Primary cilium participates in radiation-induced bystander effects through TGF-ß1 signaling.

Many studies have indicated that tumor growth factor-beta (TGF-ß) signaling mediates radiation-induced bystander effects (RIBEs). The primary cilium (PC) coordinates several signaling pathways including TGF-ß signaling to regulate diverse cellular processes. But whether the PC participates in TGF-ß induced RIBEs remains unclear. The cellular levels of TGF-ß1 were detected by western blot analysis and the secretion of TGF-ß1 was measured by ELISA kit. The ciliogenesis was altered by CytoD treatment, STIL siRNA transfection, IFT88 siRNA transfection, or KIF3a siRNA transfection, separately, and was detected by western blot analysis and immunofluorescence staining. G0 /G1 phase cells were arrested by serum starvation and S phase cells were induced by double thymidine block. The TGF-ß1 signaling was interfered by LY2109761, a TGF-ß receptor 1 (TßR1) inhibitor, or TGF-ß1 neutral antibody. The DNA damages were induced by TGF-ß1 or radiated conditional medium (RCM) from irradiated cells and were reflected by p21 expression, 53BP1 foci, and γH2AX foci. Compared with unirradiated control, both A549 and Beas-2B cells expressed and secreted more TGF-ß1 after carbon ion beam or X-ray irradiation. RCM collected from irradiated cells or TGF-ß1 treatment caused an increase of DNA damage in cocultured unirradiated Beas-2B cells while blockage of TGF-ß signaling by TßR1 inhibitor or TGF-ß1 neutral antibody alleviates this phenomenon. IFT88 siRNA or KIF3a siRNA impaired PC formation resulted in an aggravated DNA damage in bystander cells, while elevated PC formation by CytoD or STIL siRNA resulted in a decrease of DNA damage. Furthermore, TGF-ß1 induced more DNA damages in S phases cells which showed lower PC formation rate and less DNA damages in G0 /G1 phase cells which showed higher PC formation rate. This study demonstrates the particular role of primary cilia during RCM induced DNA damages through TGF-ß1 signaling restriction and thereby provides a functional link between primary cilia and RIBEs.

Potent bystander effect and tumor tropism in suicide gene therapy using stem cells from human exfoliated deciduous teeth.

Herpes simplex virus thymidine kinase (HSVTK)/ganciclovir (GCV) suicide gene therapy has a long history of treating malignant gliomas. Recently, stem cells from human exfoliated deciduous teeth (SHED), which are collected from deciduous teeth and have excellent harvestability, ethical aspects, and self-renewal, have been attracting attention mainly in the field of gene therapy. In the present study, we assessed SHED as a novel cellular vehicle for suicide gene therapy in malignant gliomas, as we have previously demonstrated with various cell types. SHED was transduced with the HSVTK gene (SHEDTK). In vitro experiments showed a significant bystander effect between SHEDTK and glioma cell lines in coculture. Furthermore, apoptotic changes caused by caspase 3/7 activation were simultaneously observed in SHEDTK and glioma cells. Mice implanted with a mixture of U87 and SHEDTK and treated with intraperitoneal GCV survived for longer than 100 days. Additionally, tumors in treatment model mice were significantly reduced in size during the treatment period. SHEDTK implanted at the contralateral hemisphere migrated toward the tumor crossing the corpus callosum. These results suggested that SHEDTK-based suicide gene therapy has potent tumor tropism and a bystander-killing effect, potentially offering a new promising therapeutic modality for malignant gliomas.

Bio-acoustic signaling; exploring the potential of sound as a mediator of low-dose radiation and stress responses in the environment.

