Response Of Normal Cells Research Articles

Manganese toxicity is targeting an early step in the dopamine signal transduction pathway that controls lateral cilia activity in the bivalve mollusc Crassostrea virginica

Manganese is a neurotoxin causing manganism, a Parkinson-like clinical disorder. Manganese has been shown to interfere with dopaminergic neurotransmission, but the neurotoxic mechanism involved is not fully resolved. In the bivalve mollusc Crassostrea virginica also known as the eastern oyster, beating rates of lateral cilia of the gill are controlled by dopaminergic-serotonergic innervation originating from their cerebral and visceral ganglia. Terminal release of dopamine activates D2-like receptors on these gill cells inhibiting adenylyl cyclase and slowing cilia beating rates. In C. virginica, manganese treatment disrupts this dopaminergic innervation of the gill, preventing the normal cilio-inhibitory response of lateral cells to dopamine. In this study an adenylyl cyclase activator (forskolin) and two different inhibitors (MDL-12,330A and SQ 22,536) were used to determine if manganese had any effects on the adenylyl cyclase step of the dopamine D2 receptor signal transduction pathway. The results showed that neither the adenylyl cyclase activator nor the inhibitors were affected by manganese in the control of lateral ciliary activity. This suggests that in C. virginica the mechanism of manganese toxicity on the dopaminergic control of lateral ciliary activity is targeting an early step in the D2R signal transduction pathway, which may involve interference with D2 receptor activation or alternatively some other downstream signaling activity that does not affect adenylyl cyclase.

Back cover: Subtle Alterations in DNA Methylation Patterns in Normal Cells in Response to Dietary Stilbenoids

Back cover: Subtle Alterations in DNA Methylation Patterns in Normal Cells in Response to Dietary Stilbenoids

PO-127 Investigating the response of normal and cancer bladder cells to radiotherapy

IntroductionBladder cancer is the fourth most commonly diagnosed cancer among males worldwide. Recent advances in radiation delivery have increased the importance of bladder preserving strategies using radiotherapy, while also improving patient’s quality of life. However, the outcome of radiotherapy depends on the radiosensitivity of both the patient and his/her tumour. Recent developments in radiobiology have highlighted the importance of the assessment of radiation-induced DNA double-strand breaks (DSB) repair kinetics in predicting radiosensitivity of both normal and cancerous tissues. Indeed, after irradiation, several proteins (ATM, γH2AX, etc.) involved in the signalling and repair of DNA DSB relocalize as nuclear foci. The purpose of this study is to characterise the radiosensitivity of bladder cell lines, based on their capacity to repair radio-induced DNA damage.Material and methodsFour human bladder cell lines were used in this study: T24, UM-UC3, RT4 and SVHUC. Clonogenic assay was performed to study cell survival after irradiation: cells were irradiated with doses ranging from 0 to 10 Gy and their capacity to form colonies was assessed. Immunofluorescent analysis using anti-pATM and anti-γH2AX antibodies was then performed to assess DNA DSB signalling and repair kinetics after a 2Gy irradiation. Finally, we performed the three-dimensional (3D) sphere formation assay to assess the effect of irradiation on cancer/stem progenitor cells. Irradiated cells with doses ranging from 0 to 10 Gy were embedded in Matrigel and their capacity to form spheres was assessed.Results and discussionsRT4 was found to have the highest survival rate after irradiation, followed by T24, UM-UC3 and SVHUC according to the clonogenic assay results. Immunofluorescence results were consistent with cell survival as RT4 showed the fastest DSB recognition and repair kinetics while SVHUC had a much slower rate. Furthermore, the capacity of cells to recognise radio-induced DNA damage was found to have the best correlation with their survival. Lastly, the sphere formation ability of the different cell lines showed a differential response to increasing irradiation doses.ConclusionWe were able to radiobiologically characterise 4 human bladder cell lines by assessing their survival and capacity to repair radio-induced DNA damage. Moreover, we showed the differential effects of radiation on cancer stem/progenitor cells. The results highlighted the importance of DNA DSB signalling through the ATM protein and its role in cell survival after irradiation.

