Increased Expression of a Nucleolar Nop5/Sik Family Member in Metastatic Melanoma Cells
2001; Elsevier BV; Volume: 159; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)62523-0
ISSN1525-2191
AutoresKen’i Nakamoto, Akihiko Ito, Kenji Watabe, Yu‐ichiro Koma, Hideo Asada, Kunihiko Yoshikawa, Yasuhisa Shinomura, Yuji Matsuzawa, Hiroshi Nojima, Yukihiko Kitamura,
Tópico(s)RNA modifications and cancer
ResumoF10 and BL6 cells of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells can metastasize to lungs after subcutaneous injection. Differences in gene expression between the two cell lines were examined, and a greater expression of the Sik-similar protein (Sik-SP) gene was found in BL6 cells. Structurally, Sik-SP belongs to the nucleolar Nop5/Sik family whose members play central roles in ribosome biogenesis; however, the function of Sik-SP has not been examined. Cytology with green fluorescent protein-fused proteins showed that Sik-SP was localized to the nucleolus. To examine whether Sik-SP is involved in ribosome biogenesis, two parameters were measured: magnitude of ribosomal RNA synthesis per nucleus and magnitude of protein production from the same amount of mRNA of an exogenous luciferase gene. Both values and, in addition, nucleolar size were larger in COS-7 monkey kidney cells overexpressing Sik-SP and BL6 cells than in mock-transfected COS-7 and F10 cells, respectively. Sik-SP seemed to promote ribosome biogenesis in the nucleolus. Furthermore, the expression of Sik-SP seemed to confer a greater cell growth response to serum, because such a response was greater in BL6 cells and F10 cells overexpressing Sik-SP than in untreated and mock-transfected F10 cells. Sik-SP may render melanoma cells more competent to survive through augmenting the activity of nucleolus. F10 and BL6 cells of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells can metastasize to lungs after subcutaneous injection. Differences in gene expression between the two cell lines were examined, and a greater expression of the Sik-similar protein (Sik-SP) gene was found in BL6 cells. Structurally, Sik-SP belongs to the nucleolar Nop5/Sik family whose members play central roles in ribosome biogenesis; however, the function of Sik-SP has not been examined. Cytology with green fluorescent protein-fused proteins showed that Sik-SP was localized to the nucleolus. To examine whether Sik-SP is involved in ribosome biogenesis, two parameters were measured: magnitude of ribosomal RNA synthesis per nucleus and magnitude of protein production from the same amount of mRNA of an exogenous luciferase gene. Both values and, in addition, nucleolar size were larger in COS-7 monkey kidney cells overexpressing Sik-SP and BL6 cells than in mock-transfected COS-7 and F10 cells, respectively. Sik-SP seemed to promote ribosome biogenesis in the nucleolus. Furthermore, the expression of Sik-SP seemed to confer a greater cell growth response to serum, because such a response was greater in BL6 cells and F10 cells overexpressing Sik-SP than in untreated and mock-transfected F10 cells. Sik-SP may render melanoma cells more competent to survive through augmenting the activity of nucleolus. It has long been known by pathologists that hypertrophy of the nucleolus is one of the most consistent cytological features of cancer cells,1Busch H Smetana K Nucleoli of tumor cells.in: Busch H Smetana K The Nucleolus. Academic Press, London, New York1970: 448-471Google Scholar, 2Pianese G Beitrag zur Histologie und Aetiologie der Carcinoma. Histologische und exprimentelle Untersuchungen.Beitr Pathol Anat Allgem Pathol. 1896; 142: S1-S193Google Scholar and is closely related to the rapidity of cancer cell proliferation.3Derenzini M Trere D Pession A Montanaro L Sirri V Ochs RL Nucleolar function and size in cancer cells.Am J Pathol. 