The Histone Deacetylase Inhibitor, Sodium Butyrate, Augments Chlorambucil Cytotoxicity in the Promyelocytic Leukaemia Cell Line, HL-60.

Treatment of various haematological malignancies such as chronic lymphocytic leukaemia (CLL) with alkylating drugs (e.g. chlorambucil, CLB) fails to extinguish the leukaemic clone and often results in chemo-resistance. A possible mechanism of drug resistance in CLL B cells is p53 inactivation. The resistance of cells to standard chemotherapy may be ameliorated by agents that alter the chromatin remodelling processes, DNA methylation and histone acetylation (e.g. sodium butyrate, NaBu). We used the HL-60 cell line (which carries a non-functional p53 protein) as a cell model for resistant CLL B cells and determined the sensitivity of HL-60 cells to a range of concentrations of CLB (5μM,10μM and 20μM) and NaBu (0.5mM, 1mM and 2mM) by tetrazolium salt-based proliferation assays of cells incubated with the drugs over 4 days (days 0, 1, 2 and 3). Cells were resistant to 5μM CLB until day 3 where 56% of cells were killed. 10μM and 20μM CLB had very similar effects (p>0.05) with both treatments killing 83.2% and 87.3% respectively of cells by the end of the incubation. The cell death induced by NaBu was both time- and dose-dependent, with 2mM NaBu being the most effective treatment, while 0.5mM NaBu did not result in significant cell death (p>0.05). To investigate if the treatment of HL-60 cells with both drugs would enhance or have an additive killing effect on the cells, we tested combined treatment involving a series of lower drug concentrations of each of the two drugs (5μM/10μM CLB ± 0.2mM/0.5mM/1mM). All combined treatments using 5μM CLB were more effective in cell killing than when the cells were treated singly with CLB or NaBu (p<0.001). The most effective combined treatment was 5μMCLB+0.2mM NaBu (62% cell death) compared to only 10% and 40% cell killing caused by CLB and NaBu alone, respectively. In contrast, the effect of 10μM CLB was not augmented by NaBu in concentrations ranging between 0.2mM and 1mM (p>0.05). In conclusion, combined treatment involving NaBu increased the cytotoxic effect of low concentrations of CLB in p53-inactivated cells, implying the potential benefit of chromatin modifying agents in cytotoxic therapy. Further studies including the use of a wider range of concentrations of CLB with NaBu or other chromatin modifying agents are underway to investigate possible synergism in the killing of HL-60 cells by these agents.