Abstract LB-169: Asbestos and anthelmintics: Repurposing drugs to prevent mesothelioma in high-risk populations

Abstract The purpose of this study is to address the rising mortality rates in mesothelioma by using repurposed drugs in a chemoprevention setting. An initial screen of 100 repurposed agents at clinically relevant concentrations was conducted across three mesothelioma cell lines using AlamarBlue assays. Having identified the top three individual and combinatorial treatments, analysis of cell-cycle and apoptosis response to these drugs was used to corroborate the findings of the initial screen. As a more representative disease model a mouse PDX was developed from a human biphasic mesothelioma, and furthermore, primary tissue obtained, both of which we have used for explant experiments to assess the efficacy of the top treatments in a more realistic and translational model. As BAP-1 mutations are highly frequent in both acquired and spontaneous mesotheliomas, a CRISPR generated knock-out cell-line will model early-stage disease, and MET-5A mesothelial cells to assess normal cell response. The three top agents, comprising zinc acetate, niclosamide, and colchicine, have proven consistently efficacious in reducing mesothelioma cell-line viability by up to 72.59% individually and 93.55% in combination. Congruent results are provided by annexin V-FITC and FACS experiments which indicate treatments induce cell cycle alterations and apoptosis. Furthermore, FACS analysis of PDX explants also shows increased apoptosis of up to 27.0% in response to 24-hour treatment of two combined drugs. Importantly, the PDX still retains original histopathological features of the human tumour, making it an important foundation for further ex vivo and ultimately in vivo study. Initial results are promising and indicate that several repurposed agents significantly reduce survival and increase death of mesothelioma both in vitro and ex vivo. Mesothelioma, being predominantly caused by long-term asbestos exposure, has been under-researched due to an overreliance on the disuse of asbestos. As such, there is no effectual treatment and average survival remain static at under one year. However, following initial asbestos exposure, a 50-year latency precedes diagnosis, which offers an opportune window for a prevention strategy. Using the CRISPR-BAP-1-knockout cell-line to model early-stage disease, we hope to translate the top candidate into a primary prevention setting. However given the increasing frequency of late stage mesothelioma diagnoses, there is also a tangible need for secondary and tertiary prevention. Repurposed drugs, which offer long term safety and side effect profiles, as well as requiring less temporal and financial investment, allow expedited translation of any potential top candidate into clinic. Through further mechanistic analyses in in vitro and ex vivo cancer models, we hope to identify a single agent or combination that could significantly impede mesothelioma progression and ultimately reduce mortality. Citation Format: Bethan G. Rogoyski, Ankur Karmokar, Sameena Khan, Hong Cai, Farhat L. Khanim, Sara Busacca, Lynne Howells, Dean A. Fennell, Anne L. Thomas, Karen Brown. Asbestos and anthelmintics: Repurposing drugs to prevent mesothelioma in high-risk populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-169.

Subtle Alterations in DNA Methylation Patterns in Normal Cells in Response to Dietary Stilbenoids.

Searching for correlations between dietary polyphenols and risk of chronic diseases has been a challenge due to the lack of quantitative evaluation methods of long-term exposure. We previously observed substantial DNA methylation changes in human cancer cells upon treatment with polyphenols of the stilbenoid class. When induced in normal cells, such molecular changes may persist and reflect chronic exposure. Illumina 450K microarray is used to delineate a genome wide DNA methylation landscape in MCF10A human immortalized mammary epithelial cells exposed to resveratrol (RSV) at noncytotoxic 15μM dose for 9 days. Subtle alterations are observed suggesting remodeling of DNA methylation patterns rather than switch on/off changes. Using pyrosequencing, DNA methylation is quantitatively measured at eight CpG sites located within KCNJ4, RNF169, BCHE, DAOA, HOXA9, RUNX3, KRTAP2-1, and TAGAP, upon exposure to RSV or pterostilbene and shows similar differences induced by both stilbenoids. Two of the probes, Runx3 and Kcnj4, are successfully verified in whole blood DNA from healthy rats on diets supplemented with stilbenoids. The study provides strong support for testing the utility of polyphenol-mediated changes in DNA methylation as quantitative measures of long-term dietary exposures in nutritional epidemiology and clinical trials.

