On the effects of 30.5 GHz sinusoidal wave exposure on glioblastoma organoids.

Introduction: Glioblastoma (grade IV) is the most aggressive primary brain tumor in adults, representing one of the biggest therapeutic challenges due to its highly aggressive nature. In this study, we investigated the impact of millimeter waves on tridimensional glioblastoma organoids derived directly from patient tumors. Our goal was to explore novel therapeutic possibilities in the fight against this challenging disease. Methods: The exposure setup was meticulously developed in-house, and we employed a comprehensive dosimetry approach, combining numerical and experimental methods. Biological endpoints included a global transcriptional profiling analysis to highlight possible deregulated pathways, analysis of cell morphological changes, and cell phenotypic characterization which are all important players in the control of glioblastoma progression. Results and discussion: Our results revealed a significant effect of continuous millimeter waves at 30.5 GHz on cell proliferation and apoptosis, although without affecting the differentiation status of glioblastoma cells composing the organoids. Excitingly, when applying a power level of 0.1 W (Root Mean Square), we discovered a remarkable (statistically significant) therapeutic effect when combined with the chemotherapeutic agent Temozolomide, leading to increased glioblastoma cell death. These findings present a promising interventional window for treating glioblastoma cells, harnessing the potential therapeutic benefits of 30.5 GHz CW exposure. Temperature increase during treatments was carefully monitored and simulated with a good agreement, demonstrating a negligible involvement of the temperature elevation for the observed effects. By exploring this innovative approach, we pave the way for improved future treatments of glioblastoma that has remained exceptionally challenging until now.

Involvement of Mitochondria in the Selective Response to Microsecond Pulsed Electric Fields on Healthy and Cancer Stem Cells in the Brain.

In the last few years, pulsed electric fields have emerged as promising clinical tools for tumor treatments. This study highlights the distinct impact of a specific pulsed electric field protocol, PEF-5 (0.3 MV/m, 40 µs, 5 pulses), on astrocytes (NHA) and medulloblastoma (D283) and glioblastoma (U87 NS) cancer stem-like cells (CSCs). We pursued this goal by performing ultrastructural analyses corroborated by molecular/omics approaches to understand the vulnerability or resistance mechanisms triggered by PEF-5 exposure in the different cell types. Electron microscopic analyses showed that, independently of exposed cells, the main targets of PEF-5 were the cell membrane and the cytoskeleton, causing membrane filopodium-like protrusion disappearance on the cell surface, here observed for the first time, accompanied by rapid cell swelling. PEF-5 induced different modifications in cell mitochondria. A complete mitochondrial dysfunction was demonstrated in D283, while a mild or negligible perturbation was observed in mitochondria of U87 NS cells and NHAs, respectively, not sufficient to impair their cell functions. Altogether, these results suggest the possibility of using PEF-based technology as a novel strategy to target selectively mitochondria of brain CSCs, preserving healthy cells.

Characterization of Early and Late Damage in a Mouse Model of Pelvic Radiation Disease.

Pelvic radiation disease (PRD), a frequent side effect in patients with abdominal/pelvic cancers treated with radiotherapy, remains an unmet medical need. Currently available preclinical models have limited applications for the investigation of PRD pathogenesis and possible therapeutic strategies. In order to select the most effective irradiation protocol for PRD induction in mice, we evaluated the efficacy of three different locally and fractionated X-ray exposures. Using the selected protocol (10 Gy/day × 4 days), we assessed PRD through tissue (number and length of colon crypts) and molecular (expression of genes involved in oxidative stress, cell damage, inflammation, and stem cell markers) analyses at short (3 h or 3 days after X-ray) and long (38 days after X-rays) post-irradiation times. The results show that a primary damage response in term of apoptosis, inflammation, and surrogate markers of oxidative stress was found, thus determining a consequent impairment of cell crypts differentiation and proliferation as well as a local inflammation and a bacterial translocation to mesenteric lymph nodes after several weeks post-irradiation. Changes were also found in microbiota composition, particularly in the relative abundance of dominant phyla, related families, and in alpha diversity indices, as an indication of dysbiotic conditions induced by irradiation. Fecal markers of intestinal inflammation, measured during the experimental timeline, identified lactoferrin, along with elastase, as useful non-invasive tools to monitor disease progression. Thus, our preclinical model may be useful to develop new therapeutic strategies for PRD treatment.

