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Poly(ADP‐ribose) polymerase (PARP) inhibitors (PARPi) are effective in cancers with defective homologous recombination DNA repair (HRR), including BRCA1/2‐related cancers. A test to identify additional HRR‐deficient tumors will help to extend their use in new indications. We evaluated the activity of the PARPi olaparib in patient‐derived tumor xenografts (PDXs) from breast cancer (BC) patients and investigated mechanisms of sensitivity through exome sequencing, BRCA1 promoter methylation analysis, and immunostaining of HRR proteins, including RAD51 nuclear foci. In an independent BC PDX panel, the predictive capacity of the RAD51 score and the homologous recombination deficiency (HRD) score were compared. To examine the clinical feasibility of the RAD51 assay, we scored archival breast tumor samples, including PALB2‐related hereditary cancers. The RAD51 score was highly discriminative of PARPi sensitivity versus PARPi resistance in BC PDXs and outperformed the genomic test. In clinical samples, all PALB2‐related tumors were classified as HRR‐deficient by the RAD51 score. The functional biomarker RAD51 enables the identification of PARPi‐sensitive BC and broadens the population who may benefit from this therapy beyond BRCA1/2‐related cancers. Synopsis Sensitive and highly specific biomarkers usable in archived formalin fixed parafin embedded (FFPE) tumour samples are needed to extend the use of PARP inhibitors beyond BRCA1/2‐related cancers. The RAD51 score may satisfy this clinical unmet need. The RAD51 score shows complete discriminative capacity in predicting PARP inhibitor response. The RAD51 score is feasible in routine breast tumor samples without prior exposure to DNA damaging agents. Carrying a PALB2 mutation is associated with a low RAD51score. Graphical Abstract Sensitive and highly specific biomarkers usable in archived formalin fixed parafin embedded (FFPE) tumour samples are needed to extend the use of PARP inhibitors beyond BRCA1/2‐related cancers. The RAD51 score may satisfy this clinical unmet need.

DNA repair by homologous recombination is essential for preserving genomic integrity. The RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) play important roles in this process. In this study, we show that human RAD51 interacts with RAD51C‐XRCC3 or RAD51B‐C‐D‐XRCC2. In addition to being critical for RAD51 focus formation, RAD51C localizes to DNA damage sites. Inhibition of RAD51C results in a decrease in cellular proliferation consistent with a role in repairing double‐strand breaks (DSBs) that occur naturally. To monitor a single DNA repair event, we developed immunofluorescence and chromatin immunoprecipitation (ChIP) methods on human cells where a unique DSB can be created in vivo . Using this system, we observed a single focus of RAD51C, RAD51 and 53BP1, which colocalized with γ‐H2AX. ChIPs revealed that endogenous human RAD51, RAD51C, RAD51D, XRCC2, XRCC3 and MRE11 proteins are recruited in the S–G2 phase of the cell cycle, while Ku80 is recruited during G1. We propose that RAD51C ensures a tight regulation of RAD51 assembly during DSB repair and plays a direct role in repairing DSBs in vivo .

Hypoxia exists in all solid tumors and leads to clinical radioresistance and adverse prognosis. We hypothesized that hypoxia and cellular localization of gold nanoparticles (AuNPs) could be modifiers of AuNP-mediated radiosensitization. The possible mechanistic effect of AuNPs on cell cycle distribution and DNA double-strand break (DSB) repair postirradiation were also studied. Clonogenic survival data revealed that internalized and extracellular AuNPs at 0.5 mg/mL resulted in dose enhancement factors of 1.39 ± 0.07 and 1.09 ± 0.01, respectively. Radiosensitization by AuNPs was greatest in cells under oxia, followed by chronic and then acute hypoxia. The presence of AuNPs inhibited postirradiation DNA DSB repair, but did not lead to cell cycle synchronization. The relative radiosensitivity of chronic hypoxic cells is attributed to defective DSB repair (homologous recombination) due to decreased (RAD51)-associated protein expression. Our results support the need for further study of AuNPs for clinical development in cancer therapy since their efficacy is not limited in chronic hypoxic cells.

