Yin Hong, Glass Jonathan
Feist-Weiller Cancer Center and Department of Medicine, Health Sciences Center, Shreveport, Louisiana 71130-3932, USA.
J Biol Chem. 2006 Apr 28;281(17):11496-505. doi: 10.1074/jbc.M511138200. Epub 2006 Feb 20.
Prostate cancer cell lines were examined for proteins that partnered with the transcription factor C/EBPalpha by use of a pull-down assay with S-tagged C/EBPalpha combined with matrix-assisted laser desorption ionization time-of-flight mass spectroscopy analysis. Ku70, Ku80, and poly(ADP-ribose) polymerase-1 (PARP-1) were identified as proteins that associated with C/EBPalpha. The physical interaction of C/EBPalpha with these partner proteins was further demonstrated by glutathione S-transferase (GST) pull-downs using purified protein expressed in Escherichia coli. The strongest binding was between C/EBPalpha and PARP-1. Immunoprecipitation of C/EBPalpha expressed in prostate cancer cells co-precipitated Ku70, Ku80, and PARP-1. Deletion analysis of C/EBPalpha indicated that the C terminus of C/EBPalpha was essential for the interaction of C/EBPalpha with Ku70, Ku80, and PARP-1. Functional analysis of the interaction between C/EBPalpha and the Ku proteins as well as PARP-1 showed that cells exhibiting these interactions had increased radiation sensitivity and decreased ability to repair double strand DNA breaks. Deficient DNA repair was dependent on the prostate cancer cell line tested, suggesting a complex process. We conclude that the association of C/EBPalpha with Ku proteins and PARP-1 raises the likelihood that C/EBPalpha-expressing prostate cancer cells may be more sensitive to DNA-damaging agents and may be important in the design of new prostate cancer therapies.
利用带有S标签的C/EBPα进行下拉实验，并结合基质辅助激光解吸电离飞行时间质谱分析，对前列腺癌细胞系中与转录因子C/EBPα相互作用的蛋白质进行了检测。Ku70、Ku80和聚（ADP - 核糖）聚合酶-1（PARP - 1）被鉴定为与C/EBPα相关的蛋白质。通过使用在大肠杆菌中表达的纯化蛋白进行谷胱甘肽S - 转移酶（GST）下拉实验，进一步证实了C/EBPα与这些伴侣蛋白之间的物理相互作用。C/EBPα与PARP - 1之间的结合最强。对前列腺癌细胞中表达的C/EBPα进行免疫沉淀，共沉淀出了Ku70、Ku80和PARP - 1。对C/EBPα的缺失分析表明，C/EBPα的C末端对于C/EBPα与Ku70、Ku80和PARP - 1的相互作用至关重要。对C/EBPα与Ku蛋白以及PARP - 1之间相互作用的功能分析表明，表现出这些相互作用的细胞辐射敏感性增加，修复双链DNA断裂的能力下降。DNA修复缺陷取决于所测试的前列腺癌细胞系，提示这是一个复杂的过程。我们得出结论，C/EBPα与Ku蛋白和PARP - 1的关联增加了表达C/EBPα的前列腺癌细胞对DNA损伤剂可能更敏感的可能性，并且可能在新的前列腺癌治疗设计中具有重要意义。