1998; 152: 1291-1297PubMed Google Scholar, 4Derenzini M Trere D Pession A Govoni M Sirri V Chieco P Nucleolar size indicates the rapidity of cell proliferation in cancer tissues.J Pathol. 2000; 191: 181-186Crossref PubMed Scopus (211) Google Scholar The nucleolus is a structural and functional unit in which ribosomal genes are located and transcribed, and where ribosomal RNA (rRNA) and ribosome synthesis occur.5Olson MOJ Dundr M Szebeni A The nucleolus: an old factory with unexpected capabilities.Trends Cell Biol. 2000; 10: 189-196Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar Therefore, hypertrophy of the nucleolus is regarded as a state in which rRNA and ribosome synthesis has increased. A dividing cell is required to duplicate its protein complement before division. It is likely that the nucleolar function is continuously up-regulated in progressively proliferating cancer cells to respond to the increased demand for ribosome biogenesis. Recently, molecular basis for the nucleolar hypertrophy has become gradually known. In cancer cells possessing larger nucleoli and a shorter cell-doubling time, several nucleolar proteins are expressed more abundantly, including the RNA polymerase I upstream-binding factor,3Derenzini M Trere D Pession A Montanaro L Sirri V Ochs RL Nucleolar function and size in cancer cells.Am J Pathol. 1998; 152: 1291-1297PubMed Google Scholar topoisomerase I,3Derenzini M Trere D Pession A Montanaro L Sirri V Ochs RL Nucleolar function and size in cancer cells.Am J Pathol. 1998; 152: 1291-1297PubMed Google Scholar fibrillarin,3Derenzini M Trere D Pession A Montanaro L Sirri V Ochs RL Nucleolar function and size in cancer cells.Am J Pathol. 1998; 152: 1291-1297PubMed Google Scholar nucleolin,6Olson MOJ The role of proteins in nucleolar structure and function.in: Strauss PR Wilson SH The Eukaryotic Nucleolus: Molecular Biochemistry and Molecular Assemblies. Telford Press, Inc., Cadwell1990: 519-559Google Scholar and protein B23.6Olson MOJ The role of proteins in nucleolar structure and function.in: Strauss PR Wilson SH The Eukaryotic Nucleolus: Molecular Biochemistry and Molecular Assemblies. Telford Press, Inc., Cadwell1990: 519-559Google Scholar These proteins have important functions in rRNA processing and synthesis.3Derenzini M Trere D Pession A Montanaro L Sirri V Ochs RL Nucleolar function and size in cancer cells.Am J Pathol. 1998; 152: 1291-1297PubMed Google Scholar, 6Olson MOJ The role of proteins in nucleolar structure and function.in: Strauss PR Wilson SH The Eukaryotic Nucleolus: Molecular Biochemistry and Molecular Assemblies. Telford Press, Inc., Cadwell1990: 519-559Google Scholar Concomitantly, rRNA synthetic activity is elevated in cancer cells with larger nucleoli.3Derenzini M Trere D Pession A Montanaro L Sirri V Ochs RL Nucleolar function and size in cancer cells.Am J Pathol. 1998; 152: 1291-1297PubMed Google Scholar In addition to tumor growth rate, nucleolar size can predict the metastatic behaviors of a tumor. In cutaneous7Gambini C Casazza S Borgiani L Canepa M Rovida S Rongioletti F Rebora A Counting the nucleolar organizer region-associated proteins is a prognostic clue of malignant melanoma.Arch Dermatol. 1992; 128: 487-490Crossref PubMed Scopus (34) Google Scholar, 8Barzilai A Goldberg I Yulash M Pavlotsky F Zuckerman A Trau H Azizi E Kopolovic J Silver-stained nucleolar organizer regions (AgNORs) as a prognostic value in malignant melanoma.Am J Dermatopathol. 1998; 20: 473-477Crossref PubMed Scopus (24) Google Scholar and uveal9McLean IW Sibug ME Becker RL McCurdy JB Uveal melanoma: the importance of large nucleoli in predicting patient outcome—an automated image analysis study.Cancer. 