Nuclear matrix proteins specific for subtypes of human hematopoietic cells

Nuclear matrices were prepared from isolated subtypes of human hematopoietic cells and from cultured leukemia cells. The nuclear matrix proteins were analyzed by high-resolution two-dimensional gel electrophoresis and computer-assisted image analysis. While more than 200 protein spots were shared among the cells, about 50 distinct spots were found characteristic for individual cells or groups of related cells. This allowed to differentiate between hematopoietic cells and nonhematopoietic cells, lymphocytes and myeloid cells, monocytes, neutrophils, and promyelocytic leukemia cells. B and T lymphocytes could not be differentiated. Myeloid cells with their polymorph nuclei were characterized by the presence of 13 and by the absence of seven distinct spots, as well as by low concentrations of nuclear lamins and of heterogeneous nuclear ribonucleoproteins. Neutrophils with multilobular nuclei displayed six additional spots, while lacking 18 nuclear matrix protein spots. The nuclear matrix of proliferating cells showed three distinct spots in addition to proliferating cell nuclear antigen, increased concentrations of numatrin (B23), and heterogeneous nuclear ribonucleoproteins. The described cell-specific nuclear matrix proteins may represent new markers for hematopoietic cells.     

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.     

A monoclonal antibody recognizes an epitope common to an avian-specific nuclear antigen and to cytokeratins

X3, a monoclonal antibody of unusual specificity, is described. This antibody reacts with one or more cytokeratin polypeptides and also reacts with an avian (chicken, quail) nuclear antigen that appears to be present in all cell types (chicken) tested, although with variable staining pattern and intensity. This antigen is distinct from the cytokeratins but does have an epitope in common with this class of proteins. It disappears from the nucleus during the early stages of cell division and reappears during anaphase as a granular cytoplasmic structure. In late telophase the antigen is relocated in the nucleus. This antigen, which we have designated as avian-specific nuclear antigen (AVNA), is not associated with chromatin or ribonucleoproteins. From immunoblotting experiments on chicken fibroblast nuclei, AVNA is probably a complex composed of one or several polypeptides, one of which has a molecular weight of approximately 60 kD. The proteins were identified as nuclear matrix proteins rather than pore complex-lamina proteins by immunoblotting experiments on the purified nuclear matrix of chicken erythrocytes. The major polypeptide had a molecular weight of 60 kD and the minor polypeptide a molecular weight of 69 kD.     

Nuclear dreams: The malignant alteration of nuclear architecture

Cancer is diagnosed by examining the architectural alterations to cells and tissues. Changes in nuclear structure are among the most universal of these and include increases in nuclear size, deformities in nuclear shape, and changes in the internal organization of the nucleus. These may all reflect changes in the nuclear matrix, a non-chromatin nuclear scaffolding determining nuclear form, higher order chromatin folding, and the spatial organization of nucleic acid metabolism. Malignancy-induced changes in this structure may have profound effects on chromatin folding, on the fidelity of genome replication, and on gene expression. Elucidating the mechanisms and the biological consequences of nuclear changes will require the identification of the major structural molecules of the internal nuclear matrix and an understanding of their assembly into structural elements. If biochemical correlates to malignant alterations in nuclear structure can be identified then nuclear matrix proteins and, perhaps nuclear matrix-associated structural RNAs, may be an attractive set of diagnostic markers and therapeutic targets. J. Cell. Biochem. 70:172–180, 1998. © 1998 Wiley-Liss, Inc.     

Immunocytochemical Detection of Structural and Regulatory Proteins in Rat Adrenal Nuclear Matrix

The nuclear matrix is a specific cell structure consisting of a residual nucleoskeleton that extends from the nucleoli to the nuclear envelope. The nuclear matrix of steroido-genic cells was isolated previously from a purified nuclear fraction. We present here an in situ extraction method, modified Lutz's method, for rat glandular adrenal cell nuclear matrix. This residual organelle was characterized and studied using immunocytochemical methods. The adrenal glands were removed, the cells prepared in suspension and deposited by cytospin onto Poly-L-lysine glass slides. The nuclear matrix was extracted with Nonidet P-40, DNase I and high and low ionic strength buffers. Structural proteins, nuclear lamins, coilin and fibrillarin were detected immunocytochemically. The adrenal fasciculata cells were easily identified by this method because of their large nuclei and abundant lipid droplets in the cytoplasm. After immunocytochemical detection by antibodies against lamins A and C, a marked brown layer at the periphery of the nucleus was observed. The intensity of the staining was lower using the antibody against nuclear lamin B. Immunocytochemical detection of the protein coilin revealed punctuated stained areas, 2-6 per nucleus, that probably correspond to the coiled bodies. The protein fibrillarin was detected at the nucleolus and coiled bodies. Our technique is simple, reveals well preserved adrenal nuclear matrices, and may be a useful method for immunocytochemical analysis and in situ hybridization.     

