Acquired cross-linker resistance associated with a novel spliced BRCA2 protein variant for molecular phenotyping of BRCA2 disruption.

BRCA2 encodes a protein with a fundamental role in homologous recombination that is essential for normal development. Carrier status of mutations in BRCA2 is associated with familial breast and ovarian cancer, while bi-allelic BRCA2 mutations can cause Fanconi anemia (FA), a cancer predisposition syndrome with cellular cross-linker hypersensitivity. Cancers associated with BRCA2 mutations can acquire chemo-resistance on relapse. We modeled acquired cross-linker resistance with an FA-derived BRCA2-mutated acute myeloid leukemia (AML) platform. Associated with acquired cross-linker resistance was the expression of a functional BRCA2 protein variant lacking exon 5 and exon 7 (BRCA2ΔE5+7), implying a role for BRCA2 splicing for acquired chemo-resistance. Integrated network analysis of transcriptomic and proteomic differences for phenotyping of BRCA2 disruption infers impact on transcription and chromatin remodeling in addition to the DNA damage response. The striking overlap with transcriptional profiles of FA patient hematopoiesis and BRCA mutation associated ovarian cancer helps define and explicate the 'BRCAness' profile.

Defective sister chromatid cohesion is synthetically lethal with impaired APC/C function.

Warsaw breakage syndrome (WABS) is caused by defective DDX11, a DNA helicase that is essential for chromatid cohesion. Here, a paired genome-wide siRNA screen in patient-derived cell lines reveals that WABS cells do not tolerate partial depletion of individual APC/C subunits or the spindle checkpoint inhibitor p31(comet). A combination of reduced cohesion and impaired APC/C function also leads to fatal mitotic arrest in diploid RPE1 cells. Moreover, WABS cell lines, and several cancer cell lines with cohesion defects, display a highly increased response to a new cell-permeable APC/C inhibitor, apcin, but not to the spindle poison paclitaxel. Synthetic lethality of APC/C inhibition and cohesion defects strictly depends on a functional mitotic spindle checkpoint as well as on intact microtubule pulling forces. This indicates that the underlying mechanism involves cohesion fatigue in response to mitotic delay, leading to spindle checkpoint re-activation and lethal mitotic arrest. Our results point to APC/C inhibitors as promising therapeutic agents targeting cohesion-defective cancers.

The iron-sulfur cluster assembly network component NARFL is a key element in the cellular defense against oxidative stress.

Aim of this study was to explore cellular changes associated with increased resistance to atmospheric oxygen using high-resolution DNA and RNA profiling combined with functional studies. Two independently selected oxygen-resistant substrains of HeLa cells (capable of proliferating at >80% O2, i.e. hyperoxia) were compared with their parental cells (adapted to growth at 20% O2, but unable to grow at >80% O2). A striking consistent alteration found to be associated with the oxygen-resistant state appeared to be an amplified and overexpressed region on chromosome 16p13.3 harboring 21 genes. The driver gene of this amplification was identified by functional studies as NARFL, which encodes a component of the cytosolic iron-sulfur cluster assembly system. In line with this result we found the cytosolic c-aconitase activity as well as the nuclear protein RTEL1, both Fe-S dependent proteins, to be protected by NARFL overexpression under hyperoxia. In addition, we observed a protective effect of NARFL against hyperoxia-induced loss of sister-chromatid cohesion. NARFL thus appeared to be a key factor in the cellular defense against hyperoxia-induced oxidative stress in human cells. Our findings suggest that new insight into age-related degenerative processes may come from studies that specifically address the involvement of iron-sulfur proteins.

Impaired repair of ionizing radiation-induced DNA damage in Cockayne syndrome cells.

Cockayne syndrome (CS) cells are defective in transcription-coupled repair (TCR) and sensitive to oxidizing agents, including ionizing radiation. We examined the hypothesis that TCR plays a role in ionizing radiation-induced oxidative DNA damage repair or alternatively that CS plays a role in transcription elongation after irradiation. Irradiation with doses up to 100 Gy did not inhibit RNA polymerase II-dependent transcription in normal and CS-B fibroblasts. In contrast, RNA polymerase I-dependent transcription was severely inhibited at 5 Gy in normal cells, indicating different mechanisms of transcription response to X rays. The frequency of radiation-induced base damage was 2 × 10(-7) lesions/base/Gy, implying that 150 Gy is required to induce one lesion/30-kb transcription unit; no TCR of X-ray-induced base damage in the p53 gene was observed. Therefore, it is highly unlikely that defective TCR underlies the sensitivity of CS to ionizing radiation. Overall genome repair levels of radiation-induced DNA damage measured by repair replication were significantly reduced in CS-A and CS-B cells. Taken together, the results do not provide evidence for a key role of TCR in repair of radiation-induced oxidative damages in human cells; rather, impaired repair of oxidative lesions throughout the genome may contribute to the CS phenotype.

v-Raf murine sarcoma viral oncogene mutation status in serous borderline ovarian tumors and the effect on clinical behavior.

