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The present investigation aimed to characterise the shape of dose-response curve and determining the frequency distribution of various aberration types as a function of dose and radiation quality in AG01522 primary human fibroblasts in the 0.1- to 1-Gy dose range. For this purpose, the cells were irradiated with 7.7 and 28.5 keV µm(-1) low-energy protons, 62 keV µm(-1 4)He(2+) ions (LNL Radiobiology facility) or X rays and samples collected for 24-colour mFISH analysis. X rays and 7.7 keV µm(-1) protons displayed a quadratic dose-response curve solely for total and simple exchanges, whereas for high-linear energy transfer radiations, a linear dose-response curve was observed for all the aberration categories, with the exception of complex exchanges.

"Telomere integrity is important for chromosome stability. The main objective of our study was to investigate the relationship between telomere length modulation and mitotic chromosome segregation induced by ionizing radiation in human primary fibroblasts. We used X-rays and low-energy protons because of their ability to induce different telomeric responses. Samples irradiated with 4 Gy were fixed at different times up to 6 days from exposure and telomere length, anaphase abnormalities, and chromosome aberrations were analyzed. We observed that X-rays induced telomere shortening in cells harvested at 96 hrs, whereas protons induced a significant increase in telomere length at short as well as at long harvesting times (24 and 96 hrs). Consistent with this, the analysis of anaphase bridges at 96 hrs showed a fourfold increase in X-ray- compared with proton-irradiated samples, suggesting a correlation between telomere length\/dysfunction and chromosome missegregation. In line with these findings, the frequency of dicentrics and rings decreased with time for protons whereas it remained stable after X-rays irradiation. Telomeric FISH staining on anaphases revealed a higher percentage of bridges with telomere signals in X-ray-treated samples than that observed after proton irradiation, thus suggesting that the aberrations observed after X-ray irradiation originated from telomere attrition and consequent chromosome end-to-end fusion. This study shows that, beside an expected "early" chromosome instability induced shortly after irradiation, a delayed one occurs as a result of alterations in telomere metabolism and that this mechanism may play an important role in genomic stability.. . "

The relationship between DNA repair failure and cancer is well established as in the case of rare, high penetrant genes in high cancer risk families. Beside this, in the last two decades, several studies have investigated a possible association between low penetrant polymorphic variants in genes devoted to DNA repair pathways and risk for developing cancer. This relationship would be also supported by the observation that DNA repair processes may be modulated by sequence variants in DNA repair genes, leading to susceptibility to environmental carcinogens. In this framework, the aim of this review is to provide the reader with the state of the art on the association between common genetic variants and cancer risk, limiting the attention to single nucleotide polymorphisms (SNPs) of the NBN gene and providing the various odd ratios (ORs). In this respect, the NBN protein, together with MRE11 and RAD50, is part of the MRN complex which is a central player in the very early steps of sensing and processing of DNA double-strand breaks (DSBs), in telomere maintenance, in cell cycle control, and in genomic integrity in general. So far, many papers were devoted to ascertain possible association between common synonymous and non-synonymous NBN gene polymorphisms and increased cancer risk. However, the results still remain inconsistent and inconclusive also in meta-analysis studies for the most investigated E185Q NBN miscoding variant.

""Long-term biological effects of low-dose radiation are little known nowadays and its carcinogenic risk is estimated on the assumption that the risk remains linearly proportional to the radiation dose down to low-dose levels. However in the last 20 years this hypothesis has gradually begun to seem in contrast with a huge collection of experimental evidences, which has shown the presence of a plethora of non-linear phenomena (including hypersensitivity and induced radioresistance, adaptive response, and non-targeted phenomena like bystander effect and genomic instability) occurring after low-dose irradiation. These phenomena might imply a non-linear behaviour of cancer risk curves in the low-dose region and question the validity of the Linear No-Threshold (LNT) model currently used for cancer risk assessment through extrapolation from existing high-dose data. Moreover only few information is available regarding the effects induced on cryopreserved cells by multi-year background radiation exposure, which might induce a radiation-damage accumulation, due to the inhibition of cellular repair mechanisms. In this framework, the multi-year EXCALIBUR (EXposure effeCts At Low doses of Ionizing radiation in Biological CultURes) experiment, funded by INFN-CNS5, has undertaken a multiscale approach investigation on the biological effects induced in in vitro and in vivo biological systems, in culture and cryopreserved conditions, as a function of radiation quality (X\\\/γ-rays, protons, He-4 ions of various energies) and dose, with particular emphasis on the low-dose region and non-linear phenomena, in terms of different biological endpoints. PACS 87.53.-j-Effects of ionizing radiation on biological systems. PACS 87.53.Ay-Biophysical mechanisms of interaction. PACS 87.53.Bn-Dosimetry\\\/exposure assessment. PACS 87.18.Gh-Cell-cell communication; collective behavior of motile cells""

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Telomeres are specialized nucleoprotein complexes that serve as protective caps of linear eukaryotic chromosomes. The loss of the ends chromosomes due to these un-rejoined DSBs may not be lethal to the cell, but may instead result in the loss of functional telomeres, chromosome fusion, and initiation of breakage/fusion/bridge cycle-induced chromosome instability.The telomeres also partecipate in process of chromosomal repair, as evidenced by “de novo” synthesis of telomere repeats at DSBs and by the capacity of telomeres to bound the essential components of the DNA repair machinery.Based on the observation that high-LET radiations efficientely induce chromosome aberrations, we tested whether low energy protons were able to affect telomere structure. Human primary fibroblasts (HFFF2) and mouse embryonic fibroblasts (MEFs) were irradiated with 4 Gy of 31 keV/um of protons at the radiobiology facility at the 7 MV Van de Graaff CN of the INFN-LNL. Experiments with X-rays were also carried out. Cells were fixed after either 24 hrs or 15 days from treatment. Before harvesting, chromosomes were allowed to condense throught 30’ incubation with Calyculin-A, an agent able to induce premature chromosome condensation. To evaluate radiation-induced alterations of telomere length, Q-FISH staining was performed on condensed chromosomes and interphase nuclei with fluorescent PNA telomeric probe and telomere size was analysed with TFL-TELO software. Preliminary results will be presented and discussed.

In this work we have studied cells derived from five Italian patients characterised by growth delay, immunodeficiency and microcephaly. Because normal development of the immune system requires the introduction of DNA double strand breaks (DSBs) during antigen receptor gene assembly, defect in the repair of these specialised breaks can lead to profound immunodeficiency. The Mre11/Rad50/Nbs1 (M/R/N) complex is a component of both the nonhomologous end-joining and homologous-recombination pathways and plays a critical role in the repair of DNA double strand breaks. In humans, mutations in the Nbs1 gene lead to the autosomic recessive genetic disorder Nijmegen breakage syndrome (NBS), characterised by a bird-like face phenotype, immunodeficiency, developmental abnormalities and high frequency of malignancies. Cells of NBS patients show spontaneous chromosome instability, high sensitivity to ionising radiation, accelerated shortening of telomeres and abnormal cell-cycle checkpoints. NBS has been shown to be associated with a mutation in Nbs1 gene, which codes for the nibrin protein. About 90% of the NBS patients are of Slavic origin and carry the major founder mutation 657del5 in exon 6. To determine whether defects in factors known to be involved in DSBs repair could contribute to the clinical immunodeficiency observed in our patients, cellular and molecular characteristics of lymphoblastoid cells (LCLs) established from them, were analysed.PCR amplification of exon 6 showed the presence of both copies of the Nbs1 gene in all the investigated individuals. Furthermore, normal levels of proteins involved in the repair of DSBs and in cellular response to DNA damage (NBS1, Mre11, Rad50, ATM, ATR, Lig IV, XRCC4) were detected. Radiosensitivity of some of the LCLs analysed was shown by the induction of chromosome aberrations in a G2-phase assay. The efficiency to rejoin DSBs was evaluated by means of PFGE.In order to ascertain the proficency of DNA-damage response pathways, LCLs have been irradiated with 2-10 Gy of X-rays and proteins subjected to immunoblot with either a p53 antibody, a specific antibody for the phosphorylated form of p53, NBS1 or SMC1. We believe that dissecting the clinical and cellular phenotype of these as well as future NBS-like patients will help to identify a subset of individuals with the NBS clinical phenotype, whose analysis will allow for more clear-cut research of new genes involved in the maintenance of genome integrity.

