Figure 4
Histone deacetylase inhibitor-induced, ATM/ATR-dependent NKG2D ligand expression sensitizes Ewing sarcoma for natural killer cell cytotoxicity. A-C. Heterogeneous induction of activating NKG2D ligands and/or inhibitory HLA class I molecules in chemotherapy-sensitive (grey) and -resistant (black) cell lines upon pre-treatment with HDI NaB (A), MS-275 (B) and SAHA (C), as assessed by flow cytometry. Results are expressed as mean ± SEM fold increase in MFI-ratio over medium control, obtained in at least three independent experiments. [HDI] = 1/5 IC50 value, except for CADO-ES (NaB 1/20 IC50 value; MS-275 and SAHA 1/10 IC50 value). D. Representative examples of flow cytometry plots for MICB for SK-ES-1 and CADO-ES upon pre-treatment with HDI MS-275 (IC50 values); untreated cells in grey. E. Pre-treatment with caffeine (5 mM) for 2 hours prior to incubation with HDI MS-275 (1/5 of IC50 value) largely abolished HDI-mediated MICB expression (similar results were observed for MICA and ULBP2-3), as assessed by flow cytometry. No such effects were observed for HLA class I expression. Results are expressed as the mean ± SEM fold increase in MFI-ratio over medium control, and are representative of at least two independent experiments. Similar results were obtained for NaB and SAHA (not shown). F. Cytotoxicity of resting natural killer cells was evaluated in 51Cr release assays. Pre-treatment of CADO-ES cells with NaB (1/20 and 1/10 IC50 value) sensitized, in a dose-dependent manner, for natural killer cell cytotoxicity. Statistical analysis (paired t-test) revealed significantly increased sensitivity of NaB pre-treated cells at effector-to-target ratio's > 5:1 (p < 0.05). Similar results were observed for CADO-ES with MS-275 and SAHA, and for SK-ES-1 with SAHA (not shown).