Radiation metabolomics. 1. Identification of minimally invasive urine biomarkers for gamma-radiation exposure in mice

Gamma-radiation exposure has both short- and long-term adverse health effects. The threat of modern terrorism places human populations at risk for radiological exposures, yet current medical countermeasures to radiation exposure are limited. Here we describe metabolomics for gamma-radiation biodosimetry in a mouse model. Mice were gamma-irradiated at doses of 0, 3 and 8 Gy (2.57 Gy/min), and urine samples collected over the first 24 h after exposure were analyzed by ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOFMS). Multivariate data were analyzed by orthogonal partial least squares (OPLS). Both 3- and 8-Gy exposures yielded distinct urine metabolomic phenotypes. The top 22 ions for 3 and 8 Gy were analyzed further, including tandem mass spectrometric comparison with authentic standards, revealing that N-hexanoylglycine and beta-thymidine are urinary biomarkers of exposure to 3 and 8 Gy, 3-hydroxy-2-methylbenzoic acid 3-O-sulfate is elevated in urine of mice exposed to 3 but not 8 Gy, and taurine is elevated after 8 but not 3 Gy. Gene Expression Dynamics Inspector (GEDI) self-organizing maps showed clear dose-response relationships for subsets of the urine metabolome. This approach is useful for identifying mice exposed to gamma radiation and for developing metabolomic strategies for noninvasive radiation biodosimetry in humans.

Radiation therapy for HIV-associated diffuse large cell non-Hodgkin lymphoma

PURPOSE: To report the clinical experience with external beam radiotherapy (RT) for AIDS-related lymphoma (ARL) with or without the involvement of the central nervous system (CNS) in HIV-infected patients. PATIENTS AND METHODS: Clinical outcome of 24 HIV-seropositive patients with ARL treated with RT from 1995 to 2004 was reviewed, testing factors associated with outcome. RESULTS: After 1 and 5 years, the overall survival was 65% and 35%, respectively. The mean RT dose was 31 Gy after normalization to fractions of daily 2 Gy (range, 7.8-47.2 Gy). Radiotherapy dose was associated with survival in univariate (P = .04) and multivariate analysis (P = .01). Other factors in univariate analysis associated with outcome were viral load (VL), highly active antiretroviral therapy (HAART), ARL stage, and CNS involvement. Patients with CNS involvement achieved complete response in 46% and improved clinical performance was seen in 73%. CONCLUSIONS: After chemotherapy, RT in combination with HAART is highly active, and RT should be encouraged especially after suboptimal responses to induction treatment.

The Molecular Crosstalk between the MET Receptor Tyrosine Kinase and the DNA Damage Response - Biological and Clinical Aspects

Radiation therapy remains an imperative treatment modality for numerous malignancies. Enduring significant technical achievements both on the levels of treatment planning and radiation delivery have led to improvements in local control of tumor growth and reduction in healthy tissue toxicity. Nevertheless, resistance mechanisms, which presumably also involve activation of DNA damage response signaling pathways that eventually may account for loco-regional relapse and consequent tumor progression, still remain a critical problem. Accumulating data suggest that signaling via growth factor receptor tyrosine kinases, which are aberrantly expressed in many tumors, may interfere with the cytotoxic impact of ionizing radiation via the direct activation of the DNA damage response, leading eventually to so-called tumor radioresistance. The aim of this review is to overview the current known data that support a molecular crosstalk between the hepatocyte growth factor receptor tyrosine kinase MET and the DNA damage response. Apart of extending well established concepts over MET biology beyond its function as a growth factor receptor, these observations directly relate to the role of its aberrant activity in resistance to DNA damaging agents, such as ionizing radiation, which are routinely used in cancer therapy and advocate tumor sensitization towards DNA damaging agents in combination with MET targeting.

Adaptive plasticity and genetic divergence in feeding efficiency during parallel adaptive radiation of whitefish (Coregonus spp.)