IL-6 counteracts IL-2-dependent suppression of T follicular helper cell responses

Abstract T follicular helper (Tfh) cells are a specialized subset of CD4+ T cells that aide in the production of high-affinity antibody (Ab) responses. In the absence of Tfh cells, there is impairment in the generation of protective Ab responses to pathogens. In autoimmune patients, however, the expansion of unwanted self-reactive Tfh cells favor auto-Ab responses and subsequent Ab-mediated pathology. Thus, understanding the factors that regulate the generation of Tfh cells is critical for developing more efficient vaccination strategies and for designing new therapeutic approaches to prevent Ab-mediated autoimmune diseases. In this regard, it is generally acknowledged that IL-2 inhibits CD4 T cells from differentiating into Tfh cells. Conversely, previous studies suggest that IL-6 signaling is required for normal Tfh cell responses. However, Tfh cells normally differentiate in the absence of IL-6 in some experimental models, suggesting that IL-6 is not absolutely necessary for Tfh cell formation. Thus, the exact role played by IL-6 in controlling Tfh cell responses remains elusive. Using an influenza infection model, we found that IL-6 is dispensable for the priming of influenza-specific Tfh cell precursors, but essential for the maintenance of the Tfh cells at the peak of the influenza immune response. Mechanistically, our results demonstrate that already differentiated Tfh cells produce large amounts of IL-2 in the GCs and that intrinsic IL-6 signaling is needed to antagonize the negative impact of IL-2 in the ongoing Tfh cell response. Collectively, our data demonstrate that IL-6 induces a program that allows for Tfh cells to produce IL-2 without succumbing to the deleterious effects of IL-2 on Tfh cell differentiation.

Molecular Insights Into Withaferin-A-Induced Senescence: Bioinformatics and Experimental Evidence to the Role of NFκB and CARF.

Withaferin-A (Wi-A) has been shown to possess anticancer activity. Molecular mechanism(s) of its action has not been fully resolved. We recruited low dose of Wi-A that caused slow growth arrest in cancer cells and was relatively safe for normal cells. Consistently, we detected nuclear translocation of nuclear factor kappa B (NFκB) and activation of p38MAPK selectively in cancer cells. Bioinformatics analyses revealed that Wi-A did not disrupt IKKα/IKKβ-Nemo complex that regulates NFκB activity. However, it caused moderate change in the conformation of IKKβ-Nemo interacting domain. Experimental data revealed increased level of phosphorylated IκBα in Wi-A-treated cells, suggesting an activation of IKK complex that was supported by nuclear translocation of NFκB. Molecular docking analysis showed that Wi-A did not disrupt; however, decreased the stability of the NFκB-DNA complex. It was supported by downregulation of DNA-binding and transcriptional activities of NFκB. Further analysis revealed that Wi-A caused upregulation of CARF (collaborator of ARF) demonstrating an activation of DNA damage oxidative stress response in both cancer and normal cells. In line with this, upregulation of p21WAF1, p16INK4A, and hypophosphorylated pRB and induction of senescence were observed demonstrating that Wi-A-induced senescence is mediated by multiple pathways in which CARF-mediated DNA damage and oxidative stress play a major role.

Response of human normal and leukemia cells to factors released by amnion fragments in vitro.