OBJECTIVES: This commentary reviews and evaluates the role of sound signals as part of the infosome of cells and organisms. Emission and receipt of sound has recently been identified as a potentially important universal signaling mechanism invoked when organisms are stressed. Recent evidence from plants, animals and microbes suggests that it could be a stimulus for specific or general molecular cellular stress responses in different contexts, and for triggering population level responses. This paper reviews the current status of the field with particular reference to the potential role of sound signaling as an immediate/early bystander effector (RIBE) during radiation-induced stress. CONCLUSIONS: While the chemical effectors involved in intercellular and inter-organismal signaling have been the subject of intense study in the field of Chemical Ecology, less appears to be known about physical signals in general and sound signals in particular. From this review we conclude that these signals are ubiquitous in each kingdom and behave very like physical bystander signals leading to regulation of metabolic pathways and gene expression patterns involved in adaptation, synchronization of population responses, and repair or defence against damage. We propose the hypothesis that acoustic energy released on interaction of biota with electromagnetic radiation may represent a signal released by irradiated cells leading to, or complementing, or interacting with, other responses, such as endosome release, responsible for signal relay within the unirradiated individuals in the targeted population.

A general theoretical framework to design base editors with reduced bystander effects.

Base editors (BEs) hold great potential for medical applications of gene therapy. However, high precision base editing requires BEs that can discriminate between the target base and multiple bystander bases within a narrow active window (4 - 10 nucleotides). Here, to assist in the design of these optimized editors, we propose a discrete-state stochastic approach to build an analytical model that explicitly evaluates the probabilities of editing the target base and bystanders. Combined with all-atom molecular dynamic simulations, our model reproduces the experimental data of A3A-BE3 and its variants for targeting the "TC" motif and bystander editing. Analyzing this approach, we propose several general principles that can guide the design of BEs with a reduced bystander effect. These principles are then applied to design a series of point mutations at T218 position of A3G-BEs to further reduce its bystander editing. We verify experimentally that the new mutations provide different levels of stringency on reducing the bystander editing at different genomic loci, which is consistent with our theoretical model. Thus, our study provides a computational-aided platform to assist in the scientifically-based design of BEs with reduced bystander effects.

Ionising Radiation Promotes Invasive Potential of Breast Cancer Cells: The Role of Exosomes in the Process.

Along with the cells that are exposed to radiation, non-irradiated cells can unveil radiation effects as a result of intercellular communication, which are collectively defined as radiation induced bystander effects (RIBE). Exosome-mediated signalling is one of the core mechanisms responsible for multidirectional communication of tumor cells and their associated microenvironment, which may result in enhancement of malignant tumor phenotypes. Recent studies show that exosomes and exosome-mediated signalling also play a dynamic role in RIBE in cancer cell lines, many of which focused on altered exosome cargo or their effects on DNA damage. However, there is a lack of knowledge regarding how these changes in exosome cargo are reflected in other functional characteristics of cancer cells from the aspects of invasiveness and metastasis. Therefore, in the current study, we aimed to investigate exosome-mediated bystander effects of 2 Gy X-ray therapeutic dose of ionizing radiation on the invasive potential of MCF-7 breast cancer cells in vitro via assessing Matrigel invasion potential, epithelial mesenchymal transition (EMT) characteristics and the extent of glycosylation, as well as underlying plausible molecular mechanisms. The findings show that exosomes derived from irradiated MCF-7 cells enhance invasiveness of bystander MCF-7 cells, possibly through altered miRNA and protein content carried in exosomes.

Role of Extracellular Vesicles in Compromising Cellular Resilience to Environmental Stressors.

Extracellular vesicles (EVs), like exosomes, are nanosized membrane-enveloped vesicles containing different bioactive cargo, such as proteins, lipids, mRNA, miRNA, and other small regulatory RNAs. Cell-derived EVs, including EVs originating from stem cells, may capture components from damaged cells or cells impacted by therapeutic treatments. Interestingly, EVs derived from stem cells can be preconditioned to produce and secrete EVs with different therapeutic properties, particularly with respect to heat-shock proteins and other molecular cargo contents. This behavior is consistent with stem cells that also respond differently to various microenvironments. Heat-shock proteins play roles in cellular protection and mediate cellular resistance to radiotherapy, chemotherapy, and heat shock. This review highlights the possible roles EVs play in mediating cellular plasticity and survival when exposed to different physical and chemical stressors, with a special focus on the respiratory distress due to the air pollution.

