The Study Of The Functional Association Between P53 And BRCA1 On The Repair Of Irradiation-induced DNA DSBs

ObjectiveDNA breaks like DNA replication fork stalling, DNA single strand break and DNA double strand breaks (DSBs) can be induced by endogenous DNA damaging agents such as metabolites, reactive oxygen species (ROS) or by exposure to exogenous damaging agents including ionizing radiation (IR), UV light and chemotherapeutic drugs. Of the many types of DNA lesions, DSBs are considered extremely deleterious chromosomal lesions which can lead to chromosome rearrangements or mutations and cause cell death and tumorigenesis. Two mechanistically major pathways are adapted by cells in response to DSBs:homologous recombination (HR) and non-homologous end joining (NHEJ).Breast cancer susceptibility gene 1 (BRCA1) is a critical mediator in the regulation of HR in response to DSBs. The tumor suppressor p53 gene, which has frequently been found to be mutated in malignancies, is involved in multiple cellular processes ranging from apoptosis, cell cycle, transcription, genomic stability, to DNA damage repair. Some studies have suggested that the association of BRCA1 and p53 is required for transcriptional regulation of genes involved in cell replication and DNA repair pathways. However, the relationship between the two proteins in molecular mechanisms of DNA repair in response to IR-induced DNA DSBs, especially HR mechanism, is still not clear.In this study, we explored the functional association between p53 and BRCA1 on the regulation of HR activity, radiosensitivity and chromosomal stability in breast cancer cells in response to IR-induced DNA DSBs. The study of the functional link between p53 and BRCA1 in the regulation of HR should help to shed further light on HR molecular mechanisms and tumorigenesis.Method1. The establishment of working modelsMCF7 cells with wild type p53 (wtp53) background were infected by the stock solution of virus particles with E6 or LXSN to obain a pair of isogenic cell lines, p53-deficient MCF7/E6 cells and wtp53-proficient MCF7/LXSN cells. In shRNA knock-down experiment, MCF7 cells were infected by virus particles with pLKO-1 and p53shRNA to obain another pair of isogenic cell lines. MCF7 cells were transfected with recombination substrate reporter pDR-GFP to obain MCF7/pDR-GFP cells.2. The detection of p53 and BRCA1 expressionMCF7 cells were infected with HPV-E6 or p53 shRNA to knock down the expression of p53, and the expression of p53, BRCA1 or p21 were analyzed by RT-qPCR and western blotting at mRNA and protein level with (at 8h after 10Gy) or without IR treatment. In addition, the dynamic change of BRCA1 and Rad51 foci formation were determined by immunofluorescent staining under the same conditions. MCF7 cells were infected with BRCA1 shRNA to knock down the expression of BRCA1, and then BRCA1 or p53 was detected by western blotting with or without IR treatment.3. The test of p53 and BRCA1 protein interactionWe used the H1299 cell line complemented with wtp53 to test potential interaction between BRCA1 and p53 by immunoprecipitation.4. HR assayMCF7/pDR-GFP cells were used to analyze relative SR and I-SceI-induced HR by flow cytometry after being infected with HPV-E6 or p53 shRNA to knock down the expression of p53, and subsequently restoring exogenous active p53 in the cells infected with p53sh RNA.5. Cell cycle analysisMCF7 cells were infected with HPV-E6 or p53 shRNA and subsequently restoring exogenous active p53 in the cells infected with p53 shRNA. At 8h after exposure of cells to 10Gy of irradiation or without IR treatment, cells were collected and treated with ethanol, and the solution containing PI and 0.1％ triton X-100 to detect cell cycle profiling by flow cytometry.6. Cell radiosensitivity was measured by clonogenic survival assay.MCF7 cells were infected with HPV-E6 or BRCA1 shRNA and exposed to 0,2,4 and 6Gy irradiation. Visible colonies were counted after 3 week incubation and were identified with methanol fixation and methylene blue staining. Survival fractions were calculated.7. FISH for chromosome aberration analysis.FISH was performed using a pan-telomeric PNA probe. FISH analysis was performed on metaphase spreads prepared 24h after 2Gy irradiation to detect chromatid, chromosomal and radial structure aberrations.Results1. Successfully established the working modelsWestern blotting assay showed that the expression of p53 and p21 were reduced significantly in MCF7 cells infected with HPV-E6 or p53 shRNA compared with p53-proficient cells. p53-dependent p21 induction increased obviously after restoring exogenous active p53 in p53-deficient MCF7 cells. These suggested that we successfully established two pairs of p53-deficient isogenic cell lines. Flow cytometry results indicated that we obtained MCF7/pDR-GFP cells stably expressing recombination substrate pDR-GFP.2. p53 suppressed hyper-recombination in response to DNA damage.The results