Safety in Rats of a Novel Nasal Spray Formulation for the Prevention of Airborne Viral Infections.

Hexedra+® is a nasal spray containing hydroxypropyl methylcellulose, beta-cyclodextrin, and usnic acid. It has been developed with the aim of reducing the risk of transmission of airborne viral infections, with particular reference to influenza and COVID-19. As part of the preclinical development of the product, we carried out a study on thirty male Wistar rats divided into three study groups and treated with Hexedra+, an alternative formulation containing a double concentration of usnic acid (0.015% instead of 0.0075%) or saline solution. Products were administered at the dose of 30 µL into each nostril, three times a day for seven consecutive days by means of a micropipette. By the end of the treatment period, no significant changes were observed in body weight. Histological examination of nasal mucosa and soft organs did not show any significant difference in the three study groups. Serum transaminase level remained in the normal limit in all the animals treated. The serum level of usnic acid was measured in order to assess the absorption of the molecule through the nasal mucosa. By the end of the study period, the usnic acid serum level was negligible in all the animals treated. In conclusion, the safety profile of Hexedra+ appears favorable in the animal model studied.

Aglianico Grape Seed Semi-Polar Extract Exerts Anticancer Effects by Modulating MDM2 Expression and Metabolic Pathways.

Grapevine (Vitis vinifera L.) seeds are rich in polyphenols including proanthocyanidins, molecules with a variety of biological effects including anticancer action. We have previously reported that the grape seed semi-polar extract of Aglianico cultivar (AGS) was able to induce apoptosis and decrease cancer properties in different mesothelioma cell lines. Concomitantly, this extract resulted in enriched oligomeric proanthocyanidins which might be involved in determining the anticancer activity. Through transcriptomic and metabolomic analyses, we investigated in detail the anticancer pathway induced by AGS. Transcriptomics analysis and functional annotation allowed the identification of the relevant causative genes involved in the apoptotic induction following AGS treatment. Subsequent biological validation strengthened the hypothesis that MDM2 could be the molecular target of AGS and that it could act in both a p53-dependent and independent manner. Finally, AGS significantly inhibited tumor progression in a xenograft mouse model of mesothelioma, confirming also in vivo that MDM2 could act as molecular player responsible for the AGS antitumor effect. Our findings indicated that AGS, exerting a pro-apoptotic effect by hindering MDM2 pathway, could represent a novel source of anticancer molecules.

Liver Steatosis and Steatohepatitis Alter Bile Acid Receptors in Brain and Induce Neuroinflammation: A Contribution of Circulating Bile Acids and Blood-Brain Barrier.

A tight relationship between gut-liver diseases and brain functions has recently emerged. Bile acid (BA) receptors, bacterial-derived molecules and the blood-brain barrier (BBB) play key roles in this association. This study was aimed to evaluate how non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) impact the BA receptors Farnesoid X receptor (FXR) and Takeda G-protein coupled receptor 5 (TGR5) expression in the brain and to correlate these effects with circulating BAs composition, BBB integrity and neuroinflammation. A mouse model of NAFLD was set up by a high-fat and sugar diet, and NASH was induced with the supplementation of dextran-sulfate-sodium (DSS) in drinking water. FXR, TGR5 and ionized calcium-binding adaptor molecule 1 (Iba-1) expression in the brain was detected by immunohistochemistry, while Zonula occludens (ZO)-1, Occludin and Plasmalemmal Vesicle Associated Protein-1 (PV-1) were analyzed by immunofluorescence. Biochemical analyses investigated serum BA composition, lipopolysaccharide-binding protein (LBP) and S100ß protein (S100ß) levels. Results showed a down-regulation of FXR in NASH and an up-regulation of TGR5 and Iba-1 in the cortex and hippocampus in both treated groups as compared to the control group. The BA composition was altered in the serum of both treated groups, and LBP and S100ß were significantly augmented in NASH. ZO-1 and Occludin were attenuated in the brain capillary endothelial cells of both treated groups versus the control group. We demonstrated that NAFLD and NASH provoke different grades of brain dysfunction, which are characterized by the altered expression of BA receptors, FXR and TGR5, and activation of microglia. These effects are somewhat promoted by a modification of circulating BAs composition and by an increase in LBP that concur to damage BBB, thus favoring neuroinflammation.