Identification of nuclear structural protein alterations associated with seminomas

Currently, there are no specific markers available for the early detection and for monitoring testicular cancer. Based upon an approach that targets nuclear structure, we have identified a set of proteins that are specific for seminomas, which may then have clinical utility for the disease. Utilizing samples obtained from men with no evidence of testicular cancer (n = 5) as well as those with seminomas (n = 6), nuclear matrix proteins were extracted and separated using a high‐resolution two‐dimensional electrophoresis gel system. The proteins were identified by mass spectrometry analysis. These analyses revealed seven nuclear matrix proteins associated with the normal testes, which did not appear in the seminomas. In the seminomas, four nuclear matrix proteins were identified to be associated with the disease that were absent in the normal testes. Mass spectrometric and immunoblot analyses of these proteins revealed that one of the proteins identified in the normal testes appears to be StAR‐related lipid transfer protein 7 (StARD7). In the non‐seminoma tissues, one of the identified proteins appears to be cell division protein kinase 10 (CDK10). Both StarD7 and CDK10 could potentially be involved in cell differentiation and growth, and thus may serve as potential targets for therapy of prognostication of seminomas. This is the first study to examine the role of nuclear structural proteins as potential biomarkers in testicular cancer. We are currently examining the roles of some of the identified proteins as potential biomarkers for the disease. J. Cell. Biochem. 108: 1274–1279, 2009. © 2009 Wiley‐Liss, Inc.     

Prereplicative increase of nuclear matrix-bound DNA polymerase-α and primase activities in HeLa S3 cells following dilution of long-term cultures

We investigated the association of DNA polymerase and DNA primase activity with the nuclear matrix in HeLa S3 cells diluted with fresh medium after having been cultured without any medium change for 7 days. Flow cytometric analysis demonstrated that just before dilution about 85% of the cells were in the G₁ phase of the cycle, whereas 8% were in the S phase. After dilution with fresh medium, 18–22 h were required for the cell population to attain a stable distribution with respect to the cell cycle. At that time, about 38% of the cells were in the S phase. DNA polymerase and DNA primase activity associated with the nuclear matrix prepared from cells just before dilution represented about 10% of nuclear activity. As judged by [³H]-thymidine incorporation and flow cytometric analysis, an increase in the number of S-phase cells was evident at least 6 h after dilution. However, as early as 2 h after dilution into fresh medium, a striking prereplicative increase of the two activitites was seen in the nuclear matrix fraction but not in cytosol or isolated nuclei. Both DNA polymerase and primase activities bound to the matrix were about 60% of nuclear activity. Overall, the nuclear matrix was the cell fraction where the highest induction (about 10-fold) of both enzymatic activities was seen at 30 h after dilution, whereas in cytosol and isolated nuclei the increase was about two- and fourfold, respectively. Typical immunofluorescent patterns given by an antibody to 5-bromodeoxyuridine were seen after dilution. These findings, which are at variance with our own previous results obtained with cell cultures synchronized by either a double thymidine block or aphidicolin exposure, strengthen the contention that DNA replication is associated with an underlying nuclear structure and demonstrate the artifacts that may be generated by procedures commonly used to synchronize cell cultures. J. Cell. Biochem. 71:11–20, 1998. © 1998 Wiley-Liss, Inc.     

Estrogen regulation of nuclear matrix-intermediate filament proteins in human breast cancer cells

