A systems radiation biology approach to unravel the role of chronic low-dose-rate gamma-irradiation in inducing premature senescence in endothelial cells.

PURPOSE: The aim of this study was to explore the effects of chronic low-dose-rate gamma-radiation at a multi-scale level. The specific objective was to obtain an overall view of the endothelial cell response, by integrating previously published data on different cellular endpoints and highlighting possible different mechanisms underpinning radiation-induced senescence. MATERIALS AND METHODS: Different datasets were collected regarding experiments on human umbilical vein endothelial cells (HUVECs) which were chronically exposed to low dose rates (0, 1.4, 2.1 and 4.1 mGy/h) of gamma-rays until cell replication was arrested. Such exposed cells were analyzed for different complementary endpoints at distinct time points (up to several weeks), investigating cellular functions such as proliferation, senescence and angiogenic properties, as well as using transcriptomics and proteomics profiling. A mathematical model was proposed to describe proliferation and senescence. RESULTS: Simultaneous ceasing of cell proliferation and senescence onset as a function of time were well reproduced by the logistic growth curve, conveying shared equilibria between the two endpoints. The combination of all the different endpoints investigated highlighted a dose-dependence for prematurely induced senescence. However, the underpinning molecular mechanisms appeared to be dissimilar for the different dose rates, thus suggesting a more complex scenario. CONCLUSIONS: This study was conducted integrating different datasets, focusing on their temporal dynamics, and using a systems biology approach. Results of our analysis highlight that different dose rates have different effects in inducing premature senescence, and that the total cumulative absorbed dose also plays an important role in accelerating endothelial cell senescence.

Targeting cellular stress in vitro improves osteoblast homeostasis, matrix collagen content and mineralization in two murine models of osteogenesis imperfecta.

Most cases of dominantly inherited osteogenesis imperfecta (OI) are caused by glycine substitutions in the triple helical domain of type I collagen α chains, which delay collagen folding, and cause the synthesis of collagen triple helical molecules with abnormal structure and post-translational modification. A variable extent of mutant collagen ER retention and other secondary mutation effects perturb osteoblast homeostasis and impair bone matrix quality. Amelioration of OI osteoblast homeostasis could be beneficial both to osteoblast anabolic activity and to the content of the extracellular matrix they deposit. Therefore, the effect of the chemical chaperone 4-phenylbutyrate (4-PBA) on cell homeostasis, collagen trafficking, matrix production and mineralization was investigated in primary osteoblasts from two murine models of moderate OI, Col1a1+/G349C and Col1a2+/G610C. At the cellular level, 4-PBA prevented intracellular accumulation of collagen and increased protein secretion, reducing aggregates within the mutant cells and normalizing ER morphology. At the extracellular level, increased collagen incorporation into matrix, associated with more mature collagen fibrils, was observed in osteoblasts from both models. 4-PBA also promoted OI osteoblast mineral deposition by increasing alkaline phosphatase expression and activity. Targeting osteoblast stress with 4-PBA improved both cellular and matrix abnormalities in culture, supporting further in vivo studies of its effect on bone tissue composition, strength and mineralization as a potential treatment for classical OI.

Predicting DNA damage foci and their experimental readout with 2D microscopy: a unified approach applied to photon and neutron exposures.

The consideration of how a given technique affects results of experimental measurements is a must to achieve correct data interpretation. This might be challenging when it comes to measurements on biological systems, where it is unrealistic to have full control (e.g. through a software replica) of all steps in the measurement chain. In this work we address how the effectiveness of different radiation qualities in inducing biological damage can be assessed measuring DNA damage foci yields, only provided that artefacts related to the scoring technique are adequately considered. To this aim, we developed a unified stochastic modelling approach that, starting from radiation tracks, predicts both the induction, spatial distribution and complexity of DNA damage, and the experimental readout of foci when immunocytochemistry coupled to 2D fluorescence microscopy is used. The approach is used to interpret γ-H2AX data for photon and neutron exposures. When foci are reconstructed in the whole cell nucleus, we obtain information on damage characteristics "behind" experimental observations, as the average damage content of a focus. We reproduce how the detection technique affects experimental findings, e.g. contributing to the saturation of foci yields scored at 30 minutes after exposure with increasing dose and to the lack of dose dependence for yields at 24 hours.

