Adámik Matej, Bažantová Pavla, Navrátilová Lucie, Polášková Alena, Pečinka Petr, Holaňová Lucie, Tichý Vlastimil, Brázdová Marie
Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic.
Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic; Department of Biology and Ecology, Faculty of Science, University of Ostrava, Chittussiho 10, 701 03 Ostrava, Czech Republic.
Biochem Biophys Res Commun. 2015 Jan 2;456(1):29-34. doi: 10.1016/j.bbrc.2014.11.027. Epub 2014 Nov 18.
Site-specific DNA recognition and binding activity belong to common attributes of all three members of tumor suppressor p53 family proteins: p53, p63 and p73. It was previously shown that heavy metals can affect p53 conformation, sequence-specific binding and suppress p53 response to DNA damage. Here we report for the first time that cadmium, nickel and cobalt, which have already been shown to disturb various DNA repair mechanisms, can also influence p63 and p73 sequence-specific DNA binding activity and transactivation of p53 family target genes. Based on results of electrophoretic mobility shift assay and luciferase reporter assay, we conclude that cadmium inhibits sequence-specific binding of all three core domains to p53 consensus sequences and abolishes transactivation of several promoters (e.g. BAX and MDM2) by 50μM concentrations. In the presence of specific DNA, all p53 family core domains were partially protected against loss of DNA binding activity due to cadmium treatment. Effective cadmium concentration to abolish DNA-protein interactions was about two times higher for p63 and p73 proteins than for p53. Furthermore, we detected partial reversibility of cadmium inhibition for all p53 family members by EDTA. DTT was able to reverse cadmium inhibition only for p53 and p73. Nickel and cobalt abolished DNA-p53 interaction at sub-millimolar concentrations while inhibition of p63 and p73 DNA binding was observed at millimolar concentrations. In summary, cadmium strongly inhibits p53, p63 and p73 DNA binding in vitro and in cells in comparison to nickel and cobalt. The role of cadmium inhibition of p53 tumor suppressor family in carcinogenesis is discussed.
位点特异性DNA识别和结合活性是肿瘤抑制蛋白p53家族所有三个成员(p53、p63和p73)的共同属性。先前的研究表明,重金属会影响p53的构象、序列特异性结合,并抑制p53对DNA损伤的反应。在此,我们首次报道,已被证明会干扰各种DNA修复机制的镉、镍和钴,也会影响p63和p73的序列特异性DNA结合活性以及p53家族靶基因的反式激活。基于电泳迁移率变动分析和荧光素酶报告基因分析的结果,我们得出结论,镉抑制所有三个核心结构域与p53共有序列的序列特异性结合,并在50μM浓度下消除几个启动子(如BAX和MDM2)的反式激活。在存在特异性DNA的情况下,所有p53家族核心结构域因镉处理而导致的DNA结合活性丧失得到部分保护。消除DNA-蛋白质相互作用的有效镉浓度,对于p63和p73蛋白而言比对p53高约两倍。此外,我们检测到EDTA对所有p53家族成员的镉抑制具有部分可逆性。二硫苏糖醇(DTT)仅能逆转镉对p53和p73的抑制作用。镍和钴在亚毫摩尔浓度下消除DNA-p53相互作用,而在毫摩尔浓度下观察到对p63和p73 DNA结合的抑制作用。总之,与镍和钴相比,镉在体外和细胞中强烈抑制p53、p63和p73的DNA结合。本文讨论了镉抑制p53肿瘤抑制家族在致癌作用中的作用。
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