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.

Micro-RNA and Proteomic Profiles of Plasma-Derived Exosomes from Irradiated Mice Reveal Molecular Changes Preventing Apoptosis in Neonatal Cerebellum.

Cell communication via exosomes is capable of influencing cell fate in stress situations such as exposure to ionizing radiation. In vitro and in vivo studies have shown that exosomes might play a role in out-of-target radiation effects by carrying molecular signaling mediators of radiation damage, as well as opposite protective functions resulting in resistance to radiotherapy. However, a global understanding of exosomes and their radiation-induced regulation, especially within the context of an intact mammalian organism, has been lacking. In this in vivo study, we demonstrate that, compared to sham-irradiated (SI) mice, a distinct pattern of proteins and miRNAs is found packaged into circulating plasma exosomes after whole-body and partial-body irradiation (WBI and PBI) with 2 Gy X-rays. A high number of deregulated proteins (59% of WBI and 67% of PBI) was found in the exosomes of irradiated mice. In total, 57 and 13 miRNAs were deregulated in WBI and PBI groups, respectively, suggesting that the miRNA cargo is influenced by the tissue volume exposed to radiation. In addition, five miRNAs (miR-99b-3p, miR-200a-3p, miR-200a, miR-182-5p, miR-182) were commonly overexpressed in the exosomes from the WBI and PBI groups. In this study, particular emphasis was also given to the determination of the in vivo effect of exosome transfer by intracranial injection in the highly radiosensitive neonatal cerebellum at postnatal day 3. In accordance with a major overall anti-apoptotic function of the commonly deregulated miRNAs, here, we report that exosomes from the plasma of irradiated mice, especially in the case of WBI, prevent radiation-induced apoptosis, thus holding promise for exosome-based future therapeutic applications against radiation injury.

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.

Role of Apolipoprotein E in the Hippocampus and Its Impact following Ionizing Radiation Exposure.

Apolipoprotein E (ApoE) is a lipid carrier in both the peripheral and the central nervous systems (CNSs). Lipid-loaded ApoE lipoprotein particles bind to several cell surface receptors to support membrane homeostasis and brain injury repair. In the brain, ApoE is produced predominantly by astrocytes, but it is also abundantly expressed in most neurons of the CNS. In this study, we addressed the role of ApoE in the hippocampus in mice, focusing on its role in response to radiation injury. To this aim, 8-week-old, wild-type, and ApoE-deficient (ApoE-/-) female mice were acutely whole-body irradiated with 3 Gy of X-rays (0.89 Gy/min), then sacrificed 150 days post-irradiation. In addition, age-matching ApoE-/- females were chronically whole-body irradiated (20 mGy/d, cumulative dose of 3 Gy) for 150 days at the low dose-rate facility at the Institute of Environmental Sciences (IES), Rokkasho, Japan. To seek for ApoE-dependent modification during lineage progression from neural stem cells to neurons, we have evaluated the cellular composition of the dentate gyrus in unexposed and irradiated mice using stage-specific markers of adult neurogenesis. Our findings indicate that ApoE genetic inactivation markedly perturbs adult hippocampal neurogenesis in unexposed and irradiated mice. The effect of ApoE inactivation on the expression of a panel of miRNAs with an established role in hippocampal neurogenesis, as well as its transcriptional consequences in their target genes regulating neurogenic program, have also been analyzed. Our data show that the absence of ApoE-/- also influences synaptic functionality and integration by interfering with the regulation of mir-34a, mir-29b, and mir-128b, leading to the downregulation of synaptic markers PSD95 and synaptophysin mRNA. Finally, compared to acute irradiation, chronic exposure of ApoE null mice yields fewer consequences except for the increased microglia-mediated neuroinflammation. Exploring the function of ApoE in the hippocampus could have implications for developing therapeutic approaches to alleviate radiation-induced brain injury.

Clinicopathologic and immunohistochemical features of ovarian clear cell carcinomas in comparison with type I and type II tumors.