The tissue matrix consists of linkages and interactions of the nuclear matrix, cytoskeleton, and extracellular matrix. This system is a dynamic structural component of the cell that organizes and processes structural and functional information to maintain and coordinate cell function and gene expression. We have studied estrogen regulation of nuclear matrix associated proteins, including the intimately connected cytoskeletal intermediate filaments, in T-47D5 human breast cancer cells. Three proteins (identified as cytokeratins 8, 18, and 19) present in the nuclear matrix-intermediate filament fraction (NM-IF) of cells grown in estrogen-replete conditions were dramatically reduced when the cells were grown in acute (1 week) estrogen-depleted conditions. Replacing estrogen in the medium of acute estrogen-depleted cells restored expression of these proteins. T-47D5 cells that are chronically depleted of estrogen (T5-PRF) are estrogen-nonresponsive in culture. These cells overexpressed these three proteins, compared to parent cells grown in the presence of estrogen. Treatment of the T5-PRF cells with estrogen did not lead to further up-regulation of these proteins. Treating T-47D5 cells in estrogen-replete conditions with the antiestrogens 4-hydroxytamoxifen and ICI 164 384 (100 nM, 3 days) resulted in a significant reduction in these proteins, while no effect was seen in long-term chronic estrogen-depleted T-47D5 cells. In conclusion, we have identified NM-IF proteins (cytokeratins 8, 18, and 19) in human breast cancer cells that are estrogen regulated and may play a role in estrogen action in human breast cancer cells. © 1996 Wiley-Liss, Inc.     

Lamins of ocular lens epithelial cells

Experiments were performed to characterize a prominent nuclear matrix (NM) protein isolated from tissue cultured mouse lens epithelial cells. This NM protein was separated by SDS-PAGE and the stained gel band was analyzed by mass spectroscopy. Blast analysis of the amino acid sequence derived by mass spectroscopy revealed the presence of Lamin C in the NM of the mouse lens epithelial cells. We also examined nuclear proteins of adult and fetal human lenses. Data collected from these experiments showed the presence of Lamin C in both adult and fetal lens cells. However fetal lens cells only show Lamin C dimers, whereas adult human lens contained dimers, monomers and degraded Lamin C. Early and late passaged tissue cultured mouse lens epithelial cells also contained Lamin C in the nucleus with a preponderance of the dimer in the early passaged cells. The biological significance of the presence of dimers in human fetal lens cells and early passaged mouse lens cells is not known. However, it could suggest an enhanced docking capability of Lamin C dimers for other physiologically important nuclear proteins.     

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.     

视黄酸诱导人神经母细胞瘤SK-N-SH细胞分化过程中核基质蛋白组成的变化

在鉴定视黄酸(retinoic acid, RA)诱导人神经母细胞瘤SK-N-SH细胞分化的基础上,应用免疫细胞化学、选择性抽提和蛋白质组学分析技术,对SK-N-SH细胞诱导分化过程中核基质蛋白组成变化进行了系统研究.实验结果显示,经1 μmol/L RA处理后SK-N-SH细胞呈极性状,伸出较长的轴突样突起,胞体逐渐变小变圆.免疫细胞化学结果显示,处理后神经细胞特异表达的蛋白synaptophysin、NSE、MAP2的表达量都较对照组有明显增强.双向凝胶电泳分析显示,在RA诱导SK-N-SH细胞分化前后存在52个差异表达的核基质蛋白,经质谱分析,鉴定了其中的41个蛋白.蛋白印迹杂交进一步确证了诱导分化差异表达核基质蛋白中nucleophosmin和prohibitin等的表达变化.研究结果表明,1 μmol/L RA对SK-N-SH细胞具有显著的诱导分化作用,在SK-N-SH细胞分化过程中,其核基质蛋白组成发生了明显变化.这些变化对于揭示人神经母细胞瘤细胞癌变与逆转机制和肿瘤细胞增殖与分化调控机理均有十分重要的意义,从而为研究神经系统正常发育过程及神经系统疾病的发病机理提供科学依据.     

Quantitative whole-cell proteome analysis of pseudorabies virus-infected cells

A quantitative proteome study using the stable isotope labeling with amino acids in cell culture technique was performed on bovine kidney cells after infection with the alphaherpesvirus pseudorabies virus (PrV), the etiological agent of Aujeszky's disease. To enhance yields of proteins to be identified, raw extracts were fractionated by affinity solid-phase extraction with a combination of a cibacron blue F3G-A and a heparin matrix and with a phosphoprotein-specific matrix. After two-dimensional gel electrophoresis in different pH ranges between pH 3 and pH 10, 2,600 proteins representing 565 genes were identified by mass spectrometry and screened for virus-induced changes in relative protein levels. Four hours after infection, significant quantitative variations were found for constituents of the nuclear lamina, representatives of the heterogeneous nuclear ribonucleoproteins, proteins involved in membrane trafficking and intracellular transport, a ribosomal protein, and heat shock protein 27. Several proteins were present in multiple charge variants that were differentially affected by infection with PrV. As a common pattern for all these proteins, a mass shift in favor of the more acidic isoforms was observed, suggesting the involvement of viral or cellular kinases.     