AIMS: To determine the incidence of activating v-raf murine sarcoma viral oncogene (BRAF) mutations in 30 serous borderline tumors (SBTs) of the ovary and the accompanying implants and to link BRAF mutation status to the clinical behavior of these tumors. METHODS AND RESULTS: Serous borderline tumors and noninvasive implants of 30 patients were analyzed for the presence of the BRAF V599E mutation, and mutation status was correlated to 70 months of clinical follow-up. Mutation status could be assessed in 27 SBTs. Eleven (41%) showed a BRAF mulation. Four (80%) of 5 patients with bilateral SBT showed a BRAF mutation in both ovaries. From the 8 implants that were analyzed for BRAF, 2 (25%) were mutated together with their primary tumor. v-Raf murine sarcoma viral oncogene mutation positive SBTs tend to present with a lower International Federation of Gynecology and Obstetrics stage and a higher tumor volume and are less frequently aneuploid. Seventy months' follow-up indicated no significant recurrence-free survival difference between these groups. CONCLUSIONS: v-Raf murine sarcoma viral oncogene mutations are common in ovarian SBT, are strongly associated with bilateral tumors, and are also found in implants. A larger number of tumors should be investigated to assess clinical importance of BRAF mutation status in SBTs.

Aberrant Epstein-Barr virus persistence in HIV carriers is characterized by anti-Epstein-Barr virus IgA and high cellular viral loads with restricted transcription.

OBJECTIVE: Epstein-Barr virus (EBV)-positive lymphomas in HIV carriers are paralleled by elevated EBV-DNA loads in the circulation. Approximately 20% of asymptomatic HIV carriers also show elevated circulating EBV-DNA loads. We aimed to characterize the nature of this EBV DNA and to determine the transcriptional phenotype of EBV in blood, in relation to serological parameters. DESIGN: A total of 197 random asymptomatic HIV carriers, representing 2% of the Dutch HIV-positive population, were sampled for blood, peripheral blood mononuclear cells and plasma. In addition, 39 EBV-DNA carriers were sampled twice, with a 5-year interval. METHODS: EBV-DNA loads were determined by LightCycler-based real-time polymerase chain reaction (PCR). EBV transcription was studied by nucleic acid sequence-based amplification and reverse transcriptase PCR. IgA and IgG antibodies to EBV antigens EBNA1 and VCA-p18 were quantified by synthetic peptide-based enzyme-linked immunosorbent assay. RESULTS: : Elevated EBV-DNA loads were found in whole blood of 19.3% of the tested HIV population, which were persistent in 82%. Plasma samples were EBV-DNA negative and circulating EBV DNA could be attributed to the B-cell compartment. Transcription of only LMP2 and (non-translated) transcripts from the BamHI-A region of the EBV genome was found, whereas EBNA1, LMP1 and lytic EBV transcripts were absent despite high cellular EBV-DNA loads. IgA-reactivity to VCA-p18 was seen in 69%. IgG to VCA-p18 was significantly higher in high EBV-DNA load carriers. CONCLUSION: Asymptomatic HIV carriers show aberrant EBV persistence in the circulation, characterized by elevated, B-cell-associated EBV-DNA loads. EBV transcription is restricted, arguing for EBV gene shutdown in circulating EBV-carrying B cells. Increased IgA and IgG reactive to VCA-p18 is indicative of increased lytic EBV replication, possibly occurring at mucosal lymphoid sites but not in the circulation.

Cisplatin and doxorubicin repress Vascular Endothelial Growth Factor expression and differentially down-regulate Hypoxia-inducible Factor I activity in human ovarian cancer cells.

Vascular Endothelial Growth Factor (VEGF) and its transcriptional regulator Hypoxia-inducible Factor 1 (HIF-1) play an important role in the process of angiogenesis in many types of cancer, including ovarian cancer. We have examined whether the DNA-damaging drugs cisplatin and doxorubicin and the microtubule inhibitors docetaxel and paclitaxel can affect VEGF expression and HIF-1 activity in three human ovarian cancer cell lines. We demonstrate that cisplatin and doxorubicin abolish hypoxia-induced VEGF mRNA expression in all cell lines, while basal VEGF mRNA expression was also downregulated. Transient transfection with a HIF-1-responsive luciferase construct indicated that cisplatin and doxorubicin inhibited hypoxic activation of HIF-1. Cisplatin repressed HIF-1alpha protein expression in all cell lines. Stimulation of HIF-1alpha protein degradation by cisplatin was observed in the only cell line expressing wild-type p53. Cisplatin also inhibited the synthesis of HIF-1alpha protein for which p53 was dispensable. Interestingly, cisplatin strongly reduced the protein levels of the HIF-1 coactivators p300 and CREB-binding protein (CBP) under hypoxia in all cell lines. Although doxorubicin inhibited hypoxic activation of HIF-1, this drug had no significant effect on the expression levels of HIF-1alpha and hypoxic expression of p300 and CBP was only weakly reduced. Docetaxel and paclitaxel did neither influence VEGF expression nor hypoxia-induced HIF-1 activity. In total, our findings indicate that cisplatin and doxorubicin can repress hypoxic induction of VEGF expression by inhibiting HIF-1 through different mechanisms. This knowledge may be useful for future treatment schedules including agents that target the HIF-1 signalling pathway.