Parallel phenotypic divergence in replicated adaptive radiations could either result from parallel genetic divergence in response to similar divergent selec- tion regimes or from equivalent phenotypically plastic response to the repeated occurrence of contrasting environments. In post-glacial fish, repli- cated divergence in phenotypes along the benthic-limnetic habitat axis is commonly observed. Here, we use two benthic-limnetic species pairs of whitefish from two Swiss lakes, raised in a common garden design, with reciprocal food treatments in one species pair, to experimentally measure whether feeding efficiency on benthic prey has a genetic basis or whether it underlies phenotypic plasticity (or both). To do so, we offered experimental fish mosquito larvae, partially burried in sand, and measured multiple feed- ing efficiency variables. Our results reveal both, genetic divergence as well as phenotypically plastic divergence in feeding efficiency, with the pheno- typically benthic species raised on benthic food being the most efficient forager on benthic prey. This indicates that both, divergent natural selection on genetically heritable traits and adaptive phenotypic plasticity, are likely important mechanisms driving phenotypic divergence in adaptive radiation.

IMPROVING QUANTITATIVE TREATMENT RESPONSE MONITORING WITH DEFORMABLE IMAGE REGISTRATION

Quantitative imaging with 18F-FDG PET/CT has the potential to provide an in vivo assessment of response to radiotherapy (RT). However, comparing tissue tracer uptake in longitudinal studies is often confounded by variations in patient setup and potential treatment induced gross anatomic changes. These variations make true response monitoring for the same anatomic volume a challenge, not only for tumors, but also for normal organs-at-risk (OAR). The central hypothesis of this study is that more accurate image registration will lead to improved quantitation of tissue response to RT with 18F-FDG PET/CT. Employing an in-house developed “demons” based deformable image registration algorithm, pre-RT tumor and parotid gland volumes can be more accurately mapped to serial functional images. To test the hypothesis, specific aim 1 was designed to analyze whether deformably mapping tumor volumes rather than aligning to bony structures leads to superior tumor response assessment. We found that deformable mapping of the most metabolically avid regions improved response prediction (P<0.05). The positive predictive power for residual disease was 63% compared to 50% for contrast enhanced post-RT CT. Specific aim 2 was designed to use parotid gland standardized uptake value (SUV) as an objective imaging biomarker for salivary toxicity. We found that relative change in parotid gland SUV correlated strongly with salivary toxicity as defined by the RTOG/EORTC late effects analytic scale (Spearman’s ρ = -0.96, P<0.01). Finally, the goal of specific aim 3 was to create a phenomenological dose-SUV response model for the human parotid glands. Utilizing only baseline metabolic function and the planned dose distribution, predicting parotid SUV change or salivary toxicity, based upon specific aim 2, became possible. We found that the predicted and observed parotid SUV relative changes were significantly correlated (Spearman’s ρ = 0.94, P<0.01). The application of deformable image registration to quantitative treatment response monitoring with 18F-FDG PET/CT could have a profound impact on patient management. Accurate and early identification of residual disease may allow for more timely intervention, while the ability to quantify and predict toxicity of normal OAR might permit individualized refinement of radiation treatment plan designs.