Effects of Ultra-Short Pulsed Electric Field Exposure on Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most common brain cancer in adults. GBM starts from a small fraction of poorly differentiated and aggressive cancer stem cells (CSCs) responsible for aberrant proliferation and invasion. Due to extreme tumor heterogeneity, actual therapies provide poor positive outcomes, and cancers usually recur. Therefore, alternative approaches, possibly targeting CSCs, are necessary against GBM. Among emerging therapies, high intensity ultra-short pulsed electric fields (PEFs) are considered extremely promising and our previous results demonstrated the ability of a specific electric pulse protocol to selectively affect medulloblastoma CSCs preserving normal cells. Here, we tested the same exposure protocol to investigate the response of U87 GBM cells and U87-derived neurospheres. By analyzing different in vitro biological endpoints and taking advantage of transcriptomic and bioinformatics analyses, we found that, independent of CSC content, PEF exposure affected cell proliferation and differentially regulated hypoxia, inflammation and P53/cell cycle checkpoints. PEF exposure also significantly reduced the ability to form new neurospheres and inhibited the invasion potential. Importantly, exclusively in U87 neurospheres, PEF exposure changed the expression of stem-ness/differentiation genes. Our results confirm this physical stimulus as a promising treatment to destabilize GBM, opening up the possibility of developing effective PEF-mediated therapies.

Microsecond Pulsed Electric Fields: An Effective Way to Selectively Target and Radiosensitize Medulloblastoma Cancer Stem Cells.

PURPOSE: Cancer stem cells constitute an endless reserve for the maintenance and progression of tumors, and they could be the reason for conventional therapy failure. New therapeutic strategies are necessary to specifically target them. In this context, microsecond pulsed electric fields have been selected to expose D283Med cells, a human medulloblastoma cell line resulted to be rich in cancer stem cells, and normal human astrocytes. METHODS: We analyzed in vitro different endpoints at different times after microsecond pulsed electric field exposure, such as permeabilization, reactive oxygen species generation, cell viability/proliferation, cell cycle, and clonogenicity, as well as the expression of different genes involved in cell cycle, apoptosis, and senescence. Furthermore, the response of D283Med cells exposed to microsecond pulsed electric fields was validated in vivo in a heterotopic mouse xenograft model. RESULTS: Our in vitro results showed that a specific pulse protocol (ie, 0.3 MV/m, 40 µs, 5 pulses) was able to induce irreversible membrane permeabilization and apoptosis exclusively in medulloblastoma cancer stem cells. In the surviving cells, reactive oxygen species generation was observed, together with a transitory G2/M cell-cycle arrest with a senescence-associated phenotype via the upregulation of GADD45A. In vivo results, after pulsed electric field exposure, demonstrated a significant tumor volume reduction with no eradication of tumor mass. In conjunction, we verified the efficacy of electric pulse pre-exposure followed by ionizing irradiation in vivo to enable complete inhibition of tumor growth. CONCLUSIONS: Our data reveal novel therapeutic options for the targeting of medulloblastoma cancer stem cells, indicating nonionizing pulsed electric field pre-exposure as an effective means to overcome the radioresistance of cancer stem cells.

