Tepper Clifford G, Gregg Jeffrey P, Shi Xu-Bao, Vinall Ruth L, Baron Colin A, Ryan Philip E, Desprez Pierre-Yves, Kung Hsing-Jien, deVere White Ralph W
Department of Biochemistry and Molecular Medicine, University of California, Davis School of Medicine, Division of Basic Sciences, UC Davis Cancer Center, Sacramento, California 95817, USA.
Prostate. 2005 Dec 1;65(4):375-89. doi: 10.1002/pros.20308.
Tumor suppressor p53 mutations are associated with the transition of prostate cancer to metastatic, hormone-refractory disease and stable expression of p53 gain-of-function (p53GOF) alleles support growth of LNCaP in androgen-depleted medium. In this study, we performed gene expression profiling of four LNCaP-p53GOF sublines to test the hypothesis that different p53GOF mutants mediated androgen independence via modulation of a common set of genes.
Expression profiling was performed using Affymetrix HG-U95Av2 arrays followed by hierarchical clustering to identify expression patterns associated with particular molecular alterations. p53GOF-mediated regulation of Id-1 expression was validated by RT-PCR and dual-luciferase reporter assays. RNA interference was used to investigate the effects of Id-1 and Id-3 suppression.
LNCaP-p53GOF sublines possessed a molecular signature consisting of 95 differentially regulated genes that could be segregated into two clusters of transcripts induced (n=50) and repressed (n=45) by p53GOF expression. To begin validating these genes as effectors of the p53 mutants, we evaluated one of the overexpressed genes, Id-1. RT-PCR confirmed the microarray results and revealed elevated Id-1 levels in LNCaP-p53-P151S (loss-of-function only mutant), thereby implicating p53 mutational inactivation, but not gain-of-function, as a basis for Id-1 deregulation. Reporter assays demonstrated enhanced Id-1 promoter activity in an LNCaP-p53GOF subline. The contribution of Id-1 to p53GOF-mediated biology was demonstrated by the ability of RNAi-mediated gene silencing to decrease both basal and androgen-independent proliferation.
While different p53GOF mutants result in overall distinct expression profiles, they share a common set of differentially-expressed genes that can be used to signify their presence and provide insight into mechanisms underlying androgen independence.
肿瘤抑制因子p53突变与前列腺癌向转移性、激素难治性疾病的转变相关，且p53功能获得性（p53GOF）等位基因的稳定表达支持LNCaP细胞在雄激素缺乏培养基中的生长。在本研究中，我们对四个LNCaP-p53GOF亚系进行了基因表达谱分析，以检验不同的p53GOF突变体通过调控一组共同基因介导雄激素非依赖性的假说。
使用Affymetrix HG-U95Av2芯片进行表达谱分析，随后进行层次聚类以识别与特定分子改变相关的表达模式。通过RT-PCR和双荧光素酶报告基因检测验证p53GOF介导的Id-1表达调控。使用RNA干扰研究Id-1和Id-3抑制的作用。
LNCaP-p53GOF亚系具有一个由95个差异调节基因组成的分子特征，这些基因可分为两个转录本簇，分别由p53GOF表达诱导（n = 50）和抑制（n = 45）。为了开始验证这些基因是p53突变体的效应器，我们评估了其中一个过表达基因Id-1。RT-PCR证实了芯片结果，并揭示了LNCaP-p53-P151S（仅功能丧失突变体）中Id-1水平升高，从而表明p53突变失活而非功能获得是Id-1失调的基础。报告基因检测表明在一个LNCaP-p53GOF亚系中Id-1启动子活性增强。RNAi介导的基因沉默能够降低基础增殖和雄激素非依赖性增殖，这证明了Id-1对p53GOF介导生物学的作用。
虽然不同的p53GOF突变体导致总体上不同的表达谱，但它们共享一组共同的差异表达基因，这些基因可用于表明它们的存在，并深入了解雄激素非依赖性的潜在机制。