THE ROLE OF E2F1 IN THE RESPONSE TO DNA DOUBLE STRAND BREAKS

The importance of E2F transcription factors in the processes of proliferation and apoptosis are well established. E2F1, but not other E2F family members, is also phosphorylated and stabilized in response to various forms of DNA damage to regulate the expression of cell cycle and pro-apoptotic genes. E2F1 also relocalizes and forms foci at sites of DNA double-strand breaks but the function of E2F1 at sites of damage is still unknown. Here I reveal that E2F1 deficiency leads to increased spontaneous DNA break and impaired recovery following exposure to ionizing radiation. In response to DNA double-strand breaks, NBS1 phosphorylation and foci formation are defective in cells lacking E2F1, but NBS1 expression levels are unaffected. Moreover, it was observed that an association between NBS1 and E2F1 is increased in response to DNA damage, suggesting that E2F1 may promote NBS1 foci formation through a direct or indirect interaction at sites of DNA breaks. E2F1 deficient cells also display impaired foci formation of RPA and Rad51, which suggests a defect in DNA end resection and formation of single-stranded DNA at DNA double-strand breaks. I also found E2F1 status affects foci formation of the histone acetyltransferase GCN5 in response to DNA double-strand breaks. E2F1 is phosphorylated at serine 31 (serine 29 in mouse) by the ATM kinase as part of the DNA damage response. To investigate the importance of this event, our lab developed an E2F1 serine 29 mutant mouse model. I find that E2F1 serine 29 mutant cells show loss of E2F1 foci formation in response to DNA double-strand breaks. Furthermore, DNA repair and NBS1 foci formation are impaired in E2f1S29A/S29A cells. Taken together, my results indicate novel roles for E2F1 in the DNA damage response, which may directly promote DNA repair and genome maintenance.

Immune response to regressor and progressor tumor variants

Most skin cancers induced in mice by Ultraviolet (UV) radiation express highly immunogenic Tumor specific transplantation antigens (TSTAs) and thus exhibit a regressor phenotype. In this study, I have used cloned genes encoding tumor antigens and oncogenes in conjunction with DNA transfection technique to isolate and characterize regressor variants from progressor tumors and vice versa. The purpose of this study was (1) to determine whether the product of a cloned gene (216) from UV-1591 tumor, which encodes a novel MHC class I antigen can function as a tumor rejection antigen when expressed on unrelated, nonantigenic, murine tumor cells or whether its function is restricted to UV-induced tumors, and (2) to determine the processes by which progressor variants derived from a regressor UV-2240 cell line by transfection with an activated Ha-ras oncogene escape the immune defenses of the normal immunocompetent host.^ To answer the first question, a spontaneously transformed, nonimmunogenic cell line (10T-1) was cotransfected with DNA from p216 and pSV2-neo plasmids. Results demonstrate that the product of a cloned TSTA gene from a UV-induced murine tumor is capable of functioning as a tumor rejection antigen when expressed on unrelated, nonantigenic tumor cells. In addition, these results indicate that this approach could be used to augment the immune response against poorly antigenic tumors.^ To answer the second question, progressor variants were isolated from a highly antigenic UV radiation-induced C3H mouse regressor fibrosarcoma cell line, UV-2240, by transfection with an activated Ha-ras oncogene. Subcutaneous injection of Ha-ras-transfected UV-2240 cells into immunocompetent C3H mice produced tumors in 4 of 36 animals. In addition, the Ha-ras-induced progressor variants produced experimental lung metastasis in both normal C3H and nude mice, although they induced more lung nodules in nude mice than in normal C3H mice. Results indicate that the progressor phenotype of the Ha-ras-induced tumor variants is not due to loss of TSTAs or MHC class I antigens. This implies that some tumors can escape the immune defenses of the normal immunocompetent host by mechanisms other than the loss of TSTAs and MHC class I antigens. (Abstract shortened with permission of author.) ^

The effect of ultraviolet radiation on the pathogenesis of {\it Candida albicans} in mice

