AKT1 is frequently up-regulated in sporadic breast cancer, whereas BRCA1 is frequently mutated in familial breast cancer. Because BRCA1 is involved in homologous recombination (HR), we addressed whether AKT1 also has an effect on this process. We showed that AKT1 repressed HR through cytoplasmic retention of BRCA1 and RAD51 proteins, resulting in a BRCA1-deficient-like phenotype. This process does not require direct BRCA1 phosphorylation by AKT1. The cytoplasmic retention of BRCA1 and RAD51 correlated with activated AKT1 in tumor cell lines and in biopsies from sporadic breast cancers. Under nonpathologic conditions, fibroblast growth factor, which activates AKT1 and stimulates proliferation in fibroblasts, impaired excessive HR without fully inhibiting it, promoting genome stability. Our study reveals that the regulation of BRCA1 and RAD51 is altered in a high frequency of sporadic breast cancers and highlights the role of extracellular AKT signaling-dependent regulation of HR and genome stability. [Cancer Res 2008;68(22):9404-12]

Histone deacetylase inhibitors (HDI) have shown promise as candidate radiosensitizers for many types of cancers. However, the mechanisms of action are not well understood, and whether they could sensitize multiple myeloma (MM) to radiation therapy is unclear. In this study, we show that suberoylanilide hydroxamic acid (SAHA) at low concentrations has minimal cytotoxic effects, yet can significantly increase radiosensitivity of MM cells. SAHA seems to block RAD51 protein response to ionizing radiation, consistent with an inhibitory effect on the formation of RAD51 focus in irradiated MM cells. These effects of SAHA on RAD51 focus are independent of cell-cycle distribution changes. Furthermore, we show that SAHA selectively inhibits the homology-directed repair (HDR) pathway. The results of this study suggest that SAHA, a recently approved HDI in clinical trials for malignancies, at lower concentrations may act as a radiosensitizer via disruption of the RAD51-dependent HDR pathway. Mol Cancer Res; 10(8); 1052-64. Ó2012 AACR.

In order to analyse the relationships between regulation of apoptosis and homologous recombination (HR), we overexpressed proapoptotic Bax or only-BH3 Bid proteins or antiapoptotic Bcl-2 or Bcl-XL, in hamster CHO cells or in SV40-transformed human fibroblasts. We measured HR induced by c-rays, UVC or a specific double-strand cleavage targeted in the recombination substrate by the meganuclease I-SceI. We show here that the induction of both recombinant cells and recombinant colonies was impaired when expressing Bcl-2 family members, in hamster as well as in human cells. Moreover, the pro-as well as antiapoptotic Bcl-2 family members inhibited HR, independently of degradation of the RAD51 recombination protein and of their impact on apoptosis. These data reveal a mechanism of HR downregulation by potentially proapoptotic proteins, distinct from and parallel to degradation of recombination proteins, a situation that should also optimize the efficiency of programmed cell death.

Homology-directed repair (HDR) is essential to limit mutagenesis, chromosomal instability (CIN) and tumorigenesis. We have characterized the consequences of HDR deficiency on anaphase, using markers for incomplete chromosome separation: DAPI-bridges and Ultra-fine bridges (UFBs). We show that multiple HDR factors (Rad51, Brca2 and Brca1) are critical for complete chromosome separation during anaphase, while another chromosome break repair pathway, non-homologous end joining, does not affect chromosome segregation. We then examined the consequences of mild versus severe HDR disruption, using two different dominant-negative alleles of the strand exchange factor, Rad51. We show that mild HDR disruption is viable, but causes incomplete chromosome separation, as detected by DAPI-bridges and UFBs, while severe HDR disruption additionally results in multipolar anaphases and loss of clonogenic survival. We suggest that mild HDR disruption favors the proliferation of cells that are prone to CIN due to defective chromosome separation during anaphase, whereas, severe HDR deficiency leads to multipolar divisions that are prohibitive for cell proliferation.

Background: RNF168 promotes chromosomal break localization of 53BP1 and BRCA1; 53BP1 loss rescues homologous recombination (HR) in BRCA1-deficient cells. Results: RNF168 depletion suppresses HR defects caused by BRCA1 silencing; RNF168 influences HR similarly to 53BP1. Conclusion: RNF168 is important for HR defects caused by BRCA1 loss. Significance: Although RNF168 promotes BRCA1 and 53BP1 localization to chromosomal breaks, RNF168 affects HR similarly to 53BP1.

Genetic stability requires coordination of a network of pathways including DNA repair/recombination and apoptosis. In addition to its canonical anti-apoptotic role, Bcl-2 negatively impacts genome stability. In this study, we identified the breast cancer tumor suppressor BRCA1, which plays an essential role in homologous recombination (HR), as a target for Bcl-2 in the repression of HR. Indeed, ionizing radiation-induced BRCA1 foci assembly was repressed when Bcl-2 was expressed ectopically, in human SV40 fibroblasts, or spontaneously, in lymphoma t(14:18) cells and in HeLa and H460 cancer cell lines. Moreover, we showed that the transmembrane (TM) domain of Bcl-2 was required for both inhibition of BRCA1 foci assembly and the inhibition of HR induced by a double-strand break targeted into an intrachromosomal HR substrate by the meganuclease I-SceI. Fluorescence confocal microscopy, proximity ligation assay, and electron microscopy analyses as well as Western blot analysis of subcellular fractions showed that Bcl-2 and BRCA1 colocalized to mitochondria and endoplasmic reticulum in a process requiring the TM domain of Bcl-2. Targeting BRCA1 to the endomembranes depletes BRCA1 from the nucleus and, thus, accounts for the inhibition of HR. Furthermore, our findings support an apoptosis-stimulatory role for the cytosolic form of BRCA1, suggesting a new tumor suppressor function of BRCA1. Together, our results reveal a new mode of BRCA1 regulation and for HR in the maintenance of genome stability. Cancer Res; 71(10); 3590-602. Ó2011 AACR.

No abstract

We have addressed the role of the F-box helicase 1 (Fbh1) protein during genome maintenance in mammalian cells. For this, we generated two mouse embryonic stem cell lines deficient for Fbh1: one with a homozygous deletion of the N-terminal F-box domain (Fbh1 f/f ), and the other with a homozygous disruption (Fbh1 −/− ). Consistent with previous reports of Fbh1-deficiency in vertebrate cells, we found that Fbh1 −/− cells show a moderate increase in Rad51 localization to DNA damage, but no clear defect in chromosome break repair. In contrast, we found that Fbh1 f/f cells show a decrease in Rad51 localization to DNA damage and increased cytoplasmic localization of Rad51. However, these Fbh1 f/f cells show no clear defects in chromosome break repair. Since some Rad51 partners and F-box-associated proteins (Skp1-Cul1) have been implicated in progression through mitosis, we considered whether Fbh1 might play a role in this process. To test this hypothesis, we disrupted mitosis using catalytic topoisomerase II inhibitors (bisdioxopiperazines), which inhibit chromosome decatenation. We found that both Fbh1 f/f and Fbh1 −/− cells show hypersensitivity to topoisomerase II catalytic inhibitors, even though the degree of decatenation stress was not affected. Furthermore, following topoisomerase II catalytic inhibition, both Fbh1-deficient cell lines show substantial defects in anaphase separation of chromosomes. These results indicate that Fbh1 is important for restoration of normal mitotic progression following decatenation stress.