Regulatory role of nucleophosmin during the differentiation of human liver cancer cells.

Nucleophosmin (NPM, also known as B23), mainly localized in the nucleolus, has been reported to be overexpressed in many types of human cancer, including colon, ovarian, prostate and gastric cancer. NPM was identified while screening the differential nuclear matrix proteins during HMBA-induced differentiation of human liver cancer cells. We investigated the aberrant expression and subcellular localization of NPM in clinical liver cancer tissues and a cell line with the aim of providing more evidence for revealing the roles of NPM on regulating liver cancer cell proliferation and differentiation. In addition, we studied the potential interaction between NPM and several important proteins. Our results revealed that NPM protein was overexpressed in cancer cells, which was in accordance with the overexpressed mRNA in cancer tissues compared to the corresponding non-cancer tissues. We also found a decrease of NPM in protein and mRNA levels upon treatment with the differentiation reagent HMBA. We focused on the aberrant localization of NPM. Immunochemistry and immunofluorescence revealed aberrant cytoplasmic and nucleoplasm localization of NPM in liver cancer tissues and its colocalization with c-Myc, c-Fos, P53 and Rb in the SMMC-7721 cell line. The interactions between NPM and the above proteins were confirmed by GST pull-down assay and co-immunoprecipitation assay. These findings indicate that NPM plays a regulatory role in liver cancer, which deserves in-depth investigation.

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.

Aberrant expression and localization of hnRNP-A2/B1 is a common event in human gastric adenocarcinoma.

BACKGROUND AND AIM: Nuclear-matrix proteins can be proteomic markers for cancer lesions. The present study aimed to determine the roles of heterogeneous nuclear ribonucleoproteins--A2 and B1 (hnRNP-A2/B1) in human gastric carcinogenesis. METHODS: Human gastric cancer and non-cancerous tissues were collected for immunohistochemical analysis. Proteomics technique, Western blot, laser confocal microscope, and real-time quantitative reverse transcription-polymerase chain reaction were performed to determine the aberrant expression of nuclear-matrix proteins. RESULTS: hnRNP-A2/B1 existed in the nuclear matrix of gastric cancer cells, and its expression was enhanced in human gastric cancer and decreased by hexamethylene bisacetamide. The colocalization of hnRNP-A2/B1 with c-myc, c-fos, p53, and Rb was translocated from the nucleolus to the cytoplasm during the differentiation of tumor cells. CONCLUSIONS: hnRNP-A2/B1 affected tumor cell differentiation through interaction with oncogenes and tumor-suppressor genes, and it was overexpressed in human gastric cancer. We postulate that hnRNP-A2/B1 could serve as a biomarker for the diagnosis of human gastric cancer.

Localization of prohibitin in the nuclear matrix and alteration of its expression during differentiation of human neuroblastoma SK-N-SH cells induced by retinoic acid.

The nuclear matrix-intermediate filament system of human neuroblastoma SK-N-SH cells before and after retinoic acid (RA) treatment was selectively extracted and the distribution of prohibitin (PHB) in the nuclear matrix, as well as its colocalization with related genes, was observed. Results of two-dimensional gel electrophoresis (2-DE), mass spectrometry (MS) identification, and protein immunoblotting all confirm that PHB was present in the components of SK-N-SH nuclear matrix proteins and was down-regulated after RA treatment. Immunofluorescence microscopy observations show that PHB was localized in the nuclear matrix and its distribution was altered due to RA treatment. Laser confocal microscopy results reveal that PHB colocalized with the expression products of c-myc, c-fos, p53, and Rb, but the colocalization region was altered after RA treatment. Our results prove that PHB is a nuclear matrix protein and is localized in nuclear matrix fibers. The distribution of PHB in SK-N-SH cells and its colocalization with related proto-oncogenes and tumor suppressor genes suggest that PHB plays pivotal roles in the differentiation of SK-N-SH cells and deserves further study.

Synthesis and characterization of a novel cationic polymer gene delivery vector.

Non-viral vectors have been widely used in gene transfection. However, its drawbacks limit its applications. In this study, a novel cationic polymer was developed as a DNA condensing agent for systemic gene delivery. Its transfection efficiency, cytotoxicity, and biocompatibility were also evaluated. Sofast, novel cationic polymer of branched polyethlenimine, was constructed by chemical methods. Its diameter, zeta potential, nucleic acid binding ability, and anti-nuclease ability were detected by electron microscopy and gel electrophoresis. In vitro, the efficiency of transfection was measured by comparing it with other gene vectors in different cell lines. MTT assay was performed to determine cytotoxicity. The compatibility of Sofast gene vector in the serum and its stability were investigated. Mouse, guinea pig and rabbit were used to process the toxic, allergenic, and pyrogenic properties of the vector in vivo. The in vivo expression was performed in the guinea pig. The results from an in vitro assay proved that the Sofast gene vector had a higher transfection efficiency than other gene vectors in a variety of primary cell cultures and transformed cell lines. The cytotoxicity assay showed a lower cytotoxicity and the cellular survival rate was >90%. The Sofast gene vector possessed compatibility with the serum and was fit to be transported at normal temperature. The results from in vivo tests indicated that the Sofast gene vector had greatly lower cytotoxicity, better biocompatibility, and higher transfection efficiency compared with other gene vectors. Because the Sofast gene vector had higher transfection efficiency, lower cytotoxicity and better compatibility than other gene vectors, it could be used for gene transfection both in vitro and in vivo.