1997; 79: 982-988Crossref PubMed Scopus (17) Google Scholar, 10Bechrakis NE Sehu KW Lee WR Damato BE Forester MH Transformation of cell type in uveal melanomas.Arch Ophthalmol. 2000; 118: 1406-1412Crossref PubMed Scopus (15) Google Scholar melanomas, the mean nucleolar area in patients who died of metastasis was significantly larger than the mean in patients who did not develop metastasis. The increase in nucleolar size is believed to reflect the increase in nuclear and nucleolar activity during the process of malignant progression from poorly to highly metastatic melanoma cells.7Gambini C Casazza S Borgiani L Canepa M Rovida S Rongioletti F Rebora A Counting the nucleolar organizer region-associated proteins is a prognostic clue of malignant melanoma.Arch Dermatol. 1992; 128: 487-490Crossref PubMed Scopus (34) Google Scholar, 8Barzilai A Goldberg I Yulash M Pavlotsky F Zuckerman A Trau H Azizi E Kopolovic J Silver-stained nucleolar organizer regions (AgNORs) as a prognostic value in malignant melanoma.Am J Dermatopathol. 1998; 20: 473-477Crossref PubMed Scopus (24) Google Scholar, 9McLean IW Sibug ME Becker RL McCurdy JB Uveal melanoma: the importance of large nucleoli in predicting patient outcome—an automated image analysis study.Cancer. 1997; 79: 982-988Crossref PubMed Scopus (17) Google Scholar, 10Bechrakis NE Sehu KW Lee WR Damato BE Forester MH Transformation of cell type in uveal melanomas.Arch Ophthalmol. 2000; 118: 1406-1412Crossref PubMed Scopus (15) Google Scholar, 11Crocker J Nucleolar organizer regions.in: Underwood JC Current Topics in Pathology. Springer-Verlag, Inc., New York1990: 91-150Google Scholar However, the genetic alterations that might be implicated in the changes in nucleolar size and function during such processes have not been studied intensively. To study the molecular mechanisms of melanoma metastasis, various types of mouse models have been developed.12Nicolson GL Cancer metastasis: organ colonization and the cell-surface properties of malignant cells.Biochim Biophys Acta. 1982; 695: 113-176PubMed Google Scholar, 13Nicolson GL Cancer metastasis: tumor cell and host organ properties important in metastasis to specific secondary sites.Biochem Biophys Acta. 1988; 948: 175-224PubMed Google Scholar Among these, the C57BL/6 mouse model using B16 melanoma cells is one of the most useful.14Poste G Fidler IJ The pathogenesis of cancer metastasis.Nature. 1980; 283: 139-146Crossref PubMed Scopus (1368) Google Scholar From B16 cells, various sublines have been established that display distinct metastatic phenotypes.15Hart IR The selection and characterization of an invasive variant of the B16 melanoma.Am J Pathol. 1979; 97: 587-600PubMed Google Scholar, 16Poste G Doll J Hart IR Fidler IJ In vitro selection of murine B16 melanoma variants with enhanced tissue-invasive properties.Cancer Res. 1980; 40: 1636-1644PubMed Google Scholar The F10 and BL6 sublines heavily metastasize to the lungs after intravenous injection.15Hart IR The selection and characterization of an invasive variant of the B16 melanoma.Am J Pathol. 1979; 97: 587-600PubMed Google Scholar, 16Poste G Doll J Hart IR Fidler IJ In vitro selection of murine B16 melanoma variants with enhanced tissue-invasive properties.Cancer Res. 1980; 40: 1636-1644PubMed Google Scholar In contrast, although BL6 cells are also metastatic after subcutaneous injection, F10 cells are only poorly so.15Hart IR The selection and characterization of an invasive variant of the B16 melanoma.Am J Pathol. 