Fig 4. HDR inactivation radiosensitizes human Notch-driven cancer.

(A) Cell cycle distribution of unirradiated and 4Gy-treated CUTLL-1 cells. DNA content was assessed by propidium iodide incorporation and FACS analysis. (B) Targeting RAD51 or inhibiting the Mre11-Rad50-Nbs1 complex radiosensitizes CUTLL-1 cells. Clonogenic survival was performed in CUTLL-1 cells expressing human RAD51 shRNA or non-silencing control shRNA (left), or in cells treated with or without 50 μM Mirin (right). Surviving colonies (>50 cells) were scored at 11–14 days post irradiation. Data represent three independent experiments for each assay. (C) Complete CUTLL-1 tumor response after single dose radiotherapy. CUTLL-1 chloromas (100–150 mm³) in flanks of NOD-SCID female mice were irradiated, and tumor volumes were measured using calipers 2x weekly for 3 months. Complete response was defined as lack of measurable tumor. Parentheses denote number of mice⁄group. The curve was fit to data by nonlinear regression analysis using the Prism Sigmoidal Curve Fit program. (D) RAD51 inactivation radiosensitizes Notch-driven tumors. NOD-SCID female mice harboring RAD51 shRNA-expressing CUTLL-1 xenografts (KD RAD51) or non-silenced control CUTLL-1 tumors (control) were treated with 12Gy and tumor size measured as in (C).