Reprogramming of microRNA expression via E2F1 downregulation promotes Salmonella infection both in infected and bystander cells.

Cells infected with pathogens can contribute to clearing infections by releasing signals that instruct neighbouring cells to mount a pro-inflammatory cytokine response, or by other mechanisms that reduce bystander cells' susceptibility to infection. Here, we show the opposite effect: epithelial cells infected with Salmonella Typhimurium secrete host factors that facilitate the infection of bystander cells. We find that the endoplasmic reticulum stress response is activated in both infected and bystander cells, and this leads to activation of JNK pathway, downregulation of transcription factor E2F1, and consequent reprogramming of microRNA expression in a time-dependent manner. These changes are not elicited by infection with other bacterial pathogens, such as Shigella flexneri or Listeria monocytogenes. Remarkably, the protein HMGB1 present in the secretome of Salmonella-infected cells is responsible for the activation of the IRE1 branch of the endoplasmic reticulum stress response in non-infected, neighbouring cells. Furthermore, E2F1 downregulation and the associated microRNA alterations promote Salmonella replication within infected cells and prime bystander cells for more efficient infection.

Role of epigenetic mechanisms in propagating off-targeted effects following radiation based therapies - A review.

Despite being an important diagnostic and treatment modality, ionizing radiation (IR) is also known to cause genotoxicity and multiple side effects leading to secondary carcinogenesis. While modern cancer radiation therapy has improved patient recovery and enhanced survival rates, the risk of radiation-related adverse effects has become a growing challenge. It is now well-accepted that IR-induced side effects are not exclusively restricted to exposed cells but also spread to distant 'bystander' cells and even to the unexposed progeny of the irradiated cells. These 'off-targeted' effects involve a plethora of molecular events depending on the type of radiation and tumor tissue background. While the mechanisms by which off-targeted effects arise remain obscure, emerging evidence based on the non-mendelian inheritance of various manifestations of them as well as their persistence for longer periods supports a contribution of epigenetic factors. This review focuses on the major epigenetic phenomena including DNA methylation, histone modifications, and small RNA mediated silencing and their versatile role in the manifestation of IR induced off-targeted effects. As short- and long-range communication vehicles respectively, the role of gap junctions and exosomes in spreading these epigenetic-alteration driven off-targeted effects is also discussed. Furthermore, this review emphasizes the possible therapeutic potentials of these epigenetic mechanisms and how beneficial outcomes could potentially be achieved by targeting various signaling molecules involved in these mechanisms.

Downregulation of NK cell activities in Apolipoprotein C-III-induced hyperlipidemia resulting from lipid-induced metabolic reprogramming and crosstalk with lipid-laden dendritic cells.

OBJECTIVE: Apolipoprotein C-III (Apoc3) is a key component of triglyceride-rich lipoproteins (TRL). The Apoc3-transgenic mice are characterized by high levels of plasma triglyceride and free fatty acids (FFAs). Apoc3 stimulates human monocytes via activation of the NLRP3 inflammasome. Considering the NK cell downregulation in obese individuals and the possible stimulatory-effects of macrophages, variations of NK cell functions and underlying mechanisms were investigated in mice with Apoc3-induced hyperlipidemia. METHODS: Variations of activities and glycolipid metabolism in NK cells of the Apoc3-transgenic mice with hyperlipidemia were detected. Molecular mechanisms of lipid-induced metabolic-reprogramming in NK cells were analyzed based on the transcriptome sequencing. Finally, effects of DCs in mice with hyperlipidemia on NK cell functions were determined. RESULTS: Impaired number and function of NK cells in Apoc3TG mice was involved with the increased fatty acid oxidation and decreased glycolysis. Increased uptake of FFAs in Apoc3TG-NK cells contributed to the peroxisome proliferator-activated receptor (PPAR) activation and the downstream PTEN-AKT-mTOR/FOXO1 signaling pathway. Inhibition of PPAR or CPT1α only partly reversed the IFN-γ production of Apoc3TG-NK cells, but completely restored IFN-γ secretion by palmitic acid-treated NK cells ex vivo, indicating that other factors contributed to the Apoc3TG-NK cell downregulation. Meanwhile, Apoc3TG-DCs, which contained more lipids in the cytoplasm, depended on reactive oxygen species (ROS) to increase the expressions PD-L1, TGF-ß1, and NKG2D ligands and suppress NK cell activities. DCs of the Apoc3TG-CD36-/+ hybrid mice with less intracellular lipids and ROS production could not inhibit NK cells, indicating that intracellular FFAs promoted the immune-modulatory function of DCs. CONCLUSIONS: The downregulation of NK cell activities in individuals with Apoc3-induced hyperlipidemia was due to the increased fatty acid oxidation in NK cells and the bystander suppression caused by lipid-laden DCs. The dual recovery function of NK cells and DCs would improve the prognosis of patients with metabolic syndrome.

