Decrease in MRE11 expression and function leads to increased sensitivity towards PARP inhibition in endometrial carcinoma cell lines.

<p>A, Efficiency of siRNAs at different time points after transfection shown by western blotting. B, Sensitivity assay (colorimetric with SRB): all cell lines were treated with PARP inhibitor (BMN673) in triplicates for 14 days. Survival is represented in percentage of the control (DMSO treated). Knock down of MRE11 with three different sets of siRNAs lead to an increased sensitivity towards PARP inhibition in the endometrial carcinoma cell lines HEC1A (8-fold increase, p<0.0001) and HEC-116 (6-fold increase, p = 0.0006). C, Cell lines were treated with the MRE11-inhibitor mirin at a final concentration of 30 µM in triplicates for 10 days. Survival was assessed by a colorimetric sensitivity assay (SRB) and is shown in percentage of the control (DMSO treated). Treatment with the MRE11-inhibitor mirin lead to increased sensitivity to PARP inhibition in both, HEC1A (2.6-fold increase, p = 0.001) and HEC-116 (17-fold increase, p<0.0001).</p

Immunohistochemical expression of MRE11, RAD50, NBS1-p95, and mismatch repair proteins.

<p>Immunohistochemical expression of MRE11, RAD50, NBS1-p95, and mismatch repair proteins.</p

MRE11 protein expression in endometrial carcinomas as assessed by immunihistochemistry.

<p>A, negative (complete loss of MRE11 protein expression); B, + (weak nuclear expression); C, ++ (intermediate nuclear expression); D, +++ (strong nuclear expression).</p

Endometrial carcinoma cell lines with loss of MRE11 show increased sensitivity to PARP inhibitors.

<p>A, Western blot demonstrating the expression status of MRE11, NBS1 and RAD50 in 10 endometrial carcinoma cell lines. Only the cell line AN3CA harbours a mutation of MRE11, which leads to a dramatically reduced expression of MRE11. Tubulin was used as loading control. B, Colony formation assays demonstrating the sensitivity to the PARP inhibitor (BMN673) in 10 endometrial carcinoma cell lines: all cell lines were treated in triplicates for 14 days. Survival is represented in percentage of the control (DMSO treated). The cell colonies were quantified using a colorimetric method (SRB).</p

Impaired RAD51 foci formation upon knockdown of MRE11.

<p>A, Immunofluorescence images showing DAPI-stained nuclei (blue) and RAD51 foci (green) in representative fields of HEC-1A and HEC-116 endometrial carcinoma cells. Both wild-type MRE11 expressing cell lines were either treated with three different sets of siRNAs for MRE11 or with siRNA for luciferase (siLuc) as a control. The transfected cells were then exposed to 10 Gy of irradiation or not exposed, as indicated. B, RAD51 foci formation shown as a percentage of RAD51 foci positive cells (more than 5 foci per cell were assessed as positive) without treatment and 6 hours after 10 Gy of irradiation. A minimum of 100 cells was counted to determine RAD51 foci formation frequency in three independent experiments. **P≤0.01, ***P≤0.001.</p

Association of MRE11 with RAD50, NBS1, mismatch repair protein status, and clinicopathological factors.

<p>*Fisher’s Exact test;</p><p>**chi-square for trends.</p

MRE11 mutation analysis in endometrioid and serous endometrial carcinoma samples.

<p>The immunohistochemistry status of p53 and MRE11 and the nuclear immunoreactivity of MRE11 are shown. Immunoreactivity of MRE11 was scored as: negative (0), weak (1), moderate (2), and strong (3).</p