Role of DNA-PK dependent NHEJ and PARP dependent BER on sensitivity of glioma cells knockdown for Rad51 towards TMZ.

<p>(<b>A</b>) Inhibition of DNA-PK activity with NU7026. DNA-PK activity was determined in cell extracts in the presence and absence of NU7026. (<b>B</b>) Clonogenic survival of stable Rad51 knocked-down glioma cells (LN-229-Rad51sh-4) compared to empty vector-transfected cells (LN-229-pS-empty) after TMZ or ionizing radiation (IR) treatment in the presence or absence of the DNA-PKcs inhibitor NU7026. Equitoxic TMZ and ionizing radiation (IR) doses producing about 30 to 40% toxicity in the absence of DNA-PK inhibition were used [TMZ/IR doses: LN-229-pS-empty: 2.7 µM/2.7Gy; LN-229-Rad51sh-4: 1.5 µM/1.9 Gy]. *, p<0.05, significance levels were calculated using the Mann-Whitney U test (n = 5). (<b>C</b>) Clonogenic survival of control (LN-229-pS-empty-2) and Rad51 knockdown (LN-229-Rad51sh-23) glioma cells as a function of olaparib concentration. Cells were co-treated with TMZ or not. TMZ doses producing 30 to 40% toxicity in the absence of PARP inhibition were selected for LN-229-Rad51sh-23 (0.8 µM) and control cells (2.5 µM).</p

Knockdown of the HR protein Rad51 sensitizes glioma cells to O⁶AA chemotherapeutics.

<p>(<b>A</b>) Rad51 protein expression in knockdown clones and control assessed by western blot, quantified, corrected for loading (Erk-2) and expressed relative to control. (<b>B</b>) Clonogenic survival for stable Rad51 knockdown glioma cells (LN229-Rad51sh) compared to empty vector transfected cells (LN-229-pS-empty) following TMZ treatment. (<b>C</b>) Clonogenic survival for stable Rad51 knockdown glioma cells compared to empty vector transfected cells following ACNU treatment. (<b>D</b>) Correlation between the relative Rad51 expression, determined from A, and TMZ concentration that kills 95% of glioma cells, determined from B. Line was fitted using the equation y = minimum + (maximum-minimum)×(1-exp(-kx)). (<b>E</b>) Clonogenic survival for stable Rad51 knockdown glioma cells compared to empty vector transfected cells following ionizing radiation (IR).</p

HR downregulation sensitizes to O⁶-alkylguanine lesions in glioma cells.

<p>(<b>A</b>) Western blot analysis of MGMT and Rad51 protein levels of glioma cells stably transfected to express MGMT (LN-229-MGMT) and cells stably expressing MGMT and knockdown for Rad51 (LN-229-MGMT+Rad51sh). Erk-2 was used for loading control. (<b>B</b>) Clonogenic survival for cells stably transfected to express MGMT (LN-229-MGMT) and cells stably expressing MGMT and knockdown for Rad51 (LN-229-MGMT+Rad51sh) after TMZ treatment. (<b>C</b>) Apoptosis in T98G glioma cells that were transiently knocked-down for Rad51 (or transfected with non-sense siRNA) treated with TMZ. Apoptosis was determined by Sub-G1 flow cytometry 144 h after TMZ addition.</p

Correlations between Rad51 expression level and γH2AX foci (A) and the amount of γH2AX foci and cell death (B).

<p>Relative Rad51 expression was determined from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027183#pone-0027183-g001" target="_blank">Fig.1A</a> and the doses required to kill 95% of glioma Rad51 knockdown cell lines cells are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027183#pone-0027183-g001" target="_blank">Fig.1B</a>.</p

Co-localization of 53BP1 and γH2AX.

<p>53BP1 was immuno-detected as a second marker for DSB. Representative micrographs are shown.</p

HR down-regulation impairs the repair of TMZ induced DNA-DSBs.

<p>Kinetics of TMZ-induced γH2AX foci formation and disappearance in Rad51 knockdown glioma cells treated with 10 µM TMZ. See (<b>A</b>) for representative micrographs and (<b>B</b>) for quantification. Each measure point represents the mean of 200 cells per experiment. Experiments were repeated twice and mean values +/− SD are shown.</p

Knockdown of the HR protein BRCA2 sensitizes glioma cells to O⁶AA chemotherapeutics.

<p>(<b>A</b>) BRCA2 protein expression following knockdown was assessed by western blot. (<b>B</b>) Clonogenic survival following TMZ treatments in transient BRCA2 or non-sense (n.s.) siRNA transfected glioma cells. (<b>C</b>) Clonogenic survival following ACNU treatments in transient BRCA2 or non-sense (n.s.) siRNA transfected glioma cells. (<b>D</b>) Clonogenic survival following ionizing radiation (IR) in transient BRCA2 or non-sense (n.s.) siRNA transfected glioma cells.</p

Apoptosis induction after TMZ treatment in Rad51 kd glioma cells.

<p>(<b>A</b>) Apoptosis determined by annexin V/PI double-staining 144 h after 10 µM TMZ in stable Rad51 knockdown clones of the cell line LN229. (<b>B</b>) Western blot analysis of transient Rad51 knockdown in U87MG and T98G cells, or transfected with non-sense siRNA. Erk-2 was used as loading control. (<b>C</b>) Apoptosis induction after TMZ treatment in Rad51 transient knocked-down glioma cells U87MG and T98G, as well as in the stable Rad51 knockdown clone LN-229-Rad51-sh8 and the empty vector control cell line LN-229-pS-empty. Apoptosis was assessed by Sub-G1 analysis performed 144 h after 10 µM TMZ treatment. For MGMT depletion 10 µM O⁶BG was added 1 h before TMZ. * p<0.05, significance level determined using the t-student test (n = 3).</p