Two types of ovarian carcinomas are distinguished with respect to morphology, biology, and clinical course, and are designated as Type I and Type II tumors. However, placement of clear cell carcinomas into one of these 2 groups has been problematic as they exhibit morphologic, molecular, and clinical features that do not entirely resemble either Type I or Type II tumors. The present study aimed at better elucidating the clinicopathologic and immunohistochemical features of clear cell carcinomas, in comparison with the 2 main broad categories. To this end, a panel of classic clinicopathologic and immunohistochemical parameters, including estrogen receptor α (ERα), ERß, progesterone receptor, Ki67, p53, and HER2/neu was evaluated in 71 Type I, 157 Type II, and 21 clear cell carcinomas. Overall, findings from the present study support the idea that ovarian clear cell carcinomas are neither Type I nor Type II carcinomas of the ovary; indeed, results obtained showed that similarities between clear cell carcinomas and Type I were limited to the patient's age, tumor dimension, incidence of lymph node and extranodal metastases, and p53 labeling index, whereas the patient's age and incidence of extranodal metastases were the only parameters comparable with the Type II group. The hormonal receptor profile of clear cell carcinomas was characterized by low expression of nuclear ERα and progesterone receptor, and by almost exclusively nuclear ERß immunopositivity, features significantly different from both Type I and II tumors. Finally, the percentage of HER2/neu-positive samples in clear cell carcinomas was 10- and 2.5-fold higher than Type I and Type II ovarian tumors, respectively. In conclusion, our study provides insights into clear cell carcinoma that could help in explaining its unique prognostic features, and, eventually, in orienting toward new therapeutic options.

Changes in the expression of oestrogen receptors and E-cadherin as molecular markers of progression from normal epithelium to invasive cancer in elderly patients with vulvar squamous cell carcinoma.

AIMS: The most common vulvar squamous cell carcinoma (conventional SCC) occurs in elderly women and develops following a human papillomavirus (HPV)-negative pathway. Because the highest incidence of conventional SCC is observed in patients with low oestrogen levels (postmenopausal women), the aim was to investigate whether hormonal factors could play a role in the development of cancer. METHODS AND RESULTS: The expression profile of oestrogen receptor α (ERα), ERß and progesterone receptor (PR) in a section containing both normal and tumour tissue, as well as the SCC-associated vulvar lesion, was evaluated in 34 elderly patients. Also, as recent studies have identified E-cadherin as a novel transcriptional target of oestrogen signalling, the modulation of this epithelial-mesenchymal transition (EMT) marker was studied. Finally, the expression of the proliferation marker Ki67 and of the apoptotic marker p53 was assessed. Results showed that changes in both ERα and ERß expression characterize the transition from normal epithelium to cancer in patients with vulvar SCC: ERα was lost in cancer while ERß decreased, mainly showing cytoplasmic localization. A reduction in the expression of E-cadherin was also observed in tumours, compared to normal epithelium. CONCLUSIONS: The data put the ER signalling pathway into the spotlight as a potentially important factor in vulvar carcinogenesis.

Cytoplasmic expression of oestrogen receptor beta (ERß) as a prognostic factor in vulvar squamous cell carcinoma in elderly women.

AIMS: To investigate the prognostic value of cytoplasmic oestrogen receptor beta (ERß) expression in a series of untreated patients with non-human papillomavirus (HPV)-related vulvar cancer. METHODS AND RESULTS: Immunohistochemistry was carried out using a polyclonal rabbit anti-human ERß antibody. The nuclear and cytoplasmic expression of ERß was evaluated in 33 patients. Cytoplasmic immunoreactivity was correlated with histopathological and molecular parameters (Ki67, p21), disease-free survival (DFS) and overall survival (OS). The expression of cytoplasmic ERß was found to be associated with grade (P=0.006), while no association was found with any of the remaining variables examined. Cases with high cytoplasmic ERß expression showed lower DFS and OS compared to cases with low cytoplasmic ERß (P=0.007, P=0.01, respectively). There was also a progressive decline in both the DFS and OS with increasing tumour size (P=0.05, P=0.07, respectively) and with increasing depth of infiltration (P=0.14, P=0.07, respectively). On multivariate analysis, only tumour size and cytoplasmic ERß staining retained an independent negative prognostic role for DFS and OS. CONCLUSIONS: The assessment of cytoplasmic ERß expression could be helpful to identify poor prognosis in elderly patients with non-HPV-related vulvar squamous cell carcinoma (SCC).

