Department of Life Sciences, Faculty of Agriculture, Meiji University, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan.
Int J Oncol. 2010 Feb;36(2):479-90.
Two crucial cell cycle regulators, p16INK4A and p14ARF, are produced from the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene locus by alternative reading frames; these regulators act as tumor suppressors during tumorigenesis. However, the molecular events incidental to the acute functional loss of CDKN2A remain a critical issue. Two pivotal regulatory pathways of cell fate determination involving p16INK4A/retinoblastoma protein (pRb)/E2F1 and p14ARF/p53 interact tightly with each other; however, novel factors with an integral or overlapping role in these two pathways remain incompletely defined. To this end, we specifically decreased the expression of p16INK4A or p14ARF proteins using RNA interference (RNAi) in HeLa cells. Using a DNA microarray approach, we showed that several genes are commonly regulated in both p16INK4A and p14ARF knockdown cells, compared with control RNA-treated cells. We focused on the FBXL16 (F-box and leucine-rich repeat protein 16) gene, the expression of which was reproducibly upregulated in p16INK4A and p14ARF knockdown cells, as evaluated using RT-PCR. Interestingly, the promoter region of FBXL16 was shown to be upregulated by activator E2Fs. Finally, RNAi-mediated knockdown of FBXL16 increased the cell proliferation rate of HeLa cells. Together, our results illustrate a unique aspect of the interdependence between the p16INK4A/pRb/E2F1 and p14ARF/p53 pathways at a molecular level.
两种关键的细胞周期调控因子，p16INK4A 和 p14ARF，是由细胞周期蛋白依赖性激酶抑制剂 2A（CDKN2A）基因座通过交替阅读框产生的；这些调节剂在肿瘤发生过程中作为肿瘤抑制因子发挥作用。然而，CDKN2A 急性功能丧失所伴随的分子事件仍然是一个关键问题。两个涉及 p16INK4A/视网膜母细胞瘤蛋白（pRb）/E2F1 和 p14ARF/p53 的细胞命运决定的关键调节途径紧密相互作用；然而，在这两个途径中具有整体或重叠作用的新因子仍然没有完全定义。为此，我们使用 RNA 干扰（RNAi）在 HeLa 细胞中特异性降低 p16INK4A 或 p14ARF 蛋白的表达。使用 DNA 微阵列方法，我们表明与对照 RNA 处理的细胞相比，几种基因在 p16INK4A 和 p14ARF 敲低细胞中通常受到共同调控。我们专注于 FBXL16（F 框和亮氨酸丰富重复蛋白 16）基因，该基因的表达在 p16INK4A 和 p14ARF 敲低细胞中被反复上调，如使用 RT-PCR 评估的那样。有趣的是，FBXL16 的启动子区域被激活剂 E2Fs 上调。最后，RNAi 介导的 FBXL16 敲低增加了 HeLa 细胞的增殖速率。总之，我们的结果说明了 p16INK4A/pRb/E2F1 和 p14ARF/p53 途径在分子水平上相互依存的独特方面。
