Redox regulation of RAD51 Cys319 and homologous recombination by peroxiredoxin 1.

RAD51 is a critical recombinase that functions in concert with auxiliary mediator proteins to direct the homologous recombination (HR) DNA repair pathway. We show that Cys319 RAD51 possesses nucleophilic characteristics and is important for irradiation-induced RAD51 foci formation and resistance to inhibitors of poly (ADP-ribose) polymerase (PARP). We have previously identified that cysteine (Cys) oxidation of proteins can be important for activity and modulated via binding to peroxiredoxin 1 (PRDX1). PRDX1 reduces peroxides and coordinates the signaling actions of protein binding partners. Loss of PRDX1 inhibits irradiation-induced RAD51 foci formation and represses HR DNA repair. PRDX1-deficient human breast cancer cells and mouse embryonic fibroblasts display disrupted RAD51 foci formation and decreased HR, resulting in increased DNA damage and sensitization of cells to irradiation. Following irradiation cells deficient in PRDX1 had increased incorporation of the sulfenylation probe DAz-2 in RAD51 Cys319, a functionally-significant, thiol that PRDX1 is critical for maintaining in a reduced state. Molecular dynamics (MD) simulations of dT-DNA bound to a non-oxidized RAD51 protein showed tight binding throughout the simulation, while dT-DNA dissociated from an oxidized Cys319 RAD51 filament. These novel data establish RAD51 Cys319 as a functionally-significant site for the redox regulation of HR and cellular responses to IR.

BMI, physical activity, and breast cancer subtype in white, black, and Sea Island breast cancer survivors.

Higher BMI, lower rates of physical activity (PA), and hormone receptor-negative breast cancer (BC) subtype are associated with poorer BC treatment outcomes. We evaluated the prevalence of high BMI, low PA level, and BC subtype among survivors with white/European American (EA) and African American (AA) ancestry, as well as a distinct subset of AAs with Sea Island/Gullah ancestry (SI). We used the South Carolina Central Cancer Registry to identify 137 (42 EAs, 66 AAs, and 29 SIs) women diagnosed with BC and who were within 6-21 months of diagnosis. We employed linear and logistic regression to investigate associations between BMI, PA, and age at diagnosis by racial/ethnic group. Most participants (82%) were overweight/obese (P=0.46). BMI was highest in younger AAs (P=0.02). CDC PA guidelines (≥150min/week) were met by only 28% of participants. The frequency of estrogen receptor (ER)-negative BC subtype was lower in EAs and SIs than in AAs (P<0.05). This is the first study to identify differences in obesity and PA rates, and BC subtype in EAs, AAs, and SIs. BMI was higher, PA rates were lower, and frequency of ER-negative BC was higher in AAs as compared to EAs and SIs. This study highlights the need to promote lifestyle interventions among BC survivors, with the goal of reducing the likelihood of a BC recurrence. Integrating dietary and PA interventions into ongoing survivorship care is essential. Future research could evaluate potential differential immune responses linked to the frequency of triple negative BC in AAs.

Rad51 overexpression rescues radiation resistance in BRCA2-defective cancer cells.

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.

Restoration of CAPAN-1 cells with functional BRCA2 provides insight into the DNA repair activity of individuals who are heterozygous for BRCA2 mutations.

Mutations in the BRCA2 gene are associated with inherited, early-onset breast cancer. CAPAN-1 cells have been useful for studying how BRCA2 mutations contribute to malignant transformation. They exhibit loss of heterozygosity (LOH), and the remaining copy of BRCA2 has a 6174delT mutation, which causes a premature C-terminal truncation that removes the domains for DNA repair and the nuclear localization signals. The DNA repair protein RAD51, which interacts with BRCA2, exhibits impaired nuclear translocation in CAPAN-1. It has been speculated that RAD51 may require BRCA2 for nuclear entry and that C-terminally truncated BRCA2 may retain RAD51 in the cytoplasm. This may cause heterozygous individuals to exhibit deficient DNA repair and cell viability comparable to individuals with LOH or biallelic BRCA2 mutations. We simulated a heterozygous condition by using stably transfected CAPAN-1 cells with wild-type BRCA2. Fusion of a nuclear localization signal to RAD51 did not increase its ability to independently enter the nuclei of CAPAN-1 cells. Furthermore, restoration of functional BRCA2 did not significantly improve DNA repair, nor did it reestablish cell viability in CAPAN-1 cells. The results imply that C-terminally truncated BRCA2 hinders RAD51 nuclear translocation, possibly contributing to genetic instabilities in homozygous as well as heterozygous individuals.

Radiation enhances caspase 3 cleavage of Rad51 in BRCA2-defective cells.

After DNA damage, caspases cleave and activate proteins involved in cell death by apoptosis but also cleave and inactivate proteins implicated in DNA repair. Here we report a rapid onset of Rad51 cleavage by caspase 3 in BRCA2-defective mouse and human cells. This rapid cleavage was reduced markedly by transfer of full-length human BRCA2 into BRCA2-defective mouse or human cells, which also blocked the association of caspase 3 and Rad51 proteins. Overall caspase 3 activity was increased in BRCA2-defective cells, but the time course was much slower than that for Rad51 cleavage. We further showed that caspase 3 cleavage of Rad51 resulted in a functional decrease in Rad51 strand exchange activity and that inhibition of caspase 3 activity increased Rad51 protein levels and Rad51 foci. These findings indicate that BRCA2 inhibits Rad51 cleavage and subsequent apoptosis.

Intensity-based signal separation algorithm for accurate quantification of clustered centrosomes in tissue sections.

Centrosomes are small organelles that organize the mitotic spindle during cell division and are also involved in cell shape and polarity. Within epithelial tumors, such as breast cancer, and some hematological tumors, centrosome abnormalities (CAs) are common, occur early in disease etiology, and correlate with chromosomal instability and disease stage. In situ quantification of CA by optical microscopy is hampered by overlap and clustering of these organelles, which appear as focal structures. CA has been frequently associated with Tp53 status in premalignant lesions and tumors. Here the authors described an approach to accurately quantify centrosome frequencies in tissue sections and tumors, independently of background or noise levels. Applying simple optical rules in nondeconvolved conventional 3D images of stained tissue sections, the authors showed that they could evaluate more accurately and rapidly centrosome frequencies than with traditional investigator-based visual analysis or threshold-based techniques. The resulting population-based frequency of centrosomes per nucleus could then be used to approximate the proportion of cells with CA in that same population. This was done by taking into account baseline centrosome amplification and proliferation rates measured in the tissue. Using this technique, the authors showed that 20-30% of cells have amplified centrosomes in Tp53 null mammary tumors.