Ultraviolet (UV) radiation produces immunological alterations in both humans and animals that include a decrease in the delayed type hypersensitivity (DTH) response to complex antigens, and to the induction of the suppressor T cell pathway. Cell-mediated immunity of the type that is altered by UV radiation has been shown to be important in host resistance against microorganisms. My dissertation addresses questions concerning the effects of UV radiation on the pathogenesis of opportunistic fungal pathogens such as Candida albicans.^ The (DTH) response of C3H mice exposed to ultraviolet (UV) radiation before (afferent arm of DTH) or after (efferent arm of DTH) infection with Candida albicans was markedly and systemically suppressed. Although suppression of both the afferent and efferent phases of DTH were caused by similar wavebands within the ultraviolet region, the dose of UV radiation that suppressed the efferent arm of DTH was 10-fold higher than the dose that suppressed the afferent arm of the DTH reaction.^ The DTH response of C57BL/6 mice was also suppressed by UV radiation; however the suppression was accomplished by exposure to significantly lower doses UV radiation compared to C3H mice. In C57BL/6 mice, the dose of UV radiation that suppressed the afferent phase of DTH was 5-fold higher than the dose that suppressed the efferent phase.^ Exposure of C3H mice to UV radiation before sensitization induced splenic suppressor T cells that upon transfer to normal recipients, impaired the induction of DTH to Candida. In contrast, the suppression caused by UV irradiation of mice after sensitization was not transferable. Spleen cells from sensitized mice exhibited altered homing patterns in animals that were exposed to UV radiation shortly before receiving cells, suggesting that UV-induced suppression of the efferent arm of DTH could result from an alteration in the distribution of effector cells.^ UV radiation decreased the survival of Candida-infected mice; however, no correlation was found between suppression of the DTH response and the course of lethal infection. This suggested that DTH was not protective against lethal disease with this organism. UV radiation also changed the persistence of the organism in the internal organs. UV-irradiated, infected animals had increased numbers of Candida in their kidneys compared to non-irradiated mice. Sensitization prior to UV irradiation aided clearance of the organism from the kidneys of UV-irradiated mice.^ These data show that UV radiation suppresses cell-mediated immunity to Candida albicans in mice and increases mortality of Candida-infected mice. Moreover, the data suggest that an increase in environmental UV radiation could increase the severity of pathogenic infections. ^

Hybridization and adaptive radiation

Whether interspecific hybridization is important as a mechanism that generates biological diversity is a matter of controversy. Whereas some authors focus on the potential of hybridization as a source of genetic variation, functional novelty and new species, others argue against any important role, because reduced fitness would typically render hybrids an evolutionary dead end. By drawing on recent developments in the genetics and ecology of hybridization and on principles of ecological speciation theory, I develop a concept that reconciles these views and adds a new twist to this debate. Because hybridization is common when populations invade new environments and potentially elevates rates of response to selection, it predisposes colonizing populations to rapid adaptive diversification under disruptive or divergent selection. I discuss predictions and suggest tests of this hybrid swarm theory of adaptive radiation and review published molecular phylogenies of adaptive radiations in light of the theory. Some of the confusion about the role of hybridization in evolutionary diversification stems from the contradiction between a perceived necessity for cessation of gene flow to enable adaptive population differentiation on the one hand [1], and the potential of hybridization for generating adaptive variation, functional novelty and new species 2, 3 and 4 on the other. Much progress in the genetics 5, 6, 7, 8 and 9 and ecology of hybridization 9, 10 and 11, and in our understanding of the role of ecology in speciation (see Glossary) 12, 13 and 14 make a re-evaluation timely. Whereas botanists traditionally stressed the diversity-generating potential of hybridization 2, 3 and 14, zoologists traditionally saw it as a process that limits diversification [1] and refer to it mainly in the contexts of hybrid zones (Box 1) and reinforcement of reproductive isolation [15]. Judging by the wide distribution of allopolyploidy among plants, many plant species might be of direct hybrid origin or descended from a hybrid species in the recent past [16]. The ability to reproduce asexually might explain why allopolyploid hybrid species are more common in plants than in animals. Allopolyploidy arises when meiotic mismatch of parental chromosomes or karyotypes causes hybrid sterility. Mitotic error, duplicating the karyotype, can restore an asexually maintained hybrid line to fertility. Although bisexual allopolyploid hybrid species are not uncommon in fish [17] and frogs [18], the difficulty with which allopolyploid animals reproduce, typically requiring gynogenesis[19], makes establishment and survival of allopolyploid animal species difficult.