MODELLING γ-H2AX FOCI INDUCTION TO MIMIC LIMITATIONS IN THE SCORING TECHNIQUE.

An approach based on track-structure calculations has been developed to take account of artefacts occurring during γ-H2AX foci detection in 2D images of samples analyzed through immunocytochemistry. The need of this works stems from the observed saturation in foci yields measured after X-ray doses higher than few grays, hindering an unambiguous quantification of DNA damage and of radiation effectiveness. The proposed modelling approach allows to simulate the observer's point of view for foci scoring, mimicking the selection of a slice Δz of the cell nucleus due to the microscope depth of field, and applying a clustering algorithm to group together damages within a resolution parameter r. Calculation results were benchmarked with experimental measurements at an early time-point for mouse breast cancer cells, irradiated with X-ray doses in the range 0-5 Gy. The model is able to reproduce the saturation in experimental data.

A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC.

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between colorectal tumor cells and the immune system. Colorectal carcinoma is among the most common type of cancer, typically treated by surgery, chemotherapy, and radiotherapy. Advantages of radiotherapy in targeting the tumor are well known. However, even limited exposures of healthy tissues are of great concern, particularly regarding the effects on the intestinal barrier and the immune system. The adopted setup allows to study the interplay between two cell populations in a condition more similar to the physiological one, when compared to normal cell cultures. For this purpose, we resort to different techniques and we used an in vitro co-culture model, based on Caco-2 cells differentiated as a monolayer and PBMC, sharing the same culture medium. This protocol has been developed to focus on both macroscopic effects, i.e. cell viability and Trans-Epithelial Electrical Resistance (TEER), and, through western blot, molecular alterations, i.e. the activation of inflammatory pathway in immune cells and the tight junction protein expression in Caco-2 cells. Initial evaluation of radiation effects on Caco-2 cell viability was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays, while TEER was measured at fixed time intervals through an ohmmeter specifically designed for co-culture systems. In this way, the effects due to radiation, the presence of Peripheral Blood Mononuclear Cells (PBMC), and eventually their synergistic effect, can be demonstrated. Through these complementary techniques, we observed a high radio-resistance of Caco-2 within the range of 2 - 10 Gy of X-rays and an increased Caco-2 monolayer permeability when PBMCs were added. In particular, PBMC presence was found to be associated with the variation in the tight junction scaffold proteins expression.

Functional analysis of a novel ENG variant in a patient with hereditary hemorrhagic telangiectasia (HHT) identifies a new Sp1 binding-site.

Hereditary Hemorrhagic Telangiectasia (HHT) is a rare disease, with an autosomal dominant inheritance and a worldwide incidence of about 1: 5000 individuals. In >80% of patients, HHT is caused by mutations in either ENG or ACVRL1, which code for ENDOGLIN and Activin A Receptor Type II-Like Kinase 1 (ALK1), belonging to the TGF-ß/BMP signalling pathway. Typical HHT clinical features are mucocutaneous telangiectases, arteriovenous malformations, spontaneous and recurrent epistaxis, as well as gastrointestinal bleedings. An additional, but less frequent, clinical manifestation in some HHT patients is the presence of Pulmonary Arterial Hypertension (PAH). The aim of this work is to describe the functional role of a novel ENG intronic variant found in a patient affected by both HHT and PAH, in order to assess whether it has a pathogenic role. We proved that the variant lies in a novel binding-site for the transcription factor Sp1, known to be involved in the regulation of ENG and ACVRL1 transcription. We confirmed a pathogenic role for this intronic variant, as it significantly reduces ENG transcription by affecting this novel Sp1 binding-site.

Ataluren-driven restoration of Shwachman-Bodian-Diamond syndrome protein function in Shwachman-Diamond syndrome bone marrow cells.

