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The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.

Purpose Whether an anticoagulant prophylaxis is needed for patients with cancer with a central venous catheter is a highly controversial subject. We designed a study to compare different prophylactic strategies over 3 months of treatment. Methods We performed a phase III prospective, open-label randomized trial. After the insertion of a central venous access device, consecutive patients with planned chemotherapy for cancer were randomized to no anticoagulant prophylaxis, low molecular weight heparin [low molecular weight heparin (LMWH); with isocoagulation doses], or warfarin 1 mg/day. Treatments were given over the first 3 months. Doppler ultrasound and venographies were performed on days 1 and 90, respectively, or sooner in case of clinical presumption of thrombosis. Results A total of 420 patients were randomized, and 407 were evaluable. Forty-two catheter-related deep vein thrombosis (DVT) occurred (10.3 %), 20 in those with no anticoagulation, 8 in those receiving warfarin, and 14 in those receiving LMWH. Nine additional non-related catheter deep vein thrombosis (CDVT) occurred. Anticoagulation significantly reduced the incidence of catheter-related DVT ( p  = 0.035) and catheter non-related DVT ( p  = 0.007), with no difference between warfarin and LMWH. Safety was good (3.4 % of attributable events) but compliance with randomized prophylaxis was lower than expected. Conclusions Prophylaxis showed a benefit regarding catheter-related and non-catheter-related DVT with no increase in serious side effects.

DNA repair is essential in maintaining genome integrity and defects in different steps of the process have been linked to cancer and aging. It is a long lasting question how DNA repair is spatially and temporarily organized in the highly compartmentalized nucleus and whether the diverse nuclear compartments regulate differently the efficiency of repair. Increasing evidence suggest the involvement of nuclear pore complexes in repair of double-strand breaks (DSBs) in yeast. Here, we show that the human nucleoporin 153 (NUP153) has a role in repair of DSBs and in the activation of DNA damage checkpoints. We explore the mechanism of action of NUP153 and we propose its potential as a novel therapeutic target in cancers.

Potentially lethal damage (PLD) repair has been defined as that property conferring the ability of cells to recover from DNA damage depending on the postirradiation environment. Using a novel cyclin dependent kinase 1 inhibitor RO-3306 to arrest cells in the G2 phase of the cell cycle, examined PLD repair in G2 in cultured Chinese hamster ovary (CHO) cells. Several CHO-derived DNA repair mutant cell lines were used in this study to elucidate the mechanism of DNA double-strand break repair and to examine PLD repair during the G2 phase of the cell cycle. While arrested in G2 phase, wild-type CHO cells displayed significant PLD repair and improved cell survival compared with cells released immediately from G2 after irradiation. Both the radiation-induced chromosomal aberrations and the delayed entry into mitosis were also reduced by G2-holding PLD recovery. The PLD repair observed in G2 was observed in nonhomologous end-joining (NHEJ) mutant cell lines but absent in homologous recombination mutant cell lines. From the survival curves, G2-NHEJ mutant cell lines were found to be very sensitive to gamma-ray exposure when compared to G2/homologous recombination mutant cell lines. Our findings suggest that after exposure to ionizing radiation during G2, NHEJ is responsible for the majority of non-PLD repair, and conversely, that the homologous recombination is responsible for PLD repair in G2.

Fluorescence in situhybridization (FISH) is an extremely effective and sensitive approach to analyzing chromosome aberrations. Until recently, this procedure has taken multiple days to complete. The introduction of telomeric and centromeric peptide nucleic acid (PNA) probes has reduced the procedure's duration to several hours, but the protocols still call for a high temperature (80–90°C) step followed by 1–3 h of hybridization. The newest method to speed up the FISH protocol is the use of a microwave to shorten the heating element to less than a minute; however this protocol still calls for a 1-h hybridization period. We have utilized PNA centromere/telomere probes in conjunction with a microwave oven to show telomere and centromere staining in as little as 30 s. We have optimized the hybridization conditions to increase the sensitivity and effectiveness of the new protocol and can effectively stain chromosomes in 2 min and 30 s of incubation. We have found that our new approach to FISH produces extremely clear and distinct signals. Radiation-induced dicentric formation in mouse and human fibroblast cells was analyzed by two individual scorers and the observed dicentrics matched very well.

The incorporation of halogenated pyrmidines such as bromo- and iodo-deoxyuridines (BrdU, IdU) into DNA as thymidine analogs enhances cellular radiosensitivity when high-linear energy transfer (LET) radiation is not used. Although it is known that high-LET ionizing radiation confers fewer biological effects resulting from halogenated pyrimidine incorporation, the exact mechanisms of reduced radiosensitivity with high-LET radiation are not clear. We investigated the radiosensitization effects of halogenated pyrimidines with high-LET radiation using accelerated carbon and iron ions. Cells synchronized into the G1 phase after unifilar (1 cell cycle) and bifilar (2 cell cycles) substitution with 10 μM BrdU were exposed to various degrees of LET with heavy ions and X-rays. We then carried out a colony formation assay to measure cell survival. The γ-H2AX focus formation assay provided a measure of DNA double-strand break (DSB) formation and repair kinetics. Chromosomal aberration formations for the first post-irradiation metaphase were also scored. For both low-LET X-rays and carbon ions (13 keV/μm), BrdU incorporation led to impaired DNA repair kinetics, a larger initial number of DNA DSBs more frequent chromosomal aberrations at the first post-irradiated metaphase, and increased radiosensitivity for cell lethality. The enhancement ratio was higher after bifilar substitution. In contrast, no such synergistic enhancements were observed after high-LET irradiation with carbon and iron ions (70 and 200 keV/μm, respectively), even after bifilar substitution. Our results suggest that BrdU substitution did not modify the number and quality of DNA DSBs produced by high-LET radiation. The incorporation of halogenated pyrimidines may produce more complex/clustered DNA damage along with radicals formed by low-LET ionizing radiation. In contrast, the severity of damage produced by high-LET radiation may undermine the effects of BrdU and account for the observed minimal radiosensitization effects.

