The human recq1 helicase is highly expressed in glioblastoma and plays an important role in tumor cell proliferation

RecQ helicases play an essential role in the maintenance of genome stability. In humans, loss of RecQ helicase function is linked to predisposition to cancer and premature ageing. Distinct molecular functions for the five human RecQ helicases, and RECQ1 in particular, in replication stress and cancer remain to be defined. Within the GLIOMA project we are studying the expression and the role of RECQ1 in replication stress response and repair in glioblastoma and Glioma Stem Cells (GSC). Different papers show that GSCs play an important role in the initiation, aggressiveness and recurrence of glioblastoma. Here, we show that RECQ1 is highly expressed in various types of solid tumours, but only in the case of glioblastoma, the expression of RECQ1 is significantly increased in tumoural tissue relative to the surrounding perilesional. We also show that the depletion of RECQ1 by RNAi results in a significant reduction of cellular proliferation, perturbation of S-phase progression, and spontaneous γ-H2AX foci formation in T98G and U87 glioblastoma cells. Moreover, RECQ1 depleted cells are hypersensitive to hydroxyurea or temozolomide treatment. Collectively, these results indicate that RECQ1 has an important role in the maintenance of genome integrity and might represent a new suitable target for anti cancer therapies aimed to arrest cell proliferation in glioma. In the framework of GLIOMA project we are also working to establish a zebrafish cancer model to characterize the role of RECQ1 in glioblastoma formation and progression in vivo

The human RECQ1 helicase is highly expressed in glioblastoma and plays an important role in tumor cell proliferation

<p>Abstract</p> <p>Background</p> <p>RecQ helicases play an essential role in the maintenance of genome stability. In humans, loss of RecQ helicase function is linked with predisposition to cancer and/or premature ageing. Current data show that the specific depletion of the human RECQ1 helicase leads to mitotic catastrophe in cancer cells and inhibition of tumor growth in mice.</p> <p>Results</p> <p>Here, we show that RECQ1 is highly expressed in various types of solid tumors. However, only in the case of brain gliomas, the high expression of RECQ1 in glioblastoma tissues is paralleled by a lower expression in the control samples due to the poor expression of RECQ1 in non-dividing tissues. This conclusion is validated by immunohistochemical analysis of a tissue microarray containing 63 primary glioblastomas and 19 perilesional tissue samples, as control. We also show that acute depletion of RECQ1 by RNAi results in a significant reduction of cellular proliferation, perturbation of S-phase progression, and spontaneous γ-H2AX foci formation in T98G and U-87 glioblastoma cells. Moreover, RECQ1 depleted T98G and U-87 cells are hypersensitive to HU or temozolomide treatment.</p> <p>Conclusions</p> <p>Collectively, these results indicate that RECQ1 has a unique and important role in the maintenance of genome integrity. Our results also suggest that RECQ1 might represent a new suitable target for anti cancer therapies aimed to arrest cell proliferation in brain gliomas.</p

Viral priming of cell intrinsic innate antiviral signaling by the unfolded protein response

The innate response to a pathogen is critical in determining the outcome of the infection. However, the interplay of different cellular responses that are activated following viral infection and their contribution to innate antiviral signalling has not been clearly established. This work shows that flaviviruses, including Dengue, Zika, West Nile and Tick-borne encephalitis viruses, activate the unfolded protein response before transcription of interferon regulatory factor 3 induced genes. Infection in conditions of unfolded protein response priming leads to early activation of innate antiviral responses and cell intrinsic inhibition of viral replication, which is interferon regulatory factor 3 dependent. These results demonstrate that the unfolded protein response is not only a physiological reaction of the cell to viral infection, but also synergizes with pattern recognition sensing to mount a potent antiviral response

Topoisomerase II\u3b2 mediates the resistance of glioblastoma stem cells to replication stress-inducing drugs

The mesenchymal state in cancer is usually associated with poor prognosis due to the metastatic predisposition and the hyper-activated metabolism. Exploiting cell glucose metabolism we propose a new method to detect mesenchymal-like cancer cells. We demonstrate that the uptake of glucose-coated magnetic nanoparticles (MNPs) by mesenchymal-like cells remains constant when the glucose in the medium is increased from low (5.5 mM) to high (25 mM) concentration, while the MNPs uptake by epithelial-like cells is significantly reduced. These findings reveal that the glucose-shell of MNPs plays a major role in recognition of cells with high-metabolic activity. By selectively blocking the glucose transporter 1 channels we showed its involvement in the internalization process of glucose-coated MNPs. Our results suggest that glucose-coated MNPs can be used for metabolic-based assays aimed at detecting cancer cells and that can be used to selectively target cancer cells taking advantage, for instance, of the magnetic-thermotherapy