AimsThe aim of this study is to investigate the expression profiles of cell cycle related proteins in nasal extranodal NK/T cell lymphoma, nasal type (ENKTCL).MethodsThe expression profiles of cell cycle related proteins were assessed with a cell cycle antibody array and validated by immunohistochemistry. Correlations between the expression levels of proteins and clinical outcomes of patients with nasal ENKTCL were evaluated.ResultsThe expression of full length ataxia telangiectasia mutated (ATM) in nasal ENKTCL significantly decreased compared with that in nasal benign lymphoid proliferative disease (NBLPD), but the expression levels of p-ATM, CHK2 and RAD51 significantly increased in nasal ENKTCL compared with that in NBLPD. Kaplan-Meier analysis showed that the expression levels of p-ATM and CHK2 in nasal ENKTCL were inversely related to overall survival (p=0.011 and p=0.025, respectively).ConclusionAbnormalities in the ATM pathway may play a crucial role in the oncogenesis and chemoradiotherapy resistance of nasal ENKTCL.

Adult T cell leukemia/lymphoma (ATLL) is a life-threatening malignancy of HTLV-1 infected Th lymphocytes. In the present study host–virus interactions were investigated by assessment of HTLV-1 proviral load (PVL) and host gene expression. A cross-sectional study was carried out on 18 ATLL, 10 HAM/TSP patients and 18 HTLV-1 asymptomatic carriers (ACs). DNA and mRNA of the peripheral blood mononuclear cells were extracted for PVL and LAT, BIM, c-FOS and RAD51 gene expression measurement using qRT-PCR. The mean PVL in ATLL patients was 11,430 ± 3770 copies/10 4 which was statistically higher than ACs, 530 ± 119 copies/10 4 , ( p  < 0.001). The expression of BIM, and c-FOS in ATLL patients were higher than HTLV-1 ACs; however, there were no statistically significant differences. The expression of RAD51 as an essential player on DNA repair showed around 160 times increase in ATLL group (166 ± 95) compared to ACs (1.04 ± 0.34) which is statistically significant ( p  < 0.001). Interestingly, there was a positive correlation between RAD51 expression and HTLV-PVL. The expression of LAT as a central adaptor in TCR signaling interestingly was around 36 times higher in ATLL group than ACs (ATLL; 41.33 ± 19.91 vs. ACs; 1.15 ± 0.22, p  < 0.001). This finding showed that TCR signaling pathway mainly provides the growth factors for transformed cells. Furthermore, the overexpression of RAD51 which has been induced in HTLV-1 infected cells as a consequence of virus replication is not able to overcome the DNA damage toward cell transformation.

TRF2 (telomeric repeat binding factor 2) is an essential component of the telomeric cap, where it forms and stabilizes the T-loop junctions. TRF2 forms the T-loops by stimulating strand invasion of the 3' overhang into duplex DNA. TRF2 also has been shown to localize to nontelomeric DNA double-strand breaks, but its functional role in DNA repair has not been examined. Here, we present evidence that TRF2 is involved in homologous recombination (HR) repair of nontelomeric double-strand breaks. Depletion of TRF2 strongly inhibited HR and delayed the formation of Rad51 foci after γ-irradiation, whereas overexpression of TRF2 stimulated HR. Depletion of TRF2 had no effect on nonhomologous end-joining, and overexpression of TRF2 inhibited nonhomologous end-joining. We propose, based on our results and on the ability of TRF2 to mediate strand invasion, that TRF2 plays an essential role in HR by facilitating the formation of early recombination intermediates.

In fission yeast, meiotic prophase nuclei develop structures known as linear elements (LinEs), instead of a canonical synaptonemal complex. LinEs contain Rec10 protein. While Rec10 is essential for meiotic recombination, the precise role of LinEs in this process is unknown. Using in situ immunostaining, we show that Rec7 (which is required for meiosis-specific DNA double-strand break (DSB) formation) aggregates in foci on LinEs. The strand exchange protein Rad51, which is known to mark the sites of DSBs, also localizes to LinEs, although to a lesser degree. The number of Rec7 foci corresponds well with the average number of genetic recombination events per meiosis suggesting that Rec7 marks the sites of recombination. Rec7 and Rad51 foci do not co-localize, presumably because they act sequentially on recombination sites. The localization of Rec7 is dependent on Rec10 but independent of the DSB-inducing protein Rec12/Spo11. Neither Rec7 nor Rad51 localization depends on the LinE-associated proteins Hop1 and Mek1, but the formation of Rad51 foci depends on Rec10, Rec7, and, as expected, Rec12/Spo11. We propose that LinEs form around designated recombination sites before the induction of DSBs and that most, if not all, meiotic recombination initiates within the setting provided by LinEs.