1979; 97: 587-600PubMed Google Scholar, 17Fidler IJ Selection of successive tumour lines for metastasis.Nat New Biol. 1973; 242: 148-149Crossref PubMed Scopus (1280) Google Scholar We have attempted to reveal the difference in gene expression between the two sublines by using the library subtraction method, and were able to isolate several genes that are up- or down-regulated in BL6 cells.18Nakaji T Kataoka TR Watabe K Nishiyama K Nojima H Shimada Y Sato F Matsushima H Endo Y Kuroda Y Kitamura Y Ito A Maeda S A new member of the GTPase superfamily that is upregulated in highly metastatic cells.Cancer Lett. 1999; 147: 139-147Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 19Ito A Katoh F Kataoka TR Okada M Tsubota N Asada H Yoshikawa K Maeda S Kitamura Y Yamasaki H Nojima H A role for heterologous gap junctions between melanoma and endothelial cells in metastasis.J Clin Invest. 2000; 105: 1189-1197Crossref PubMed Scopus (167) Google Scholar, 20Kataoka TR Ito A Asada H Watabe K Nishiyama K Nakamoto K Itami S Yoshikawa K Ito M Nojima H Kitamura Y Annexin VII as a novel marker for invasive phenotype of malignant melanoma.Jpn J Cancer Res. 2000; 91: 75-83Crossref PubMed Scopus (35) Google Scholar, 21Ito A Kataoka TR Watanabe M Nishiyama K Mazaki Y Sabe H Kitamura Y Nojima H A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (144) Google Scholar, 22Watabe K Ito A Asada H Endo Y Kobayashi T Nakamoto K Itami S Takao S Shinomura Y Aikou T Yoshikawa K Matsuzawa Y Kitamura Y Nojima H Structure, expression and chromosome mapping of MLZE, a novel gene which is preferentially expressed in metastatic melanoma cells.Jpn J Cancer Res. 2001; 92: 140-151Crossref PubMed Scopus (83) Google Scholar The differentially expressed genes we analyzed had relevance to the clinical phenotypes of invasion and metastasis. The connexin 2619Ito A Katoh F Kataoka TR Okada M Tsubota N Asada H Yoshikawa K Maeda S Kitamura Y Yamasaki H Nojima H A role for heterologous gap junctions between melanoma and endothelial cells in metastasis.J Clin Invest. 2000; 105: 1189-1197Crossref PubMed Scopus (167) Google Scholar and MLZE22Watabe K Ito A Asada H Endo Y Kobayashi T Nakamoto K Itami S Takao S Shinomura Y Aikou T Yoshikawa K Matsuzawa Y Kitamura Y Nojima H Structure, expression and chromosome mapping of MLZE, a novel gene which is preferentially expressed in metastatic melanoma cells.Jpn J Cancer Res. 2001; 92: 140-151Crossref PubMed Scopus (83) Google Scholar genes were up-regulated in BL6 cells, and were intensively expressed in the invasive descent of skin melanoma lesions. The annexin VII gene was down-regulated in BL6 cells, and its expression in human melanoma was related to a better prognosis.20Kataoka TR Ito A Asada H Watabe K Nishiyama K Nakamoto K Itami S Yoshikawa K Ito M Nojima H Kitamura Y Annexin VII as a novel marker for invasive phenotype of malignant melanoma.Jpn J Cancer Res. 2000; 91: 75-83Crossref PubMed Scopus (35) Google Scholar Genetic differences between F10 and BL6 cells seemed to reflect the process by which human melanomas progress to more malignant phenotypes. If there is a difference in nucleolar function between the two sublines, the comparative approach we used could provide genes that are responsible for changes in the nucleolus during the malignant progression process. In the present study, we first compared the nucleolar size in F10 and BL6 cells, and found that BL6 cells contain nucleoli that are larger than those of F10 cells. We therefore assumed that BL6 cells express particular genes involved in nucleolar sizing at higher levels than F10 cells do. With this assumption in mind, we screened the cDNA clone pool obtained after the subtraction of a BL6 cDNA library with F10-derived mRNA. We isolated the Sik-similar protein (Sik-SP) gene, a member of the Nop5/Sik family that consists of highly conserved nucleolar proteins.23Vorbruggen G Onel1 S Jackle H