The effects of ultraviolet B radiation on immune responses to {\it Borrelia burgdorferi}, the causative agent of Lyme disease

Ultraviolet B (UVB) radiation, in addition to being carcinogenic, is also immunosuppressive. Immunologically, UVB induces suppression locally, at the site of irradiation, or systemically, by inducing the production of a variety of immunosuppressive cytokines. Systemic effects include suppression of delayed-type hypersensitivity (DTH) responses to a variety of antigens (e.g. haptens, proteins, bacterial antigens, or alloantigens). One of the principal mediators of UV-induced immune suppression is the T helper-2 (Th2) cytokine interleukin-10 (IL-10); this suggests that UV irradiation induces suppression by shifting the immune response from a Th1 (cellular) to a Th2 (humoral) response. These "opposing" T helper responses are usually mutually exclusive, and polarized Th1 or Th2 responses may lead to either protection from infection or increased susceptibility to disease, depending on the infectious agent and the route of infection.^ This study examines the effects of UVB irradiation on cellular and humoral responses to Borrelia burgdorferi (Bb), the causative agent of Lyme disease (LD) in both immunization and infectious disease models; in addition, it examines the role of T cells in protection from and pathology of Bb infection. Particular emphasis is placed on the Bb-specific antibody responses following irradiation since UVB effects on humoral immunity are not fully understood. Mice were irradiated with a single dose of UV and then immunized (in complete Freund's adjuvant) or infected with Bb (intradermally at the base of the tail) in order to examine both DTH and antibody responses in both systems. UVB suppressed the Th1-associated antibodies IgG2a and IgG2b in both systems, as well as the DTH response to Bb in a dose dependent manner. Injection of anti-IL-10 antibody into UV-irradiated mice within 24 h after UV exposure restored the DTH response, as well as the Th1 antibody (IgG2a and IgG2b) response. In addition, injecting recombinant IL-10 mimicked some of the effects of UV radiation.^ Bb-specific Th1 T cell lines (BAT2.1-2.3) were generated to examine the role of T cells in Lyme borreliosis. All lines were CD4$\sp+,$ $\alpha\beta\sp+$ and proliferated specifically in response to Bb. The BAT2 cell lines not only conferred a DTH response to naive C3H recipients, but reduced the number of organisms recovered from the blood and tissues of mice infected with Bb. Furthermore, BAT2 cell lines protected mice from Bb-induced periarthritis. ^

Immunology and HIV expression in skin, in vitro, and in response to ultraviolet light

HIV can enter the body through Langerhans cells, dendritic cells, and macrophages in skin mucosa, and spreads by lysis or by syncytia. Since UVL induces of HIV-LTR in transgenic mice mid in cell lines in vitro, we hypothesized that UVB may affect HIV in people and may affect HIV in T cells in relation to dose, apoptosis, and cytokine expression. To determine whether HIV is induced by UVL in humans, a clinical study of HIV+ patients with psoriasis or pruritus was conducted during six weeks of UVB phototherapy, Controls were HIV-psoriasis patients receiving UVB and HIV+ KS subjects without UVB.Blood and skin biopsy specimens were collected at baseline, weeks 2 and 6, and 4 weeks after UVL. AIDS-related skin diseases showed unique cytokine profiles in skin and serum at baseline. In patients and controls on phototherapy, we observed the following: (1) CD4+ and CD8+ T cell numbers are not significantly altered during phototherapy, (2) p24 antigen levels, and also HIV plasma levels increase in patients not on antiviral therapy, (3) HIV-RNA levels in serum or plasma. (viral load) can either increase or decrease depending on the patient's initial viral load, presence of antivirals, and skin type, (4) HIV-RNA levels in the periphery are inversely correlated to serum IL-10 and (5) HIV+ cell in skin increase after UVL at 2 weeks by RT-PCR in situ hybridization mid we negatively correlated with peripheral load. To understand the mechanisms of UVB mediated HIV transcription, we treated Jurkat T cell lines stably transfected with an HIV-LTR-luciferase plasmid only or additionally with tat-SV-40 early promoter with UVB (2 J/m2 to 200 J/m2), 50 to 200 ng/ml rhIL-10, and 10 μg/ml PHA as control. HIV promoter activity was measured by luciferase normalized to protein. Time points up to 72 hours were analyzed for HIV-LTR activation. HIV-LTR activation had the following properties: (1) requires the presence of Tat, (2) occurs at 24 hours, and (3) is UVB dose dependent. Changes in viability by MTS (3-(4,5-dimethyhhiazol-2-y1)-5-(3-carboxymethoxyphonyl)-2-(4-sulfophenyl)-2H-tetrazolium) mixed with PMS (phenazine methosulfate) solution and apoptosis by propidium iodide and annexin V using flow cytometry (FC) were seen in irradiated Jurkat cells. We determined that (1) rhIL-10 moderately decreased HIV-LTR activation if given before radiation and greatly decreases it when given after UVB, (2) HIV-LTR activation was low at doses of greater than 70 J/m2, compared to activation at 50 J/m2. (Abstract shortened by UMI.)^

