ABSTRACT
Bcl-2/adenovirus E1B 19 kDa interacting protein 2 like-1 (BNIPL-1) is a novel human protein identified in our laboratory, which can interact with Bcl-2 and Cdc42GAP and induce apoptosis via the BNIP-2 and Cdc42GAP homology (BCH) domain. In the present study, we established the Hep3B-Tet-on stable cell line in which expression of BNIPL-1 can be induced by doxycycline. The cell proliferation activity assay showed that the overexpression of BNIPL-1 suppresses Hep3B cell growth in vitro. The differential expression profiles of 588 known genes from BNIPL-1-transfected Hep3B-Tet-on and vector control cells were determined using the Atlas human cDNA expression array. Fifteen genes were differentially expressed between these two cell lines, among which seven genes were up-regulated and eight genes were down-regulated by BINPL-1. Furthermore, the differential expression result was confirmed by semiquantitative RT-PCR. Among these differentially expressed genes, p16INK4, IL-12, TRAIL and the lymphotoxin beta gene involved in growth suppression or cell apoptosis were up-regulated, and PTEN involved in cell proliferation was down-regulated by BNIPL-1. These results suggest that BNIPL-1 might inhibit cell growth though cell cycle arrest and/or apoptotic cell death pathway(s).
Subject(s)
Carcinoma, Hepatocellular/genetics , Carrier Proteins/physiology , Gene Expression Regulation, Neoplastic , Liver Neoplasms/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Carrier Proteins/genetics , Cell Line, Tumor , Cell Proliferation , Down-Regulation , Doxycycline/pharmacology , Gene Expression Profiling , Genetic Vectors , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Up-RegulationABSTRACT
AIM: Bcl-2/adenovirus E1B 19 ku interacting protein 2-like (BNIPL-2) is a novel protein recently identified in our laboratory. BNIPL-2 is homologous to human BNIP-2, a potentially proapoptotic protein, and can interact with Bcl-2 and Cdc42GAP and promote apoptosis in BEL-7402 cells. Here we report the gene-expression profile regulated by BNIPL-2 in human hepatocarcinoma Hep3B cells and the analysis of its potential roles in cell apoptosis. METHODS: BNIPL-2 was overexpressed in Hep3B cells using tetracycline inducible or Tet-on system. Screened by Western blot, the cells with low background and high induction fold of BNIPL-2 were obtained. We performed Atlas human cDNA expression array hybridization on these cells and analyzed the data with Quantarray software to identify BNIPL-2-regulated genes and their expression profile. RT-PCR was used to confirm the altered expression level of part of genes identified by the Atlas array hybridization. RESULTS: Fifteen of 588 genes spotted on the Atlas membrane showed altered expression levels in BNIPL-2-transfected Hep3B-Tet-on cells, in which 8 genes involved in cell apoptosis or growth inhibition were up-regulated and 7 genes involved in cellular proliferation were down-regulated following overexpression of BNIPL-2. CONCLUSION: cDNA array is a powerful tool to explore gene expression profiles under inducible conditions. The data obtained using the cDNA expression microarray technology indicates that BNIPL-2 may play its roles in apoptosis through regulating the expression of genes associated with cell apoptosis, growth inhibition and cell proliferation.