Proteomic tools in clinical tissues: unlocking the pathology archives

2009/2010Background: Clinical proteomics aims to characterize the protein networks altered by pathologic processes or therapeutic treatment, and to develop biomarker profiling technologies to promptly detect diseases and treat them more effectively. The challenge of translating proteomic profiling to the bedside lies in applying technologies for the analysis of human tissues, which are routinely obtained by biopsy or surgery, without substantially modifying the clinical workflow. Formalin-fixed and paraffin-embedded (FFPE) tissues are the most widely available archive material suitable to discover new biomarkers or therapy targets, for their validation and for their implementation in clinical practice. However, the lack of standardised guidelines for protein analysis of archive tissues has hampered their regular use in the daily practice. Furthermore, methods to extract proteins and to identify and analyse them also quantitatively have only recently been developed. This research was carried out to develop, standardise and apply qualitative and quantitative proteomic methodologies to archive tissues. Objectives: 1. Development, standardisation and validation of protein extraction methods and protocols from fixed and paraffin-embedded tissues with different pre-analytical conditions. 2. Application of protein analysis in two different research settings: a. at diagnostic level: screening of monoclonal antibodies against Human papillomaviruses (HPVs) in cervix uteri lesions suitable for early detection and progression risk assessment; b. at translational research level: identification of a new therapy target for glioblastoma, deepening the functions of human RECQ1 helicase, an enzyme involved in the maintenance of chromosome stability. Methods: 1. In order to develop, standardise and validate protein extraction methods and protocols from fixed and paraffin-embedded tissues, lung, colon and breast cancer specimens were collected by three groups from the European consortium IMPACTS (Integration of Molecular Pathology and Cell and Tissue Structure). Each specimen was divided into two equivalent sets: one was fixed in formalin and paraffin-embedded; the other one was fixed in a new alcohol-based fixative (FineFix) and paraffin-embedded. In order to compare the fixation procedures and the protein extraction efficiency, a total of 81 protein lysates were prepared in five different laboratories of the consortium. Selected marker proteins were compared, at quantitative level, between the two fixation procedures using the reverse phase protein array technology. Tests were applied to determine whether any of the fixation methods caused differences in protein lysate microarray measurements. Pairwise differences between fixation methods were tested. 2. (a) In diagnostics, protein analysis was applied to 138 FFPE cervix uteri squamous lesions provided by a multicentric retrospective study. They were characterized at molecular level for the presence and type of HPV by using PCR-based systems or the Hybrid Capture assay. Thirty-nine monoclonal antibodies directed against the E7 viral oncoprotein were supplied by a private company. They were validated by generating different tissue microarrays in which multiple punches for each bioptic sample were done, taking different cervical lesions (from low grade to invasive squamous cancer). The antibody providing better staining and specificity results was selected and validated with other tissue microarrays, which included a total of 138 tissue cores from different cervical lesions, and with different HPV-types infections. HPV capsid protein L1, the surrogate biomarkers p16, hTERT, p53 and Ubiquitin, and the proliferative marker Ki67 were also tested in the same way. For the selected E7 antibody and the six biomarkers, one-way analysis of variance was performed to detect significant differences in the mean number of positive cells and in the mean staining intensity (evaluated by a score from 0 to 3+). The analysis was carried out among different types of cervical lesions, both at cytoplasmatic and nuclear level, by epithelial layer. We made use of repeated-measure analysis of variance to ascertain significant differences in the mean number of positive cells between E7 monoclonal antibody and each biomarker. Spearman’s rank correlation test and linear regression analysis were employed in order to detect correlations between E7 and the other biomarkers. 