Metabolomics Reveals Aging-associated Attenuation of Noninvasive Radiation Biomarkers in Mice: Potential Role of Polyamine Catabolism and Incoherent DNA Damage-repair

Development of methods for rapid screening and stratification of subjects after exposure is an integral part of countermeasures against radiation. The potential demographic and exposure history-related heterogeneity of exposed populations warrants robust biomarkers that withstand and reflect such differences. In this study, the effect of aging and repeated exposure on the metabolic response to sublethal irradiation was examined in mice using UPLC-ESI-QTOF mass spectrometry. Aging attenuated postexposure elevation in excretions of DNA damage biomarkers as well as N(1)-acetylspermidine. Although N(1)-acetylspermidine and 2'-deoxyuridine elevation was highly correlated in all age groups, xanthine and N(1)-acetylspermidine elevation was poorly correlated in older mice. These results may reflect the established decline in DNA damage-repair efficiency associated with aging and indicate a novel role for polyamine metabolism in the process. Although repeated irradiation at long intervals did not affect the elevation of N(1)-acetylspermidine, 2'-deoxyuridine, and xanthine, it did significantly attenuate the elevation of 2'-deoxycytidine and thymidine compared to a single exposure. However, these biomarkers were found to identify exposed subjects with accuracy ranging from 82% (xanthosine) to 98% (2'-deoxyuridine), irrespective of their age and exposure history. This indicates that metabolic biomarkers can act as robust noninvasive signatures of sublethal radiation exposure.

Characterization of the potential role for RNA-binding protein FUS/TLS in DNA damage response: A quantitative proteomic approach

FUS/TLS (fused in sarcoma/translocated in liposarcoma) is a ubiquitously expressed RNA-binding protein of the hnRNP family, that has been discovered as fused to transcription factors, through chromosomal translocations, in several human sarcomas and found in protein aggregates in neurons of patients with an inherited form of Amyotrophic Lateral Sclerosis (ALS) [1]. To date, FUS/TLS has been implicated in a variety of cellular processes such as gene expression control, transcriptional regulation, pre-mRNA splicing and miRNA processing [2]. In addition, some evidences link FUS/TLS to genome stability control and DNA damage response. In fact, mice lacking FUS/TLS are hypersensitive to ionizing radiation (IR) and show high levels of chromosome instability and in response to double-strand breaks, FUS/TLS gets phosphorylated by the protein kinase ATM [3,4,5]. Furthermore, the inducible depletion of FUS/TLS in a neuroblastoma cell line (SH-SY5Y FUS/TLS TET-off iKD) subjected to genotoxic stress (IR) resulted in an increased phosphorylation of γH2AX respect to control cells, suggesting an higher activation of the DNA damage response. The study aims to investigate the specific role of FUS/TLS in DNA damage response through the characterization of the proteomic profile of SH-SY5Y FUS/TLS iKD cells subjected to DNA damage stress, by mass spectrometry-based quantitative proteomics (e.g. SILAC). Preliminary results of mass spectrometric identification of FUS/TLS interacting proteins in HEK293 cells, expressing a recombinant flag-tagged FUS/TLS protein, highlighted the interactions with several proteins involved in DNA damage response, such as DNA-PK, XRCC-5/-6, and ERCC-6, raising the possibilities that FUS/TLS is involved in this pathway, even thou its exact role still need to be addressed.