Restricted expression and subnuclear localization of the Drosophila gene Dnop5, a member of the Nop/Sik family of the conserved rRNA processing factors.Mech Dev. 2000; 90: 305-308Crossref PubMed Scopus (14) Google Scholar The Nop5/Sik family members bind box C/D small nucleolar RNA.24Newman DR Kuhn JF Shanab GM Maxwell ES Box C/D snoRNA-associated proteins: two pairs of evolutionarily ancient proteins and possible links to replication and transcription.RNA. 2000; 6: 861-879Crossref PubMed Scopus (113) Google Scholar, 25Yang Y Isaac C Wang C Dragon F Meier UT Conserved composition of mammalian box H/ACA and box C/D small nucleolar ribonucleoprotein particles and their interaction with the common factor Nopp140.Mol Biol Cell. 2000; 11: 567-577Crossref PubMed Scopus (95) Google Scholar, 26Lafontaine DL Tollervey D Synthesis and assembly of the box C+D small nucleolar RNPs.Mol Cell Biol. 2000; 20: 2650-2659Crossref PubMed Scopus (128) Google Scholar The resulting complexes, named small nucleolar ribonucleoproteins, play a pivotal role in rRNA processing necessary for ribosome assembly.27Tollervey D Trans-acting factors in ribosome synthesis.Exp Cell Res. 1996; 229: 226-232Crossref PubMed Scopus (62) Google Scholar, 28Maxwell ES Fournier MJ The small nucleolar RNAs.Annu Rev Biochem. 1995; 64: 897-934Crossref PubMed Scopus (548) Google Scholar In yeast, at least two members, Nop56 and Nop58, are present,29Gautier T Berges T Tollervey D Hurt E Nucleolar KKE/D repeat proteins Nop56p and Nop58p interact with Nop1p and are required for ribosome biogenesis.Mol Cell Biol. 1997; 17: 7088-7098Crossref PubMed Scopus (234) Google Scholar and yeast deficient in either undergoes growth arrest because of defects in protein synthesis.29Gautier T Berges T Tollervey D Hurt E Nucleolar KKE/D repeat proteins Nop56p and Nop58p interact with Nop1p and are required for ribosome biogenesis.Mol Cell Biol. 1997; 17: 7088-7098Crossref PubMed Scopus (234) Google Scholar, 30Wu P Brockenbrough JS Metcalfe AC Chen S Aris JP Nop5p is a small nucleolar ribonucleoprotein component required for pre-18 S rRNA processing in yeast.J Biol Chem. 1998; 273: 16453-16463Crossref PubMed Scopus (119) Google Scholar, 31Lafontaine DL Tollervey D Nop58p is a common component of the box C+D snoRNPs that is required for snoRNA stability.RNA. 1999; 5: 455-467Crossref PubMed Scopus (139) Google Scholar In mammalian cells, the presence of counterparts to the yeast Nop5/Sik family members has already been confirmed,24Newman DR Kuhn JF Shanab GM Maxwell ES Box C/D snoRNA-associated proteins: two pairs of evolutionarily ancient proteins and possible links to replication and transcription.RNA. 2000; 6: 861-879Crossref PubMed Scopus (113) Google Scholar, 25Yang Y Isaac C Wang C Dragon F Meier UT Conserved composition of mammalian box H/ACA and box C/D small nucleolar ribonucleoprotein particles and their interaction with the common factor Nopp140.Mol Biol Cell. 2000; 11: 567-577Crossref PubMed Scopus (95) Google Scholar, 32Lyman SK Gerace L Baserga SJ Human Nop5/Nop58 is a component common to the box C/D small nucleolar ribonucleoproteins.RNA. 1999; 5: 1597-1604Crossref PubMed Scopus (60) Google Scholar but their roles in nucleolar function are not yet well understood. Such is the case with Sik-SP: the cDNA sequence of Sik-SP is in the GenBank database, but no functional analysis has been done on this molecule yet. We assumed that Sik-SP might have a role similar to that of the yeast Nop5/Sik family members. In this study, we used transient expression experiments to show that Sik-SP is located primarily at the nucleolus, and increased the area of the nucleolus recognized by toluidine blue stain. Functional analysis indicated that Sik-SP increases