Amount Of Nucleolin Research Articles

Cell-surface nucleolin acts as a central mediator for carcinogenic, anti-carcinogenic, and disease-related ligands

Purpose Cell-surface nucleolin in human gastric cancer cell lines is a receptor for TNF-α-inducing protein (Tipα) of Helicobacter pylori. The binding complex of nucleolin and Tipα is internalized into the cells and then induces tumor progression of human gastric cancer. Surface nucleolin is also a receptor of human immunodeficiency virus-1, and the anti-HIV pseudopeptide (HB-19) showed anti-carcinogenic activity in vivo. Surface nucleolin has dual functions depending on the ligands: In order to understand the mechanisms of surface nucleolin, it is necessary to review surface nucleolin and its relation to carcinogenic ligands and anti-carcinogenic ligands. Other ligands can be grouped among disease-related ligands, which is an important new topic for the prevention of various ailments. Results and discussionThis paper mainly deals with two ligands of surface nucleolin, Tipα and pseudopeptide HB-19. The binding complex of nucleolin and Tipα induces expression of TNF-α and chemokine genes and activates NF-κB in gastric cancer cells of humans and mice. However, when human gastric cancer cell line MKN-1 was transfected with nucleolin-targeted siRNA, the result was inhibition of cell migration and elongation induced by Tipα. The amount of surface nucleolin was reduced in membrane fraction of the nucleolin knockdown MKN-1 cells, but the amount of nucleolin in the cytosol or nuclear fractions of the cells did not change. The results indicate that surface nucleolin acts as a carcinogenic mediator for Tipα of H. pylori. In contrast, both the viral external envelop glycoprotein gp120 of HIV and the anti-HIV pseudopeptide HB-19 bind to surface nucleolin. Through this binding, treatment with HB-19 inhibited tumor development in human breast cancer cell line MDA-MB-231 and rhabdoid tumor cell line derived from Wilms’s tumor in xenograft nude mouse models. The results show that surface nucleolin acts as an anti-carcinogenic mediator for HB-19.ConclusionBased on these discrete functions of surface nucleolin, the binding complex of carcinogenic ligands and surface nucleolin seems to be competing with that of anti-carcinogenic ligands and surface nucleolin. Moreover, carcinogenic ligands derived from endogenous sources play a significant role in human cancer development, and the interaction of surface nucleolin with disease-related ligands will be a new research subject for the prevention and treatment of various ailments.

Abstract 1934: Molecular mechanism of abnormal localization of nucelolin on cell-surface of gastric cancer cells, with regard to Ras signaling pathway

Abstract Our intensive study of TNF-α inducing protein (Tipα), a carcinogenic factor secreted from H. pylori, revealed that nucleolin acts as a receptor of Tipα in a mouse gastric cancer cell line (MGT-40). Although nucleolin is a major nucleolar protein, nucleolin was found in the cell membrane by cell fractionation and on the cell-surface by flow cytometrical analysis. We further found that nucleolin shuttles Tipα from cell surface to the cytosol, resulting in expression of TNF-α gene, an endogenous tumor promoter. Moreover, a large amount of nucleolin was found in the membrane fraction of MGT-40 cells, but not in that of mouse normal glandular stomach. These results suggest that abnormal localization of nucleolin occures in gastric cancer, as an associated process of carcinogenesis. In the light of this evidence, we think that the abnormal localization of nucleolin on cell surface produces the optimal carcinogenic environment for Tipα binding in gastric cancer development by H. pylori infection. Experimentally we found that large amounts of nucleolin were expressed on the surface of four human gastric cancer cell lines (MKN-45, MKN-74, AGS, and KATOIII), with smaller amounts on that of another cell line (MKN-1), although the total amounts of nucleolin were almost the same among these five cell lines. It is important to note that the amounts of cell-surface nucleolin are correlated well with those of Ras-GTP form, the active form of Ras protein in these cell lines, and we confirmed that MKN-1 cells had the lowest amounts of Ras-GTP form among the five lines. Moreover, the treatment of AGS cells with okadaic acid, a tumor promoter and inhibitor of protein phosphatases 1 and 2A, reduced the amount of nucleolin localized on cell-surface, indicating that abnormal localization of nucleolin on cell surface can be induced by Ras signaling pathway and post-translational modifications. Thus, we discuss the relationship between the abnormal localization of nucleolin and the disorder in the Ras signaling pathway in gastric cancer development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1934. doi:10.1158/1538-7445.AM2011-1934

Nucleolin is regulated both at the level of transcription and translation

Nucleolin is a multi-functional protein that is located to the nucleolus. In tissue culture cells, the stability of nucleolin is related to the proliferation status of the cell. During development, rat cardiomyocytes proliferate actively with increases in the mass of the heart being due to both hyperplasia and hypertrophy. The timing of this shift in the phenotype of the myocyte from one capable of undergoing hyperplasia to one that can grow only by hypertrophy occurs within 4 days of post-natal development. Thus, cardiomyocytes are an ideal model system in which to study the regulation of nucleolin during growth in vivo. Using Western blot and quantitative RT-PCR (TaqMan) we found that the amount of nucleolin is regulated both at the level of transcription and translation during the development of the cardiomyocyte. However, in cells which had exited the cell cycle and were subsequently given a hypertrophic stimulus, nucleolin was regulated post-transcriptionally.

