Rapid isolation of nucleoli from detergent purified nuclei of various tumor and tissue culture cells

Muramatsu, Masami; Hayashi, Yujiro; Onishi, Toshio; Sakai, Minako; Takai, Kazuyuki; Kashiyama, Taku

doi:10.1016/0014-4827(74)90250-x

Cited by 105 publications

(41 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Nucleoli were prepared from purified cell nuclei, following the published method (34); purity of the preparations was monitored by microscopic analysis, after staining with Azure C, as described (34). Cell nuclei were homogeneously pure, and the purity of nucleoli was 95%.…”

Section: Methodsmentioning

confidence: 99%

Cell Cycle-controlled Interaction of Nucleolin with the Retinoblastoma Protein and Cancerous Cell Transformation

Grinstein

Shan

Karawajew

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

Retinoblastoma protein (Rb) is a multifunctional tumor suppressor, frequently inactivated in certain types of human cancer. Nucleolin is an abundant multifunctional phosphoprotein of proliferating and cancerous cells, recently identified as cell cycle-regulated transcription activator, controlling expression of human papillomavirus type 18 (HPV18) oncogenes in cervical cancer. Here we find that nucleolin is associated with Rb in intact cells in the G 1 phase of the cell cycle, and the complex formation is mediated by the growth-inhibitory domain of Rb. Association with Rb inhibits the DNA binding function of nucleolin and in consequence the interaction of nucleolin with the HPV18 enhancer, resulting in Rb-mediated repression of the HPV18 oncogenes. The intracellular distribution of nucleolin in epithelial cells is Rb-dependent, and an altered nucleolin localization in human cancerous tissues results from a loss of Rb. Our findings suggest that deregulated nucleolin activity due to a loss of Rb contributes to tumor development in malignant diseases, thus providing further insights into the molecular network for the Rb-mediated tumor suppression.Nucleolin is an abundant multifunctional phosphoprotein of proliferating and cancerous cells (1, 2) (reviewed in Ref.3). High levels of nucleolin expression are related to poor clinical prognosis for certain cancer types (2, 4). A role of nucleolin in activation and repression of gene transcription as well as in regulation of RNA metabolism has been reported (5-7). Nucleolin is a subunit of the transcription factor LR1, which activates expression of the c-myc gene and the EBNA-1 (Epstein-Barr virus nuclear antigen 1) gene in B-cell lymphomas (7-9). Nucleolin is also directly involved in post-transcriptional inhibition of the p53 gene expression (10). As activator of transcription of HPV18 2 oncogenes in cervical cancer cells, nucleolin has oncogenic potential (11). Nucleolin controls the chromatin structure of the HPV18 enhancer in vivo and is directly linked to HPV18-induced cervical cancer formation (11). Nucleolin is a substrate of cyclin-dependent kinases (CDKs), CDK1, CDK4, and CDK2 (12, 13), and its DNA binding activity is most prominent in the S phase of the cell cycle (11). Here we report a functional interaction of nucleolin with Rb, related to cell transformation.Rb is a prototypical tumor suppressor, frequently inactivated in certain types of human cancer, and it controls cell proliferation, differentiation, and survival (14). Therefore, it is important to work out molecular mechanisms of Rb-mediated tumor suppression to understand the cancer disease process (14, 15). Rb functions to constrain cell proliferation by providing a cell cycle checkpoint between the G 1 and S phases (16,17). Phosphorylation of Rb by cyclin-dependent kinases is initiated in mid-G 1 phase, and Rb phosphorylation is associated with G 1 /S transition and S phase progression (18,19). Hypophosphorylated Rb restrains cell proliferation, in part by targeting E2F transcription factors (20 -23)...

show abstract

Section: Methodsmentioning

confidence: 99%

Cell Cycle-controlled Interaction of Nucleolin with the Retinoblastoma Protein and Cancerous Cell Transformation