Amnion is a membrane surrounding the embryo/fetus which determine growth factors and interleukins with angiogenic, immunogenic, and anti-inflammatory properties. The aim of the present study was to investigate the effects of conditioned culture medium from 24-h cultures of human amnion (hAM CCM) on migration and proliferation of human umbilical vein endothelial primary cells (HUVECs), freshly isolated bone marrow mononuclear cells (BM MNCs), and Jurkat leukemia cell line. Amnion membrane was freshly isolated from healthy placenta and its fragments cultured in vitro to produce hAM CCM. Members of the IGFBP protein family made up one third of all assayed proteins present in the hAM medium. The hAM CCM did not affect the proliferation rate of HUVECs or MNCs, but we observed more intensive migration of those cells, and lower expression of CD31 surface antigen on HUVECs as compared to control cultures. In contrast, Jurkat cells did not respond to hAM CCM treatment by proliferation or mobility change. The conditioned medium from 24-h cultures of human amnion is easy to obtain and is a convenient source of various growth and other factors that may be useful in practical medicine.

Small Molecule Catalysts with Therapeutic Potential.

Catalysts are employed in many areas of research and development where they combine high efficiency with often astonishing selectivity for their respective substrates. In biology, biocatalysts are omnipresent. Enzymes facilitate highly controlled, sophisticated cellular processes, such as metabolic conversions, sensing and signalling, and are prominent targets in drug development. In contrast, the therapeutic use of catalysts per se is still rather limited. Recent research has shown that small molecule catalytic agents able to modulate the redox state of the target cell bear considerable promise, particularly in the context of inflammatory and infectious diseases, stroke, ageing and even cancer. Rather than being “active” on their own in a more traditional sense, such agents develop their activity by initiating, promoting, enhancing or redirecting reactions between biomolecules already present in the cell, and their activity therefore depends critically on the predisposition of the target cell itself. Redox catalysts, for instance, preferably target cells with a distinct sensitivity towards changes in an already disturbed redox balance and/or increased levels of reactive oxygen species. Indeed, certain transition metal, chalcogen and quinone agents may activate an antioxidant response in normal cells whilst at the same time triggering apoptosis in cancer cells with a different pre-existing “biochemical redox signature” and closer to the internal redox threshold. In pharmacy, catalysts therefore stand out as promising lead structures, as sensor/effector agents which are highly effective, fairly selective, active in catalytic, i.e., often nanomolar concentrations and also very flexible in their structural design.

Direct and Intestinal Epithelial Cell-Mediated Effects of TLR8 Triggering on Human Dendritic Cells, CD14+CD16+ Monocytes and γδ T Lymphocytes.

Toll-like receptor (TLR)7/8 plays a crucial role in host recognition/response to viruses and its mucosal expression directly correlates with intestinal inflammation. The aim of this study was to investigate the role of TLR7/8 stimulation of intestinal epithelium in shaping the phenotype and functions of innate immunity cell subsets, and to define direct and/or epithelial cell-mediated mechanisms of the TLR7/8 agonist R848 immunomodulatory activity. We describe novel, TLR8-mediated, pro- and anti-inflammatory effects of R848 on ex vivo cultured human blood monocytes and γδ T lymphocytes, either induced by direct immune cell stimulation or mediated by intestinal epithelial cells (IEC). Apical stimulation with R848 led to its transport across normal polarized epithelial cell monolayer and resulted in the inhibition of monocyte differentiation toward immunostimulatory dendritic cells and Th1 type response. Furthermore, γδ T lymphocyte activation was promoted following direct exposure of these cells to the agonist. Conversely, a selective enrichment of the CD14+CD16+ monocyte subpopulation was observed, which required a CCL2-mediated inflammatory response of normal epithelial cells to R848. Of note, a TLR-mediated activation of control γδ T lymphocytes was promoted by inflamed intestinal epithelium from active Crohn’s disease patients. This study unravels a novel regulatory mechanism linking the activation of the TLR8 pathway in IEC to the monocyte-mediated inflammatory response, and highlights the capacity of the TLR7/8 agonist R848 to directly enhance the activation of γδ T lymphocytes. Overall these results expand the range of cell targets and immune responses controlled by TLR8 triggering that may contribute to the antiviral response, to chronic inflammation, as well as to the adjuvant activity of TLR8 agonists, highlighting the role of intestinal epithelium microenvironment in shaping TLR agonist-induced responses.