Objectives: Martini et al. developed a nomogram to predict significant (>25%) renal function loss after robot-assisted partial nephrectomy and identified four risk categories. We aimed to externally validate Martini’s nomogram on a large, national, multi-institutional data set including open, laparoscopic, and robot-assisted partial nephrectomy. Methods: Data of 2584 patients treated with partial nephrectomy for renal masses at 26 urological Italian centers (RECORD2 project) were collected. Renal function was assessed at baseline, on third postoperative day, and then at 6, 12, 24, and 48 months postoperatively. Multivariable models accounting for variables included in the Martini’s nomogram were applied to each approach predicting renal function loss at all the specific timeframes. Results: Multivariable models showed high area under the curve for robot-assisted partial nephrectomy at 6- and 12-month (87.3% and 83.6%) and for laparoscopic partial nephrectomy (83.2% and 75.4%), whereas area under the curves were lower in open partial nephrectomy (78.4% and 75.2%). The predictive ability of the model decreased in all the surgical approaches at 48 months from surgery. Each Martini risk group showed an increasing percentage of patients developing a significant renal function reduction in the open, laparoscopic and robot-assisted partial nephrectomy group, as well as an increased probability to develop a significant estimated glomerular filtration rate reduction in the considered time cutoffs, although the predictive ability of the classes was

We present an investigation of the effects on BxPC3 pancreatic cancer cells of proton therapy combined with hyperthermia, assisted by magnetic fluid hyperthermia performed with the use of magnetic nanoparticles. The cells’ response to the combined treatment has been evaluated by means of the clonogenic survival assay and the estimation of DNA Double Strand Breaks (DSBs). The Reactive Oxygen Species (ROS) production, the tumor cell invasion and the cell cycle variations have also been studied. The experimental results have shown that the combination of proton therapy, MNPs administration and hyperthermia gives a clonogenic survival that is much smaller than the single irradiation treatment at all doses, thus suggesting a new effective combined therapy for the pancreatic tumor. Importantly, the effect of the therapies used here is synergistic. Moreover, after proton irradiation, the hyperthermia treatment was able to increase the number of DSBs, even though just at 6 h after the treatment. Noticeably, the magnetic nanoparticles’ presence induces radiosensitization effects, and hyperthermia increases the production of ROS, which contributes to cytotoxic cellular effects and to a wide variety of lesions including DNA damage. The present study indicates a new way for clinical translation of combined therapies, also in the vision of an increasing number of hospitals that will use the proton therapy technique in the near future for different kinds of radio-resistant cancers.

Actin-related protein 2/3 complex subunit 1B (ARPC1B) deficiency is a recently described inborn error of immunity (IEI) presenting with combined immunodeficiency and characterized by recurrent infections and thrombocytopenia. Manifestations of immune dysregulation, including colitis, vasculitis, and severe dermatitis, associated with eosinophilia, hyper-IgA, and hyper-IgE are also described in ARPC1B-deficient patients. To date, hematopoietic stem cell transplantation seems to be the only curative option for patients. ARPC1B is part of the actin-related protein 2/3 complex (Arp2/3) and cooperates with the Wiskott–Aldrich syndrome protein (WASp) in the regulation of the actin cytoskeleton remodeling and in driving double-strand break clustering for homology-directed repair. In this study, we aimed to investigate radiosensitivity (RS) in ARPC1B-deficient patients to assess whether it can be considered an additional disease trait. First, we performed trio-based next-generation-sequencing studies to obtain the ARPC1B molecular diagnosis in our index case characterized by increased RS, and then we confirmed, using three different methods, an increment of radiosensitivity in all enrolled ARPC1B-deficient patients. In particular, higher levels of chromatid-type aberrations and γH2AX foci, with an increased number of cells arrested in the G2/M-phase of the cell cycle, were found in patients’ cells after ionizing radiation exposition and radiomimetic bleomycin treatment. Overall, our data suggest increased radiosensitivity as an additional trait in ARPC1B deficiency and support the necessity to investigate this feature in ARPC1B patients as well as in other IEI with cytoskeleton defects to address specific clinical follow-up and optimize therapeutic interventions.

Introduction We aimed to test the hypothesis that the immune-modulatory effect of statins may improve survival outcomes in patients with non-muscle invasive bladder cancer (NMIBC). We focused on a cohort of patients diagnosed with high risk NMIBC, that were treated with intravesical BCG immunotherapy. Patients and methods We included patients at first diagnosis of T1 high grade NMIBC after transurethral resection of bladder (TURB). All procedures were performed at 18 different tertiary institutions between January 2002 and December 2012. Univariable and multivariable models were used to test differences in terms of residual tumour, disease recurrence, disease progression and overall mortality (OM) rates. Results Overall, 1510 patients with T1 high grade NMIBC at TURB were included in our analyses. Of these, 402 (26.6%) were statin users. At multivariable analysis, statin use was associated with a higher rates of high grade BC at re-TURB (OR: 1.37, 95%CI: 1.04-1.78; p=0.022), while at follow-up it was not independently associated with OM (HR: 0.71, 95%CI: 0.50-1.03; p=0.068) and disease progression rates (HR: 0.97, 95%CI: 0.79-1.19; p=0.753). Conversely, statin use has been shown to be independently associated with a lower risk of recurrence (HR:0.80, 95%CI: 0.67-0.95; p=0.009). The median recurrence-free survival was 47 (95%CI 40-49) months for those classified as non-statin users vs. 53 (95%CI 48-68) months in those classified as statin users. Conclusions Statin daily intake do not compromise oncological outcomes in high risk NMIBC patients treated with BCG. Moreover, statin may have a beneficial effect on recurrence rates in this cohort of patients.

Most human tumors maintain telomere lengths by telomerase, whereas a portion of them (10%&ndash, 15%) uses a mechanism named alternative lengthening of telomeres (ALT). The telomeric G-quadruplex (G4) ligand RHPS4 is known for its potent antiproliferative effect, as shown in telomerase-positive cancer models. Moreover, RHPS4 is also able to reduce cell proliferation in ALT cells, although the influence of G4 stabilization on the ALT mechanism has so far been poorly investigated. Here we show that sensitivity to RHPS4 is comparable in ALT-positive (U2OS, SAOS-2) and telomerase-positive (HOS) osteosarcoma cell lines, unlinking the telomere maintenance mechanism and RHPS4 responsiveness. To investigate the impact of G4 stabilization on ALT, the cardinal ALT hallmarks were analyzed. A significant induction of telomeric doublets, telomeric clusterized DNA damage, ALT-associated Promyelocytic Leukaemia-bodies (APBs), telomere sister chromatid exchanges (T-SCE) and c-circles was found exclusively in RHPS4-treated ALT cells. We surmise that RHPS4 affects ALT mechanisms through the induction of replicative stress that in turn is converted in DNA damage at telomeres, fueling recombination. In conclusion, our work indicates that RHPS4-induced telomeric DNA damage promotes overactivation of telomeric recombination in ALT cells, opening new questions on the therapeutic employment of G4 ligands in the treatment of ALT positive tumors.

