title: Polypyrimidine tract-binding protein induces p19(Ink4d) expression and inhibits the proliferation of H1299 cells.
abstract: The expression of polypyrimidine tract-binding protein (PTB) is up-regulated in many types of cancer. Here, we studied the role of PTB in the growth of non small cell lung cancer cells. Data showed that PTB overexpression inhibited the growth of H1299 cells at least by inhibiting DNA synthesis. Quantitative real-time PCR and Western blot analyses showed that PTB overexpression in H1299 cells specifically induced the expression of p19(Ink4d), an inhibitor of cyclin-dependent kinase 4. Repression of p19(Ink4d) expression partially rescued PTB-caused proliferation inhibition. PTB overexpression also inhibited the growth and induced the expression of p19(Ink4d) mRNA in A549 cells. However, Western blot analyses failed to detect the presence of p19(Ink4d) protein in A549 cells. To address how PTB induced p19(Ink4d) in H1299 cells, we showed that PTB might up-regulate the activity of p19(Ink4d) gene (CDKN2D) promoter. Besides, PTB lacking the RNA recognition motif 3 (RRM3) was less effective in growth inhibition and p19(Ink4d) induction, suggesting that RNA-binding activity of PTB plays an important role in p19(Ink4d) induction. However, immunoprecipitation of ribonuclearprotein complexes plus quantitative real-time PCR analyses showed that PTB might not bind p19(Ink4d) mRNA, suggesting that PTB overexpression might trigger the other RNA-binding protein(s) to bind p19(Ink4d) mRNA. Subsequently, RNA electrophoretic mobility-shift assays revealed a 300-base segment (designated as B2) within the 3'UTR of p19(Ink4d) mRNA, with which the cytoplasmic lysates of PTB-overexpressing cells formed more prominent complexes than did control cell lysates. Insertion of B2 into a reporter construct increased the expression of the chimeric luciferase transcripts in transfected PTB-overexpressing cells but not in control cells; conversely, overexpression of B2-containing reporter construct in PTB-overexpressing cells abolished the induction of p19(Ink4d) mRNA. In sum, we have shown that PTB plays as a negative regulator in H1299 cell proliferation at least by inducing p19(Ink4d) expression at transcriptional and post-transcriptional levels.
authors.affiliation: Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan Town, Miaoli, Taiwan, Republic of China. shankung@nhri.org.tw
authors.forename: Shankung
authors.initials: S
authors.lastname: Lin
authors.forename: Ming Jen
authors.initials: MJ
authors.lastname: Wang
authors.forename: Kuo-Yun
authors.initials: KY
authors.lastname: Tseng
mesh_terms: D020413:3' Untranslated Regions; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019938:Cyclin D1 / Q000235:genetics; D050764:Cyclin-Dependent Kinase Inhibitor p19 / Q000235:genetics*; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism; D038941:Polypyrimidine Tract-Binding Protein / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D015533:Transcriptional Activation
chemical_list: D020413:3' Untranslated Regions; D050764:Cyclin-Dependent Kinase Inhibitor p19; D019938:Cyclin D1; D038941:Polypyrimidine Tract-Binding Protein
references.citation: Mol Med. 2002 May;8(5):263-72
references.pmid: 12359957
references.citation: Nucleic Acids Res. 2005 May 31;33(10):3095-108
references.pmid: 15928332
references.citation: Biochem Soc Trans. 2005 Jun;33(Pt 3):457-60
references.pmid: 15916540
references.citation: Mol Cell. 1999 Sep;4(3):431-7
references.pmid: 10518224
references.citation: Oncogene. 2007 Jul 26;26(34):4961-8
references.pmid: 17310993
references.citation: J Biol Chem. 2006 Oct 27;281(43):32294-302
references.pmid: 16950790
references.citation: FEBS J. 2009 Nov;276(22):6658-68
references.pmid: 19843185
references.citation: Mol Cell Biol. 2004 May;24(10):4174-83
references.pmid: 15121839
references.citation: Cancer Res. 2010 Nov 15;70(22):8977-80
references.pmid: 20978194
references.citation: J Biol Chem. 2008 Jul 18;283(29):20277-87
references.pmid: 18499661
references.citation: J Immunol. 2003 Jan 15;170(2):979-88
references.pmid: 12517964
references.citation: Cell Mol Life Sci. 2008 Feb;65(4):516-27
references.pmid: 17975705
references.citation: Biol Reprod. 2007 Jun;76(6):1025-33
references.pmid: 17329592
references.citation: J Biol Chem. 2009 Dec 25;284(52):36500-36510
references.pmid: 19887373