Cellular responses of normal (HL-7702) and cancerous (HepG2) hepatic cells to dust extract exposure

Cancerous human liver cell line has been used to test the hepatic toxicity of indoor dust, showing its organic extract decreases cell viability. However, little is known about its impact on normal human liver cell line. In the present study, we compared the cellular responses between carcinoma cell line (HepG2) and normal cell line (HL-7702) after exposing to 10–640 μg/100 μL organic dust extract for 24 h. The dust extract caused cytotoxicity, oxidative damage, inflammatory response and loss of mitochondrial transmembrane potential (MMP) in both cells. The inhibition of cell viability in HL-7702 cells was stronger than that in HepG2 cells, with HL-7702 cells having lower LC50. Higher production of oxidative stress, more loss of MMP and stronger suppression of antioxidant enzymes mRNA level occurred in HepG2 cells, while mRNA expression and hepcidin secretion were enhanced in HL-7702 cells at 40/100 μL, indicating the dust extract probably perturbed their liver Fe homeostasis. Our data showed considerable differences in cellular responses between normal and cancerous cell lines. To obtain accurate data, normal hepatocytes should be employed as they better match with the in vivo tissue than cancerous cell lines.

Mass Cytometric Analysis Reveals Viable Activated Caspase-3+ Luminal Progenitors in the Normal Adult Human Mammary Gland

The normal adult human female mammary gland isabilayered structure consisting of an outer basal layer and two readily distinguished subsets of cells within the inner luminal layer. We now present a validated methodology for undertaking large-scale multi-parameter mass cytometric analyses of these cell types at single-cell resolution. In addition, we show how combining this approach with invitro clonogenic assays of the proliferative and signaling responses of normal human mammary cells to epidermal growth factor (EGF) allows additional subsets with different EGF responses to be discerned. This included the identification of a subset of cells within the phenotypically defined luminal progenitor fraction that displays an elevated content of active caspase-3, including some that generate clones invitro in response to EGF, with immunohistochemical evidence of their presence insitu in fixed preparations of normal human breast tissue.

Radiobiological Response of Normal Human Prostate Cells Located Outside of the Primary Beam Path in a Humanoid Phantom Model Treated with a Conventional SBRT Dose Fractionation Scheme

Radiobiological Response of Normal Human Prostate Cells Located Outside of the Primary Beam Path in a Humanoid Phantom Model Treated with a Conventional SBRT Dose Fractionation Scheme

Expression of ALCAM (CD166) and PD-L1 (CD274) independently predicts shorter survival in malignant pleural mesothelioma

Diffuse malignant mesothelioma of the pleura is a highly aggressive tumor typically associated with short survival. ALCAM (CD166), a type I transmembrane protein, is a member of the immunoglobulin superfamily. In normal cells, ALCAM regulates physiological processes such as angiogenesis and immune response. In cancer, it is associated with neoplastic progression, including invasion, migration, and metastasis. Furthermore, ALCAM is considered one of the cancer stem cell markers such as ALDH1 (ALDH1A1) and SALL4. The PD-L1 (CD274)/PD-1 (PDCD1, CD279) pathway is crucial for the modulation of immune responses in normal cells. Nevertheless, pathologic activation of the PD-L1/PD-1 pathway participates in immune evasion by tumor cells. Many PD-L1-expressing tumor cells have been identified in different types of cancer, including malignant mesothelioma. In this study, 175 well-characterized primary diffuse pleural mesotheliomas, including the epithelioid (n = 148), biphasic (n = 15), and sarcomatoid (n = 12) histotypes, were evaluated immunohistochemically for cancer stem cell markers (ALCAM, ALDH1, and SALL4) and PD-L1 expression. Twenty-five percent of the mesotheliomas (43/175) expressed ALCAM, whereas ALDH1 and SALL4 positivity was seen in 1% to 2% of cases. Thirty-three percent of the analyzed tumors (57/175) contained PD-L1-positive cells. Overall survival was significantly decreased in the cohort of patients with ALCAM- or PD-L1-positive tumors (both P < .01). Furthermore, the multivariate Cox hazards regression analysis identified ALCAM and PD-L1 (both P < 0.01) as potential independent risk factors. Thus, a combination of these 2 markers might be useful for prognostication and planning the treatment of patients with malignant pleural mesothelioma.