Locally advanced or metastatic renal cell carcinomas (mRCCs) account for up to 15% of all kidney cancer diagnoses. Systemic therapies (with or without surgery) represent gold standard treatments, mostly based on tyrosine kinase inhibitors in association with immunotherapy. We provide an overview of the current knowledge of miRNAs as predictors of treatment resistance. A systematic review of the literature was carried out in January 2022 following the PICO methodology. Overall, we included seven studies—four testing plasmatic miRNAs, two exosomal miRNAs, and one urinary miRNA. A total of 789 patients were included (354 for plasmatic miRNAs, 366 for urinary miRNAs, and 69 for exosomal miRNAs). Several miRNAs were tested within the included studies, but six plasmatic (miR9-5-p¸ miR-192, miR193-3p, miR-501-3p¸ miR-221, miR-376b-3p) one urinary (miR-30a-5p), and three exosomal (miR-35-5p, miR-301a-3p, miR-1293) were associated with resistance to systemic treatments or treatment failure in mRCC patients. Results showed a fair accuracy of these biomarkers in predicting treatment resistance and overall survival. However, to date, the biomarkers tested have not been validated and their clinical uses are not recommended. Nevertheless, the literature results are encouraging; future large clinical trials are warranted to validate the effectiveness of these tools in clinical decision-making.

Objective: To examine the effect of diagnosis year, defined as contemporary (2010–2014), intermediate (2006–2009) and historical (2001–2005) on cancer-specific mortality (CSM) in patients with metastatic renal cell carcinoma (mRCC). Methods: Within Surveillance, Epidemiology, and End Results registry (2001–2014), we identified patients with mRCC. Cumulative incidence and competing risks regression (CRR) models examined CSM, after accounting for other-cause mortality. Finally, we performed subgroup analyses according to histological subtype: clear-cell mRCC (ccmRCC) versus non-ccmRCC. Results: We identified 15,444 patients with mRCC. Of those, 41.0, 28.7 and 30.3% were diagnosed, respectively, in the contemporary, intermediate and historical years. Of all, 47.1, 5.3 and 47.6% were, respectively, ccmRCC, non-ccmRCC and other mRCC histological variants [sarcomatoid mRCC, cyst-associated mRCC, collecting duct carcinoma and mRCC not otherwise specified (NOS)]. Overall, 24-month CSM rates were, respectively, 61.0, 63.7 and 67.3% in contemporary, intermediate and historical patients. In all patients, multivariable CRR models exhibited higher CSM in intermediate (HR 1.11; pÂ

A combination of carbon ions/photons irradiation and hyperthermia as a novel therapeutic approach for the in-vitro treatment of pancreatic cancer BxPC3 cells is presented. The radiation doses used are 0&ndash, 2 Gy for carbon ions and 0&ndash, 7 Gy for 6 MV photons. Hyperthermia is realized via a standard heating bath, assisted by magnetic fluid hyperthermia (MFH) that utilizes magnetic nanoparticles (MNPs) exposed to an alternating magnetic field of amplitude 19.5 mTesla and frequency 109.8 kHz. Starting from 37 &deg, C, the temperature is gradually increased and the sample is kept at 42 &deg, C for 30 min. For MFH, MNPs with a mean diameter of 19 nm and specific absorption rate of 110 &plusmn, 30 W/gFe3o4 coated with a biocompatible ligand to ensure stability in physiological media are used. Irradiation diminishes the clonogenic survival at an extent that depends on the radiation type, and its decrease is amplified both by the MNPs cellular uptake and the hyperthermia protocol. Significant increases in DNA double-strand breaks at 6 h are observed in samples exposed to MNP uptake, treated with 0.75 Gy carbon-ion irradiation and hyperthermia. The proposed experimental protocol, based on the combination of hadron irradiation and hyperthermia, represents a first step towards an innovative clinical option for pancreatic cancer.

Cancer cells need to acquire telomere maintenance mechanisms in order to counteract progressive telomere shortening due to multiple rounds of replication. Most human tumors maintain their telomeres expressing telomerase whereas the remaining 15%&ndash, 20% utilize the alternative lengthening of telomeres (ALT) pathway. Previous studies have demonstrated that ionizing radiations (IR) are able to modulate telomere lengths and to transiently induce some of the ALT-pathway hallmarks in normal primary fibroblasts. In the present study, we investigated the telomere length modulation kinetics, telomeric DNA damage induction, and the principal hallmarks of ALT over a period of 13 days in X-ray-exposed primary cells. Our results show that X-ray-treated cells primarily display telomere shortening and telomeric damage caused by persistent IR-induced oxidative stress. After initial telomere erosion, we observed a telomere elongation that was associated to the transient activation of a homologous recombination (HR) based mechanism, sharing several features with the ALT pathway observed in cancer cells. Data indicate that telomeric damage activates telomeric HR-mediated repair in primary cells. The characterization of HR-mediated telomere repair in normal cells may contribute to the understanding of the ALT pathway and to the identification of novel strategies in the treatment of ALT-positive cancers.

Objective: To compare the outcomes of robotic radical nephrectomy (RRN) to those of laparoscopic radical nephrectomy (LRN) for large renal masses. Methods: This was a retrospective analysis of RRN and LRN cases performed for large (≥ cT2) renal masses from 2004 to 2017 and collected in the multi-institutional international database (ROSULA: RObotic SUrgery for LArge renal masses). Peri-operative, functional, and oncologic outcomes were compared between each approach. Descriptive analyses were performed and presented as medians with interquartile ranges. Inverse probability of treatment weighting-adjusted multivariable analyses were used to identify predictors of peri-operative complications. Kaplan–Meier analysis and Cox regression models were used to assess survival outcomes. Results: A total of 941 patients (RRN = 404, LRN = 537) were identified. There was no difference in terms of gender, age, and clinical tumor size. Over the study period, RRN had an annual increase of 11.75% (95% CI [7.34, 17.01] p < 0.001) and LRN had an annual decline of 5.39% (95% CI [−6.94, −3.86] p < 0.001). Patients undergoing RRN had higher BMI (27.6 [IQR 24.8–31.1] vs. 26.5 [24.1–30.0] kg/m 2 , p < 0.01). Operative duration was longer for RRN (185.0 [150.0–237.2] vs. 126 [90.8–180.0] min, p < 0.001). Length of stay was shorter for RRN (3.0 [2.0–4.0] vs. 5.0 [4.0–7.0] days, p < 0.001). RRN cases presented more advanced disease (higher pathologic staging [pT3–4 52.5 vs. 24.2%, p < 0.001], histologic grade [high grade 49.3 vs. 30.4%, p < 0.001], and rate of nodal disease [pN1 5.4 vs. 1.9%, p < 0.01]). Surgical approach did not represent an independent risk factor for peri-operative complications (OR 1.81 95% CI [0.97–3.39], adjusted p = 0.2). The main study limitation is the retrospective design. Conclusions: This study represents the largest known multi-center comparison between RRN and LRN. The two procedures seem to offer similar peri-operative outcomes. Notably, RRN has been increasingly utilized, especially in the setting of more advanced and surgically challenging disease without increasing the risk of peri-operative complications.

Telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) constitute the core telomerase enzyme that maintains the length of telomeres. Telomere maintenance is affected in a broad range of cancer and degenerative disorders. Taking advantage of gain‐ and loss‐of‐function approaches, we show that Argonaute 2 (AGO2) promotes telomerase activity and stimulates the association between TERT and TERC. AGO2 depletion results in shorter telomeres as well as in lower proliferation rates in vitro and in vivo. We also demonstrate that AGO2 interacts with TERC and with a newly identified sRNA (terc‐sRNA), arising from the H/ACA box of TERC. Notably, terc‐sRNA is sufficient to enhance telomerase activity when overexpressed. Analyses of sRNA‐Seq datasets show that terc‐sRNA is detected in primary human tissues and increases in tumors as compared to control tissues. Collectively, these data uncover a new layer of complexity in the regulation of telomerase activity by AGO2 and might lay the foundation for new therapeutic targets in tumors and telomere diseases.

Background The benefit of cytoreductive nephrectomy (CNT) for cancer-specific mortality (CSM)-free survival is unclear in contemporary metastatic non-clear cell renal cell carcinoma (non-ccmRCC) patients. Objective To assess the role of CNT in non-ccmRCC patients. Design, setting, and participants Within Surveillance, Epidemiology, and End Results registry (2001–2014), we identified patients with non-ccmRCC. Intervention CNT versus no CNT in non-ccmRCC patients. Outcome measurements and statistical analysis Multivariable logistic regression, cumulative incidence, competing-risks regression models, incremental survival benefit (ISB), conditional survival, and landmark analyses were performed. Sensitivity analyses focused on histological subtypes and most contemporary patients (2010–2014). Results and limitations Of 851 patients with non-ccmRCC, 67.6% underwent CNT. In multivariable logistic regression, year of diagnosis in contemporary ( p 0.001) and intermediate ( p =0.008) tertiles, as well as age ≥75 yr ( p 0.001) yielded lower CNT rates. Cumulative incidence showed 2-yr CSM of 52.6% versus 77.7%, respectively, after CNT versus no CNT. CSM after CNT versus no CNT was invariably lower in all histologic subtypes and in contemporary patients. Multivariable competing-risks regression models predicting CSM favored CNT (hazard ratio [HR]: 0.38, confidence interval: 0.30–0.47, p 0.001) in all patients and in all subgroups defined by histologic subtypes (HR: 0.14–0.43, all p ≤0.02), as well as in contemporary patients (HR: 0.32, p 0.001). The ISB analyses yielded statistically significant and clinically meaningful CSM-free survival benefit of +3 mo after CNT versus no CNT in individuals with observed CSM-free survival ≤24 mo. The 2-yr CSM-free survival increased from baseline of 46.1% versus 19.4% (Δ=26.7%, p 0.001) to 70.3% versus 54.4% (Δ=15.9%, p =0.005) after CNT versus no CNT, in patients that survived 12 mo, respectively. Landmark analyses rejected bias favoring CNT. Data were retrospective. Conclusions CSM is lower after CNT for non-ccmRCC in all histologic subtypes and in contemporary patients except for unproven ISB in collecting duct patients. This observation should encourage greater CNT consideration in non-ccmRCC. Patient summary Cytoreductive nephrectomy appears to improve survival in metastatic non-clear cell renal cell carcinoma, but it is used infrequently.

Purpose: The European Association of Urology (EAU) guidelines for penile cancer (PC) are exclusively based on retrospective studies and have low grades of recommendation. The aim of this study was to assess the adherence to guidelines by investigating the management strategies for primary tumours and inguinal lymph nodes. Methods: We retrospectively reviewed the clinical charts of 176 PC patients who underwent surgery in eight European centres from 2010 to 2016. The stage and grade were assessed according to the 2009 AJCC–UICC TNM classification system. To assess adherence rates, we compared theoretical and practical adherence to the EAU guidelines. Results: Overall, 176 patients were enrolled. Partial amputation was the most frequent surgical approach (39%). 53.7% of tumours were stage Tis-T1b and the remaining 46.3% were stage T2-T4. Palpable lymph nodes were detected in 30.1% of patients and 45.1% underwent lymphadenectomy (LY). A sizeable group of tumours (43.2%) were N0. For primary treatment, adherence to the EAU guidelines was good (66%). In non-adherent cases, reasons for discrepancy were patient’s choice (17%), surgeon’s preference (36%), and other causes (47%). For LY, the guideline adherence was 70%, with either patient’s or surgeon’s choice or other causes accounting for discrepancy in 28, 20, and 52% of non-adherent cases, respectively. Conclusion: Adherence to the EAU guidelines for PC was quite high across the eight European centres involved in the study. This notwithstanding, strategies for further improvement should be developed and evenly adopted.

Background While partial nephrectomy (PN) represents the standard surgical management for cT1 renal masses, its role for cT2 tumors is controversial. Robot-assisted PN (RAPN) is being increasingly implemented worldwide. Objective To analyze perioperative, functional, and oncological outcomes of RAPN for cT2 tumors. Design, setting, and participants Retrospective analysis of a large multicenter, multinational dataset of patients with nonmetastatic cT2 masses treated with robotic surgery (ROSULA: RObotic SUrgery for LArge renal mass). Intervention Robotic-assisted PN. Outcome measurements and statistical analysis Patients’ demographics, lesion characteristics, perioperative variables, renal functional data, pathology, and oncological data were analyzed. Univariable and multivariable regression analyses assessed the relationships with the risk of intra-/postoperative complications, recurrence, and survival. Results and limitations A total of 298 patients were analyzed. Median tumor size was 7.6 (7–8.5) cm. Median RENAL score was 9 (8–10). Median ischemia time was 25 (20–32) min. Median estimated blood loss was 150 (100–300) ml. Sixteen patients had intraoperative complications (5.4%), whereas 66 (22%) had postoperative complications (5% were Clavien grade ≥3). Multivariable analysis revealed that a lower RENAL score (odds ratio [OR] 0.46, 95% confidence interval [CI] 0.21–0.65, p = 0.02) and pathological pT2 stage (OR 0.51, 95% CI 0.12–0.86, p = 0.001) were protective against postoperative complications. A total of 243 lesions (82%) were malignant. Twenty patients (8%) had positive surgical margins. Ten deaths and 25 recurrences/metastases occurred at a median follow-up of 12 (5–35) mo. At univariable analysis, higher pT stage was predictive of a likelihood of recurrences/metastases (p = 0.048). While there was a significant deterioration of renal function at discharge, this remained stable over time at 1-yr follow-up. The main limitation of this study is its retrospective design. Conclusions RAPN in the setting of select cT2 renal masses can safely be performed with acceptable outcomes. Further studies are warranted to corroborate our findings and to better define the role of robotic nephron sparing for this challenging indication. Patient summary This report shows that robotic surgery can be used for safe removal of a large renal tumor in a minimally invasive fashion, maximizing preservation of renal function, and without compromising cancer control.

