Leaner Virna D, Kinoshita Ichiro, Birrer Michael J
Cell and Cancer Biology Department, National Cancer Institute, National Institutes of Health, Rockville, MD 20850, USA.
Oncogene. 2003 Aug 28;22(36):5619-29. doi: 10.1038/sj.onc.1206644.
To investigate the role of individual Jun proteins in cell growth and transformation, we have used a doxycycline-inducible retroviral vector to regulate their expression in rat fibroblasts. AP-1 complexes enriched with cJun and JunB result in morphological alterations and anchorage-independent cell growth consistent with a transformation-like phenotype, whereas complexes enriched with JunD had an antiproliferative effect. These results suggest that genes regulated by both cJun and JunB are potentially involved in transformation and that they can be distinguished from those regulated by AP-1 complexes containing JunD. To identify genes regulated by cJun and JunB that may have a role in anchorage-independent growth, we investigated differential gene expression by each of the Jun family members using the Affymetrix Rat oligonucleotide microarray, RG_U34A containing approximately 8000 genes. Differentially regulated genes were identified and grouped for correlation with regulation by the different Jun proteins. A total of 33 candidate genes were found to be differentially regulated by both cJun and JunB and not by JunD. These genes have roles in cell metabolism, growth, signal transduction, migration and adhesion. We validated the differential regulation by cJun and JunB of 10 candidate genes by Northern blot analysis. Of these, eight were further characterized as potential direct targets of AP-1 regulation based on Northern blot results showing differential regulation that correlate with cJun expression. Our results show that inducible cJun and JunB expression result in anchorage-independent growth of Rat1a cells, distinct from JunD-expressing cells. This model system and a functional genomic approach enabled us to differentiate AP-1-regulated genes involved in transformation from AP-1-regulated genes known as bystander genes. This approach significantly reduces the number of bystanders and allows for the targeting of genes specifically involved in transformation.
为了研究单个Jun蛋白在细胞生长和转化中的作用，我们使用了一种强力霉素诱导型逆转录病毒载体来调节其在大鼠成纤维细胞中的表达。富含cJun和JunB的AP-1复合物导致形态改变和不依赖贴壁的细胞生长，这与转化样表型一致，而富含JunD的复合物具有抗增殖作用。这些结果表明，由cJun和JunB共同调控的基因可能参与转化，并且它们可以与由含有JunD的AP-1复合物调控的基因区分开来。为了鉴定可能在不依赖贴壁生长中起作用的、受cJun和JunB调控的基因，我们使用包含约8000个基因的Affymetrix大鼠寡核苷酸微阵列RG_U34A，研究了每个Jun家族成员的差异基因表达。鉴定出差异调控的基因并进行分组，以与不同Jun蛋白的调控进行相关性分析。总共发现33个候选基因受cJun和JunB共同差异调控，而不受JunD调控。这些基因在细胞代谢、生长、信号转导、迁移和黏附中发挥作用。我们通过Northern印迹分析验证了10个候选基因受cJun和JunB的差异调控。其中，基于Northern印迹结果显示的与cJun表达相关的差异调控，有8个进一步被表征为AP-1调控的潜在直接靶标。我们的结果表明，诱导型cJun和JunB表达导致Rat1a细胞不依赖贴壁生长，这与表达JunD的细胞不同。这个模型系统和功能基因组学方法使我们能够区分参与转化的AP-1调控基因和被称为旁观者基因的AP-1调控基因。这种方法显著减少了旁观者的数量，并允许靶向特异性参与转化的基因。