Human Medulloblastoma Cell Lines: Investigating on Cancer Stem Cell-Like Phenotype.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Despite the progress of new treatments, the risk of recurrence, morbidity, and death remains significant and the long-term adverse effects in survivors are substantial. The fraction of cancer stem-like cells (CSCs) because of their self-renewal ability and multi-lineage differentiation potential is critical for tumor initiation, growth, and resistance to therapies. For the development of new CSC-targeted therapies, further in-depth studies are needed using enriched and stable MB-CSCs populations. This work, aimed at identifying the amount of CSCs in three available human cell lines (DAOY, D341, and D283), describes different approaches based on the expression of stemness markers. First, we explored potential differences in gene and protein expression patterns of specific stem cell markers. Then, in order to identify and discriminate undifferentiated from differentiated cells, MB cells were characterized using a physical characterization method based on a high-frequency dielectrophoresis approach. Finally, we compared their tumorigenic potential in vivo, through engrafting in nude mice. Concordantly, our findings identified the D283 human cell line as an ideal model of CSCs, providing important evidence on the use of a commercial human MB cell line for the development of new strategic CSC-targeting therapies.

ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of ßTrCP.

The Hedgehog (Hh) pathway is essential for embryonic development and tissue homeostasis. Aberrant Hh signaling may occur in a wide range of human cancers, such as medulloblastoma, the most common brain malignancy in childhood. Here, we identify endoplasmic reticulum aminopeptidase 1 (ERAP1), a key regulator of innate and adaptive antitumor immune responses, as a previously unknown player in the Hh signaling pathway. We demonstrate that ERAP1 binds the deubiquitylase enzyme USP47, displaces the USP47-associated ßTrCP, the substrate-receptor subunit of the SCFßTrCP ubiquitin ligase, and promotes ßTrCP degradation. These events result in the modulation of Gli transcription factors, the final effectors of the Hh pathway, and the enhancement of Hh activity. Remarkably, genetic or pharmacological inhibition of ERAP1 suppresses Hh-dependent tumor growth in vitro and in vivo. Our findings unveil an unexpected role for ERAP1 in cancer and indicate ERAP1 as a promising therapeutic target for Hh-driven tumors.

Alterations in Morphology and Adult Neurogenesis in the Dentate Gyrus of Patched1 Heterozygous Mice.

Many genes controlling neuronal development also regulate adult neurogenesis. We investigated in vivo the effect of Sonic hedgehog (Shh) signaling activation on patterning and neurogenesis of the hippocampus and behavior of Patched1 (Ptch1) heterozygous mice (Ptch1+/- ). We demonstrated for the first time, that Ptch1+/- mice exhibit morphological, cellular and molecular alterations in the dentate gyrus (DG), including elongation and reduced width of the DG as well as deregulations at multiple steps during lineage progression from neural stem cells to neurons. By using stage-specific cellular markers, we detected reduction of quiescent stem cells, newborn neurons and astrocytes and accumulation of proliferating intermediate progenitors, indicative of defects in the dynamic transition among neural stages. Phenotypic alterations in Ptch1+/- mice were accompanied by expression changes in Notch pathway downstream components and TLX nuclear receptor, as well as perturbations in inflammatory and synaptic networks and mouse behavior, pointing to complex biological interactions and highlighting cooperation between Shh and Notch signaling in the regulation of neurogenesis.

Synthetic lethal genetic interactions between Rad54 and PARP-1 in mouse development and oncogenesis.

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.

Age-related effects of X-ray irradiation on mouse hippocampus.

Therapeutic irradiation of pediatric and adult patients can profoundly affect adult neurogenesis, and cognitive impairment manifests as a deficit in hippocampal-dependent functions. Age plays a major role in susceptibility to radiation, and younger children are at higher risk of cognitive decay when compared to adults. Cranial irradiation affects hippocampal neurogenesis by induction of DNA damage in neural progenitors, through the disruption of the neurogenic microenvironment, and defective integration of newborn neurons into the neuronal network. Our goal here was to assess cellular and molecular alterations induced by cranial X-ray exposure to low/moderate doses (0.1 and 2 Gy) in the hippocampus of mice irradiated at the postnatal ages of day 10 or week 10, as well as the dependency of these phenomena on age at irradiation. To this aim, changes in the cellular composition of the dentate gyrus, mitochondrial functionality, proteomic profile in the hippocampus, as well as cognitive performance were evaluated by a multidisciplinary approach. Our results suggest the induction of specific alterations in hippocampal neurogenesis, microvascular density and mitochondrial functions, depending on age at irradiation. A better understanding of how irradiation impairs hippocampal neurogenesis at low and moderate doses is crucial to minimize adverse effects of therapeutic irradiation, contributing also to radiation safety regulations.

