Figure 2.

High levels of RAD51 decrease HR-frequency and sensitize cells to MMC. (A) Representative examples for I-SceI-induced recombination frequencies in U2OS cells. Cells were transfected with the I-Sce-I linearized pGC substrate (bottom) and monitored as the number of GFP positive cells by FACS analysis. Transfection without linearized plasmids served as control (top). (B) Relative HR-frequency in U2OS cells with normal and high RAD51 expression after stable (left) or transient (right) transfection of the GFP-reporter. For both experiments a high level of RAD51 was induced for 24 h before transfection and HR-frequencies were calculated 24 h later. Columns depict the percentage of recombination events and bars represent the standard deviation of at least 3 experiments. Statistical analyses were performed using Student's t-test. (C) Relative HR-frequency in breast cancer cell lines with low, normal and high RAD51 expression. Cells were transiently transfected and HR-frequencies were calculated 24 h after transfection. Symbols depict the percentage of recombination events and bars represent the standard deviation of at least 3 experiments. Statistical analyses were performed using Student's t-test. (D) Influence of RAD51 protein expression in different breast cancer cell lines on HR frequency. Data were taken from Figures 1B and 2C and fitted by linear regression analysis. (E) Cellular sensitivity to MMC in U2OS and (F) breast cancer cell lines. Exponentially growing cells were treated with increasing doses up to 60 nM MMC for 24h (U2OS) or with 1.5 µM (breast cancer cell lines) for 1 h and cellular sensitivity was monitored by colony formation. Data of at least 3 independent experiments were fitted by linear regression analysis (A) or statistical analysis was performed using Student's t-test (B).