rRNA synthesis and the translational efficiency of the exogenous luciferase (Luc) gene. In addition, we found that expression of a human Nop5/Sik family member correlated well with the metastatic phenotypes of clinical melanomas. The Nop5/Sik family members may therefore promote nucleolar function in metastatic melanoma cells. Mouse melanoma (B16, F10, BL6, and K1735-P12Nicolson GL Cancer metastasis: organ colonization and the cell-surface properties of malignant cells.Biochim Biophys Acta. 1982; 695: 113-176PubMed Google Scholar) and melanocytic (melan-a33Bennett CD Cooper JP Hart RI A line of nontumorigenic mouse melanocytes, syngeneic with the B16 melanoma and requiring a tumor promoter for growth.Int J Cancer. 1987; 39: 414-418Crossref PubMed Scopus (402) Google Scholar) cells were kindly provided by Dr. I. J. Fidler (The University of Texas, Houston, TX) and Dr. D. C. Bennett (St. Georges Hospital Medical School, London, UK), respectively. NIH3T3 and Balb3T3 fibroblastic cells, MC/9 and P-815 mastocytoma cells, and COS-7 kidney cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). All cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS). Two hundred nmol/L of 12-O-tetradecanoylphorbol-13-acetate was added to grow melan-a cells. For the experiment of serum starvation and stimulation, F10 and BL6 cells (2 × 104) were plated onto 35- or 100-mm culture dishes with DMEM/10% FCS (day 0). On the next day (day 1), some dishes of F10 cells were used for transfection. On day 2, the medium was replaced with DMEM, and on day 4, the medium was replaced with DMEM/10% FCS. Cells were harvested by trypsin-ethylenediaminetetraacetic acid solution (Sigma, St. Louis, MO) from 35-mm dishes on every day from days 1 to 6, and from 100-mm dishes on each of days 7 and 8, and the cell number was counted. A portion of the cells was stained with trypan blue (Life Technologies, Inc., Gaithersburg, MD), and the proportion of dead cells was determined. It was always <0.1%. Experiments were performed in triplicate, and repeated three times. The cell-doubling time was determined by counting cells in triplicate samples at 24-, 48-, and 72-hour intervals according to the method described by Patterson.34Patterson Jr, MK Measurement of growth and viability of cells in culture.in: Jokoby WB Pastran IH Methods in Enzymology. Academic Press, New York1979: 141-167Crossref Scopus (516) Google Scholar The method for constructing a subtracted cDNA library has been described previously.19Ito A Katoh F Kataoka TR Okada M Tsubota N Asada H Yoshikawa K Maeda S Kitamura Y Yamasaki H Nojima H A role for heterologous gap junctions between melanoma and endothelial cells in metastasis.J Clin Invest. 2000; 105: 1189-1197Crossref PubMed Scopus (167) Google Scholar, 21Ito A Kataoka TR Watanabe M Nishiyama K Mazaki Y Sabe H Kitamura Y Nojima H A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (144) Google Scholar, 35Ito A Morii E Maeyama K Jippo T Kim DK Lee YM Ogihara H Hashimoto K Kitamura Y Nojima H Systematic method to obtain novel genes that are regulated by mi transcription factor: impaired expression of granzyme B and tryptophan hydroxylase in mi/mi cultured mast cells.Blood. 1998; 91: 3210-3221Crossref PubMed Google Scholar After four rounds of subtraction, ∼1400 clones were rescued and subjected to Northern blot analysis with the RNA of F10 and BL6 cells. Clones of interest were sequenced. The DNA sequences were used to search the National Center for Biotechnology Information database using the BLASTN algorithm. The TNT T7 quick-coupled transcription/translation system (Promega, Madison, WI) was used according to the manufacturer's instructions. We previously described two modified pEGFP-C1 (Novagen, Madison, WI) vectors, termed pEGFP3B and pEGFP3.36Fujii T Tamura K Copeland NG Gilbert DJ Jenkins NA Yomogida K Tanaka H Nishimune Y Nojima H Abiko Y Sperizin is a murine RING zinc-finger protein specifically expressed in haploid germ cells.Genomics. 