The pentacyclic acridine RHPS4 is a highly potent and specific G-quadruplex (G4) ligand, which binds and stabilizes telomeric G4 leading to the block of the replication forks at telomeres and consequently to telomere dysfunctionalization. In turn, the cell recognizes unprotected telomeres as DNA double-strand breaks with consequent activation of DNA repair response at telomeres, cellular growth impairment, and death. Data from the literature showed the capability of this compound to sensitize U251MG glioblastoma radioresistant cell line to X-rays sparsely ionizing radiations. In the present paper, it was investigated whether RHPS4 is also able to increase the effect of clinical carbon ion beams (cells irradiated in the middle of a spread-out Bragg peak, in the energy range of 246-312 MeV·μm-1 and a dose-averaged linear energy transfer of 46 keV·μm-1 ). Interestingly, also for charged particles whose damage inflicted to DNA is more complex than that of sparsely ionizing radiations and results in higher Relative Biological Effectiveness (RBE), RHPS4 significantly potentiated the radiation effect in terms of cell killing, delayed rejoining of DNA double-strand breaks (γ-H2AX and 53BBP1 immunofluorescence staining), chromosome aberrations (pan-centromeric/telomeric FISH and multicolor FISH), and G2 /M-phase accumulation in GBM cells. Overall, the results provide the first evidence that the combined administration of the G4-ligand RHPS4 with charged particles interfere with cellular processes involved in cell survival leading to radiosensitization of highly radioresistant tumor cells.

Reversine is a small synthetic molecule that inhibits multiple mitotic kinases, including MPS1 as well as Aurora kinase A and B (AURKA and AURKB). Here, we investigated the effects of reversine on p53-deficient vs p53-proficient cancer cells. We found that low doses (~0.5 µM) of reversine, which selectively inhibit MPS1 and hence impair the spindle assembly checkpoint, kill human TP53 (-/-) colon carcinoma cells less efficiently than their wild-type counterparts. In sharp contrast, high doses (~5 µM) of reversine induced hyperploidization and apoptosis to a much larger extent in TP53 (-/-) than in TP53 (+/+) cells. Such a selective cytotoxicity could not be reproduced by the knockdown of MPS1, AURKA and AURKB, neither alone nor in combination, suggesting that it involves multiple (rather than a few) molecular targets of reversine. Videomicroscopy-based cell fate profiling revealed that, in response to high-dose reversine, TP53 (-/-) (but not TP53 (+/+) ) cells undergo several consecutive rounds of abortive mitosis, resulting in the generation of hyperpolyploid cells that are prone to succumb to apoptosis upon the activation of mitotic catastrophe. In line with this notion, the depletion of anti-apoptotic proteins of the BCL-2 family sensitized TP53 (-/-) cells to the toxic effects of high-dose reversine. Moreover, the knockdown of BAX or APAF-1, as well as the chemical inhibition of caspases, limited the death of TP53 (-/-) cells in response to high-dose reversine. Altogether, these results suggest that p53-deficient cells are particularly sensitive to the simultaneous inhibition of multiple kinases, including MPS1, as it occurs in response to high-dose reversine.

// Mirella Tanori 1 , Arianna Casciati 1 , Francesco Berardinelli 2 , Simona Leonardi 1 , Emanuela Pasquali 1 , Francesca Antonelli 1 , Barbara Tanno 1 , Paola Giardullo 2,3 , Alessandro Pannicelli 4 , Gabriele Babini 5 , Ilaria De Stefano 3 , Antonella Sgura 2 , Mariateresa Mancuso 1 , Anna Saran 1 and Simonetta Pazzaglia 1 1 Laboratory of Biomedical Technologies, Agenzia Nazionale per le Nuove Tecnologie, l’Energia e lo Sviluppo Economico Sostenibile (ENEA), CR-Casaccia, Rome, Italy 2 Department of Science, University Roma Tre, Rome, Italy 3 Department of Radiation Physics, Universita degli Studi Guglielmo Marconi, Rome, Italy 4 Technical Unit of Energetic Efficiency, ENEA, Rome, Italy 5 Department of Physics, University of Pavia, Pavia, Italy Correspondence to: Simonetta Pazzaglia, email: // Keywords : cerebellum, expression profiles, medulloblastoma, apoptosis, senescence Received : June 14, 2016 Accepted : June 26, 2016 Published : July 07, 2016 Abstract Mutations in DNA repair pathways are frequent in human cancers. Hence, gaining insights into the interaction of DNA repair genes is key to development of novel tumor-specific treatment strategies. In this study, we tested the functional relationship in development and oncogenesis between the homologous recombination (HR) factor Rad54 and Parp-1 , a nuclear enzyme that plays a multifunctional role in DNA damage signaling and repair. We introduced single or combined Rad54 and Parp-1 inactivating germline mutations in Ptc1 heterozygous mice, a well-characterized model of medulloblastoma, the most common malignant pediatric brain tumor. Our study reveals that combined inactivation of Rad54 and Parp-1 causes a marked growth delay culminating in perinatallethality, providing for the first time evidence of synthetic lethal interactions between Rad54 and Parp-1 in vivo . Although the double mutation hampered investigation of Rad54 and Parp-1 interactions in cerebellum tumorigenesis, insights were gained by showing accumulation of endogenous DNA damage and increased apoptotic rate in granule cell precursors (GCPs). A network-based approach to detect differential expression of DNA repair genes in the cerebellum revealed perturbation of p53 signaling in Rad54 -/- / Parp-1 -/- / Ptc1 +/- , and MEFs from combined Rad54/Parp-1 mutants showed p53/p21-dependent typical senescent features. These findings help elucidate the genetic interplay between Rad54 and Parp-1 by suggesting that p53/p21-mediated apoptosis and/or senescence may be involved in synthetic lethal interactions occurring during development and inhibition of tumor growth.

One of the hallmarks of cancer consists in the ability of tumor cells to divide indefinitely, and to maintain stable telomere lengths throughout the activation of specific telomere maintenance mechanisms (TMM). Therefore in the last fifteen years, researchers proposed to target telomerase or telomeric structure in order to block limitless replicative potential of cancer cells providing a fascinating strategy for a broad-spectrum cancer therapy. In the present review, we report in vitro and in vivo evidence regarding the use of chemical agents targeting both telomerase or telomere structure and showing promising antitumor effects when used in combination with ionizing radiation (IR). RNA interference, antisense oligonucleotides (e.g., GRN163L), non-nucleoside inhibitors (e.g., BIBR1532) and nucleoside analogs (e.g., AZT) represent some of the most potent strategies to inhibit telomerase activity used in combination with IR. Furthermore, radiosensitizing effects were demonstrated also for agents acting directly on the telomeric structure such as G4-ligands (e.g., RHPS4 and Telomestatin) or telomeric-oligos (T-oligos). To date, some of these compounds are under clinical evaluation (e.g., GRN163L and KML001). Advantages of Telomere/Telomerase Targeting Compounds (T/TTCs) coupled with radiotherapy may be relevant in the treatment of radioresistant tumors and in the development of new optimized treatment plans with reduced dose adsorbed by patients and consequent attenuation of short- end long-term side effects. Pros and cons of possible future applications in cancer therapy based on the combination of T/TCCs and radiation treatment are discussed.

Many and varied are the proposed mechanisms that lead to resistance to ionizing radiation treatment. Among them, an inverse relationship between telomere length and radioresistance has been recently advanced. Investigating such a relationship in TK6 lymphoblasts, we found that clones originating from cells survived to 4 Gy of X-rays showed a significantly higher telomere length when compared with clones grown from untreated cells. The lengthening observed was not attributable to a radiation-induced increase in telomerase activity, as demonstrated by TRAP assay performed in the dose range of 1–10 Gy. Given the evidence that TK6 whole population was characterized by heterogeneity in cellular mean telomere length and telomere loss, we tested the hypothesis that a process of selection may favour cells with longer telomeres (more radioresistant cells) following exposure to irradiation. In order to do this 15 independent TK6 clones were selected and characterized for telomere length and loss on the basis of q-FISH and flow-FISH analysis. Among the screened clones four characterized by long telomeres and four characterized by short telomeres were tested for their radiosensitivity by means of clonogenic assay. The results obtained showed that, in our experimental conditions (cellular model, radiation doses) no significant correlation was observed between radiosensitivity and mean telomere lengths, whereas a positive correlation was observed with respect to telomere loss. Overall, these results indicate that telomere loss and not mean telomere length plays a critical role in the phenomenon of radiosensitivity/radioresistance.

