Abstract

Post-translational modifications of histone proteins including acetylation and methylation are known to be important in the regulation of gene expression, and histone modification patterns are found to be altered in many tumor types. We have established a liquid chromatography/mass spectrometry (LC/MS) analysis technique that allows for detailed interrogation of histone protein post-translational patterns. Application of this technique to histone proteins isolated from Chronic Lymphocytic Leukemia (CLL) patient cells and CD19-selected normal B cells has demonstrated that primary CLL cells have a pattern of H2A modification distinct from normal B cells, and that changes in each of the histone proteins H2A, H2B, H3 and H4 occur following treatment with HDAC inhibitors. We have used this and other techniques to understand how factors that influence apoptotic threshold, such as Bcl-2, can also cause changes in histone post-translational modifications induced by depsipeptide and other HDAC inhibitors. To initially explore this, we utilized the 697 lymphoblastoid cell line transfected either with an empty vector (697-neo) or a vector driving a ten-fold overexpression of Bcl-2 protein (697-Bcl-2). Cells were exposed to varying concentrations of depsipeptide for 4 or 24 hours and analyzed for growth inhibition, cell death, HDAC inhibition, and p21 induction. The results of these studies are summarized in the following table:

Effects of Depsipeptide Treatment on 697 Cells at 24 Hours

Relative % Cell GrowthApoptotic Cells (%)Relative HDAC Inhibition (%)p21, fold increase
697-neo untreated 100 8.2 1.0 
697-neo 3.8 nM DDP 66.3 9.0 74.0 1.5 
697-neo 38 nM DDP 41.0 57.5 97.0 2.3 
697-Bcl-2 untreated 100 7.6 1.0 
697-Bcl-2 3.8 nM DDP 90.4 7.5 77.3 1.9 
697-Bcl-2 38 nM DDP 80.6 10.4 94.6 3.9 
Relative % Cell GrowthApoptotic Cells (%)Relative HDAC Inhibition (%)p21, fold increase
697-neo untreated 100 8.2 1.0 
697-neo 3.8 nM DDP 66.3 9.0 74.0 1.5 
697-neo 38 nM DDP 41.0 57.5 97.0 2.3 
697-Bcl-2 untreated 100 7.6 1.0 
697-Bcl-2 3.8 nM DDP 90.4 7.5 77.3 1.9 
697-Bcl-2 38 nM DDP 80.6 10.4 94.6 3.9 

Over-expression of Bcl-2 completely blocked apoptosis induced by depsipeptide, as compared to the empty vector cell line. Despite this, growth inhibition, HDAC inhibition and p21 protein induction was observed in the Bcl-2 over-expressing cell line. Specific acetylation of H4 was analyzed by LC/MS and demonstrated that baseline histone acetylation patterns were identical between the 697-neo and 697-Bcl-2 cell lines. With depsipeptide treatment, however, distinct differences were noted in the pattern of acetylation between the two cell lines at 24 hours. Specifically, 697-neo showed increases in the di-, tri-, and tetra-acetylated H4 forms, with the tri-acetylated form predominating. In the 697-Bcl-2 cells the di-acetylated form was the most abundant followed closely by tri- and tetra- acetylated forms, and in contrast to the 697-neo cells, a penta-acetylated form is strongly detected. Our data suggest that molecular characteristics of tumor cells that alter apoptotic thresholds may influence changes in histone acetylation following HDAC inhibitor treatment. Exploration of the relationship between histone acetylation and Bcl-2 over-expression is currently under study.

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