MK-4101, a Potent Inhibitor of the Hedgehog Pathway, Is Highly Active against Medulloblastoma and Basal Cell Carcinoma.

Aberrant activation of the Hedgehog (Hh) signaling pathway is implicated in the pathogenesis of many cancers, including medulloblastoma and basal cell carcinoma (BCC). In this study, using neonatally irradiated Ptch1(+/-) mice as a model of Hh-dependent tumors, we investigated the in vivo effects of MK-4101, a novel SMO antagonist, for the treatment of medulloblastoma and BCC. Results clearly demonstrated a robust antitumor activity of MK-4101, achieved through the inhibition of proliferation and induction of extensive apoptosis in tumor cells. Of note, beside antitumor activity on transplanted tumors, MK-4101 was highly efficacious against primary medulloblastoma and BCC developing in the cerebellum and skin of Ptch1(+/-) mice. By identifying the changes induced by MK-4101 in gene expression profiles in tumors, we also elucidated the mechanism of action of this novel, orally administrable compound. MK-4101 targets the Hh pathway in tumor cells, showing the maximum inhibitory effect on Gli1 MK-4101 also induced deregulation of cell cycle and block of DNA replication in tumors. Members of the IGF and Wnt signaling pathways were among the most highly deregulated genes by MK-4101, suggesting that the interplay among Hh, IGF, and Wnt is crucial in Hh-dependent tumorigenesis. Altogether, the results of this preclinical study support a therapeutic opportunity for MK-4101 in the treatment of Hh-driven cancers, also providing useful information for combination therapy with drugs targeting pathways cooperating with Hh oncogenic activity. Mol Cancer Ther; 15(6); 1177-89. ©2016 AACR.

A multidisciplinary approach unravels early and persistent effects of X-ray exposure at the onset of prenatal neurogenesis.

BACKGROUND: In humans, in utero exposure to ionising radiation results in an increased prevalence of neurological aberrations, such as small head size, mental retardation and decreased IQ levels. Yet, the association between early damaging events and long-term neuronal anomalies remains largely elusive. METHODS: Mice were exposed to different X-ray doses, ranging between 0.0 and 1.0 Gy, at embryonic days (E) 10, 11 or 12 and subjected to behavioural tests at 12 weeks of age. Underlying mechanisms of irradiation at E11 were further unravelled using magnetic resonance imaging (MRI) and spectroscopy, diffusion tensor imaging, gene expression profiling, histology and immunohistochemistry. RESULTS: Irradiation at the onset of neurogenesis elicited behavioural changes in young adult mice, dependent on the timing of exposure. As locomotor behaviour and hippocampal-dependent spatial learning and memory were most particularly affected after irradiation at E11 with 1.0 Gy, this condition was used for further mechanistic analyses, focusing on the cerebral cortex and hippocampus. A classical p53-mediated apoptotic response was found shortly after exposure. Strikingly, in the neocortex, the majority of apoptotic and microglial cells were residing in the outer layer at 24 h after irradiation, suggesting cell death occurrence in differentiating neurons rather than proliferating cells. Furthermore, total brain volume, cortical thickness and ventricle size were decreased in the irradiated embryos. At 40 weeks of age, MRI showed that the ventricles were enlarged whereas N-acetyl aspartate concentrations and functional anisotropy were reduced in the cortex of the irradiated animals, indicating a decrease in neuronal cell number and persistent neuroinflammation. Finally, in the hippocampus, we revealed a reduction in general neurogenic proliferation and in the amount of Sox2-positive precursors after radiation exposure, although only at a juvenile age. CONCLUSIONS: Our findings provide evidence for a radiation-induced disruption of mouse brain development, resulting in behavioural differences. We propose that alterations in cortical morphology and juvenile hippocampal neurogenesis might both contribute to the observed aberrant behaviour. Furthermore, our results challenge the generally assumed view of a higher radiosensitivity in dividing cells. Overall, this study offers new insights into irradiation-dependent effects in the embryonic brain, of relevance for the neurodevelopmental and radiobiological field.