The aim of this study was to estimate a general pattern of meiotic recombination in the domestic dog (Canis familiaris) using immunolocalization of MLH1, a mismatch repair protein of mature recombination nodules. We prepared synaptonemal complex (SC) spreads from 124 spermatocytes of three male dogs and mapped 4959 MLH1 foci along 4712 autosomes. The mean number of MLH1 foci for all autosomes was 40.0 foci per cell. Total recombination length of the male dog autosomal genome map was estimated as 2000 cM. A global pattern of MLH1 foci distribution along the autosomal bivalents was rather similar to that found in the mammals studied: a high frequency near the telomeres and a low frequency near the centromeres. An obligate MLH1 focus in the X-Y pairing region was usually located very close to Xp-Yq telomeres. The distances between MLH1 foci at autosomal bivalents were consistent with crossover interference. A comparison of the interference estimates coming from the distribution of MLH1 interfocus distances and RAD51/MLH1 focus ratio indicated a substantial variation between species in the strength of interference.

In order to study the role of BRCA2 protein in homologous recombination repair and radio‐sensitization, we utilized RNA interference strategy in vitro and in vivo with human tumor cells. HeLa cells transfected with small‐interfering BRCA2 NA (BRCA2 siRNA) (Qiagen) as well as negative‐control siRNA for 48 h were irradiated, and several critical end points were examined. The radiation cell survival level was significantly reduced in HeLa cells with BRCA2 siRNA when compared with mock‐ or negative‐control siRNA transfected cells. DNA double strand break repair as measured by constant field gel‐electrophoresis showed a clear inhibition in cells with BRCA2 siRNA, while little inhibition was observed in cells with negative control siRNA. Our immuno‐staining experiments revealed a significant delay in Rad51 foci formation in cells with BRCA2 siRNA when compared with the control populations. However, none of the non‐homologous end joining proteins nor the phosphorylation of DNA‐dependent protein kinase catalytic subunit was affected in cells transfected with BRCA2 siRNA. In addition, the combined treatment with radiation and BRCA2 siRNA in xenograft model with HeLa cells showed an efficient inhibition of in vivo tumor growth. Our results demonstrate down‐regulation of BRCA2 leads to radio‐sensitization mainly through the inhibition of homologous recombination repair type double‐strand break repair; a possibility of using BRCA2 siRNA as an effective radiosensitizer in tumor radiotherapy may arise. (Cancer Sci 2008; 99: 810–815)

The RAD51 recombinase is central to repair of DNA damage arising from stalled or collapsed replication forks and DNA double strand breaks. Its essential role is revealed by the fact that this function evolved in bacteria but was retained in eukaryotes. In humans some of the RAD51 functions have been relegated to several paralogues which evolved by gene duplication. In addition to mutations, most cancers are also characterized by increased chromosomal instability manifesting as translocations, deletions, insertions, and other more complex forms of chromosomal re-arrangements. Given the central role of RAD51 in protecting against chromosomal instability it stands to reason that RAD51 mutations that alter its function should register in cancer cells. However, pan-cancer analyses of analyzed cancer genomes show a marked absence of RAD51 loss of function mutations leading to a so-called “RAD51 paradox”: increased chromosomal instability despite normal RAD51 function. One hypothesis is that mutations in the RAD51 paralogues may contribute to the genomic instability, meaning that a lack of mutations in RAD51 may be compensated by an increase of mutations in the paralogues. We queried analyzed cancer genomes from COSMIC and mapped all mutations in RAD51 and its paralogues. This revealed an increase in RAD51B, RAD51C and RAD51D paralogue mutations in human cancers. We used established algorithms to determine the probability that any mutation may affect enzyme function. Although, we did not find many “driver” mutations, numerous paralogue mutations were pathogenic or likely to destabilize enzyme function. In silico 3D structure analysis was then used to analyze the potential effect of some of these mutations on protein structure. Gene expression analysis did not reveal any changes in paralogue expression levels. Further, an evolutionary analysis did not uncover any selective pressure for mutations in RAD51 and its paralogues. A comparison of mutations reported on COSMIC with those reported on ClinVar revealed that many mutations primarily in RAD51C and RAD51D are also hereditary. Thus, it appears that an apparent low level of RAD51 mutations in cancer cells is compensated by an increase in paralogues mutations.