Development of a colloidal gold-immunochromatography assay to detect immunoglobulin G antibodies to Treponema pallidum with TPN17 and TPN47.

Syphilis remains a worldwide public health problem; it is necessary to develop a new diagnostic approach that is easier and faster than conventional tests. Here, we report a new testing method named colloidal gold-immunochromatography assay (GICA) to detect syphilis instead of fluorescent treponemal antibody-absorption (FTA-Abs). Syphilis-specific immunoglobulin G (IgG) antibody was detected with GICA established on syphilis-specific recombinant proteins, TPN17 and TPN47. FTA-Abs Treponema pallidum (TP)-IgG was set as the gold standard. A GICA test was performed to detect the serum of 14 967 subjects who took a serologic test for syphilis at the Xiamen Center of Clinical Laboratory, Fujian, China, from March 2009 to February 2010, among which 1326 cases were diagnosed as syphilitic. The results showed that the sensitivity, specificity, and positive predictive value were 99.38% (1279/1287), 99.96% (12,975/12,980), and 99.61% (1279/1284), respectively. The positive rate between the 2 test methods had no significant difference (χ(2) = 0.003, P > 0.05). Detection on 500 interference specimens indicated that the biologic false-positive rate of the GICA test was extremely low and free from other biologic and chemical factors. The characteristics of GICA TP-IgG correspond to that of FTA-Abs TP-IgG (EUROIMMUN Medizinische Labordiagnostika, Germany). The GICA test is convenient, fast, and inexpensive, and it can be used both as a confirmatory test and a screening indicator, instead of FTA-Abs TP-IgG.

Development and evaluation of a novel nano-scale vector for siRNA.

To synthesize a lipid-cationic polymer (LCP) containing brassidic acid side chain and to investigate its transfection efficiency and characteristics as a siRNA gene vector. The LCP was chemically synthesized and its nucleic acid binding capacity was determined by gel electrophoresis. HeLa-EGFP and TH1080-EGFP cell lines were transfected with siRNA against enhanced green fluorescent protein (EGFP) gene using a LCP to investigate the transfection efficiency. An MTT assay was performed to evaluate the cellular toxicity of the LCP vector. Its degradability and stability under acidic conditions were also investigated. The LCP vector possessed high DNA binding capacity. More than 73% of the cellular fluorescence was inhibited by the LCP-mediated transfection of siRNA against EGFP gene, indicating that vector had high transfection efficiency. Cellular viability was about 95% at the optimum transfection efficiency of LCP, suggesting that the cellular toxicity of LCP was very low. The LCP was also observed to be degradable; moreover, it could be easily stored at normal temperature. A gene vector used for the transfection of siRNA was successfully fabricated from synthesized LCP. Its numerous excellent properties entitle values for further scientific research.

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.

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 HMBA-induced 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.

The aberrant expressions of nuclear matrix proteins during the apoptosis of human osteosarcoma cells.

The objective of this study was to investigate altered expressions of nuclear matrix proteins (NMPs) of human osteosarcoma (OS) MG-63 cells during curcumin-induced apoptosis of human OS MG-63 cells. MG-63 cells were cultured with curcumin (7.5 mg/L) for 72 hr. Morphological alterations of cells were captured using light microscopy and transmission electron microscopy, and cell cycle distribution was estimated by flow cytometry. NMPs were selectively extracted and subjected to two-dimensional gel electrophoresis (2-DE) analysis. Western blots were performed to determine changes in the expression levels of specific NMPs. The results demonstrated that typical characteristics of apoptosis were observed. Cellular chromatin agglutinated, cell nuclei condensed, and apoptotic bodies were formed after treatment with curcumin. The 2-DE results displayed 27 NMPs, 21 of which were identified to have change in expression levels significantly during apoptosis. The altered expressions of three of these NMPs (nucleophosmin, prohibitin, and vimentin) were further confirmed by immunoblotting. These findings indicated that the apoptosis of MG-63 cells was accompanied by the expression alteration of NMPs. Our results might help to reveal the relationship between NMPs and the regulation of gene expression in the process of apoptosis, as well as provide the basic concepts for future studies on the mechanisms of apoptosis and the therapy for bone diseases.

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.