The Patched 1 tumor-suppressor gene protects the mouse lens from spontaneous and radiation-induced cataract.

Age-related cataract is the most common cause of visual impairment. Moreover, traumatic cataracts form after injury to the eye, including radiation damage. We report herein that sonic hedgehog (Shh) signaling plays a key role in cataract development and in normal lens response to radiation injury. Mice heterozygous for Patched 1 (Ptch1), the Shh receptor and negative regulator of the pathway, develop spontaneous cataract and are highly susceptible to cataract induction by exposure to ionizing radiation in early postnatal age, when lens epithelial cells undergo rapid expansion in the lens epithelium. Neonatally irradiated and control Ptch1(+/-) mice were compared for markers of progenitors, Shh pathway activation, and epithelial-to-mesenchymal transition (EMT). Molecular analyses showed increased expression of the EMT-related transforming growth factor ß/Smad signaling pathway in the neonatally irradiated lens, and up-regulation of mesenchymal markers Zeb1 and Vim. We further show a link between proliferation and the stemness property of lens epithelial cells, controlled by Shh. Our results suggest that Shh and transforming growth factor ß signaling cooperate to promote Ptch1-associated cataract development by activating EMT, and that the Nanog marker of pluripotent cells may act as the primary transcription factor on which both signaling pathways converge after damage. These findings highlight a novel function of Shh signaling unrelated to cancer and provide a new animal model to investigate the molecular pathogenesis of cataract formation.

Developmental and oncogenic radiation effects on neural stem cells and their differentiating progeny in mouse cerebellum.

Neural stem cells are highly susceptible to radiogenic DNA damage, however, little is known about their mechanisms of DNA damage response (DDR) and the long-term consequences of genotoxic exposure. Patched1 heterozygous mice (Ptc1(+/-)) provide a powerful model of medulloblastoma (MB), a frequent pediatric tumor of the cerebellum. Irradiation of newborn Ptc1(+/-) mice dramatically increases the frequency and shortens the latency of MB. In this model, we investigated the mechanisms through which multipotent neural progenitors (NSCs) and fate-restricted progenitor cells (PCs) of the cerebellum respond to DNA damage induced by radiation, and the long-term developmental and oncogenic consequences. These responses were assessed in mice exposed to low (0.25 Gy) or high (3 Gy) radiation doses at embryonic day 13.5 (E13.5), when NSCs giving rise to the cerebellum are specified but the external granule layer (EGL) has not yet formed, or at E16.5, during the expansion of granule PCs to form the EGL. We found crucial differences in DDR and apoptosis between NSCs and fate-restricted PCs, including lack of p21 expression in NSCs. NSCs also appear to be resistant to oncogenesis from low-dose radiation exposure but more vulnerable at higher doses. In addition, the pathway to DNA repair and the pattern of oncogenic alterations were strongly dependent on age at exposure, highlighting a differentiation-stage specificity of DNA repair pathways in NSCs and PCs. These findings shed light on the mechanisms used by NSCs and PCs to maintain genome integrity during neurogenesis and may have important implications for radiation risk assessment and for development of targeted therapies against brain tumors.

Oncogenic radiation abscopal effects in vivo: interrogating mouse skin.

