title: Induction of G1 cell cycle arrest and P15INK4b expression by ECRG1 through interaction with Miz-1.
abstract: ECRG1 is a novel candidate of tumor suppressor gene identified from human esophagus. To study the biological role of ECRG1 gene, we performed a GAL4-based yeast two-hybrid screen of a human fetal liver cDNA library. Using the ECRG1 cDNA as bait, we identified two putative clones as associated proteins, Miz-1 and FLNA (Filamin A). The interaction of ECRG1 and Miz-1 was confirmed by glutathione-S-transferase (GST)-pull-down assays in vitro and co-immunoprecipitation experiments in vivo. ECRG1 was co-localized with Miz-1 in nucleus, as shown by confocal microscopy. Transfection of ECRG1 gene into the esophageal cancer (EC) cells inhibited cell proliferation and induced G1 phase arrest of cell cycle. In the co-transfection of ECRG1 and Miz-1 assays, we found inhibition of cell proliferation and G1/S phase in EC cells, but the levels of cell proliferation inhibition and G1/S phase arrest were more strongly compared with the transfection of ECRG1 or Miz-1 alone. In addition, the interaction of ECRG1 and Miz-1 could induce expression of P15(INK4b) gene in esophageal cancer 9706 (EC9706) cells. However, the transfection of ECRG1 or Miz-1 alone was not revealed the expressions of P15(INK4b) gene. When antisense ECRG1 interdicted expression of endogenous ECRG1 in Balb/c-3T3 cells, Transfection of Miz-1 couldn't induce P15(INK4b) expression. The results provide evidences that ECRG1 and Miz-1 in EC cells may be acting as a co-functional protein associated with regulation of cell cycle and induction of P15(INK4b) expression. It suggests that ECRG1 may inhibit tumor cell growth by affecting cell cycle, and that expression of P15(INK4b) may be likely to enhance G1 cell cycle arrest during the interaction of ECRG1 and Miz-1. The physical interaction of ECRG1 and Miz-1 may play an important role in carcinogenesis of EC.
authors.affiliation: Department of Etiology and Carcinogenesis, Cancer Institute, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100021, P.R. China.
authors.forename: Nianxi
authors.initials: N
authors.lastname: Zhao
authors.forename: Jianbo
authors.initials: J
authors.lastname: Wang
authors.forename: Yongping
authors.initials: Y
authors.lastname: Cui
authors.forename: Liping
authors.initials: L
authors.lastname: Guo
authors.forename: Shih-Hsin
authors.initials: SH
authors.lastname: Lu
mesh_terms: D000818:Animals; D018797:Cell Cycle Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000648:ultrastructure; D003285:Contractile Proteins / Q000378:metabolism; D050762:Cyclin-Dependent Kinase Inhibitor p15; D003593:Cytoplasm / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism*; D064448:Filamins; D016193:G1 Phase; D015723:Gene Library; D006801:Humans; D047468:Immunoprecipitation; D051741:Kruppel-Like Transcription Factors; D008565:Membrane Proteins; D008840:Microfilament Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D057057:Serine Proteases; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D020798:Two-Hybrid System Techniques; D015854:Up-Regulation / Q000235:genetics
chemical_list: C493966:CDKN2B protein, human; D018797:Cell Cycle Proteins; D003285:Contractile Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D004268:DNA-Binding Proteins; D064448:Filamins; D051741:Kruppel-Like Transcription Factors; D008565:Membrane Proteins; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; C446704:TMPRSS11A protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C107961:ZBTB17 protein, human; D057057:Serine Proteases