Shwachman-Diamond syndrome (SDS) is a rare inherited recessive disease mainly caused by mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene, which encodes for the homonymous protein SBDS, whose function still remains to be fully established. SDS affects several organs causing bone marrow failure, exocrine pancreatic insufficiency, skeletal malformations, and cognitive disorders. About 15% of SDS patients develop myelodysplastic syndrome (MDS) and are at higher risk of developing acute myeloid leukemia (AML). Deficiency in SBDS expression has been associated with increased apoptosis and lack of myeloid differentiation in bone marrow hematopoietic progenitors. Importantly, most SDS patients carry nonsense mutations in SBDS. Since ataluren is a well-characterized small molecule inhibitor that can suppress nonsense mutations, here, we have assessed the efficacy of this drug in restoring SBDS expression in hematopoietic cells obtained from a cohort of SDS patients. Remarkably, we show that ataluren treatment readily restores SBDS protein expression in different cell types, particularly bone marrow stem cells. Furthermore, ataluren promotes myeloid differentiation in hematopoietic progenitors, reduces apoptotic rate in primary PBMCs, and brings mammalian target of rapamycin phosphorylation levels back to normal in both lymphoblasts and bone marrow mesenchymal stromal cells (BM-MSCs). Since a specific therapy against SDS is currently lacking, these results provide the rationale for ataluren repurposing clinical trials.

The Interplay between Radioresistant Caco-2 Cells and the Immune System Increases Epithelial Layer Permeability and Alters Signaling Protein Spectrum.

Colorectal cancer is one of the most frequent type of cancer, with a higher incidence in the developed countries. Colorectal cancer is usually managed with both surgeries, chemotherapy and radiotherapy. Radiotherapy has the well-known advantage of targeting the tumor, minimizing normal tissue exposure. Nevertheless, during radiation treatment, exposure of healthy tissues is of great concern, in particular because of the effects on the intestinal barrier functions and on cells belonging to the immune system. The functional role of intestinal barrier in avoiding paracellular trafficking and controlling bacterial spread from gut it is well known and it is due to the presence of tight junction complexes. However, intestinal barrier is fundamental in participating to the interplay with immune system, especially considering the gut-associated lymphoid tissue. Until few years ago, radiotherapy was considered to bear only a depressive action on the immune system. However, it is now recognized that the release of pro-inflammatory signals and phenotypic changes in tumoral cells due to ionizing radiation could trigger the immune system against the tumor. In this work, we address how intestinal barrier functions are perturbed by X-ray doses in the range 0-10 Gy, focusing on the interplay between tumoral cells and the immune system. To this aim, we adopted a coculture model in which Caco-2 cells can be grown in presence/absence of peripheral blood mononuclear cells (PBMC). We focused our attention on changes in the proliferation, trans-epithelial electrical resistance (TEER), cytokine release, and proteins of the junctional complexes. Our results indicate a high radioresistance of Caco-2 in the investigated dose range, and an increased permeability of the tumoral cell layer due to the presence of PBMC. This is found to be correlated with activation of PBMC, inhibiting the apoptotic pathway, with the enhancement of cytokine release and with variation of tight junction scaffold protein expression levels, assumed to be related to IFN-γ- and TNF-α-mediated signaling.

Parental origin of the deletion del(20q) in Shwachman-Diamond patients and loss of the paternally derived allele of the imprinted L3MBTL1 gene.

Shwachman-Diamond syndrome (SDS) (OMIM 260400) is a rare autosomal recessive disease characterized by exocrine pancreatic insufficiency, skeletal, and hematological abnormalities and bone marrow (BM) dysfunction. Mutations in the SBDS gene cause SDS. Clonal chromosome anomalies are often present in BM, i(7)(q10) and del(20q) being the most frequent ones. We collected 6 SDS cases with del(20q): a cluster of imprinted genes, including L3MBTL1 and SGK2 is present in the deleted region. Only the paternal allele is expressed for these genes. Based on these data, we made the hypothesis that the loss of this region, in relation to parental origin of deletion, may be of relevance for the hematological phenotype. By comparing hematological data of our 6 cases with a group of 20 SDS patients without evidence of del(20q) in BM, we observed a significant difference for Hb levels (P < 0.012), and a difference slightly above the significance level for RBC counts (P < 0.053): in both cases the values were higher in patients with del(20q). We also report preliminary evidence for an increased number of BFU-E colonies in cases with paternal deletion, data on the presence of the deletion in colonies and in mature circulating lymphocytes. © 2016 Wiley Periodicals, Inc.