Histone Deacetylase Inhibitor Depsipeptide (FK228) Induces Apoptosis through Histone Acetylation and Upregulation of Annexin A1 in AML1-ETO Expressing Kasumi-1 Cells.

The t(8;21) translocation, found in 12% of AML, creates the chimeric fusion protein, AML-ETO, which recruits the HDAC complex to AML-1-dependent promoters resulting in transcription repression. Here, we studied the effects of HDAC inhibitor FK228 in the AML1-ETO positive AML cell line, Kasumi-1 and investigated molecular mechanisms of apoptosis induction in these cells. FK228 (5nM, 24 hours) caused marked growth inhibition, apoptosis induction (annexinV+/PI-; control 4.5±0.5%, FK228 24.6±2.6%), and cell cycle arrest (G1 phase; control 59.5%, FK228 76.7%, S phase; control 32.1%, FK228 17.8%). To investigate the molecular changes induced by FK228 we used cDNA array technology. After 24 hour exposure of Kasumi-1 cells, FK228 down-regulated (≤2-fold) 14 genes and up-regulated (≥2-fold) 123 genes including apoptosis and cell cycle regulating genes (Annexin A1; 3.5 fold, serglycin; 2.6 fold, Hes1; 2.2 fold, and TNFα; 2.1 fold increase). Among these genes, we focused on the pro-apoptotic protein Annexin A1, one of the calcium-dependent phospholipid binding proteins. First, we confirmed induction of Annexin A1 mRNA by FK228 using quantitative TaqMan RT-PCR (7.2±1.7 fold). As demonstrated by Western blot analysis, FK228 upregulated the expression of both, full-length Annexin A1 protein and a cleaved isoform of Annexin A1. To identify the critical histone residue modified by FK228 on the Annexin A1 promoter, we performed chromatin immunoprecipitation (ChIP) assays quantified by TaqMan PCR. FK228 increased H4 acetylation (6.7 ±1.8 fold) and H3-K9 acetylation (3.8 ±0.4 fold compared with control at 24 hours) in the Annexin A1 promoter. Next, we investigated effects of FK228 on apoptosis and signal transduction pathways that may modulate Annexin A1 function. As cleavage of the ubiquitous protein Annexin A1 is reported to be associated with caspase activation, the effect of caspase3 inhibitor Z-DEVD-cfm was examined. Z-DEVD pretreatment of Kasumi-1 cells resulted in increased viability and partial inhibition of apoptosis by FK228 (45% decrease in apoptosis with 20μM Z-DEVD). Z-DEVD partially inhibited FK228 induced Annexin A1 cleavage isoform indicating that cleavage of Annexin A1 involves both, caspase-dependent and independent mechanisms. Since activation of the MAPK/ERK pathway has recently been linked to histone de-acetylation, we examined the effects of FK228 on ERK phosphorylation. FK228 used at 5nM inhibited ERK signaling. Further experiments are ongoing to elucidate the possible association of MAPK inhibition, histone acetylation and Annexin A1 gene expression in Kasumi-1 cells.

In summary, these findings suggest that FK228 induces apoptosis and growth inhibition in Kasumi-1 cells in part by inducing Annexin A1 expression through histone acetylation of the promoter in association with caspase-dependent Annexin 1 cleavage and inhibition of MAPK signaling.