Background: EoL-1 cells have a FIP1L1-PDGFRA fusion gene which causes the transformation of eosinophilic precursor cells into leukemia cells. Recently, we suggested that the induction of differentiation of EoL-1 cells into eosinophils by the HDAC inhibitors apicidin and n-butyrate is due to the continuous inhibition of HDACs. However, neither apicidin nor n-butyrate inhibited the expression of FIP1L1-PDGFRA mRNA, although both these inhibitors suppressed cell proliferation. Therefore, in this study, we analyzed whether the levels of FIP1L1-PDGFRα protein and phosphorylated-Stat5 involved in the signaling for the proliferation of EoL-1 cells are attenuated by HDAC inhibitors. Methods: EoL-1 cells were incubated in the presence of apicidin, TSA or n-butyrate. FIP1L1-PDGFRα and phosphorylated-Stat5 were detected by Western blotting. Results: Treatment of EoL-1 cells with apicidin at 100 nM or n-butyrate at 500 µM decreased the levels of FIP1L1-PDGFRα protein and phosphorylated-Stat5, while that with trichostatin A at 30 nM did not. Conclusions: The decrease in the level of FIP1L1-PDGFRα protein caused by apicidin and n-butyrate might be one of the mechanisms by which EoL-1 cells are induced to differentiate into eosinophils by these HDAC inhibitors.
Carrier‐mediated delivery of small interfering RNAs (siRNAs) into the living cells is important for the realization of siRNA therapeutics that can silence target genes through RNA interference. We recently proposed a new strategy for analyzing the siRNA delivery process based on affinity labeling with a peptide nucleic acid (PNA)‐based fluorescent probe (PyAATO; Py: pyrene, A: adenine; TO: thiazole orange) capable of selectively binding to the overhanging structures of siRNAs. We have prepared new probes with improved binding affinity by conjugation with a cationic oligopeptide. The probe, carrying six lysine residues (PyAATO‐Lys6 (Lys6)), displayed a 39‐fold increase in affinity, compared with that of the parent probe containing no oligopeptides. Thermodynamic characterization revealed that enhanced affinity resulted from the favorable polyelectrolyte effect, due to the electrostatic interaction between lysine residues and phosphate anions of the RNA duplexes near the overhanging structure. Lys6 showed the improved imaging ability of the carrier‐mediated siRNA delivery process in living cells, in which 20 nm siRNA could be analyzed and was considered to show the minimal off‐target effects.
Background: 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-PDGFRα without affecting the mRNA level for FIP1L1-PDGFRA. In this study, we analyzed the mechanism by which the protein level of FIP1L1-PDGFRα is decreased by apicidin and n-butyrate. Methods: EoL-1 cells were incubated in the presence of the HDAC inhibitors apicidin, trichostatin A or n-butyrate. The protein levels of FIP1L1-PDGFRα and phosphorylated eIF-2α 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-PDGFRα protein. Results: 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-PDGFRα 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-2α for up to 8 days. Conclusions: The decrease in the level of FIP1L1-PDGFRα protein by continuous inhibition of HDAC may be due to the decrease in the translation rate of FIP1L1-PDGFRA.
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