Cytoplasmic expression of estrogen receptor beta (ERß) predicts poor clinical outcome in advanced serous ovarian cancer.

OBJECTIVE: In this study we investigated the prognostic value of estrogen receptor α (ERα), ERß and progesterone receptor (PR) expression in 58 untreated advanced serous ovarian cancer patients. The study also included 12 macroscopically and histopathologically normal ovaries. MATERIALS AND METHODS: Protein expression was evaluated by immunohistochemistry, and antibody staining detected in both the nuclear and cytoplasmic compartments was taken into account. Immunopositivity was analyzed in relation to tumor clinicopathological variables, disease-free survival (DFS), and overall survival (OS). RESULTS: Epithelial cells in ovarian cancer tissue showed significantly lower levels of nuclear ERß and PR, but not ERα, than in normal ovarian tissue. In the case of ERß, however, while normal ovarian epithelium exhibited almost exclusively strong nuclear staining, ovarian cancer tissue mostly showed cytoplasmic immunopositivity. Nuclear ERα and ERß expression were not associated with clinical outcome. Conversely, any cytoplasmic ERß expression was an independent unfavorable prognostic factor for DFS, a finding approaching statistical significance also for OS. These data suggest that, in advanced serous ovarian cancer, cytoplasmic ERß signaling may be more important for patient survival than its nuclear signaling. In the case of PR, positivity was an independent favorable prognostic factor for DFS. CONCLUSIONS: These novel findings, that need to be confirmed in a large prospective trial, suggest that additional prognostic, and possibly therapeutic opportunities may be available in advanced serous ovarian cancer.

Protective role of 17 ß-estradiol on medulloblastoma development in Patched 1 heterozygous mice.

Medulloblastoma (MB) is the most common pediatric tumor of the CNS, representing ∼20% of all childhood CNS tumors. Although in recent years many molecular mechanisms that control MB development have been clarified, the effects of biological factors such as sex on this tumor remain to be explained. Epidemiological data, in fact, indicate a significant difference in the incidence of MB between the 2 sexes, with considerably higher susceptibility of males than females. Besides this different susceptibility, female sex is also a significant favorable prognostic factor in MB, with girls having a much better outcome. Despite these literature data, there has been little investigation into estrogen influence on MB development. In our study, we evaluated how hormone deficiency resulting from ovariectomy and hormone replacement influences the development of early and advanced MB stages in Patched1 heterozygous mice, a well-characterized mouse model of radiation-induced MB. Susceptibility to MB development was significantly increased in ovariectomized Ptch1(+/-) females and restored to levels observed in control mice after estrogen replacement. We next investigated the molecular mechanisms by which estrogen might influence tumor progression and show that ERß, but not ERα, is involved in modulation of MB development by estrogens. Finally, our study shows that a functional interaction between estrogen- and IGF-I-mediated pathways may be responsible for the effects observed.

17beta-Estradiol and soy phytochemicals selectively induce a type 2 polarization in mesenteric lymph nodes of ovariectomized rats.