from flow cytometry indicated that, SR and I-Scel-induced HR frequency significantly increased as compared with consh group after knockdown of p53 by its shRNA or HPV-E6 (p<0.01), and the data further indicated that I-Scel-induced HR frequency can obviously go down again after restoring wtp53 expression in p53-defective cells (p<0.01)..3. p53 inhibited the recruitment of more Rad51 to DNA breaks.The results from immunofluorescence assay showed that in non-irradiated cells, comparing with p53-proficient cells, the percentage of cells containing Rad51 foci formation in p53-deficient cells was significantly elevated to 21.9％from 5.33％(p< 0.05). In response to DNA damage, the percentage of cells carrying IR-induced Rad51 foci formation in p53-defective cells was significantly increased to 71％compared with p53-proficient cells (45.7%) (p<0.05). The dynamic change of Rad51 foci formation at various time intervals over an 8 h period after exposure of cells to 10 Gy irradiation suggested that the induction of Rad51 foci formation in p53-defective cells was significantly increased as compared with p53-proficient cells.4. p53 mediated HR through the regulation of BRCA1 activity.Western blotting assay showed that BRCA1shRNA successfully knocked down BRCA1 expression in MCF7 cells and silencing BRCAl did not affect p53 expression, while inactivation of p53 by infection of HPV-E6 virus caused a significant increase in BRCA1 protein level as compared to p53-proficient cells. There was a significant reduction of BRCA1 expression exhibited after the complementation of wtp53 in H1299 cells. The above results indicated that p53 inhibited BRCA1 over-function at protein level.The results from immunofluorescent staining suggested that in untreated cells, the percentage of cells containing BRCA1 foci was significantly increased to 50.8％from 31.4％ after p53 loss as compared to p53-proficient cells (p<0.05). In response to irradiation, p53 loss further increased the percentage of cells with BRCA1 foci to 78.6% from 51.4％ as compared with p53-proficient cells (p<0.05). The percentage of BRCA1 foci formation in cells increased obviously at various time intervals over an 8 h period after exposure of cells to 10 Gy irradiation (p<0.05). All the data suggested that p53 inhibits over-recruitment of BRCA1 to DNA breaks.Co-immunoprecipitation showed that BRCA1 was present in p53-precipitated protein complex implying there was a physical interaction between p53 and BRCA1.RT-qPCR analysis indicated that mRNA level of BRCA1 tested by all of its primers was significantly increased after p53 inactivation using p53 shRNA (p<0.01), and BRCA1 mRNA level can be further inhibited obviously after restoring wtp53 expression in p53-dificient cells(p<0.01) indicating that p53 inhibited BRCA1 over-function transcription level.5. p53 loss revert HR in BRCAl-deficient cells.HR assay showed that in BRCA1-proficient cells, inactivation of p53 increased the frequencies of SR or I-Scel-induced HR by two or five folds (p<0.01). After restoring the expression of p53 in BRCA1-deficient cells, the reduced HR increased significantly.6. The impact of the functional link between p53 and BRCA1 on cell resistance to IR treatment.Clonogenic survival assay indicated that loss of p53 in BRCA1-proficient cells significantly elevated radioresistance as compared with control group at doses of 2,4 and 6 Gy (p<0.01), while knocking down the expression of BRCA1 using shRNA significantly increased radioresistance (p<0.01). Inactivation of p53 in BRCA1-deficient cells caused a significant increase in radioresistance and cells which were sensitive to IR became resistant again at the dose of 2 Gy and 4 Gy (p<0.01), however, the cell sensitivity had no difference once the dose increased to 6 Gy (p> 0.01).7. The disruption of p53 enhanced chromosomal stability following IR treatment.Loss of p53 in BRCA1-proficient cells, chromatid, chromosomal and radial structure aberrations had no difference and present with zero in above situations (p< 0.01); and p53 deletion obviously rescued the formation of chromosome and radial structure aberrations caused by BRCA1 disruption (p<0.01). In response to DNA damage, IR-induced chromosomal and radial structure aberrations were reduced after p53 disruption in both BRCA1-proficient and-deficient cells. Chromosomal structure aberrations increased significantly after knocking down BRCA1 expression. However, double-defective BRCA1 and p53 cells showed that chromatid breaks were alleviated with or without IR treatment.Conclusion1. p53 can suppress Rad51-mediated hyper-recombinational repair.2. p53 can inhibit BRCA1 over-function in HR regulation at transcription level. There is a functional link between p53 and BRCA1 on the regulation of HR molecular mechanisms.3. The functional association between p53 and BRCA1 affected cell radioresistance and chromosomal stability.4. p53 is also involved in the HR-inhibition independently of BRCA1.