Response of Normal Cells Following Multiple Radiation Exposure under Radiotherapy Setting

Radiotherapy is an established approach for killing of tumour cells. During the process, most of the normal cells also get affected due to direct exposure or by bystander effects. To measure the damage pattern in healthy cells, a pilot study was designed under radiotherapy settings. Right leg region of Strain ‘A’ male mice was locally exposed to Cobalt60 gamma radiation with a dose of 2 Gy/ day for 5 consecutive days. After completion of each fraction, blood haematology and γH2AX studies were performed at 1 h time point in blood and bone marrow cells. Chromosomal aberration study in bone marrow was carried out at 24 h post irradiation of each fraction for evaluation of DNA damage. γH2AX and chromosomal aberration were found significantly (p&amp;lt;0.001) increased with each consecutive dose upto 4th fractions. Blood hematology showed a linear reduction in total WBC counts which included the reduction in lymphocytes and increased granulocytes with each passing dose up to 4th fraction. However, non significant damage (p&amp;gt;0.05) for all parameters have been observed for 4th and 5th split doses. The study indicated that repeated exposure leads to damage fixation in normal cells, possibly indicating a state of adaptation.

Ionizing radiation response of primary normal human lens epithelial cells.

Whilst the cataractogenic potential of ionizing radiation has been known for over the past 120 years, little is known about radiation responses of lens cells. Our previous work was the first to evaluate the radiosensitivity of lens cells with the clonogenic assay, documenting that the survival of HLEC1 human lens epithelial cells is comparable to that of WI-38 human lung fibroblasts. Moreover, HLEC1 cells were found to contain subsets where irradiation stimulates proliferation or facilitates formation of abortive colonies with fewer cells than human fibroblasts. This study aims to gain insights into these mechanisms. Irradiation of HLEC1 cells with 10% survival dose caused a growth delay but did not reduce viability. HLEC1 cells at high cumulative population doubling level were more susceptible to radiogenic premature senescence than WI-38 cells. Concerning p53 binding protein 1 (53BP1) foci, HLEC1 cells harbored less spontaneous foci but more radiogenic foci than in WI-38 cells, and the focus number returned to spontaneous levels within 48 h postirradiation both in HLEC1 and WI-38. The chemical inhibition of DNA repair kinases ataxia telangiectasia mutated, DNA-dependent protein kinase or both delayed and attenuated the appearance and disappearance of radiogenic 53BP1 foci, increased radiogenic premature senescence and enhanced clonogenic inactivation. The DNA microarray analysis suggested both radiogenic stimulation and inhibition of cell proliferation. Treatment with conditioned medium from irradiated cells did not change growth and the plating efficiency of nonirradiated cells. These results partially explain mechanisms of our previous observations, such that unrepaired or incompletely repaired DNA damage causes a growth delay in a subset of HLEC1 cells without changing viability through induction of premature senescence, thereby leading to clonogenic inactivation, but that growth is stimulated in another subset via as yet unidentified mechanisms, warranting further studies.