Role of MicroRNA-145 in DNA Damage Signalling and Senescence in Vascular Smooth Muscle Cells of Type 2 Diabetic Patients.

Increased cardiovascular morbidity and mortality in individuals with type 2 diabetes (T2DM) is a significant clinical problem. Despite advancements in achieving good glycaemic control, this patient population remains susceptible to macrovascular complications. We previously discovered that vascular smooth muscle cells (SMC) cultured from T2DM patients exhibit persistent phenotypic aberrancies distinct from those of individuals without a diagnosis of T2DM. Notably, persistently elevated expression levels of microRNA-145 co-exist with characteristics consistent with aging, DNA damage and senescence. We hypothesised that increased expression of microRNA-145 plays a functional role in DNA damage signalling and subsequent cellular senescence specifically in SMC cultured from the vasculature of T2DM patients. In this study, markers of DNA damage and senescence were unambiguously and permanently elevated in native T2DM versus non-diabetic (ND)-SMC. Exposure of ND cells to the DNA-damaging agent etoposide inflicted a senescent phenotype, increased expression of apical kinases of the DNA damage pathway and elevated expression levels of microRNA-145. Overexpression of microRNA-145 in ND-SMC revealed evidence of functional links between them; notably increased secretion of senescence-associated cytokines and chronic activation of stress-activated intracellular signalling pathways, particularly the mitogen-activated protein kinase, p38α. Exposure to conditioned media from microRNA-145 overexpressing cells resulted in chronic p38α signalling in naïve cells, evidencing a paracrine induction and reinforcement of cell senescence. We conclude that targeting of microRNA-145 may provide a route to novel interventions to eliminate DNA-damaged and senescent cells in the vasculature and to this end further detailed studies are warranted.

Exosomes and exosomal microRNA in non-targeted radiation bystander and abscopal effects in the central nervous system.

Localized cranial radiotherapy is a dominant treatment for brain cancers. After being subjected to radiation, the central nervous system (CNS) exhibits targeted effects as well as non-targeted radiation bystander effects (RIBE) and abscopal effects (RIAE). Radiation-induced targeted effects in the CNS include autophagy and various changes in tumor cells due to radiation sensitivity, which can be regulated by microRNAs. Non-targeted radiation effects are mainly induced by gap junctional communication between cells, exosomes containing microRNAs can be transduced by intracellular endocytosis to regulate RIBE and RIAE. In this review, we discuss the involvement of microRNAs in radiation-induced targeted effects, as well as exosomes and/or exosomal microRNAs in non-targeted radiation effects in the CNS. As a target pathway, we also discuss the Akt pathway which is regulated by microRNAs, exosomes, and/or exosomal microRNAs in radiation-induced targeted effects and RIBE in CNS tumor cells. As the CNS-derived exosomes can cross the blood-brain-barrier (BBB) into the bloodstream and be isolated from peripheral blood, exosomes and exosomal microRNAs can emerge as promising minimally invasive biomarkers and therapeutic targets for radiation-induced targeted and non-targeted effects in the CNS.