1999; 57: 94-101Crossref PubMed Scopus (41) Google Scholar Using clone 3-30 as a template, the polymerase chain reaction (PCR) was done with two sense primers (F-Asc-full and F-Asc-ΔN) containing an Asc I site and two antisense primers (R-Not-full and R-Not-ΔC) containing a Not I site: F-Asc-full, 5′-CATGGCGCGCCGATGGAAGGCAAAATCAATAAACAG-3′; F-Asc-ΔN, 5′-CATGGCGCGCCGGAACAGGTAGTAGAAGAGGAGCCG-3′; R-Not-full, 5′-ATAGCGGCCGCTCACTAGCTCCCAACTGAATCGTAAGC-ATG-3′; R-Not-ΔC, 5′-ATAGCGGCCGCTCACTATTCCTCCGCCTCCTCCTCCTCCAT-3′. Primer sets and the amplified cDNA regions of Sik-SP are: F-Asc-full and R-Not-full, the full-length coding region (amino acid residues 1 to 474, Sik-SP); F-Asc-full and R-Not-ΔC, the C-terminal deletion form (amino acid residues 1 to 418, Sik-SPΔC); F-Asc-ΔN and R-Not-full, the N-terminal deletion form (amino acid residues 419 to 474, Sik-SPΔN). The PCR-amplified DNA fragments were digested with Asc I and Not I, and then ligated into the pEGFP3B vector by way of the Asc I-Not I sites (pEGFP3B-Sik-SP, pEGFP3B-Sik-SPΔC, and pEGFP3B-Sik-SPΔN). The plasmid constructs encode various forms of Sik-SP fused to the 3′ end of the green fluorescent protein (GFP). The pEGFP3 vector contains an in-frame stop codon between the GFP sequence and the Asc I site, and was used as a control for the pEGFP3B vector. The pcDNA3 expression vector (Invitrogen, San Diego, CA) was modified to contain an Asc I site as described previously,21Ito A Kataoka TR Watanabe M Nishiyama K Mazaki Y Sabe H Kitamura Y Nojima H A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation.EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (144) Google Scholar and the Asc I-Not I cDNA fragment of wild-type Sik-SP was subcloned from the pEGFP3B vector into the modified pcDNA3 vector (pcDNA3-Sik-SP). The Fugene 6 transfection reagent (Roche Diagnostics Co., Indianapolis, IN) was used according to the manufacturer's instructions. A 100-μl DMEM mixture containing 3 μl of Fugene 6 and 1 μg of plasmid DNA was added to COS-7 or F10 cells that were grown in 35-mm culture dishes at a confluency of 70%, unless otherwise indicated. For co-transfection, 1 μg of each of plasmid constructs was added to the mixture. When 100-mm culture dishes were used, the quantity of each component was scaled up. Subsequently, cells were cultured in DMEM/10% FCS. Fibronectin (Sigma)-coated coverslips (25 ng/cm2) were placed at the bottom of 35-mm culture dishes, and cells were plated onto them. The next day, cells were transfected with pEGFP3B, pEGFP3B-Sik-SP, pEGFP3B-Sik-SPΔC, or pEGFP-3B-Sik-SPΔN. After the cells were cultured for 24 hours, the coverslips were fixed in 3% glutaraldehyde, and mounted in an aqueous medium. Cells were observed through an Axioplan2 microscope (Carl Zeiss, OberKochen, Germany) equipped with epifluorescence and a charge-coupled device camera (Power HAD; Sony, Tokyo, Japan), and images were created by IPLab Spectrum (Scanalytics Inc., Fairfax, VA) software on a Macintosh computer. For subsequent cell staining, the mounting medium was washed out with distilled water. To stain with toluidine blue, F10 and BL6 cells were cultured on fibronectin-coated coverslips (25 ng/cm2) for 3 hours, and fixed in 3% glutaraldehyde. Sections of paraffin-embedded tissues were deparaffinized with xylene, and hydrated in ethanol series. Cells and sections were stained for 10 minutes with 0.05% toluidine blue in a buffer (pH 4.1) containing 60 mmol/L citric acid and 80 mmol/L sodium phosphate. The