Keeping Cancer Cells At Bay Cancer cells are often aneuploid; that is, they have an abnormal number of chromosomes. But to what extent this contributes to the tumorigenic phenotype is not clear. Senovilla et al. (p. 1678 ; see the Perspective by Zanetti and Mahadevan ) found that tetraploidization of cancer cells can cause them to become immunogenic and thus aid in their clearance from the body by the immune system. Cells with excess chromosomes put stress on the endoplasmic reticulum, which leads to movement of the protein calreticulin to the cell surface. Calreticulin exposure in turn caused recognition of cancer cells in mice by the host immune system. Thus, the immune system appears to serve a protective role in eliminating hyperploid cells that must be overcome to allow unrestricted growth of cancer cells.

Proteins involved in DNA double-strand break (DSB) repair localize within the promyelocytic leukemia nuclear bodies (PML-NBs), whose disruption is at the root of the acute promyelocytic leukemia (APL) pathogenesis. All-trans-retinoic acid (RA) treatment induces PML-RARα degradation, restores PML-NB functions, and causes terminal cell differentiation of APL blasts. However, the precise role of the APL-associated PML-RARα oncoprotein and PML-NB integrity in the DSB response in APL leukemogenesis and tumor suppression is still lacking. Primary leukemia blasts isolated from APL patients showed high phosphorylation levels of H2AX (γ-H2AX), an initial DSBs sensor. By addressing the consequences of ionizing radiation (IR)-induced DSB response in primary APL blasts and RA-responsive and -resistant myeloid cell lines carrying endogenous or ectopically expressed PML-RARα, before and after treatment with RA, we found that the disruption of PML-NBs is associated with delayed DSB response, as revealed by the impaired kinetic of disappearance of γ-H2AX and 53BP1 foci and activation of ATM and of its substrates H2AX, NBN, and CHK2. The disruption of PML-NB integrity by PML-RARα also affects the IR-induced DSB response in a preleukemic mouse model of APL in vivo. We propose the oncoprotein-dependent PML-NB disruption and DDR impairment as relevant early events in APL tumorigenesis.

Objective: To evaluate contemporary international trends in the implementation of minimally invasive adrenalectomy and to assess contemporary outcomes of different minimally invasive techniques performed at urologic centers worldwide. Methods: A retrospective multinational multicenter study of patients who underwent minimally invasive adrenalectomy from 2008 to 2013 at 14 urology institutions worldwide was included in the analysis. Cases were categorized based on the minimally invasive adrenalectomy technique: conventional laparoscopy (CL), robot-assisted laparoscopy (RAL), laparoendoscopic single-site surgery (LESS), and mini-laparoscopy (ML). The rates of the four treatment modalities were determined according to the year of surgery, and a regression analysis was performed for trends in all surgical modalities. Results: Overall, a total of 737 adrenalectomies were performed across participating institutions and included in this analysis: 337 CL (46 % of cases), 57 ML (8 %), 263 LESS (36 %), and 80 RA (11 %). Overall, 204 (28 %) operations were performed with a retroperitoneal approach. The overall number of adrenalectomies increased from 2008 to 2013 (p = 0.05). A transperitoneal approach was preferred in all but the ML group (pÂ

G-quadruplex (G4) interacting agents are a class of ligands that can bind to and stabilise secondary structures located in genomic G-rich regions such as telomeres. Stabilisation of G4 leads to telomere architecture disruption with a consequent detrimental effect on cell proliferation, which makes these agents good candidates for chemotherapeutic purposes. RHPS4 is one of the most effective and well-studied G4 ligands with a very high specificity for telomeric G4. In this work, we tested the in vitro efficacy of RHPS4 in astrocytoma cell lines, and we evaluated whether RHPS4 can act as a radiosensitising agent by destabilising telomeres. In the first part of the study, the response to RHPS4 was investigated in four human astrocytoma cell lines (U251 MG, U87MG, T67 and T70) and in two normal primary fibroblast strains (AG01522 and MRCS). Cell growth reduction, histone H2AX phosphorylation and telomere-induced dysfunctional foci (TIF) formation were markedly higher in astrocytoma cells than in normal fibroblasts, despite the absence of telomere shortening. In the second part of the study, the combined effect of submicromolar concentrations of RHPS4 and X-rays was assessed in the U251 MG glioblastoma radioresistant cell line. Long-term growth curves, cell cycle analysis and cell survival experiments, clearly showed the synergistic effect of the combined treatment. Interestingly the effect was greater in cells bearing a higher number of dysfunctional telomeres. DNA double-strand breaks rejoining after irradiation revealed delayed repair kinetics in cells pre-treated with the drug and a synergistic increase in chromosome-type exchanges and telomeric fusions. These findings provide the first evidence that exposure to RHPS4 radiosensitizes astrocytoma cells, suggesting the potential for future therapeutic applications.

High-resolution multicolour banding FISH (mBAND) and multiplex FISH (mFISH) were used to analyse the aberrations of chromosome 1 in irradiated-AG01522 human primary fibroblasts. The cells were exposed to 1Gy of a panel of radiation of different qualities, such as X-rays, low-energy protons (28keV/μm), helium-ions (62keV/μm) and carbon-ions (96 and 252keV/μm). mBAND and mFISH analysis in calyculin-A G2-condensed chromosome spreads allowed us to detect intra- and interchromosome aberrations involving chromosome 1, including simple and complex-type exchanges, inversions (both para- and pericentric ones), deletions and rings. The data indicate that the induction of chromosomal exchanges was influenced by both Linear energy transfer (LET) and particle types. Moreover, the complex-to-simple exchanges ratio (C-ratio) and interchromosome to intrachromosome exchanges ratio (F-ratio) were evaluated by mFISH and mBAND techniques, respectively. Our results indicate that the C-ratio is a more reliable marker of radiation quality, with values that increased linearly in an LET-dependent manner. In addition, by means of mBAND analysis, the distribution of radiation-induced breakpoints along chromosome 1 was analyzed and compared with the expected distributions of the breaks. The expected values were calculated assuming a random distribution of the breakpoints. The data indicate that, irrespective of the radiation that was used, the breakpoints were non-randomly distributed along chromosome 1. In particular, breaks in the pericentromeric region were encountered at a higher frequency than expected. A deeper analysis revealed that breaks were not located in the constitutive heterochromatin (G-bands 1p11/1q11 and 1q12), but rather in a region comprised between 1p11.2 and 1p22.1, which includes G-light and G-dark bands.

