The Induction of Apoptosis by Histone Deacetylase Inhibition in Chronic Lymphocytic Leukemia (CLL) Cells Is Mediated through the TNF-Related Apoptosis-Inducing Ligand (TRAIL) Pathway.

Background and Objectives: Histone Deacetylase Inhibitors (HDIs) have been shown to induce cell cycle arrest, differentiation and apoptosis in a variety of myeloid and lymphoid malignancies while being relatively nontoxic to normal cells. HDIs have also been shown to sensitize CLL cells to TRAIL-induced apoptosis. In this study we investigated the effects of two HDIs, trichostatin (TSA) and valproic acid (VPA), alone and in combination with fludarabine (FLU) on apoptosis in the BJAB lymphoblastoma B-cell line and in primary CLL cells.

Materials and Methods: Cell viability was assessed by the MTT (3-(3,5-dimethylthiazol-2yl)- 2,5 diphenyl tetrazolium bromide) colorimetric assay, following 4 days of continuous drug treatment. Apoptosis was assessed by staining cell suspensions with acridine orange and quantitating apoptotic cell populations using fluorescence microscopy. Protein expression for the TRAIL receptors, DR4 and DR5, was determined by western blotting.

Results: As determined by the MTT assay, the mean concentrations of TSA and VPA to reduce cell viability by 50% (IC50) in CLL cells were 0.23 uM and 650uM, respectively. TSA at 0.5uM and VPA at 2mM induced 60% apoptosis following 48 hours of drug exposure in both BJAB and CLL cells, as measured by acridine orange staining. Addition of DR4:FC, which sequesters TRAIL away from its receptors, decreased TSA- and VPA-induced apoptosis in BJAB and CLL cells by 9 and 20% respectively, indicating activation of the TRAIL apoptotic pathway by these HDIs. In addition, exposure to TSA at a concentration of 1uM for 24 hours, up regulated the expression of DR5 in BJAB cells, but this was not observed with VPA at a concentration of 1.5mM, after up to 48 hours of treatment. Neither HDI influenced DR4 protein expression in these cells. Preliminary results show that VPA potentiated FLU-induced apoptosis by 20% in both BJAB and CLL cells.

Conclusions: Both TSA and VPA induce apoptosis in BJAB and CLL cells by activation of the TRAIL apoptotic pathway. For TSA, this effect may be potentiated by up regulation of DR5 expression. VPA potentiates FLU-induced apoptosis in BJAB and CLL cells, suggesting a therapeutic role for the combination of HDIs and nucleoside analogues in the management of CLL. Ongoing studies are determining whether this effect is specific for CLL cells, as compared to normal lymphocytes, and the optimum scheduling for synergy.