samples were washed in water, dehydrated in ethanol, and mounted in Permount medium (Fisher Scientific, Fair Lawn, NJ). Stained cells were observed with an image processing system, which consisted of a charge-coupled device camera (HC-2500; Fuji Photo Film Co., Tokyo, Japan), a microscope (AX80; Olympus, Tokyo, Japan), a color monitor, a Macintosh computer (Power Mac G4), image processing software (Adobe Photoshop Ver 5.0; Adobe System Inc., Mountain View, CA), and a digital cursor. Images were created on the computer monitor screen at a magnification of ×7250. This magnification was achieved by an intermediate zoom magnification system (Provis, Olympus). On the monitor, the nucleolar diameters were between 7 and 38 mm. A cursor was used manually to delineate the margins of the toluidine blue-stained nucleoli in at least 30 fields for each sample. The area occupied by the delineation was expressed in a pixel unit, and converted into a μm2 unit by the magnification rate on the monitor. The mean nucleolar area was calculated by observing 200 nucleoli for F10 and BL6 cells, and 50 nucleoli for COS-7 and human melanoma cells. The pRL-SV40, pRL-CMV, and pRL-null vectors were purchased from Promega. The Xho I-Eco RI DNA fragment including the modified chicken β-actin promoter was subcloned from the pAG-2 vector37Miyazaki J Takaki S Araki K Tashiro F Tominaga A Takatsu K Yamamura K Expression vector system based on the chicken beta-actin promoter directs efficient production of interleukin-5.Gene. 1989; 79: 269-277Crossref PubMed Scopus (370) Google Scholar, 38Niwa H Yamamura K Miyazaki J Efficient selection for high-expression transfectants with a novel eukaryotic vector.Gene. 1991; 108: 193-199Crossref PubMed Scopus (4626) Google Scholar into the pRL-null by way of the Xho I-Eco RI sites (pRL-actin). The three vectors express the Renilla Luc gene under the control of the simian virus 40 enhancer/early promoter, cytomegalovirus enhancer/immediate early promoter, and chicken β-actin promoter, respectively. Cells were transfected with each of the three vectors. In addition, cells were co-transfected with pcDNA3 or pcDNA3-Sik-SP. After the cells were cultured for 24 hours, the Renilla Luc activity was measured by using the Dual luciferase assay system (Promega) according to the manufacturer's instructions. The level of Luc activity was normalized against the amount of Luc plasmid DNA introduced by transfection, as determined by Southern blot analysis. After the cells were extracted in the passive lysis buffer of the Luc assay kit, a half volume of the lysate was subjected to phenol/chloroform extraction, followed by ethanol precipitation. The DNA obtained was blotted with an [α-32P] dCTP-labeled Nhe I-Xba I DNA fragment of the pRL-null vector, which contained the Renilla Luc gene. Hybridized bands were quantified by measuring the peak area of the densitometric tracing (BAS 2000 system, Fuji Photo Film Co.). Values of Luc activity were divided by values of the band intensity, and expressed as relative Luc activity in arbitrary units. Experiments were performed in triplicate, and repeated three times. In vitro rRNA synthetic activity was evaluated essentially as described previously.39Stirpe F Novello F Factors affecting the activity of ribonucleic acid polymerase solubilized from rat liver nuclei: effect on ionic strength, spermine and divalent ions with native and denatured deoxyribonucleic acid.Eur J Biochem. 1970; 15: 505-512Crossref PubMed Scopus (29) Google Scholar, 40Janne O Bullock LP Bardin CW Jacob ST Early androgen action in kidney of normal and androgen-insensitive (tfm/y) mice. Changes in RNA polymerase and chromatin template a
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