Ren Chengzhen, Li Likun, Goltsov Alexei A, Timme Terry L, Tahir Salahaldin A, Wang Jianxiang, Garza Laura, Chinault A Craig, Thompson Timothy C
Scott Department of Urology, Baylor College of Medicine, Houston, Texas 77030, USA.
Mol Cell Biol. 2002 May;22(10):3345-57. doi: 10.1128/MCB.22.10.3345-3357.2002.
We identified a novel mouse gene, mRTVP-1, as a p53 target gene using differential display PCR and extensive promoter analysis. The mRTVP-1 protein has 255 amino acids and differs from the human RTVP-1 (hRTVP-1) protein by two short in-frame deletions of two and nine amino acids. RTVP-1 mRNA was induced in multiple cancer cell lines by adenovirus-mediated delivery of p53 and by gamma irradiation or doxorubicin both in the presence and in the absence of endogenous p53. Analysis of RTVP-1 expression in nontransformed and transformed cells further supported p53-independent gene regulation. Using luciferase reporter and electrophoretic mobility shift assays we identified a p53 binding site within intron 1 of the mRTVP-1 gene. Overexpression of mRTVP-1 or hRTVP-1 induced apoptosis in multiple cancer cell lines including prostate cancer cell lines 148-1PA, 178-2BMA, PC-3, TSU-Pr1, and LNCaP, a human lung cancer cell line, H1299, and two isogenic human colon cancer cell lines, HCT116 p53(+/+) and HCT116 p53(-/-), as demonstrated by annexin V positivity, phase-contrast microscopy, and in selected cases 4',6'-diamidino-2-phenylindole staining and DNA fragmentation. Deletion of the signal peptide from the N terminus of RTVP-1 reduced its apoptotic activities, suggesting that a secreted and soluble form of RTVP-1 may mediate, in part, its proapoptotic activities.
我们利用差异显示PCR和广泛的启动子分析，鉴定出一个新的小鼠基因mRTVP-1，它是一种p53靶基因。mRTVP-1蛋白有255个氨基酸，与人类RTVP-1（hRTVP-1）蛋白的区别在于有两个短的框内缺失，分别缺失两个和九个氨基酸。在多种癌细胞系中，通过腺病毒介导的p53传递、γ射线照射或阿霉素处理，无论有无内源性p53，RTVP-1 mRNA均被诱导表达。对非转化细胞和转化细胞中RTVP-1表达的分析进一步支持了p53非依赖性基因调控。我们利用荧光素酶报告基因和电泳迁移率变动分析，在mRTVP-1基因的第1内含子中鉴定出一个p53结合位点。mRTVP-1或hRTVP-1的过表达在多种癌细胞系中诱导凋亡，包括前列腺癌细胞系148-1PA、178-2BMA、PC-3、TSU-Pr1和LNCaP、人肺癌细胞系H1299，以及两个同基因人结肠癌细胞系HCT116 p53(+/+)和HCT116 p53(-/-)，通过膜联蛋白V阳性、相差显微镜观察，在某些情况下通过4',6'-二脒基-2-苯基吲哚染色和DNA片段化得以证实。从RTVP-1的N端缺失信号肽降低了其凋亡活性，这表明RTVP-1的分泌型和可溶性形式可能部分介导其促凋亡活性。