OBJECTIVE: This study was designed to compare the effects of 17beta-estradiol (17beta-E2) and a phytoestrogen-containing soy extract on the immune system in an ovariectomized rat model of menopause. Specifically, T- and B-lymphocyte subsets, the balance of type 1 and 2 immune responses in the mesenteric lymph nodes, and serum levels of different classes of immunoglobulin were examined as study endpoints. DESIGN: Ovariectomized rats were treated with either the phytoestrogen-containing soy extract (50 or 100 mg/kg/day PO), 17beta-E2 (0.5 mg/kg/day PO), or vehicle; a sham control was included in the study. After the rats were killed, mesenteric lymph nodes and blood samples were collected. B- and T (CD4 and CD8)-lymphocyte subsets in mesenteric lymph nodes were evaluated by flow cytometry analysis. Cytokine-producing T lymphocytes were identified within each T-lymphocyte subset as TH1 (interferon-gamma CD4), TH2 (interleukin-4 CD4), TC1 (interferon-gamma CD8), and TC2 (interferon-4 CD8) lymphocytes. Serum levels of immunoglobulin classes were determined by enzyme-linked immunosorbent assay. RESULTS: There were no differences in the proportions of B lymphocytes and CD4 and CD8 T lymphocytes among groups. Treatment with 17beta-E2 and phytoestrogen-containing soy extract induced a reduction in TH1 and TC1 lymphocytes paralleled by a slight, nonsignificant, increase in the frequency of TH2. Data expressed as TH1/TH2 and TC1/TC2 ratios depicted a significant polarization of local immunity toward a humoral response. Evaluation of immunoglobulin serum levels did not show any significant difference among groups. CONCLUSIONS: Here we show that estrogens and soy phytochemicals similarly polarize the immune system toward a type 2 immune response in a preclinical model of menopause; our data draw attention to the crucial need to evaluate in clinical studies the potential side effects on the immune system of the complex soy products that are actually consumed in the postmenopausal setting.

Soy phytochemicals decrease nonsmall cell lung cancer growth in female athymic mice.

This study investigated the effects of a phytoestrogen-containing standardized soy extract (SSE) on the growth of nonsmall cell lung cancer (NSCLC; A549) xenografts in female athymic mice. Tumor-bearing mice received either sterile water or SSE [50 or 100 mg/(kg x d), per os], 5 d/wk, until the mean tumor weight in each group was at least 900 mg. Treatment with SSE reduced tumor growth in the high-dose group compared with control (P < 0.01); tumors in both treated groups had reduced proliferation and greater apoptosis compared with controls (P < 0.05). SSE treatment also induced diffuse central necrosis, reducing the viable tissue mass within the tumor. Whereas tumor levels of epidermal growth factor receptor were comparable in control and treated mice, the expression of phosphorylated protein kinase B (p-Akt) was lower in tumors of mice treated with 100 mg SSE/(kg x d) than in controls (P < 0.01). The protein level of phosphorylated mitogen-activated protein kinase also tended to be lower (P = 0.07) in this group than in controls. Estrogen receptor (ER)alpha and ERbeta were present in tumors, but their expression levels did not differ among groups. Serum insulin-like growth factor-1 concentrations also were not affected by the treatments. In conclusion, we found that soy phytochemicals slow the in vivo growth of NSCLC xenografts; the modulation of the Akt-signaling pathway observed in tumors of SSE-treated mice may have a role in the activity observed. Our research provides further support for the concept that consumption of phytoestrogens may be effective in delaying lung cancer progression.

Effects of a phytoestrogen-containing soy extract on the growth-inhibitory activity of ICI 182 780 in an experimental model of estrogen-dependent breast cancer.

The study reported here was designed to determine whether a phytoestrogen-containing soy extract (SSE) could negate/overwhelm the inhibitory effects of ICI 182 780 on the growth of estrogen-sustained human breast cancer xenografts (MCF-7), in ovariectomized athymic mice. As expected, estradiol-supplemented tumors did not grow over the study period in ICI 182 780-treated females; concomitant administration of 50 mg/kg per day SSE slightly potentiated the inhibitory activity of the drug, while at 100 mg/kg per day, SSE partially negated ICI 182 780 activity. In keeping with these in vivo outcomes, we observed that the level of cyclin D1 (and progesterone receptor) in MCF-7 xenografts was considerably reduced by ICI 182 780, an effect enhanced by concomitant treatment with 50 SSE, but reduced by the higher dosage (i.e. 100 mg/kg per day). Thrombospondin-1 (TSP-1) and kallikrein 6 (KLK6) levels were also reduced following ICI 182 780, although to a lesser degree; again, combined anti-estrogen and SSE produced a dose-dependent regulation in TSP-1 and KLK6 tumor level, with a further reduction in the mRNA gene expression at 50 SSE (compared with ICI 182 780) and a partial reversion of the drug-induced down-regulation at 100 mg/kg per day. No modulation was detected in the serum concentration of IGF-1 (a potent mitogen for estrogen receptor-positive breast cancer cell lines) either upon treatment with ICI 182 780 or concomitant administration of the anti-estrogen with SSE. In conclusion, results from this study raise concerns about the consumption of isoflavone supplements in conjunction with ICI 182 780 therapy, in postmenopausal women with estrogen-dependent breast cancer.

