Abstract：
Transcriptomic analyses have revealed hundreds of p53-regulated genes; however, these studies used a limited number of cell lines and p53-activating agents. Therefore, we searched for candidate p53-target genes by employing stress factors and cell lines never before used in a high-throughput search for p53-regulated genes. We performed RNA-Seq on A549 cells exposed to camptothecin, actinomycin D, nutlin-3a, as well as a combination of actinomycin D and nutlin-3a (A + N). The latter two substances synergise upon the activation of selected p53-target genes. A similar analysis was performed on other cell lines (U-2 OS, NCI-H460, A375) exposed to A + N. To identify proteins in cell lysates or those secreted into a medium of A549 cells in control conditions or treated with A + N, we employed mass spectrometry. The expression of selected genes strongly upregulated by A + N or camptothecin was examined by RT-PCR in p53-deficient cells and their controls. We found that p53 participates in the upregulation of: ACP5, APOL3, CDH3, CIBAR2, CRABP2, CTHRC1, CTSH, FAM13C, FBXO2, FRMD8, FRZB, GAST, ICOSLG, KANK3, KCNK6, KLRG2, MAFB, MR1, NDRG4, PTAFR, RETSAT, TMEM52, TNFRSF14, TRANK1, TYSND1, WFDC2, WFDC5, WNT4 genes. Twelve of these proteins were detected in the secretome and/or proteome of treated cells. Our data generated new hypotheses concerning the functioning of p53. Many genes activated by A + N or camptothecin are also activated by interferons, indicating a noticeable overlap between transcriptional programs of p53 and these antiviral cytokines. Moreover, several identified genes code for antagonists of WNT/β-catenin signalling pathways, which suggests new connections between these two cancer-related signalling systems. One of these antagonists is DRAXIN. Previously, we found that its gene is activated by p53. In this study, using mass spectrometry and Western blotting, we detected expression of DRAXIN in a medium of A549 cells exposed to A + N. Thus, this protein functions not only in the development of the nervous system, but it may also have a new cancer-related function.
摘要：
转录组学分析揭示了数百个p53调控基因；然而，这些研究使用了有限数量的细胞系和p53激活剂.因此,我们通过使用以前从未用于高通量搜索p53调节基因的应激因子和细胞系来搜索候选p53靶基因.我们对暴露于喜树碱的A549细胞进行RNA-Seq，放线菌素D,nutlin-3a,以及放线菌素D和nutlin-3a（AN）的组合。后两种物质协同作用于所选择的p53靶基因的激活。对其他细胞系进行了类似的分析(U-2OS，NCI-H460，A375）暴露于AN。为了鉴定细胞裂解物中的蛋白质或在对照条件或用AN处理的A549细胞培养基中分泌的蛋白质，我们采用了质谱法.通过RT-PCR在p53缺陷细胞及其对照中检查了由AN或喜树碱强烈上调的所选基因的表达。我们发现p53参与了ACP5，APOL3，CDH3，CIBAR2，CRABP2，CTHRC1，CTSH，FAM13C,FBXO2,FRMD8,FRZB,GAST,ICOSLG，KANK3,KCNK6,KLRG2,MAFB,MR1,NDRG4,PTAFR,RETSAT,TMEM52、TNFRSF14、TRANK1、TYSND1、WFDC2、WFDC5、WNT4基因。在处理的细胞的分泌组和/或蛋白质组中检测到12种这些蛋白质。我们的数据产生了有关p53功能的新假设。许多被A+N或喜树碱激活的基因也被干扰素激活，表明p53的转录程序与这些抗病毒细胞因子之间存在明显的重叠。此外，几个确定的基因编码WNT/β-连环蛋白信号通路拮抗剂，这表明这两个与癌症相关的信号系统之间有新的联系。这些拮抗剂之一是DRAXIN。以前,我们发现它的基因被p53激活。在这项研究中,使用质谱和蛋白质印迹,我们在暴露于A+N的A549细胞培养基中检测到DRAXIN的表达。这种蛋白质不仅在神经系统的发育中起作用，但它也可能具有新的癌症相关功能。
