Loss of BRCA1-A complex function in RAP80 null tumor cells

Abstract

Receptor Associated Protein 80 (RAP80) is a subunit of the BRCA1-A complex and targets BRCA1 to DNA damage sites in response to DNA double strand breaks. Since mutations of BRCA1 are associated with familial ovarian cancers, we screened 26 ovarian cancer-derived cell lines for RAP80 mutations and found that TOV-21G cells harbor a RAP80 mutation (c.1107G >A). This mutation generates a stop codon at Trp369, which deletes the partial AIR region and the C-terminal zinc fingers of RAP80. Interestingly, both the mutant and wild type alleles of RAP80 lose their expression due to promoter hypermethylation, suggesting that TOV-21G is a RAP80-null cell line. In these cells, not only is the BRCA1-A complex disrupted, but the relocation of the remaining subunits in the BRCA1-A complex including BRCA1, CCDC98, NBA1, BRCC36 and BRE is significantly suppressed. Moreover, TOV-21G cells are hypersensitive to ionizing radiation, which is due to the compromised DNA damage repair capacity in these cells. Reconstitution of TOV-21G cells with wild type RAP80 rescues these cellular defects in response to DNA damage. Thus, our results demonstrate that RAP80 is a scaffold protein in the BRCA1-A complex. Identification of TOV-21G as a RAP80 null tumor cell line will be very useful for the study of the molecular mechanism in DNA damage response.

Figure 1. Screening RAP80 mutations in ovarian cancers.

(A) RAP80 mutations in ovarian cancer cells. (B) The DNA sequences of mutant RAP80. (C) Sketch of RAP80 mutations.

Figure 2. TOV-21G is a RAP80-null cell line.

(A) The expression of RAP80 in 293T, HBL100 and TOV-21G cells was examined by Western blotting. β-actin was used as the protein loading control. (B) The transcription of RAP80 gene was examined by RT-PCR. (C) Q-PCR shows the different mRNA level of RAP80 in HBL100 and TOV-21G cells. Results are shown as plus standard deviation (s.d.) from three independent experiments. *P values<0.01. (D) DNA methylation status of the CpG islands at the RAP80 promoter region was examined by bisulfit sequencing. 5-AZA treatment significantly decreased the methylation level at the RAP80 promoter in TOV-21G cells. (E, F) 5-AZA treatment increases the transcription of RAP80 gene in TOV-21G cells. *P values<0.01. (G) 5-AZA treatment induces the protein expression of RAP80 in TOV-21G cells. The wild type and truncated mutant of RAP80 are indicated. β-actin was used as the protein loading control.

Figure 3. Loss of RAP80 suppresses the IRIF of the BRCA1-A complex.

(A) The IRIF of BRCA1 is suppressed in TOV-21G cells. HBL100 and TOV-21G cells were treated with 10 Gy of IR. Cells were fixed and examined by indicated antibodies. Bar: 10 µm. (B) The IRIF of the BRCA1-A complex is impaired when loss of RAP80. The IRIF of endogenous CCDC98 and NBA1 was examined by indicated antibodies. The IRIF of BRCC36 and BRE was examined using cells stably expressing Flag-tagged BRCC36 and BRE. Bar: 10 µm. (C) Foci positive cells are summarized. Results are averaged (±s.d.) from three independent experiments. *P values<0.01. (D) The BRCA1-A complex is dissembled in TOV-21G cells. The interaction between endogenous BRCA1 and NBA1 and interaction between CCDC98 and NBA1 were examined by IP and Western blotting using indicated antibodies.

Figure 4. Loss of RAP80 abrogates DNA damage repair.

(A) Expression of exogenous RAP80 in TOV-21G cells. (B) Cells loss of RAP80 are hypersensitive to IR. TOV-21G and TOV-21G-RAP80 cells were treated with indicated dose of IR. Survival cell colonies were calculated. (C) Comet assays show that loss of RAP80 impairs DNA damage repair. Representative images of neutral comet assays are shown. Bar: 10 µm. (D) The moment of comet tail were quantitatively measured. *P values<0.01; NS means no statistical significance. (E) Exogenous RAP80 restores the IRIF of the BRCA1-A complex in TOV-21G cells. Bar: 10 µm. (F) Foci positive cells are summarized. Data are from three independent experiments and error bars stand for standard deviation. *P values<0.01.