DNA damage and the bystander response in tumor and normal cells exposed to X-rays

Monolayer and suspension cultures of tumor (BMG-1, CCRF-CEM), normal (AG1522, HADF, lymphocytes) and ATM-mutant (GM4405) human cells were exposed to X-rays at doses used in radiotherapy (high dose and high dose-rate) or radiological imaging (low dose and low dose-rate). Radiation-induced DNA damage, its persistence, and possible bystander effects were evaluated, based on DNA damage markers (γ-H2AX, p53ser15) and cell-cycle-specific cyclins (cyclin B1 and cyclin D1). Dose-dependent DNA damage and a dose-independent bystander response were seen after exposure to high dose and high dose-rate radiation. The level of induced damage (expression of p53ser15, γ-H2AX) depended on ATM status. However, low dose and dose-rate exposures neither increased expression of marker proteins nor induced a bystander response, except in the CCRF-CEM cells. Bystander effects after high-dose irradiation may contribute to stochastic and deterministic effects. Precautions to protect unexposed regions or to inhibit transmission of DNA damage signaling might reduce radiation risks.

Abstract LB-335: Induction of Ribosomal Checkpoint Induced Senescence (RCIS) for the treatment of liver cancer

Abstract While ribosomal proteins are generally considered to be essential genes, the existence of ribosomopathies suggests that a therapeutic window for exploiting ribosomal proteins as targets for cancer therapy may exist. We report distinct stress responses of tumor cells and normal cells upon knockdown of Rpl15, a ribosomal protein which was identified in a direct in vivo shRNA screen for new therapeutic targets in liver cancer. While normal cells undergo a reversible cell cycle arrest, we found that in tumor cells, the knockdown of Rpl15 triggers a ribosomal stress response followed by induction of cellular senescence, designated ribosomal checkpoint induced senescence (RCIS). Importantly, Rpl15 suppression triggered RCIS independent of any reduction in global protein translation. Using well established therapy resistant murine HCC models, we show that shRNAs targeting Rpl15 potently suppress tumor development in genetically diverse murine HCCs. Using Rpl15 shRNA transgenic mice, allowing for ubiquitous shRNA mediated suppression of Rpl15, we show that systemic Rpl15 suppression can be tolerated for up to 5 days, thus revealing a therapeutic window for metronomic Rpl15 inhibitory therapies. However, molecular modeling analyses revealed that Rpl15 is not druggable by small molecule inhibitors and we thus set out to explore whether RCIS can be induced via interference with other ribosomal proteins or other factors involved in ribosome biogenesis. We generated and screened a focused shRNA library targeting 41 ribosomal proteins and 19 ribosome biogenesis factors and found that apart from Rpl15, only a small subset of shRNAs scored. From a druggability point of view it was interesting, that shRNAs targeting components of the RNA polymerase I complex had scored, as recently a pharmacological RNA polymerase I inhibitor became available. Both genetic and pharmacological inhibition of RNA polymerase I induced RCIS and mediated an excellent prolongation of survival (far superior to the clinically used standard therapy Sorafenib), however in contrast to targeting Rpl15, targeting RNA polymerase I failed to achieve full long term tumor suppression. Mechanistically we found a less efficient immune mediated clearance of senescent cells as a possible explanation. In line with the idea that distinct secretory profiles underlie differences in immune mediated clearance of RCIS cells, cytokine arrays revealed distinct secretory profiles of RCIS induced by Rpl15 suppression and RCIS induced via shRNA or pharmacological RNA polymerase I inhibition. Citation Format: Katharina Wolter, Marina Pesic, Sabrina Klotz, Nicolas Herranz, Torsten Wuestefeld, Tae-Won Kang, Marco Seehawer, Rishabh Chawla, Stefan Zwirner, Jonathan Cotton, Benyuan Zhou, Marcel Krüger, Frank Klawonn, Thomas Longerich, Bence Sipos, Bernd Pichler, Jesus Gil, Martin Eilers, Prem K. Premsrirut, Antti Poso, Lars Zender. Induction of Ribosomal Checkpoint Induced Senescence (RCIS) for the treatment of liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-335. doi:10.1158/1538-7445.AM2017-LB-335

Abstract 5458: Oncostatin M induces epithelial-mesenchymal transition and stem cell properties in senescent human mammary epithelial cells

