Changes in H3/H4 acetylation and the DNA damage marker γ-H2AX in peripheral blood (PB) during therapy with 5AC and entinostat. (A) Western blotting of H3/H4 acetylation results from 3 representative patient samples (patients 10, 11, and 24) during the first cycle of treatment. H2A was used as a loading control. AcH3 and AcH4 indicate acetylated H3 and acetylated H4, respectively. Note that day-3 samples were procured before any administration of the HDAC inhibitor. (B) Three-dimensional representation of interaction between doses of 5AC and entinostat (denoted MS) administered and median maximal H4 acetylation in peripheral blood mononuclear cells. The median values of the intensity index of acetylated H4 after normalization with nonacetylated H2A (loading control) are shown. The maximal increase in H4 acetylation was observed with the sequential administration of 5AC (40 and 50 mg/m2) and entinostat (6 and 8 mg). (C) DNA damage induction in both clinical responders and nonresponders during the first cycle of treatment. Western blotting showing up-regulation of γ-H2AX in 2 clinical responders (patients 4 and 17) and 2 clinical nonresponders (patients 6 and 21). Actin was used as a loading control. Day-15 (empty lane) and day-29 samples were not available for patients 17 and 21, respectively. Day-4, -11, and -12 samples were not available for patient 6. (D) Three-dimensional representation of relationship between doses of 5AC and entinostat (denoted MS) administered and median maximal γ-H2AX induction in peripheral blood mononuclear cells. The median values of the intensity index of γ-H2AX after normalization with actin (loading control) are shown.
