Abstract
The nucleosome is the basic structure of chromatin. Changes in the biochemical composition of nucleosome-associated histone tails are associated with specific gene activation states, and are the target of several antineoplastic agents such as histone deacetylase inhibitors (HDI). Nucleosomes are constrained into loops that are flanked by domains known as matrix-attached regions (MARs). MARs contain DNA topoisomerase II (Topo II) consensus sequences. Topo II is responsible for regulating and maintaining DNA topology and is the target of several antineoplastic agents such as the anthracycline IDA, an effect mediated by the induction of double strand DNA breaks (DSB). We hypothesized that the combination of a Topo II inhibitor and a HDI will have synergistic antileukemia activity. VPA and SAHA are two HDIs currently studied in several clinical trials with known antileukemia activity and tolerable toxicity. To test our hypothesis and to develop future clinical studies, we have analyzed the effect of the combination of IDA, a potent Topo II inhibitor, with VPA or SAHA. We treated the leukemic cells lines MOLT4 and HL60 with increasing doses of IDA (0.5-20nM), SAHA (0.3-3μM) or VPA (0.25-3mM) daily for 3 days. First, using trypan blue viability assays, we identified the IC10 of IDA to be 0.5nM for MOLT4 and 1.5nM for HL60. Doses in excess of 2μM of SAHA or 3mM of VPA resulted in more than 90% decrease in cell viability in both cell lines. Subsequently, SAHA at doses of 0.075-1μM and VPA at 1-3mM were used for the combination experiments with IDA at its specific cell line IC10. At low doses of SAHA (0.075-0.45 μM) and VPA (0.25-1 mM) the combination was shown to have synergistic antileukemia activity by the Fractional Product Method of Webb. These results were confirmed using Annexin V assays. Of importance, growth inhibition was independent of the sequence used. To analyze the effects of this combination on DSB generation, we analyzed using immonocytochemistry and western blot, the induction of γH2AX, a histone variant that has been identified as an early event after the DSBs. SAHA alone induced a modest increase in γH2AX compared to baseline, whereas IDA alone had a significant effect that was not potentiated by the addition of SAHA. Histone H3 and H4 acetylation increased in a dose-dependent manner (2.4–15 fold) with both SAHA and VPA, starting at 0.3μM of SAHA and 0.25mM of VPA. The addition of IDA had no significant effect on histone acetylation. Because of previous data indicating that HDIs may down-regulate the expression of Topo II-alpha, the target of IDA, we have studied using real-time PCR its levels prior and during exposure to the different combinations. SAHA or VPA had no effect on Topo II-alpha mRNA levels whereas IDA induced 2.0–3.5 fold its expression in a dose-independent manner, an effect no altered by the addition of SAHA or VPA. Expression of p21CIP1, that is silenced in both cell lines, was restored by single agent VPA, SAHA or IDA. The combination of these drugs resulted in an additive effect in terms of p21CIP1 induction. Despite this phenomenon, no changes in cell cycle status were observed in these cells. In summary, the combination of IDA and SAHA or VPA has potent in vitro antileukemia effect, and should be studied in clinical trials.
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