Nanog-driven cell-reprogramming and self-renewal maintenance in Ptch1 +/- granule cell precursors after radiation injury.

Medulloblastoma (MB) is the most common pediatric brain tumor, comprising four distinct molecular variants, one of which characterized by activation of the Sonic Hedgehog (SHH) pathway, driving 25-30% of sporadic MB. SHH-dependent MBs arise from granule cell precursors (GCPs), are fatal in 40-70% of cases and radioresistance strongly contributes to poor prognosis and tumor recurrence. Patched1 heterozygous (Ptch1 +/-) mice, carrying a germ-line heterozygous inactivating mutation in the Ptch1 gene, the Shh receptor and negative regulator of the pathway, are uniquely susceptible to MB development after radiation damage in neonatal cerebellum. Here, we irradiated ex-vivo GCPs isolated from cerebella of neonatal WT and Ptch1 +/- mice. Our results highlight a less differentiated status of Ptch1-mutated cells after irradiation, influencing DNA damage response. Increased expression levels of pluripotency genes Nanog, Oct4 and Sal4, together with greater clonogenic potential, clearly suggest that radiation induces expansion of the stem-like cell compartment through cell-reprogramming and self-renewal maintenance, and that this mechanism is strongly dependent on Nanog. These results contribute to clarify the molecular mechanisms that control radiation-induced Shh-mediated tumorigenesis and may suggest Nanog as a potential target to inhibit for adjuvant radiotherapy in treatment of SHH-dependent MB.

The relevance of prelamin A and RAD51 as molecular biomarkers in cervical cancer.

Along with their role in the maintenance of nuclear architecture, nuclear lamins also control genomic stability, DNA damage repair, transcription, cell proliferation, differentiation and senescence. Recent reports reveal that prelamin A-processing defects play a role in cancer development by impacting on transcription of key players in the maintenance of the genome stability, including RAD51. Here, we performed a 'proof of concept' study evaluating the role of prelamin A and RAD51 expression in clinical outcome of cervical cancer patients. We analyzed biomarker expression by immunohistochemistry in tumor material from locally advanced cervical cancer (LACC) patients (n=66) and correlated data with clinicopathological parameters and with response to neoadjuvant chemoradiation (CT/RT). In LACC patients who underwent neoadjuvant CT/RT the percentage of cases showing high prelamin A levels was greater in patients who completely responded to treatment (25 of 40, 62.5%) than in patients with macroscopic residual tumor (6 of 26, 23.1%, p=0.0024). Conversely, patients showing high RAD51 expression were less likely to respond to treatment (14 of 26, 53.8%) than were those with low protein levels (12 of 40, 30%, p=0.072). Only prelamin A retained an independent role in predicting response to treatment (p=0.003), while RAD51 approached statistical significance (p=0.07). Notably, high RAD51 expression highly significantly predicted poor outcome, emerging as an independent prognostic factor for disease free survival (p=0.038), while approaching statistical significance for overall survival (p=0.09). Our findings provide a framework for future prospective studies investigating molecular predictors of response to neoadjuvant chemoradiotherapy in LACC patients.

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.

Nonlinear Radiation-Induced Cataract Using the Radiosensitive Ptch1(+/-) Mouse Model.

While most of the evidence for radiation-induced late health effects relates to cancer, there has been increasing interest recently in the development of non-cancer diseases, including lens opacity, observed in populations exposed to low-dose radiation. In a recent study, we reported that mice heterozygous for the Patched1 (Ptch1) gene represented a novel and powerful animal model for this disorder, and a useful tool for investigating the mechanisms of radiogenic cataract development. Given the ongoing and considerable uncertainty in allowable lens dose levels and the existence of a threshold for the development of cataracts, we tested the effects of a decreasing range of radiation doses (2 Gy, 1 Gy and 0.5 Gy X rays) by irradiating groups of Ptch1(+/-) mice at 2 days of age. Our findings showed that at this dose range, acute exposure of this highly susceptible mouse model did not induce macroscopically detectable cataracts, and only the 2 Gy irradiated mice showed microscopic alterations of the lens. Molecular analyses performed to evaluate the induction of epithelial-mesenchymal transition (EMT) and subsequent fibrotic alterations in mouse lens cells also indicated the existence of a dose threshold for such effects in the mouse model used. The mechanisms of cataractogenesis remain unclear, and further experimental studies are essential to elucidate those mechanisms specific for cataract initiation and development after irradiation, as well as the underlying genetic factors controlling cataract susceptibility.

