title: Apoptotic pathway induced by transduction of RUNX3 in the human gastric carcinoma cell line MKN-1.
abstract: The human runt-related transcription factor 3 gene (RUNX3) is considered to be a candidate tumor suppressor gene in gastric carcinoma. However, the role of RUNX3 in the regulation of cell proliferation remains unclear. In the present study, we constructed an adenoviral vector encoding human RUNX3 cDNA under the control of a Tet-responsive promoter (Ad-Tet-FLAG-RUNX3), which regulates the expression of RUNX3 in the presence or absence of doxycycline. A recombinant adenoviral expression vector encoding LacZ (Ad-Tet-LacZ) was used as a negative control. The effect of the transduction of RUNX3 on cell growth was examined using the Tet-On system in a human gastric carcinoma cell line, MKN-1. Exogenous RUNX3 expression was induced successfully by Ad-Tet-FLAG-RUNX3, but not Ad-Tet-LacZ, in the presence of doxycycline in the MKN-1 cells. At 72 h after infection, the proliferative activity in RUNX3-expressing cells was 55% or less of that of the control cells. Flow cytometry revealed that the sub-G(1) peak was increased in cells expressing RUNX3 (34.11%), indicating that the inhibition of cell growth was due to apoptosis, which was confirmed based on Hoechst 33258 staining, the release of cytochrome c from mitochondria into the cytosol, and detection of cleaved caspase-3 by western blotting in MKN-1 cells. Comprehensive analysis using a cDNA microarray showed that RUNX3 upregulated 17 apoptosis-related genes (including FADD, TRAF6, caspase-2, ING1, ING4, Calpain 10, and DNase1) and downregulated 135 apoptosis-related genes (including FLIP, PEA15, TXN2, HSPD1, IKK, and TIAL1) in MKN-1 cells. Pathway analyses to generate functional networks of the genes suggested that promotion of the formation of the death-inducing signaling complex and activation of the mitochondria-mediated pathway were associated with RUNX3-induced apoptosis. In conclusion, our findings suggest that exogenous RUNX3 expression suppressed cell proliferation by inducing apoptosis via the death-receptor mitochondria-mediated pathway in MKN-1 cells.
authors.affiliation: Division of Organ Pathology, Department of Microbiology and Pathology, Faculty of Medicine, Tottori University, Yonago, Tottori 683-8503, Japan.
authors.forename: Yumi
authors.initials: Y
authors.lastname: Nagahama
authors.forename: Mika
authors.initials: M
authors.lastname: Ishimaru
authors.forename: Mitsuhiko
authors.initials: M
authors.lastname: Osaki
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Inoue
authors.forename: Akihiro
authors.initials: A
authors.lastname: Maeda
authors.forename: Chisato
authors.initials: C
authors.lastname: Nakada
authors.forename: Masatsugu
authors.initials: M
authors.lastname: Moriyama
authors.forename: Kenzo
authors.initials: K
authors.lastname: Sato
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Oshimura
authors.forename: Hisao
authors.initials: H
authors.lastname: Ito
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D017209:Apoptosis / Q000235:genetics*; D048708:Cell Growth Processes / Q000235:genetics; D045744:Cell Line, Tumor; D050677:Core Binding Factor Alpha 3 Subunit / Q000096:biosynthesis / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D013274:Stomach Neoplasms / Q000235:genetics* / Q000473:pathology; D013752:Tetracycline / Q000494:pharmacology; D014161:Transduction, Genetic
chemical_list: D050677:Core Binding Factor Alpha 3 Subunit; D018076:DNA, Complementary; C493729:Runx3 protein, human; D013752:Tetracycline