Effects of Sex Steroids on Silver Stained Proteins of Nucleolar Organizer Regions (Ag-NOR) in the Rabbit Uterus

Silver staining of argyrophilic proteins of nucleolar organizer regions (Ag-NOR) has been used to evaluate a hyperactive state of cells. We studied the effects of sex steroids on the Ag-NOR proteins in the rabbit uterus. Estradiol-17β (E2) caused uterine hypertrophy and increased the number of Ag-NOR dots in epithelial and stromal cells of the endometrium and smooth muscles cells in a dose dependent manner without proliferative change in the endometrium. Progestins including progesterone, medroxyprogesterone acetate (MPA), and norethindrone following E2 caused progestational proliferation of the endometrium with an increase in the Ag-NOR number to various extents, whereas methyltestosterone, a synthetic androgen, caused only a minimal proliferative change in the endometrium without an increase in the Ag-NOR number. Western blot analysis revealed that progesterone and MPA increased the amounts of a 100 kDa protein (nucleolin) and a 37 kDa protein (B23 protein) in the endometium and a marked proliferative change, whereas E2 did not. These results suggest that although the Ag-NOR number was enhanced by both progestin and estrogen, only progestins substantially increase the Ag-NOR protein in the rabbit uterus. This implies that an increase in the amount of nucleolin and B23 protein is associated with the proliferative potential of the cells.

Quantification of Ag-NOR proteins using Ag-NOR staining on western blots.

Ribosomal genes are associated with a set of silver-stained nucleolar proteins, the Ag-NOR proteins, whose amount is directly related to the duration of the cell cycle. Quantification of Ag-NOR proteins by image analysis is presently used to evaluate the rate of proliferation of cancer cells and nucleolar activity. Our objective was to establish a procedure to quantify independently each major Ag-NOR protein in cell extracts. Computerized densitometry established that the specific silver staining of Ag-NOR proteins (Ag-NOR staining) performed on Western blots makes it possible to quantify Ag-NOR proteins. Using purified Ag-NOR proteins, nucleolin, and protein B23, we observed that the intensity of Ag-NOR staining is proportional to the amount of protein. A linear relationship exists between the intensity of Ag-NOR staining and the amount of nucleolin, in the range of 0.2-1.6 micrograms. Using total nuclear extracts prepared from mammalian cells, the proportionality was maintained for total Ag-NOR-stained proteins or for a particular protein. We also determined the levels of nuclear proteins suitable for quantitative analysis. Individual Ag-NOR proteins can be quantified by computerized densitometry in nuclear extracts after Ag-NOR staining on Western blots. This procedure can be applied to establish the contribution of each Ag-NOR protein in general staining, estimate the variability of each Ag-NOR protein in normal and pathological conditions, and quantify each Ag-NOR protein contained per cell.

Synergistic stimulatory effect of glucocorticoid, EGF and insulin on the synthesis of ribosomal RNA and phosphorylation of nucleolin in primary cultured rat hepatocytes

Effects of dexamethasone, EGF and insulin on the synthesis of rRNA and phosphorylation of nucleolin in primary cultures of adult rat hepatocytes were studied. Hepatocytes were incubated for 8 h with EGF (20 ng/ml) plus insulin (0.1 μM) and/or for 20 h with dexamethasone (1 μM) before the end of incubation. The incorporation of [ 3H]uridine into acid-insoluble materials and the nuclear activity of RNA polymerase I were stimulated approx. 2-fold with EGF plus insulin and these were further enhanced 2–3-times by dexamethasone, although dexamethasone alone exerted no stimulation. When hepatocytes were incubated with [ 32P]orthophosphate, similar enhancement by these hormones was also observed in the phosphorylation of a nucleolar protein, nucleolin, which was detected by immunoprecipitation with anti-nucleolin antibodies. The amount of nucleolin was slightly increased by EGF plus insulin in the presence of dexamethasone, but scarcely changed by treatment with EGF plus insulin or dexamethasone alone. Cycloheximide inhibited RNA synthesis to a greater or lesser degree in the case of all hepatocytes which were cultured with or without these hormonal treatments. These results indicate that the in vivo effect of glucocorticoid on rRNA synthesis and nucleolin phosphorylation in liver is primarily a direct action on parenchymal cells and requires other growth factors such as EGF and insulin.