Grinstein

Shan

Karawajew

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The coding sequences for the p55 and p50 proteins were PCR amplified from the I+M+A+G+E+ clones using gene-specific primers that added an NdeI site at the start codon and a Sal I site downstream from the stop codon+ The p55/p50 PCR products were digested with NdeI and Sal I, gel-purified, and ligated into the expression vector pET15b (Novagen) at the NdeI and XhoI sites+ Selected clones were sequenced to confirm that the encoded p55/p50 sequences were correct and in frame with the N-terminal histidine (ϫ6) tag+ Cloned p55/p50 proteins were expressed in E. coli [strain BL21(DE3)] and his-tagged protein purified by nickel chelation chromatography+ Purified p55 and p50 proteins were used to produce polyclonal antibodies in New Zealand rabbits (Cocalico)+ Harvested Archaeoglobus fulgidus, Saccharomyces cerevisiae, and HeLa whole cells, isolated X. laevis oocyte germinal vesicles (nuclei), and isolated mouse Taper ascites cell nuclei (Maxwell & Martin, 1986) and nucleoli (Muramatsu & Onishi, 1977) were resuspended in SDS sample buffer, boiled for 5 min, and then stored at Ϫ80 8C+ For western blots, whole cell, nuclear, and nucleolar protein preparations were resolved by SDS-polyacrylamide gel electrophoresis and blotted to PVDF membranes (BioRad) by electrophoretic transfer+ After membrane washing and incubation with p55 or p50 antibodies, the secondary antibody [alkaline phosphatase-conjugated goat anti-rabbit IgG (Promega)] was incubated with the membranes, and the presence of p55/p50 proteins assessed by colorimetric detection of alkaline phosphatase activity+…”

Section: Affinity Chromatographic Isolation Of Snornp Proteinsmentioning

confidence: 99%

Box C/D snoRNA-associated proteins: Two pairs of evolutionarily ancient proteins and possible links to replication and transcription

Newman

Kuhn

Shanab

et al. 2000

RNA

117

125

View full text Add to dashboard Cite

show abstract

“…Mock-and HVJ-infected LLC-MK2 cells were cultured in standard or low Ca2+ media for 24 hr after infection. The nuclei of the cells were then isolated with the method by Muramatsu et al (18). The nuclear fraction was lysed as described previously (22), and M protein was detected by immunoblot using BA9.1.…”

Section: Methodsmentioning

confidence: 99%

Further Analysis of The Effect of Ca2+ on Morphogenesis of HVJ (Sendai Virus) in LLC-MK2 Cells: Effects on Phosphorylated M Protein Associated with Viral Morphogenesis.

Kim

1994

Cell Struct. Funct.

View full text Add to dashboard Cite

Keywords: HVJ(Sendai virus)/effect of Ca2+/Mprotein/HVJ morphogenesis/phosphorylation ABSTRACT. Previously we have reported that Ca2+concentration in culture mediumaffects the morphogenesis of HVJfrom infected cells. Wefurther investigated the effects of Ca2+ on HVJmorphogenesis in LLC-MK2 cells, focusing on M(matrix) protein which plays an important role in the assembly of viral components. On culture in standard medium(containing 1.8 mMCa2+), most of the Mprotein accumulated on the apical surface in patches. On double immunofluorescent staining with anti-M protein monoclonal antibody and polyclonal antibody against HVJglycoproteins, Mprotein and viral glycoproteins were observed in the same patches at the cell surface. By contrast, in low Ca2+ medium, Mprotein was evenly dispersed over the cell surface, especially in the regions of cell-cell contact, and was not seen in patches. Immunoblot analysis for Mprotein in the infected cells showed that the total amount of Mprotein was almost the same irrespective of Ca2+ concentration in the culture medium.But, there was a distinct difference in the forms of Mprotein found in the cells: most of the M protein in the cells cultured in standard mediumwas of the non-phosphorylated form, whereas the phosphorylated form of Mprotein was distinctly increased in the cells cultured in low Ca2+medium. These results suggest that Ca2+-deficiency in the mediumblocks dephosphorylation of phosphorylated Mprotein necessary for patch formation, resulting in disturbance of viral morphogenesis.

show abstract

Rapid isolation of nucleoli from detergent purified nuclei of various tumor and tissue culture cells

Cited by 105 publications

References 12 publications

Cell Cycle-controlled Interaction of Nucleolin with the Retinoblastoma Protein and Cancerous Cell Transformation

Cell Cycle-controlled Interaction of Nucleolin with the Retinoblastoma Protein and Cancerous Cell Transformation

Box C/D snoRNA-associated proteins: Two pairs of evolutionarily ancient proteins and possible links to replication and transcription

Further Analysis of The Effect of Ca2+ on Morphogenesis of HVJ (Sendai Virus) in LLC-MK2 Cells: Effects on Phosphorylated M Protein Associated with Viral Morphogenesis.

Contact Info

Product

Resources

About