Davis Joanne N, McCabe Michael T, Hayward Simon W, Park John M, Day Mark L
Department of Urology, Michigan Urology Center, University of Michigan, Ann Arbor, Michigan 48109-0944, USA.
Cancer Res. 2005 May 1;65(9):3633-42. doi: 10.1158/0008-5472.CAN-04-3129.
The loss of the retinoblastoma tumor suppressor gene (RB) is common in many human cancers, including prostate. We previously reported that engineered deletion of RB in prostate epithelial cells results in sustained cell growth in serum-free media, a predisposition to develop hyperplasia and dysplasia in prostate tissue recombinant grafts, and sensitization to hormonal carcinogenesis. Examining the molecular consequence of RB loss in this system, we show that cyclooxygenase-2 (COX-2) is significantly up-regulated following RB deletion in prostate tissue recombinants. To study the effect of RB deletion on COX-2 regulation, we generated wild-type (PrE) and Rb-/- (Rb-/-PrE) prostate epithelial cell lines rescued by tissue recombination. We show elevated COX-2 mRNA and protein expression in Rb-/-PrE cell lines with increased prostaglandin synthesis. We also find that loss of Rb leads to deregulated E2F activity, with increased expression of E2F target genes, and that exogenous expression of E2F1 results in elevated COX-2 mRNA and protein levels. COX-2 promoter studies reveal that E2F1 transcriptionally activates COX-2, which is dependent on the transactivation and DNA-binding domains of E2F1. Further analysis revealed that the E2F1 target gene, c-myb, is elevated in Rb-/-PrE cells and E2F1-overexpressing cells, whereas ectopic overexpression of c-myb activates the COX-2 promoter in prostate epithelial cells. Additionally, cotransfection with E2F1 and a dominant-negative c-myb inhibited E2F1 activation of the COX-2 promoter. Taken together, these results suggest activation of a transcriptional cascade by which E2F1 regulates COX-2 expression through the c-myb oncogene. This study reports a novel finding describing that deregulation of the Rb/E2F complex results in increased COX-2 expression and activity.
视网膜母细胞瘤肿瘤抑制基因（RB）的缺失在包括前列腺癌在内的许多人类癌症中都很常见。我们之前报道过，在前列腺上皮细胞中通过基因工程手段删除RB会导致其在无血清培养基中持续生长，在前列腺组织重组移植中易发生增生和发育异常，并且对激素致癌作用敏感。在这个系统中研究RB缺失的分子后果时，我们发现前列腺组织重组体中RB缺失后，环氧合酶-2（COX-2）显著上调。为了研究RB缺失对COX-2调控的影响，我们通过组织重组构建了野生型（PrE）和Rb-/-（Rb-/-PrE）前列腺上皮细胞系。我们发现Rb-/-PrE细胞系中COX-2 mRNA和蛋白表达升高，前列腺素合成增加。我们还发现Rb缺失导致E2F活性失调，E2F靶基因表达增加，并且外源性表达E2F1会导致COX-2 mRNA和蛋白水平升高。COX-2启动子研究表明，E2F1转录激活COX-2，这依赖于E2F1的反式激活和DNA结合结构域。进一步分析显示，E2F1靶基因c-myb在Rb-/-PrE细胞和E2F1过表达细胞中升高，而c-myb的异位过表达激活前列腺上皮细胞中的COX-2启动子。此外，与E2F1和显性负性c-myb共转染可抑制E2F1对COX-2启动子的激活。综上所述，这些结果表明E2F1通过c-myb癌基因激活了一个转录级联反应来调节COX-2表达。本研究报告了一项新发现，即Rb/E2F复合物失调导致COX-2表达和活性增加。