Ultrastructure of nuclear aggregates formed by expressing an expanded polyglutamine

Intranuclear inclusions have been observed in the brains of patients affected with Huntington's disease (HD). Neuro 2A cells that transiently expressed HD exon 1 bearing 74 glutamine repeats linked to the green fluorescent protein (GFP) and the nuclear localization sequence (NLS) contained aggregates in nuclei. The aggregates were purified by fractionation with centrifugation followed by fluorescence-activated cell sorting (FACS). Heat treatment of the aggregate in an SDS sample buffer caused the dense aggregate cores to disappear and generated a basket-like structure composed of fibrils. Biochemical analysis of the aggregates revealed that the HD exon 1-GFP fusion protein was the major component. The heterogeneous nuclear ribonucleoproteins F and H, histones and ubiquitin were found to be associated with the aggregates. Our observations suggest that the N-terminal fragment of huntingtin may organize the skeletal structure of the aggregates and may disturb normal cellular functions by trapping other proteins within the aggregates.     

Identification of c-myc-dependent proteins in the medulloblastoma cell line D425Med

High c-myc levels are linked to poor prognosis in medulloblastoma (MB), and it was the aim of the current study to search for c-myc-dependent proteins in the MB cell line D425Med. For this purpose D425Med cells and cells with knocked-down c-myc (by siRNA) were analysed by a gel-based differential proteomics study using mass spectrometry. Heterogeneous nuclear ribonucleoproteins C1/C2, heterogeneous nuclear ribonucleoprotein A/B, stathmin, endoplasmic reticulum protein ERp29 precursor and guanidinoacetate N-methyltransferase were c-myc dependently expressed. Signalling, the protein machinery, metabolism and endoplasmic reticulum function may be affected and these results enable studying tumour tissue for these proteins as potential dignity markers or pharmacological targets.     

A method for analysis of the detergent-resistant cytoskeleton of cells within organs

We describe a method for the preparation of the detergent-resistant cytoskeleton and nuclear matrix of cells within organs and tissues. Such cells were previously inaccessible to study because the three-dimensional organization of cells in organs prevented uniform distribution of the detergent throughout the multiple cell layers. We use the method presented here to compare the proteins present in the cytoskeleton, nuclear matrix and soluble fractions of cells from different histotypes. SDS-gel analysis demonstrates that soluble and nuclear matrix proteins differ greatly between histotypes while cytoskeletal proteins are relatively similar. Immunocytochemical analysis of tissue prepared using this procedure also demonstrates that the intracellular structure of cells within organs differs from that of in vitro cultured cells.     

Selected nuclear matrix proteins are targets for poly(ADP-ribose)-binding

Recent evidence suggests that poly(ADP-ribose) may take part in DNA strand break signalling due to its ability to interact with and affect the function of specific target proteins. Using a poly(ADP-ribose) blot assay, we have found that several nuclear matrix proteins from human and murine cells bind ADP-ribose polymers with high affinity. The binding was observed regardless of the procedure used to isolate nuclear matrices, and it proved resistant to high salt concentrations. In murine lymphoma LY-cell cultures, the spontaneous appearance of radiosensitive LY-S sublines was associated with a loss of poly(ADP-ribose)-binding of several nuclear matrix proteins. Because of the importance of the nuclear matrix in DNA processing reactions, the targeting of matrix proteins could be an important aspect of DNA damage signalling via the poly ADP-ribosylation system. J. Cell. Biochem. 70:596–603. © 1998 Wiley-Liss, Inc.     

hnRNP proteins and B23 are the major proteins of the internal nuclear matrix of HeLa S3 cells

The nuclear matrix is the structure that persists after removal of chromatin and loosely bound components from the nucleus. It consists of a peripheral lamina-pore complex and an intricate internal fibrogranular structure. Little is known about the molecular structure of this proteinaceous internal network. Our aim is to identify the major proteins of the internal nuclear matrix of HeLa S3 cells. To this end, a cell fraction containing the internal fibrogranular structure was compared with one from which this structure had been selectively dissociated. Protein compositions were quantitatively analyzed after high-resolution two-dimensional gel electrophoresis. We have identified the 21 most abundant polypeptides that are present exclusively in the internal nuclear matrix. Sixteen of these proteins are heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. B23 (numatrin) is another abundant protein of the internal nuclear matrix. Our results show that most of the quantitatively major polypeptides of the internal nuclear matrix are proteins involved in RNA metabolism, including packaging and transport of RNA. © 1996 Wiley-Liss, Inc.