2. (b) The application of proteomics to FFPE tissues at translational research level concerned the expression of the RECQ1 helicase in glioblastoma and in perilesional brain tissue, and its expression in lung, colon and thyroid carcinomas and in their perilesional tissues. All specimens were submitted to immunohistochemistry. The expression pattern of RECQ1 was further analysed on a tissue microarray containing 63 glioblastomas and 19 perilesional brain tissues. Furthermore, to test the function of RECQ1 in glioblastoma growth, RNA interference experiments were performed in different glioblastoma cell lines and the effect of gene silencing on cell growth and proliferation was monitored. The Wilcoxon test for matched-samples was performed to evaluate differences in immunohistochemistry outcome measures, i.e. the proportion of positive cells for RECQ1, the intensity of the signal and the number of positive cells for Ki67, between tumour and perilesional tissues. Spearman’s rank correlation test was applied to assess the relationship between intensity score of RECQ1 and the number of positive cells in tumour specimens. Kruskal-Wallis test was carried out to investigate differences in the proportion of positive cells for RECQ1 and Ki67 among groups defined by patient gender and by RECQ1 intensity score. Results: 1. Quantitative comparison between fixatives for the tested antibody panel (β-Actin, E-cadherin, EGFR, HER2 and ER) yielded a higher immunostaining intensity for the FineFix lysates compared to the FFPE ones (p = 0.04). Signal intensities for EGFR (p = 0.007) and HER2 (p = 0.02) were significantly higher in the FineFix samples compared to the FFPE samples, whereas no differences were observed for β-Actin, E-cadherin and ER. 2. (a) The E7 immunostaining was similar to that of p16, which is the most widely-used surrogate marker in clinical practice for all types of cervical squamous lesions. Furthermore, in high grade squamous intraepithelial lesions (HSILs) E7 was similar to hTERT. In the detection of squamous cell carcinoma, E7 was similar to hTERT and Ubiquitin. A comparison between the mean number of E7 and L1 positive cells showed a significant difference in low grade squamous intraepithelial lesions (p = 0.002), among HSILs (p = 0.01) and in squamous cell carcinoma (p = 0.01). Correlation analysis between the two viral proteins, E7 and L1, allows to identify two possible groups of lesions, both in the low grade squamous intraepithelial lesions (LSILs) and in the HSILs. One group was charcharterized by high level of E7 and low level of L1, the other group was characterized by high level of both marker. This groups were also ri-evalueted at morphological level and lead us to hypothesize two possible models in the progression of LSILs: the viral replicative model and the cell proliferative one. 2. (b) RECQ1 was highly expressed both in the perilesional sections and in lesional sections of all tumours analyzed, with the exception of glioblastoma where its expression was significantly higher in tumour (p = 0.001). RECQ1 expression was confined in the nuclei of the tumour cells, thus suggesting that this enzyme might play an important role in glioblastoma growth. As expected, immunohistochemical analyses against Ki67 showed that protein expression was higher in glioblastoma than in the surrounding normal tissue (p = 0.0008). No correlation between the proportion of positive cells for RECQ1 and Ki67 was found (Spearman’s rho = 0.2, p = 0.2), suggesting that the high expression of RECQ1 in brain tumours is not simply related to the higher degree of proliferation of these cells. An essential role of RECQ1 in tumour growth and proliferation was confirmed by RNA interference experiments and clonogenic assay on cell lines. Conclusions: During my doctorate, all the ojectives set were obtained. The goals achieved trough this research are the following: The standardization of a suitable protein extraction protocol from FFPE tissues. This study demonstrated that it is possible to harmonize protein analysis in archive tissues in different European laboratories by using the same protocols for tissue processing and protein extraction. The optimization of an appropriate protocol for molecular analysis of tissues fixed with new formalin-free fixative FineFix. The possibility of using proteomic approaches also on tissues fixed with reagents alternative to formalin has great potential for future pathology, when the use of formalin will be banned due to its carcinogenetic effects. Furthermore, FineFix has proven a suitable formalin substitute in clinical practice, because it preserves both specimens’ morphology and immunoreactivity. The validation of a new antibody against the E7 oncoviral protein for the detection f HPV in cervical squamous lesions. This leads us to conclude that E7 might be a suitable specific marker for the diagnosis of cervical lesions and could be used also for others HPV-linked diseases. The analysis of a battery of E7 monoclonal antibodies has highlighted their great usefulness in differential diagnosis. An in-depth knowledge of the function of RECQ1 in glioblastoma. RECQ1 expression has been studied in glioblastoma cell lines for the first time ever. This research has demonstrated its important role in glioblastoma growth and proliferation, and in maintaining genome stability. The recommendation of RECQ1 helicase as therapy target in glioblastoma, based on the evidence that the enzyme expression in this tumour type is higher than in perilesional normal tissues or in cancers of different origins. These findings lead us to consider RECQ1 a reliable target for the development of new anti-cancer therapies to eliminate proliferating tumour cells. Finally, this research demonstrated that archive tissues could be a valuable source of material for proteomics research.XXIII Cicl