Ex vivo miRNome analysis in Ptch1+/- cerebellum granule cells reveals a subset of miRNAs involved in radiation-induced medulloblastoma.

It has historically been accepted that incorrectly repaired DNA double strand breaks (DSBs) are the principal lesions of importance regarding mutagenesis, and long-term biological effects associated with ionizing radiation. However, radiation may also cause dysregulation of epigenetic processes that can lead to altered gene function and malignant transformation, and epigenetic alterations are important causes of miRNAs dysregulation in cancer.Patched1 heterozygous (Ptch1+/-) mice, characterized by aberrant activation of the Sonic hedgehog (Shh) signaling pathway, are a well-known murine model of spontaneous and radiation-induced medulloblastoma (MB), a common pediatric brain tumor originating from neural granule cell progenitors (GCPs). The high sensitivity of neonatal Ptch1+/- mice to radiogenic MB is dependent on deregulation of the Ptch1 gene function. Ptch1 activates a growth and differentiation programme that is a strong candidate for regulation through the non-coding genome. Therefore we carried out miRNA next generation sequencing in ex vivo irradiated and control GCPs, isolated and purified from cerebella of neonatal WT and Ptch1+/- mice. We identified a subset of miRNAs, namely let-7 family and miR-17~92 cluster members, whose expression is altered in GCPs by radiation alone, or by synergistic interaction of radiation with Shh-deregulation. The same miRNAs were further validated in spontaneous and radiation-induced MBs from Ptch1+/- mice, confirming persistent deregulation of these miRNAs in the pathogenesis of MB.Our results support the hypothesis that miRNAs dysregulation is associated with radiosensitivity of GCPs and their neoplastic transformation in vivo.

Gender effect in experimental models of human medulloblastoma: does the estrogen receptor ß signaling play a role?

BACKGROUND: The male-to-female sex ratio for medulloblastoma (MB) is approximately 1.5∶1, female gender being also a favorable prognostic factor. This study aimed at evaluating the impact of gender on MB tumorigenesis. METHODS: In vitro activity of 17ß-estradiol (E2), DPN [2,3-bis(4-hydroxyphenyl)-propionitrile, a selective estrogen receptor ß (ERß)-agonist], PPT [4,4',4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol, a selective ERα-agonist] or DHT (5 alpha-dihydrotestosterone) was evaluated in three human MB cell lines. D283 Med cells were transplanted into athymic mice. RESULTS: A significant expression of ERß, with little or no ERα, and low AR (androgen receptor) was found in MB cell lines. The compounds tested did not affect cell proliferation. In vivo, we observed a significantly lower growth of D283 Med in nude female mice compared to males. At microscopic examination, tumors from females showed a shift towards differentiation, as evaluated by lower nestin, and higher NSE (neuron-specific enolase) and GFAP (glial fibrillary acidic protein) expression compared to males. Tumors from females also showed lower Ki67 and p53 expression. The wild-type ERß protein (ERß1) was lost in male tumors, while it was a permanent feature in females, and a strong negative correlation was found between Ki67 and ERß1 expression. Conversely, tumor levels of ERß2 and ERß5 did not significantly differ between genders. Increased levels of cyclin-dependent kinase inhibitor p21 were observed in females, suggesting that estrogen may decrease tumor growth through blocking cell cycle progression. An inhibition of the insulin-like growth factor I (IGF-I) signaling was also evident in females. CONCLUSION: We provides mechanistic evidence supporting the idea that ERß1 signaling may have pro-differentiation and tumor suppressive function in medulloblastomas.

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.