The Mre11/Rad50/Nbs1 complex interacts with the mismatch repair system and contributes to temozolomide-induced G₂ arrest and cytotoxicity

Time course of temozolomide-induced nuclear foci formation by Mre11 (A), 53BP1 (B), and γ-H2AX (C) in U87 cells and in mismatch repair–deficient U87 cells (stably expressing Mlh1 siRNA). Cells plated on coverslips were treated with temozolomide (0 or 100 μmol/L, 3 h) and fixed at indicated time points after drug removal. Nuclear foci were detected by immunofluorescence as described in Materials and Methods. Two hundred nuclei were scored for each data point to calculate the percentage of cells displaying foci. Points, mean of triplicates; bars, SD. Representative of three independent experiments. *, first time point when number of cells displaying foci was significantly different from control (untreated cells); P < 0.05, Student's t test.

Temozolomide-induced Mre11 foci formation in MGMT-positive T98G cells in the presence or absence of MGMT inhibitor O⁶-benzylguanine. Cells grown on coverslips were pretreated with O⁶-benzylguanine (BG) 2 h before temozolomide addition and O⁶-benzylguanine was present in the media throughout the course of the experiment. Cells were fixed at indicated time points after temozolomide removal and processed for Mre11 immunofluorescent detection as described in Materials and Methods and in legend to Fig. 1. A, photomicrographs of T98G cells showing temozolomide-induced DNA damage Mre11 foci formation in cells pretreated with MGMT inhibitor O⁶-benzylguanine and the absence of such foci in MGMT-proficient cells (in the absence of O⁶-benzylguanine). B, time course of Mre11 foci formation in T98G cells in the presence or absence of O⁶-benzylguanine. Two hundred nuclei were scored for each data point to calculate the percentage of cells displaying foci. Points, mean of triplicate cultures; bars, SD.

Temozolomide treatment results in enhanced accumulation of Nbs1 and Mlh1 on chromatin (A), colocalization of Mre11 with PCNA (B), and interaction of Nbs1 with Mlh1 (C). A, Nbs1 and Mlh1 accumulated on chromatin in temozolomide-treated cells. U87 cells treated with temozolomide were trypsinized, lysed, and fractionated in soluble and chromatin-associated fractions as described in Materials and Methods. Protein levels of Nbs1, Mlh1, α-tubulin (fractionation control), and β-actin (loading control) in soluble and chromatin fractions of U87 cells were assessed by Western blot and measured by densitometry relatively to actin expression. B, colocalization of Mre11 and PCNA in DNA damage foci 24 h post temozolomide exposure. Representative pictures of cells costained with Mre11 antiserum (green) and PCNA monoclonal antibody (red). Untreated cells displayed similar PCNA-Mre11 foci colocalization in S-phase cells (∼25% of cells); in nonreplicating cells (75% of cells), PCNA staining was negative and Mre11 foci were absent (pictures not shown). C, Nbs1 associates with Mlh1 in chromatin fractions of temozolomide-treated cells. U87 cells were treated with temozolomide, harvested at time points after temozolomide exposure, lysed, and fractionated for soluble and chromatin fractions as described in Materials and Methods. Top, protein levels of Nbs1 and Mlh1 in the soluble and chromatin extracts prepared from the cells. Bottom, Nbs1 and Mlh1 proteins were immunoprecipitated from the same extracts, resolved by 8% SDS-PAGE, and immunoblotted sequentially with Nbs1 and Mlh1 antibodies. Representative immunoblots of day 4 post temozolomide exposure.

Suppression of Mre11 protein expression inhibits the ability of U87 cells to undergo temozolomide-induced G₂ arrest. siRNA targeting Mre11 (SMARTpool, Dharmacon) or negative control siRNA (siControl nontargeting siRNA pool, Dharmacon) were transfected into U87 cells using Lipofectamine reagent (Invitrogen). Three days after transfection, the cells were treated with temozolomide (100 μmol/L, 3 h) and fixed at indicated time points post temozolomide treatment. A, immunoblot showing suppression of Mre11 protein level after siRNA transfection. Lanes 1, 3, 5, 7, and 9, cells transfected with negative control siRNA. Lanes 2, 4, 6, 8, and 10, cells transfected with Mre11-targeting siRNA and harvested at indicated days post temozolomide exposure. Temozolomide was added 3 d after siRNA transfection. The level of Mre11 expression was suppressed in U87 cells by ∼90% at 3 d post siRNA transfection as determined by densitometry of Western blot. B, cell cycle profiles of siRNA-treated U87 cells 2, 3, and 4 d post temozolomide treatment as determined by fluorescence-activated cell sorting analysis of 20,000 cells. The percentages of cells in G₁, G₂, and S phases were determined by DNA ModFit software. C, G₂/G₁ ratio versus time of post temozolomide exposure is plotted for U87 cells transfected with either Mre11 or negative control siRNA.

Mre11 deficiency results in increased survival of U87 cells after temozolomide exposure. U87 cells transfected with either Mre11 or negative control (nontargeting siRNA) were exposed to temozolomide (0, 12, 25, and 50 μmol/L, 3 h) 3 d after transfection. Cells were then plated at 2,000 per 10-cm culture dish in triplicate dishes and allowed to grow for 14 d. Colonies were stained with Coomassie blue and counted (containing >50 cells). Cell survival was calculated as a percentage of number of colonies grown in temozolomide-treated cultures versus number of colonies in untreated cultures. Points, mean of triplicates; bars, SD. *, P < 0.05, values differing statistically from controls (Student's t test). Each experiment was reproduced at least thrice.