Saigusa K, Imoto I, Tanikawa C, Aoyagi M, Ohno K, Nakamura Y, Inazawa J
Department of Molecular Cytogenetics, Medical Research Institute and Graduate School of Biomedical Science, Tokyo Medical and Dental University, Tokyo, Japan.
Oncogene. 2007 Feb 22;26(8):1110-21. doi: 10.1038/sj.onc.1210148. Epub 2006 Dec 4.
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-/- 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 tumor-suppressor for glioma, that it is directly induced by p53, and that it mediates the arrest of mitotic progression.
为了鉴定在恶性胶质瘤中经常观察到的13号染色体半合子缺失的靶基因，我们使用基于芯片的比较基因组杂交技术进行了全基因组DNA拷贝数分析，并使用寡核苷酸芯片进行了基因表达分析。位于13q14.11的补体32反应基因（RGC32）被确定为一个缺失靶点，与正常脑组织相比，其在胶质瘤细胞系中的表达经常沉默。RGC32 mRNA水平在原发性星形细胞瘤分级越高时往往越低，尤其是在p53发生突变的肿瘤中。在p53突变的胶质瘤细胞系中，外源性p53可显著增加RGC32 mRNA的表达，在p53+/+结肠癌细胞中，内源性p53对DNA损伤的反应也可增加RGC32 mRNA的表达，但在同基因p53-/-细胞中则无此现象。染色质免疫沉淀和报告基因分析表明内源性p53蛋白与RGC32基因的启动子区域结合，这意味着存在p53依赖性转录活性。瞬时和稳定过表达RGC32可抑制胶质瘤细胞的生长，这可能是由于诱导了G2/M期阻滞。免疫细胞化学分析显示，在有丝分裂期间，RGC32蛋白集中在中心体。RGC32与polo样激酶1形成蛋白复合物，并在体外被磷酸化。这些观察结果暗示了一种新机制，即p53可能通过这个新发现的转录靶点对细胞周期进程进行负调控。我们的结果首次证明，RGC32可能是胶质瘤的一个潜在肿瘤抑制因子，它直接由p53诱导，并介导有丝分裂进程的阻滞。
