Ishihara Kenji, Kaneko Motoko, Kitamura Hajime, Takahashi Aki, Hong Jang Ja, Seyama Toshio, Iida Koji, Wada Hiroshi, Hirasawa Noriyasu, Ohuchi Kazuo
Laboratory of Pathophysiological Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
Int Arch Allergy Immunol. 2008;146 Suppl 1:7-10. doi: 10.1159/000126053. Epub 2008 May 27.
Acetylation and deacetylation of proteins occur in cells in response to various stimuli, and are reversibly catalyzed by histone acetyltransferase and histone deacetylase (HDAC), respectively. EoL-1 cells have an FIP1L1-PDGFRA fusion gene that causes transformation of eosinophilic precursor cells into leukemia cells. The HDAC inhibitors apicidin and n-butyrate suppress the proliferation of EoL-1 cells and induce differentiation into eosinophils by a decrease in the protein level of FIP1L1-PDGFRalpha without affecting the mRNA level for FIP1L1-PDGFRA. In this study, we analyzed the mechanism by which the protein level of FIP1L1-PDGFRalpha is decreased by apicidin and n-butyrate.
EoL-1 cells were incubated in the presence of the HDAC inhibitors apicidin, trichostatin A or n-butyrate. The protein levels of FIP1L1-PDGFRalpha and phosphorylated eIF-2alpha were determined by Western blotting. Actinomycin D and cycloheximide were used to block RNA synthesis and protein synthesis, respectively, in the chasing experiment of the amount of FIP1L1-PDGFRalpha protein.
When apicidin- and n-butyrate-treated EoL-1 cells were incubated in the presence of actinomycin D, the decrease in the protein level of FIP1L1-PDGFRalpha was significantly enhanced when compared with controls. In contrast, the protein levels were not changed by cycloheximide among these groups. Apicidin and n-butyrate induced the continuous phosphorylation of eIF-2alpha for up to 8 days.
The decrease in the level of FIP1L1-PDGFRalpha protein by continuous inhibition of HDAC may be due to the decrease in the translation rate of FIP1L1-PDGFRA.
蛋白质的乙酰化和去乙酰化在细胞中响应各种刺激而发生，分别由组蛋白乙酰转移酶和组蛋白去乙酰化酶（HDAC）可逆催化。EoL-1细胞具有FIP1L1-PDGFRA融合基因，该基因导致嗜酸性粒细胞前体细胞转化为白血病细胞。HDAC抑制剂阿皮西丁和丁酸盐可抑制EoL-1细胞的增殖，并通过降低FIP1L1-PDGFRα的蛋白质水平诱导其分化为嗜酸性粒细胞，而不影响FIP1L1-PDGFRA的mRNA水平。在本研究中，我们分析了阿皮西丁和丁酸盐降低FIP1L1-PDGFRα蛋白质水平的机制。
将EoL-1细胞在HDAC抑制剂阿皮西丁、曲古抑菌素A或丁酸盐存在的情况下进行培养。通过蛋白质印迹法测定FIP1L1-PDGFRα和磷酸化eIF-2α的蛋白质水平。在FIP1L1-PDGFRα蛋白量的追踪实验中，分别使用放线菌素D和环己酰亚胺来阻断RNA合成和蛋白质合成。
当用放线菌素D处理阿皮西丁和丁酸盐处理过的EoL-1细胞时，与对照组相比，FIP1L1-PDGFRα的蛋白质水平下降显著增强。相比之下，这些组中环己酰亚胺对蛋白质水平没有影响。阿皮西丁和丁酸盐诱导eIF-2α持续磷酸化长达8天。
持续抑制HDAC导致FIP1L1-PDGFRα蛋白水平下降可能是由于FIP1L1-PDGFRA的翻译速率降低所致。