Abstract The Interleukin-6 (IL-6) cytokine family member Oncostatin M (OSM) induces epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties in transformed cancer cells. Paradoxically, OSM induces a growth-inhibiting senescence response in normal human mammary epithelial cells (HMEC). Senescence is induced in normal cells by various stresses, such as aberrant oncogene signaling, and serves as a major tumor-suppressive barrier that must be bypassed for cellular transformation in vitro and tumorigenesis in vivo. Previous reports show that aberrant reactivation of EMT-inducing transcription factors (EMT-TFs) promotes senescence bypass. Thus, aberrant EMT may drive tumor initiation in addition to providing cancer cells with mesenchymal cell and stem cell properties that drive tumor progression, metastasis, and tumor recurrence. Conversely, our studies here indicate that much like aberrant oncogene signaling, aberrant EMT and CSC-properties induced by OSM may actually engage senescence in normal cells. Infecting Primary HMEC isolated from reduction mammoplasty tissue with lentivirus encoding shRNA to p16 created indefinite lifespan “Normal” HMEC. Subsequent infections with viruses encoding shRNA to p53, then constitutive expression of MYC and RAS created “Transformed” HMEC. After treating HMEC with recombinant OSM, proliferation and senescence were assessed by growth assays and qRT-PCR analysis of senescence-associated β-galactosidase (GLB1) expression. EMT and CSC-properties were assessed by western and qRT-PCR analysis of an epithelial marker E-cadherin (CDH1), a mesenchymal marker Vimentin (VIM), the EMT-TF Snail (SNAI1), and the CSC markers CD44 and HAS2. Flow cytometry examining BrdU-incorporation and CD24/CD44 surface expression also assessed proliferation and a CSC phenotype. OSM reduced growth by 80% and induced GLB1 expression in Normal HMEC, but not in Transformed HMEC. OSM-induced senescence was accompanied by Snail induction, changes in EMT-associated markers (reduced E-cadherin/increased Vimentin), and CSC marker (CD44 and HAS2) induction. Notably, OSM induced immediate SNAI1 and HAS2 expression; and a majority of the OSM-induced CD44+ population consisted of non-proliferating (BrdU-) cells. Unexpectedly, Snail and CD44 were also induced during oncogenic RAS-induced senescence; and Snail expression alone reduced growth and induced CD44 in normal HMEC. These results suggest that much like aberrant oncogene signaling, aberrantly induced EMT and CSC-properties may engage senescence in normal cells rather than promoting senescence bypass. Defining the mechanism linking senescence, EMT, and CSC-properties will identify targets for novel “pro-senescence” therapies that may inhibit metastasis and tumor recurrence, and expand our repertoire of therapies that enhance cancer patient outcomes. Citation Format: Benjamin L. Bryson, Damian J. Junk, Mark W. Jackson. Oncostatin M induces epithelial-mesenchymal transition and stem cell properties in senescent human mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5458. doi:10.1158/1538-7445.AM2017-5458

Possible role of polyphosphatases in yeast sensitivity to DCS-1800 electromagnetic fields

The ability of living organisms to perceive electromagnetic radiation is one of the most intriguing issues that concern the fundamental problem of interaction of living matter with the factors of physical nature. Polyphosphates can be possible receptors. The purpose of the study was to evaluate the role of the polyphosphatases PPN1 and PPX1 in the cell response to exposure to electromagnetic fields (EMFs) of 1871 MHz that were generated by DCS-1800 base stations. Six-week exposure at energy flux densities of 0.1–10 W/m2 was used. The corresponding values of specific adsorption rate (SAR) were 0.0075–1.5 W/kg. Electromagnetic radiation was found to lead to the impairment of a number of physiological and metabolic functions of cells, change their resistance to antibiotics, and result in irreversible changes in their genome. Low doses of the EMF caused the strongest biological responses. It was demonstrated that the deficiency in the ppn1 and ppx1 genes made the strains less adaptive, which resulted in an increase in their sensitivity to EMF exposure. Both polyphosphatases PPN1 and PPX1 were shown to be necessary for the normal cell response to the nonionizing electromagnetic radiation of 1871 MHz.