Mitochondria are the main cellular source of Reactive Oxygen Species (ROS). Alterations of mitochondrial metabolism and consequent loss of mitochondrial membrane potential may lead to redox imbalance and in turn to DNA damage, chromosomal instability and apoptosis. On the other hand, impaired mitochondrial functions may either exacerbate the detrimental effects of geno- and cytotoxic agents or may bring beneficial cellular responses. To study the role of mitochondria within this framework, AG01522 human primary fibroblasts were incubated with the mitochondrial polymerase γ inhibitor 2',3'-dideoxycytidine (ddC), leading to mitochondrial DNA (mtDNA) depletion and to mitochondrial dysfunctions. The successful treatment toward mtDNA depletion was confirmed by Complex-IV subunit I (COX-I) immunofluorescence and western blot assays. mtDNA-depleted cells and their counterparts were ultrastructurally characterized by transmission electron microscopy. mtDNA-depleted cells showed dramatic mitochondrial alterations such as fragmentation and cristae disruption along with a reduction of the mitochondrial membrane potential and elevated levels of ROS. Despite increased ROS levels, we did not find any difference in telomere length between ddC-treated and untreated cells. The spontaneous rate of DNA double-strand breaks (DSBs) and chromosome aberrations was significantly enhanced in mtDNA-depleted cells whereas the induction of DSBs by low-Linear Energy Transfer (LET) (X-rays; 7.7keV/μm protons) and high-LET radiations (28.5keV/μm protons) did not differ when compared with normal cells. However, in irradiated cells impaired mitochondrial functions seemed to bring beneficial cellular responses to the detrimental effect of radiations. In fact, after X-irradiation mtDNA-depleted cells show less remaining unrejoined DSBs than normal cells and furthermore a lower induction of cytogenetic damage. Overall, these data show that active mitochondrial functions are required for the proper maintenance of cellular genome stability in primary fibroblasts.

""Purpose: Biological effects produced by low doses of ionizing radiations, though relevant for the risk assessment, have not been fully elucidated. The aim of the present work was to evaluate cytogenetic endpoints, as telomere dysfunctions and chromosome instability in the low-dose range as a function of radiation quality. In particular, we analyzed whether the telomere length was modulated, as well as the involvement of telomeres in chromosomal alterations at anaphase, and the yield of stable simple and complex chromosome aberrations. Materials and methods: AG01522 human primary fibroblasts were irradiated with 0.1-1 Gy of X-rays, protons (28.5 keV\\\/μm), and 4He2+ ions (62 keV\\\/μm). Frequency of chromosome bridges carrying or not telomeric signals and telomere length were measured in irradiated samples up to 72 h. Moreover, chromosome instability was measured using multicolor fluorescence in situ hybridization (mFISH). Results: The results evidenced a linear energy transfer (LET)- and dose-dependent response in the frequency of anaphase bridges induction and in their persistence as a function of time. However, neither variation in telomere length and telomere loss, nor in the proportion of bridges bearing telomeric signals, was detected, thus indicating a minor role of telomeres in the generation of the radiation-induced chromosome bridges. Chromosome instability followed a linear-dependence with dose and LET, showing a far higher extent of complex translocations in helium-ion-irradiated cells than in proton- or X-ray-irradiated samples. Conclusions: Altogether, the results indicated the lack of telomere involvement in cytogenetic effects induced by low-dose ionizing radiation. On the contrary, chromosome aberration yield and spectrum were LET- and dose-dependent.. . ""

This work is about the setup of an in vitro system to report low-dose of X-rays as measured as cytogenetic damage. Q- and multicolor FISH (m-FISH), for telomere length and chromosome instability analysis, respectively, were compared to evaluate their sensitivity in the low-dose range in human primary fibroblasts. No telomere length modulation was observed up to 1 Gy in cycling fibroblasts, though reported for high doses, by that frustrating the purpose of using it as a low-exposure marker. To date the m-FISH is the best setup for the assessment of the chromosome structural damage: it allows stable and instable aberrations to be detected all over the karyotype. Stable ones such as balanced translocations, are not eliminated due to cell-cycle as unstable ones, so they are considered transmissible markers for retrospective dosimetry. The induction of chromosome damage showed a clear dependence on dose delivered; unstable aberrations were demonstrated after doses of 0.1 Gy, and stable aberrations after doses higher than 0.5 Gy. Summarizing, q-FISH is unfit to report low exposures while m-FISH provides better results: unstable aberrations are sensible short-term reporters, while stable ones long report exposures but with a higher induction threshold.

Biallelic mutations in the NBN gene are responsible for the Nijmegen breakage syndrome (NBS), a rare autosomal recessive disorder characterized by chromosome instability and hypersensitivity to ionising radiation (IR). Epidemiological data evidence that the NBN gene can be considered a susceptibility factor for cancer development, as demonstrated by the fact that almost 40% of NBS patients have developed a malignancy before the age of 21. Interestingly, also NBN heterozygotes, which are clinically asymptomatic, display an elevated risk to develop some types of malignant tumours, especially breast, prostate and colorectal cancers, lymphoblastic leukaemia, and non-Hodgkin’s lymphoma (NHL). So far, nine mutations in the NBN gene have been found, at the heterozygous state, in cancer patients. Among them, the 657del5, the I171V and the R215W mutations are the most frequently described. The pathogenicity of these mutations is presumably connected with their occurrence in the highly conserved BRCT tandem domains of the NBN protein, which are present in a large superfamily of proteins and are recognized as major mediators of processes related to cell-cycle checkpoints and DNA repair. This review will focus on the current state-of-knowledge regarding the correlation between carriers of NBN gene mutations and the proneness to the development of malignant tumours.

The Nijmegen breakage syndrome (NBS) is a genetic disorder. caused by mutations in NBN gene and characterized by chromosomal. instability and hypersensitivity to ionizing radiations (IR).. The N-terminus of nibrin (NBN) contains a tandem breast cancer. 1 (BRCA1) carboxy-terminal (BRCT) domain that represents one. of the major mediators of phosphorylation-dependent protein-protein. interactions in processes related to cell cycle checkpoint and. DNA repair functions. Patients with NBS compound heterozygous. for the 657del5 hypomorphic mutation and for the Arg215Trp. missense mutation (corresponding to the 643C[T gene mutation). display a clinical phenotype more severe than that of patients. homozygous for the 657del5 mutation. Here, we show that both. the 657del5 and Arg215Trp mutations, occurring within the tandem. BRCT domains of NBN, although not altering the assembly. of the MRE11\\\/RAD50\\\/NBN (MRN) complex, affect the MRE11. IR-induced nuclear foci (IRIF) formation and the DNA doublestrand. break (DSB) signaling via the phosphorylation of both. ataxia-telangiectasia-mutated (ATM) kinase and ATM downstream. targets (e.g., SMC1 and p53). Remarkably, data obtained. indicate that the cleavage of the BRCT tandem domains of NBN. by the 657del5 mutation affects the DNA damage response less. than the Arg215Trp mutation. Indeed, the 70-kDa NBN fragment,. arising from the 657del5 mutation, maintains the capability. to interact with MRE11 and c-H2AX and to form IRIF. Altogether,. the role of the tandem BRCT domains of NBN in the. localization of the MRN complex at the DNA DSB and in the. activation of the damage response is highlighted.

