Rad51 overexpression rescues radiation resistance in BRCA2-defective cancer cells

Abstract

Breast cancers with BRCA2 mutations exhibit DNA repair defects and are particularly sensitive to radiation. BRCA2 interacts with Rad51 in a complex manner involving internal BRC and C-terminal TR2 domains which play a key role in homologous recombination. BRCA2 expression also modulates Rad51 protein levels such that Rad51 protein is relatively decreased in BRCA2-defective cancer cells. This is mediated in part through BRCA2's capacity to protect Rad51 from caspase-3 proteolytic degradation. In order to distinguish between functional and expression related roles for BRCA2 we studied the results of Rad51 overexpression in mouse and human cells with inactivating BRCA2 mutations. The results show that overexpression of wild-type Rad51 partially rescues BRCA2 deficiency but that overexpression of a caspase-3 resistant Rad51 completely complements the BRCA2 defect in radiation responsiveness. These results indicate that Rad51 can compensate for some aspects of a BRCA2 gene defect and suggest that Rad51 expression levels may be an important modifier of the BRCA2 defective genotype.

Figure 1

Expression of Rad51 protein in BRCA2-defective Capan-1 cells. (A) Western blot of Rad51 protein in untransfected Capan-1 (upper panel) and BRCA2-transfected Capan1 (lower panel) in unirradiated cells (UN) or cells either 10 min (10) or 60 min (60) after irradiation with 10 Gy. (B) Quantitation of Western blots of Rad51 protein standardized to tubulin protein levels using relative expression units. Four separate Western blots were quantitated and standard error is shown by error bars.

Figure 2

Cellular localization of RAD51. Endogenous Rad51 immunofluorescence shown in the first two columns: First column is Capan-1 and second column is BRCA2-transfected Capan-1. Composite overlay of endogenous RAD51 and DAPI-stained nuclei. Exogenous GFP-Rad51 fluorescence shown in the last two columns: 3rd column is GFP-Rad51 wild-type transfected Capan-l and 4th column is Rad51D-A transfected Capan-1. The upper row represents untreated cells and the lower row are cells that were fixed 10 min after 10 Gy ionizing radiation. The white arrow in first panel points to a cell in which RAD51 is primarily nuclear.

Figure 3

Capan-1 Cell survival after irradiation. (A) Western blot of GFP-RAD51 expression. Lane 1: Mock transfection. Lane 2: Transfection with construct expressing 62 kDa wild-type GFP-RAD51. Lane 3: Transfection with construct expressing 62 kDa noncleavable GFP-RAD51 (D-A RAD51). (B) Plates of Capan-l cells transfected with either wild-type BRCA2, wild-type RAD51 or non-cleavable RAD51 (D-A RAD51) were stained and counted for cell survival at 14 d after irradiation (N = 3). Note that the error bars of means near 0.01 are wide due to the use of a log scale. (C) Western blot of GFP-Rad51 transfected cells showing presence of caspase-3 cleavage product (arrow) compared with 62 kDa GFP-Rad51 (bar). Time after irradiation is shown in hours: Un represents unirradiated and 0.1 h represents cells frozen 6 min after 10 Gy radiation.

Figure 4

Mouse brca2−/− Cell survival after Irradiation. Plates of mouse brca2−/− cells transfected with either wild-type BRCA2, wild-type RAD51 or non-cleavable RAD51 (D-A RAD51) were stained and counted for cell survival at 14 d after irradiation (N = 3).