Phytotoxic Ozone Dose–Response Relationships for Durum Wheat (<i>Triticum durum</i>, Desf.)

Ozone (O3) pollution poses a significant threat to global crop productivity, particularly for wheat, one of the most important staple foods. While bread wheat (Triticum aestivum) is unequivocally considered highly sensitive to O3, durum wheat (Triticum durum) was often found to be more tolerant. This study investigated the O3 dose–response relationships for durum wheat in the Mediterranean region, focusing mainly on grain yield losses, and utilizing the phytotoxic ozone dose (POD) metric to describe the intensity of the stressor. The results from two experiments with Open-Top Chambers performed in 2013 and 2014 on two relatively sensitive durum wheat cultivars confirmed that this wheat species is far more tolerant than bread wheat. The use of a local parameterization of a stomatal conductance model based on field measurements did not significantly improve the dose–response relationships obtained in comparison to the generic parameterization suggested by the Mapping Manual of the United Nations Economic Commission for Europe (UNECE). The POD6 critical level of 5 mmolO3 m−2 for 5% grain yield loss was remarkably higher than the one established for bread wheat with analogous experiments, highlighting that O3 risk assessments based on bread wheat may largely overestimate the damage in the Mediterranean region where durum wheat cultivation prevails

Chitosan Nanoparticles Loaded with N-Acetyl Cysteine to Mitigate Ozone and Other Possible Oxidative Stresses in Durum Wheat

Modern durum wheat cultivars are more prone to ozone stress because of their high photosynthetic efficiency and leaf gas exchanges that cause a greater pollutant uptake. This, in turn, generates an increased reactive oxygen species (ROS) production that is a challenge to control by the antioxidant system of the plant, therefore affecting final yield, with a reduction up to 25%. With the aim of mitigating oxidative stress in wheat, we used chitosan nanoparticles (CHT-NPs) either unloaded or loaded with the antioxidant compound N-acetyl cysteine (NAC), on plants grown either in a greenhouse or in an open field. NAC-loaded NPs were prepared by adding 0.5 mg/mL NAC to the CHT solution before ionotropic gelation with tripolyphosphate (TTP). Greenhouse experiments evidenced that CHT-NPs and CHT-NPs-NAC were able to increase the level of the leaf antioxidant pool, particularly ascorbic acid (AsA) content. However, the results of field trials, while confirming the increase in the AsA level, at least in the first phenological stages, were less conclusive. The presence of NAC did not appear to significantly affect the leaf antioxidant pool, although the grain yield was slightly higher in NAC-treated parcels. Furthermore, both NAC-loaded and -unloaded CHT-NPs partially reduced the symptom severity and increased the weight of 1000 seeds, thus showing a moderate mitigation of ozone injury

Oxidative Stress Mitigation by Chitosan Nanoparticles in Durum Wheat Also Affects Phytochemicals and Technological Quality of Bran and Semolina

In our previous work, durum wheat cv. Fabulis was grown over two consecutive seasons (2016&ndash;2017 and 2017&ndash;2018) in an experimental field in the north of Italy. With the aim of mitigating oxidative stress, plants were subjected to four treatments (deionized water, CHT 0.05 mg/mL, CHT-NPs, and CHT-NPs-NAC) three times during the experiment. Chitosan nanoparticles (CHT-NPs) reduced symptom severity on wheat leaves and positively influenced the final grain yield. The present work aimed at investigating whether CHT treatments and particularly N-acetyl cysteine (NAC)-loaded or -unloaded CHT-NPs, while triggering plant defense mechanisms, might also vary the nutritional and technological quality of grains. For this purpose, the grains harvested from the previous experiment were analyzed for their content in phytochemicals and for their technological properties. The results showed that CHT increased the polyphenol and tocopherol content and the reducing capacity of bran and semolina, even if the positive effect of the nano-formulation remained still unclear and slightly varied between the two years of cultivation. The positive effect against oxidative stress induced by the chitosan treatments was more evident in the preservation of both the starch pasting properties and gluten aggregation capacity, indicating that the overall technological quality of semolina was maintained. Our data confirm the role of chitosan as an elicitor of the antioxidant defense system in wheat also at the grain level

A method for the detection of virus infectivity in single cells and real time: towards an automated fluorescence neutralization test

Virus neutralizing antibodies are critical correlates of protection in vaccine development and are discriminatory in the plaque reduction neutralization test when used for the diagnosis of viral infections. However, neutralization assays are time consuming, labor intensive and highly variable, thus limiting their use. Advances in automated live imaging of cells opened new possibilities for standard virus diagnostic techniques such as neutralization assays. To this end, a reporter cell line based on the translocation of the transcription factor IRF3 in response to infection is proposed. Image acquisition of signal in a microplate format allowed the setup of a rapid, semi-automated and high-throughput fluorescent neutralization test. The study is extended to the live imaging of IRF3 translocations that could potentially cut the time of analysis to few hours. The fluorescent neutralization test is suitable for high-throughput assays and expandable to other viruses of global importance such as Zika virus