Characterization of the potential role for RNA-binding protein FUS in DNA damage response: A quantitative proteomic approach

FUS/TLS (fused in sarcoma/translocated in liposarcoma) is a ubiquitously expressed protein of the hnRNP family, that has been discovered as fused to transcription factors in several human sarcomas and found in protein aggregates in neurons of patients with an inherited form of Amyotrophic Lateral Sclerosis [Vance C. et al., 2009]. FUS is a 53 kDa nuclear protein that contains structural domains, such as a RNA Recognition Motif (RRM) and a zinc finger motif, that give to FUS the ability to bind to both RNA and DNA sequences. It has been implicated in a variety of cellular processes, such as pre-mRNA splicing, miRNA processing, gene expression control and transcriptional regulation [Fiesel FC. and Kahle PJ., 2011]. Moreover, some evidences link FUS to genome stability control and DNA damage response: mice lacking FUS are hypersensitive to ionizing radiation (IR) and show high levels of chromosome instability and, in response to double-strand breaks, FUS is phosphorylated by the protein kinase ATM [Kuroda M. et al., 2000; Hicks GG. et al., 2000; Gardiner M. et al., 2008]. Furthermore, preliminary results of mass spectrometric identification of FUS interacting proteins in HEK293 cells, expressing a recombinant flag-tagged FUS protein, highlighted the interactions with proteins involved in DNA damage response, such as DNA-PK, XRCC-5/-6, and ERCC-6, raising the possibilities that FUS is involved in this pathway, even though its role still needs to be clarified. This study aims to investigate the biological roles of FUS in human cells and in particular the putative role in DNA damage response through the characterization of the proteomic profile of the neuroblastoma cell line SH-SY5Y upon FUS inducible depletion, by a quantitative proteomic approach. The SH-SY5Y cell line that will be used in this study expresses, in presence of tetracycline, a shRNA that targets FUS mRNA, leading to FUS protein depletion (SH-SY5Y FUS iKD cells). To quantify changes in proteins expression levels a SILAC strategy (Stable Isotope Labeling by Amino acids in Cell culture) will be conducted on SH-SY5Y FUS iKD cells and a control SH-SY5Y cell line (that expresses a mock shRNA) and the relative changes in proteins levels will be evaluated after five and seven days upon FUS depletion, by nanoliquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS) and bioinformatics analysis. Preliminary experiments demonstrated that the SH-SY5Y FUS iKD cells, when subjected to genotoxic stress (high dose of IR), upon inducible depletion of FUS, showed a increased phosphorylation of gH2AX with respect to control cells, suggesting an higher activation of the DNA damage response.

Characterization of the role of the RNA-binding protein FUS in the DNA damage response

FUS/TLS (fused in sarcoma/translocated in liposarcoma), a ubiquitously expressed RNA-binding protein, has been linked to a variety of cellular processes, including RNA metabolism, microRNA biogenesis and DNA repair. However, the precise cellular function of FUS remains unclear. Recently, mutations in the FUS gene have been found in ∼5% of familial Amyotrophic Lateral Sclerosis, a neurodegenerative disorder characterized by the dysfunction and death of motor neurons. Since MEFs and B-lymphocytes derived from FUS knockdown mice display major sensitivity to ionizing radiation and chromosomal aberrations [1,2], we are investigating the effects of DNA damage both in the presence or in the absence of FUS. To this purpose, we have generated a SH-SY5Y human neuroblastoma cell line expressing a doxycycline-induced shRNA targeting FUS, which specifically depletes the protein. We have found that FUS depletion induces an activation of the DNA damage response (DDR). However, treatment with genotoxic agents did not induce any strong changes in ATM (Ataxia Telangiectasia Mutated)-mediated DDR signaling. Interestingly, genotoxic treatment results in changes in the subcellular localization of FUS in normal cells. We are currently exploring on one hand the mechanism by which FUS depletion leads to DNA damage, and on the other the functional significance of FUS relocalization after genotoxic stress.