RGC32, a novel p53-inducible gene, is located on centrosomes during mitosis and results in G2/M arrest

Abstract

To identify target genes for the hemizygous deletions of chromosome 13 that are recurrently observed in malignant gliomas, we performed genome-wide DNA copy-number analysis using array-based comparative genomic hybridization and gene expression analysis using an oligonucleotide-array. The response gene to complement 32 (RGC32) at 13q14.11 was identified as a deletion target, and its expression was frequently silenced in glioma cell lines compared with normal brain. Levels of RGC32 mRNA tended to decrease toward higher grades of primary astrocytomas, especially in tumors with mutations of p53. Expression of RGC32 mRNA was dramatically increased by exogenous p53 in a p53-mutant glioma cell line, and also by endogenous p53 in response to DNA damage in p53 +/+ colon-cancer cells, but not in isogenic p53 À/À cells. Chromatin immunoprecipitation and reporter assays demonstrated binding of endogenous p53 protein to the promoter region of the RGC32 gene, implying p53-dependent transcriptional activity. Transiently and stably overexpressed RGC32 suppressed the growth of glioma cells, probably owing to induction of G2/M arrest. Immunocytochemical analysis revealed a concentration of RGC32 protein at the centrosome during mitosis. RGC32 formed a protein complex with polo-like kinase 1 and was phosphorylated in vitro. These observations implied a novel mechanism by which p53 might negatively regulate cell-cycle progression by way of this newly identified transcriptional target. Our results provide the first evidence that RGC32 might be a possible tumorsuppressor for glioma, that it is directly induced by p53, and that it mediates the arrest of mitotic progression.

Toxicogenomics directory of chemically exposed human hepatocytes

“…The phase II metabolizing enzyme SULT1C2, which was upregulated by at least 20 chemicals in vitro (fold change >3) and revealed significantly increased expression levels in diseased liver tissue, provides one example of a gene that is deregulated in both the in vitro and the in vivo situation. Similarly, CYP3A7, the predominant cytochrome P450 in human fetal liver (Pang et al 2012) and the p53-induced gene RGCC (Huang et al 2009;Saigusa et al 2007) were increased by chemical exposure in vitro and in at least two of the studied human liver conditions. Genes that were downregulated by at least 20 chemicals (more than threefold compared to controls) and showed significantly lower expression levels in at least two liver diseases include the aldehyde dehydrogenase family members ALDH8A1 and ADH4, the steroland fatty acid-metabolizing cytochrome P450 isoenzymes CYP8B1 and CYP4A11, the urea cycle enzyme CPS1, the gluconeogenesis key enzyme PCK1, the membrane-associated ATP-binding cassette transporter ABCA8, and the glucose transporter SLC2A2.…”

“…a The genes in the overlap are listed below the corresponding Venn diagrams. b The genes in the overlap are listed in Table S18 A (Saigusa et al 2007). ALDH8A1 is one example of a gene that belongs to the downregulated chemical consensus (SV20) genes which is also decreased in NASH, cirrhosis, and HCC.…”

PRTFDC1, a possible tumor-suppressor gene, is frequently silenced in oral squamous-cell carcinomas by aberrant promoter hypermethylation

“…Each siRNA (50 nM) was transfected into OSCC cells (St. Louis, MO, USA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The numbers of viable cells 24-96 h after transfection were assessed by a colorimetric water-soluble tetrazolium salt (WST) assay as described elsewhere (Saigusa et al, 2007). The cell cycle was analysed using FACS as described elsewhere (Saigusa et al, 2007).…”

Promoter Hypermethylation Contributes to Frequent Inactivation of a Putative Conditional Tumor Suppressor Gene Connective Tissue Growth Factor in Ovarian Cancer

“…All cell lines were maintained in appropriate medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 Ag/mL streptomycin. The status of the TP53 gene (exons 5-8) mutation was determined as described previously (24). To analyze restoration of genes of interest, cells were cultured with or without various concentrations of 5-aza 2 ¶-deoxycytidine (5-aza-dCyd) for 5 days and/or 100 ng/mL trichostatin A (TSA) for the last 12 h.…”

“…For reverse transcription-PCR (RT-PCR), PCR products were electrophoresed in 3% agarose gels (9). Quantitative real-time RT-PCR experiments were done with an ABI Prism 7900 Sequence Detection System (Applied Biosystems) as described previously (24). Each assay was done in triplicate.…”