PURPOSE: To investigate the tissue dependence in transmission of abscopal radiation signals and their oncogenic consequences in a radiosensitive mouse model and to explore the involvement of gap junction intercellular communication (GJIC) in mediating radiation tumorigenesis in off-target mouse skin. METHODS AND MATERIALS: Patched1 heterozygous (Ptch1(+/-)) mice were irradiated at postnatal day 2 (P2) with 10 Gy of x-rays. Individual lead cylinders were used to protect the anterior two-thirds of the body, whereas the hindmost part was directly exposed to radiation. To test the role of GJICs and their major constituent connexin43 (Cx43), crosses between Ptch1(+/-) and Cx43(+/-) mice were similarly irradiated. These mouse groups were monitored for their lifetime, and skin basal cell carcinomas (BCCs) were counted and recorded. Early responses to DNA damage - Double Strand Breaks (DSBs) and apoptosis - were also evaluated in shielded and directly irradiated skin areas. RESULTS: We report abscopal tumor induction in the shielded skin of Ptch1(+/-) mice after partial-body irradiation. Endpoints were induction of early nodular BCC-like tumors and macroscopic infiltrative BCCs. Abscopal tumorigenesis was significantly modulated by Cx43 status, namely, Cx43 reduction was associated with decreased levels of DNA damage and oncogenesis in out-of-field skin, suggesting a key role of GJIC in transmission of oncogenic radiation signals to unhit skin. CONCLUSIONS: Our results further characterize the nature of abscopal responses and the implications they have on pathologic processes in different tissues, including their possible underlying mechanistic bases.

Dose and spatial effects in long-distance radiation signaling in vivo: implications for abscopal tumorigenesis.

PURPOSE: To investigate the dose and spatial dependence of abscopal radiation effects occurring in vivo in the mouse, along with their tumorigenic potential in the central nervous system (CNS) of a radiosensitive mouse model. METHODS AND MATERIALS: Patched1 (Ptch1)(+/-) mice, carrying a germ-line heterozygous inactivating mutation in the Ptch1 gene and uniquely susceptible to radiation damage in neonatal cerebellum, were exposed directly to ionizing radiation (1, 2, or 3 Gy of x-rays) or treated in a variety of partial-body irradiation protocols, in which the animals' head was fully protected by suitable lead cylinders while the rest of the body was exposed to x-rays in full or in part. Apoptotic cell death was measured in directly irradiated and shielded cerebellum shortly after irradiation, and tumor development was monitored in lifetime groups. The same endpoints were measured using different shielding geometries in mice irradiated with 3 or 10 Gy of x-rays. RESULTS: Although dose-dependent cell death was observed in off-target cerebellum for all doses and shielding conditions tested, a conspicuous lack of abscopal response for CNS tumorigenesis was evident at the lowest dose of 1 Gy. By changing the amount of exposed body volume, the shielding geometry could also significantly modulate tumorigenesis depending on dose. CONCLUSIONS: We conclude that interplay between radiation dose and exposed tissue volume plays a critical role in nontargeted effects occurring in mouse CNS under conditions relevant to humans. These findings may help understanding the mechanisms of long-range radiation signaling in harmful effects, including carcinogenesis, occurring in off-target tissues.

The estrogen receptor beta agonist diarylpropionitrile (DPN) inhibits medulloblastoma development via anti-proliferative and pro-apototic pathways.

Gender-related differences in medulloblastoma (MB) development have been reported with a higher incidence in males (slightly above 60%) than in females, female gender being also a significantly favorable prognostic factor in MB. The present study focused on the evaluation of the mechanisms by which estrogens protect against MB formation. To this end, we used a well characterized mouse model of MB - the Patched1 heterozygous mice. Ovariectomized mice were treated with 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), a highly potent ERß agonist, or 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT), a highly potent ERα agonist. Our results show that the ERß selective agonist DPN significantly inhibits development of MB preneoplastic lesions when compared with untreated ovariectomized mice, restoring the final incidence to that observed in the intact controls, and that these effects were achieved via activation of anti-proliferative and pro-apototic pathways. On the other hand, the ERα selective agonist PPT did not influence MB tumorigenesis relative to untreated ovariectomized mice.