of the stem cell microenvironment also control differentiation with precise spatial and temporal organization. The stem cell microenvironment is dynamically regulated by the cellular component, including stem cells themselves. Thus, a mechanism exists whereby stem cells modify the ECM, which in turn affects the fate of the stem cell. In this study, we investigated whether the type of ECM initially adsorbed to the culture substrate can influence the composition of the ECM deposited by human embryonic stem cells (hESCs) differentiating in embryoid bodies, and whether different ECM composition and deposition profiles elicit distinct differentiation fates. We have shown that the initial ECM environment hESCs are exposed to affects the fate decisions of those cells and that this initial ECM environment is constantly modified during the differentiation process. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:212–219, 2015     

TGF-β1 modifications in nuclear matrix proteins of osteoblasts during differentiation

Nuclear matrix protein (NMP) composition of osteoblasts shows distinct two-dimensional gel electrophoretic profiles of labeled proteins as a function of stages of cellular differentiation. Because NMPs are involved in the control of gene expression, we examined modifications in the representation of NMPs induced by TGF-β1 treatment of osteoblasts to gain insight into the effects of TGF-β on development of the osteoblast phenotype. Exposure of proliferating fetal rat calvarial derived primary cells in culture to TGF-β1 for 48 h (day 4–6) modifies osteoblast cell morphology and proliferation and blocks subsequent formation of mineralized nodules. Nuclear matrix protein profiles were very similar between control and TGF-β–treated cultures until day 14, but subsequently differences in nuclear matrix proteins were apparent in TGF-β–treated cultures. These findings support the concept that TGF-β1 modifies the final stage of osteoblast mineralization and alters the composition of the osteoblast nuclear matrix as reflected by selective and TGF-β–dependent modifications in the levels of specific nuclear matrix proteins. The specific changes induced by TGF-β in nuclear matrix associated proteins may reflect specialized mechanisms by which TGF-β signalling mediates the alterations in cell organization and nodule formation and/or the consequential block in extracellular mineralization. J. Cell. Biochem. 69:291–303, 1998. © 1998 Wiley-Liss, Inc.     

Responses of human MG-63 osteosarcoma cell line and human osteoblast-like cells to pulsed electromagnetic fields

We have studied the effects of low-energy, low-frequency pulsed electromagnetic fields (PEMF) on cell proliferation, in both human osteoblast-like cells obtained from bone specimens and in human MG-63 osteosarcoma cell line. Assessment of osteoblastic phenotype was performed both by immunolabeling with antiosteonectin antibody and by verifying the presence of parathyroid hormone receptors. The cells were placed in multiwell plates and set in a tissue culture incubator between a pair of Helmholtz coils powered by a pulse generator (1.3 ms, 75 Hz) for different periods of time. [³H]-Thymidine incorporation was used to evaluate cell proliferation. Since it had previously been observed that the osteoblast proliferative response to PEMF exposure may also be conditioned by the presence of serum in the medium, experiments were carried out at different serum concentrations. [³H]-thymidine incorporation increases in osteoblast-like cells, when they are exposed to PEMF in the presence of 10% fetal calf serum (FCS). The greatest effect is observed after 24 hours of PEMF exposure. No effects on cell proliferation are observed when osteoblast-like cells are exposed to PEMF in the presence of 0.5% FCS or in a serum-free medium. On the other hand, PEMF-exposed MG-63 cells show increased cell proliferation either at 10% FCS, 0.5% FCS and in serum-free medium. Nevertheless, the maximum effect of PEMF exposure on MG-63 cell proliferation depends on the percentage of FCS in the medium. The higher the FCS concentration, the faster the proliferative response to PEMF exposure. Our results show that, although MG-63 cells display some similarity with human bone cells, their responses to PEMF's exposure are quite different from that observed in normal human bone cells. Bioelectromagnetics 18: 541–547, 1997. © 1997 Wiley-Liss, Inc.     

Inactivated Sendai virus strain Tianjin induces apoptosis and autophagy through reactive oxygen species production in osteosarcoma MG-63 cells

Sendai virus strain Tianjin, a novel genotype of Sendai virus, has been proven to possess potent antitumor effect on certain cancer cell types although inactivated by ultraviolet (UV). This study was carried out to investigate the in vitro anticancer properties of UV-inactivated Sendai virus strain Tianjin (UV-Tianjin) on human osteosarcoma cells and the underlying molecular mechanism. Our studies demonstrated UV-Tianjin significantly inhibited the viability of human osteosarcoma cell lines and triggered apoptosis through activation of both extrinsic and intrinsic pathways in MG-63 cells. Meanwhile, autophagy occurred in UV-Tianjin-treated cells. Blockade of autophagy with 3-methyladenine remarkably attenuated the inhibition of cell proliferation by UV-Tianjin, suggesting that UV-Tianjin-induced autophagy may be contributing to cell death. Furthermore, UV-Tianjin induced reactive oxygen species (ROS) production, which was involved in the execution of MG-63 cell apoptosis and autophagy, as evidenced by the result that treatment of N-acetyl-L-cysteine, a ROS scavenger, attenuated both apoptosis and autophagy. In addition, inhibition of apoptosis promoted autophagy, whereas suppression of autophagy attenuated apoptosis. Our results suggest that UV-Tianjin triggers apoptosis and autophagic cell death via generation of the ROS in MG-63 cells, which might provide important insights into the effectiveness of novel strategies for osteosarcoma therapy.     

A nuclear matrix protein related to intermediate filaments proteins is a member of the complex binding alphoid DNA in vitro

A complex of three proteins (of 80, 70, 58 kDa-p80, p70, and p58, respectively) with the ability to bind alphoid DNA (alpha-satDNA) was revealed by gel mobility shift assay (GMSA) in human nuclear matrix. The probes of the alpha-satDNA bound in the GMSA with the greatest specificity, but the complex was capable of binding human satellite 3 fragment. According to ion exchange and affinity chromatography, the complex includes two DNA-binding proteins, p70 and p80, and a non-DNA-binding one, p58, which enhances the specificity of binding to the alpha-satDNA. GMSA, SDS-PAGE and immunoblotting showed that the lamins, as well as constitutive centromeric proteins (CENP-A, CENP-B, CENP-C, CENP-G), were not incorporated into the complex. It was demonstrated by immunoprecipitation assay that p70 and, probably p58, share a common antigen determinant with the rod domain of intermediate filaments (IF) proteins. The results obtained indicate that the nuclear matrix contains at least one IF-related protein that is able to bind specifically to alpha-satDNA in vitro and that this protein is distinct from the lamins.