Background Fluorescence in situ Hybridization (FISH) utilizes peptide nucleic acid (PNA) probes to identify specific DNA sequences. Traditional techniques have required the heat denaturing of the DNA in formamide followed by multiple hours at moderated temperatures to allow the probe to hybridize to its specific target. Over the past 30 years, advancements in both protocols and probes have made FISH a more reliable technique for both biological research and medical diagnostics, additionally the protocol has been shortened to several minutes. These PNA probes were designed to target and hybridize to both DNA and RNA, and PNA-protein interactions still remain unclear. Results In this study we have shown that a telomeric single stranded specific PNA probe is able to bind to its target without heat denaturing of the DNA and without formamide. We have also identified a centromere specific probe, which was found to bind its target with only incubation with formamide. Conclusions Certain PNA probes are able to hybridize with their targets with minimal to no denaturing of the DNA itself. This limited denaturing preserves the chromosome structure and may lead to more effective and specific staining.

The present study investigated the effect of targeted mutations in the DNA-dependent protein kinase catalytic subunit and phosphorylation domains on the survival of cells in response to different qualities of ionizing radiation. Mutated Chinese hamster ovary V3 cells were exposed to 500 MeV/nucleon initial energy and 200 keV/μm monoenergetic Fe ions; 290 MeV/nucleon initial energy and average 50 keV/μm spread-out Bragg peak C ions; 70 MeV/nucleon initial energy and 1 keV/μm monoenergetic protons; and 0.663 MeV initial energy and 0.3 keV/μm Cs137 γ radiation. The results demonstrated that sensitivity to high linear energy transfer radiation is increased when both S2056 and T2609 clusters each contain a point mutation or multiple mutations are present in either cluster, whereas the phosphoinositide 3 kinase cluster only requires a single mutation to induce the sensitized phenotype of V3 cells. Additionally, the present study demonstrated that sensitivity to DNA cross-linking damage by cisplatin only requires a single mutation in one of the three clusters and that additional point mutations do not increase cell sensitivity.

The aim of this study was to evaluate with a long follow-up the efficacy of concomitant chemoradiotherapy in non-metastatic inflammatory breast cancer (IBC) and to evaluate the breast conservation rate. Between 1990 and 2000, 66 non-metastatic patients with IBC were treated with chemotherapy and concomitant irradiation. The induction chemotherapy consisted of epirubicine, cyclophosphamide and vindesine, in association with split-course bi-fractionated irradiation to a total dose of 65 Gy with concomitant cisplatin and 5-fluorouracil. Maintenance chemotherapy consisted of high-dose methotrexate and six cycles of epirubicine, cyclophosphamide and fluorouracil. Hormonal treatment was given if indicated. Mastectomy was not systemic. Among 65 evaluable patients, 57 (87.6%) achieved a complete clinical response and had a breast conservation. Only six loco regional relapses were noted in six patients with a delay of 20 months and with concomitant metastatic dissemination in four cases. Median disease-free survival (DFS) was 28 months. Median overall survival (OS) was 63 months and median follow-up was 55.5 months. Induction chemotherapy and concomitant irradiation is feasible in patients with IBC, permitting a breast conservation with a high rate of local control with an OS comparable to that of the best recent series.

Radioactive copper (II) (diacetyl-bis N4-methylthiosemicarbazone) (Cu-ATSM) isotopes were originally developed for the imaging of hypoxia in tumors. Because the decay of a (64)Cu atom is emitting not only positrons but also Auger electrons, this radionuclide has great potential as a theranostic agent. However, the success of (64)Cu-ATSM internal radiation therapy would depend on the contribution of Auger electrons to tumor cell killing. Therefore, we designed a cell culture system to define the contributions to cell death from Auger electrons to support or refute our hypothesis that the majority of cell death from (64)Cu-ATSM is a result of high-LET Auger electrons and not positrons or other low-LET radiation. Chinese hamster ovary (CHO) wild type and DNA repair-deficient xrs5 cells were exposed to (64)Cu-ATSM during hypoxic conditions. Surviving fractions were compared with those surviving gamma-radiation, low-LET hadron radiation, and high-LET heavy ion exposure. The ratio of the D(10) values (doses required to achieve 10% cell survival) between CHO wild type and xrs5 cells suggested that (64)Cu-ATSM toxicity is similar to that of high-LET Carbon ion radiation (70 keV/μm). γH2AX foci assays confirmed DNA double-strand breaks and cluster damage by high-LET Auger electrons from (64)Cu decay, and complex types of chromosomal aberrations typical of high-LET radiation were observed after (64)Cu-ATSM exposure. The majority of cell death was caused by high-LET radiation. This work provides strong evidence that (64)Cu-ATSM damages DNA via high-LET Auger electrons, supporting further study and consideration of (64)Cu-ATSM as a cancer treatment modality for hypoxic tumors.