Background: Telomeres are potential markers of mitotic cellular age and are associated with physical ageing process. Long-term endurance training and higher aerobic exercise capacity (VO2max) are associated with improved survival, and dynamic effects of exercise are evident with ageing. However, the association of telomere length with exercise training and VO2max has so far been inconsistent. Our aim was to assess whether muscle telomere length is associated with endurance exercise training and VO2max in younger and older people. Methods: Twenty men; 10 young (22–27 years) and 10 old (66–77 years), were studied in this cross-sectional study. Five out of 10 young adults and 5 out of 10 older were endurance athletes, while other halves were exercising at a medium level of activity. Mean telomere length was measured as telomere/single copy gene-ratio (T/S-ratio) using quantitative real time polymerase chain reaction. VO2max was measured directly running on a treadmill. Results: Older endurance trained athletes had longer telomere length compared with older people with medium activity levels (T/S ratio 1.12±0.1 vs. 0.92±0.2, p = 0.04). Telomere length of young endurance trained athletes was not different than young non-athletes (1.47±0.2 vs. 1.33±0.1, p = 0.12). Overall, there was a positive association between T/S ratio and VO2max (r = 0.70, p = 0.001). Among endurance trained athletes, we found a strong correlation between VO2max and T/S ratio (r = 0.78, p = 0.02). However, corresponding association among non-athlete participants was relatively weak (r = 0.58, p = 0.09). Conclusion: Our data suggest that VO2max is positively associated with telomere length, and we found that long-term endurance exercise training may provide a protective effect on muscle telomere length in older people. © 2012 Østhus et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The genetic or functional inactivation of p53 is highly prevalent in human cancers. Using high-content videomicroscopy based on fluorescent TP53(+/+) and TP53(-/-) human colon carcinoma cells, we discovered that SP600125, a broad-spectrum serine/threonine kinase inhibitor, kills p53-deficient cells more efficiently than their p53-proficient counterparts, in vitro. Similar observations were obtained in vivo, in mice carrying p53-deficient and -proficient human xenografts. Such a preferential cytotoxicity could be attributed to the failure of p53-deficient cells to undergo cell cycle arrest in response to SP600125. TP53(-/-) (but not TP53(+/+) ) cells treated with SP600125 became polyploid upon mitotic abortion and progressively succumbed to mitochondrial apoptosis. The expression of an SP600125-resistant variant of the mitotic kinase MPS1 in TP53(-/-) cells reduced SP600125-induced polyploidization. Thus, by targeting MPS1, SP600125 triggers a polyploidization program that cannot be sustained by TP53(-/-) cells, resulting in the activation of mitotic catastrophe, an oncosuppressive mechanism for the eradication of mitosis-incompetent cells.

BACKGROUND: Ring chromosome 17 syndrome is a rare disease that arises from the breakage and reunion of the short and long arms of chromosome 17. Usually this abnormality results in deletion of genetic material, which explains the clinical features of the syndrome. Moreover, similar phenotypic features have been observed in cases with complete or partial loss of the telomeric repeats and conservation of the euchromatic regions. We studied two different cases of ring 17 syndrome, firstly, to clarify, by analyzing gene expression analysis using real-time qPCR, the role of the telomere absence in relationship with the clinical symptoms, and secondly, to look for a new model of the mechanism of ring chromosome transmission in a rare case of familial mosaicism, through cytomolecular and quantitative fluorescence in-situ hybridization (Q-FISH) investigations. RESULTS: The results for the first case showed that the expression levels of genes selected, which were located close to the p and q ends of chromosome 17, were significantly downregulated in comparison with controls. Moreover, for the second case, we demonstrated that the telomeres were conserved, but were significantly shorter than those of age-matched controls; data from segregation analysis showed that the ring chromosome was transmitted only to the affected subjects of the family. CONCLUSIONS: Subtelomeric gene regulation is responsible for the phenotypic aspects of ring 17 syndrome; telomere shortening influences the phenotypic spectrum of this disease and strongly contributes to the familial transmission of the mosaic ring. Together, these results provide new insights into the genotype-phenotype relationships in mild ring 17 syndrome.

Mutations at the protein-coding region of TERT gene (chromosome 5p15.33) have been well characterized in patients affected by a constitutional telomere syndrome, including diskeratosis, aplastic anemia and pulmonary fibrosis (Calado RT, Hematology, ASH 2009:338). In rare cases somatic mutations may occur in de novo MDS/AML (Calado RT, Young NS. Blood 2008;111:4446). Mutations involving the regulatory region of TERT gene have been recently identified in a consistent proportion of familial and sporadic melanoma and in a subset of tumors originating from tissues with low rate of self-renewal (glioblastoma, liposarcoma, oligodendroglioma bladder cancer, upper urinary tract cancer). These mutations create new binding motifs for Ets/TCF transcription factors, thus increasing TERT gene transcription (Killela PJ et al, PNAS 2013;110:6021; Horn S et al, Science 2013;339:959). As far as we know TERT promoter mutations were never described in MDS. Material and Methods Index Case. A 52 years old woman was selected because of a complex phenotype including abnormal skin pigmentation, familial pulmonary fibrosis, osteoarthritis, and a refractory cytopenia with multilineage dysplasia (RCMD, WHO 2008) with a 47,XX,+8 karyotype. Screening Cases. Mutational analysis was extended to a cohort of 115 patients (72 males; 44 females, median age 69) referred to the Laboratory of Cytogenetics, Hematology Department,University of Perugia, Italy. Cytogenetics was normal in 49 cases (43%). Abnormalities included: isolated del(5q) (seven cases, 6%); del(5q) plus one additional change (three cases, 3%); isolated del(20q) (fourteen cases, 12%); trisomy 8 (five cases, 4%); monosomy 7 (two cases, 2%); -Y (three cases, 3%); del(11q) (two cases, 2%); complex karyotype (twenty-four cases, 21%); other aberrations (six cases, 5%). Genomic DNA was extracted from bone marrow (BM) of all cases and from peripheral blood T lymphocytes of index case using Salting out method. In all cases TERT promoter was screened using PCR based DHPLC assay (Wave system; MD Transgenomic Inc. Omaha, NE). The 274bp amplicon was amplified with forward primer 5'GTCCTGCCCCTTCACCTTC3' and reverse primer 5'AGCACCTCGCGGTAGTGG3' using Robust Start Taq KAPA2G (Biosystems, Woburn, MA). DNA from abnormal elution profiles were re-amplified and sequenced by Sanger method (ABI 3500 Genetic Analyzer, Applied Biosystems). Variations were detected using Finch TV v. 1.4.0. In the index case 23 amplicons encompassing all 16 exons of TERT gene were also screened (NC_000005.9). Mutations cloning was carried out after RNA extraction (Trizol, Invitrogen, Life Technologies, Paisley, UK), reverse transcription (ThermoscriptTM RT-PCR System, Invitrogen) and amplification (TERT_2CF (5'-CAGCGCTACTGGCAAATGCG-3' Ta-61,4°C; and TERT_2543R (5'-GGCACTGGACGTAGGACTTG-3' Ta-61,4°C). PCR products were sub-cloned into pGEM-T easy vector (Promega, Madison, WI, USA) and sequenced. Results Index Case. Patient was a compound heterozygous for two germline variations: a nonsense mutation c.1209C>A p.C403* (exon 2) and a missense mutation c.2455C>T p.R819C (exon 8). A putative somatic A>C transition 57 bp before the ATG start codon was detected only in BM cells (Table). Screening Cases. DHPLC analysis showed three patients (2.6%) with a two-peak elution profile. Sequencing revealed a 10 bp duplication (c.1-110_1-101) in case 2; a c.1-124 C>T point mutation in case 3; a point mutation c.1-78 C>T in case 4 (Table). Comment We showed that TERT gene promoter variations are recurrent events in 2.6% of MDS patients. Only low/int1 risk MDS (IPSS) were affected in this series. The c.1-57A>C, previously reported as a germline activating variation in familial melanoma (Horn S et al, Science 2013;339:959), was likely a somatic mutation in our index case, thus supporting its role in clonal MDS proliferation. We first found the melanoma activating c.1-124 C>T point mutation (Huang FW et al, Science 2013;339:957; Killela PJ et al, PNAS 2013;110:6021) in a MDS with isolated del(5q). The new variation of our case n.4 does not appear to introduce a new transcription factor binding site (http://www.cbrc.jp/research/db/TFSEARCH.html), whereas the 10bp duplication of case 2 indicates an hypothetical binding site for Ikaros. Further results from ongoing functional studies in these cases will be presented. Disclosures: No relevant conflicts of interest to declare.