Regulation of XPA could play a role in inhibition of radiation-induced bystander effects in QU-DB cells at high doses.

INTRODUCTION: Radiation-induced bystander effects (RIBE) is the radiobiological effects detected in nonirradiated cells that have received signals from neighboring irradiated cells. In some studies, there are observations that RIBE unexpectedly reduces at high doses. In this study, the expression of two selected apoptotic and repair genes and their possible role in the formation of this unexpected reduction is examined. MATERIALS AND METHODS: The QU-DB cells were irradiated with gamma rays of a60 Co teletherapy unit at doses of 2, 4, 6, and 8 Gy. One hour following irradiation, their culture media were transferred to bystander cells to induced RIBE. After 24 h incubation, the RNA of cells was isolated and cDNA synthesized. Expression levels of BAX, XPA, and XPA/BAX ratio were examined by relative quantitative reverse transcription-polymerase chain reaction. RESULTS: In target cells, up-regulation of both genes was observed at all doses. In bystander cells, at the low dose (2 Gy), the expression of BAX was more than XPA; at 4 Gy, the ratio was balanced. A significant correlation was found between the XPA/BAX ratio and the dose, at high doses pattern of gene expression dominated by DNA repair gene. CONCLUSION: Gene expression profile was distinctive in bystander cells compared to target cells. The observed linear increasing of the ratio of XPA/BAX could support the hypothesis that the DNA repair system is stimulated and causes a reduction in RIBE at high doses.

Exploiting loss of heterozygosity for allele-selective colorectal cancer chemotherapy.

Cancer chemotherapy targeting frequent loss of heterozygosity events is an attractive concept, since tumor cells may lack enzymatic activities present in normal constitutional cells. To find exploitable targets, we map prevalent genetic polymorphisms to protein structures and identify 45 nsSNVs (non-synonymous small nucleotide variations) near the catalytic sites of 17 enzymes frequently lost in cancer. For proof of concept, we select the gastrointestinal drug metabolic enzyme NAT2 at 8p22, which is frequently lost in colorectal cancers and has a common variant with 10-fold reduced activity. Small molecule screening results in a cytotoxic kinase inhibitor that impairs growth of cells with slow NAT2 and decreases the growth of tumors with slow NAT2 by half as compared to those with wild-type NAT2. Most of the patient-derived CRC cells expressing slow NAT2 also show sensitivity to 6-(4-aminophenyl)-N-(3,4,5-trimethoxyphenyl)pyrazin-2-amine (APA) treatment. These findings indicate that the therapeutic index of anti-cancer drugs can be altered by bystander mutations affecting drug metabolic genes.

Coupling of cell fate selection model enhances DNA damage response and may underlie BE phenomenon.

Double-strand break-induced (DSB) cells send signal that induces DSBs in neighbour cells, resulting in the interaction among cells sharing the same medium. Since p53 network gives oscillatory response to DSBs, such interaction among cells could be modelled as an excitatory coupling of p53 network oscillators. This study proposes a plausible coupling model of three-mode two-dimensional oscillators, which models the p53-mediated cell fate selection in globally coupled DSB-induced cells. The coupled model consists of ATM and Wip1 proteins as variables. The coupling mechanism is realised through ATM variable via a mean-field modelling the bystander signal in the intercellular medium. Investigation of the model reveals that the coupling generates more sensitive DNA damage response by affecting cell fate selection. Additionally, the authors search for the cause-effect relationship between coupled p53 network oscillators and bystander effect (BE) endpoints. For this, they search for the possible values of uncertain parameters that may replicate BE experiments' results. At certain parametric regions, there is a correlation between the outcomes of cell fate and endpoints of BE, suggesting that the intercellular coupling of p53 network may manifest itself as the form of observed BEs.

Effect direction meta-analysis of GWAS identifies extreme, prevalent and shared pleiotropy in a large mammal.

In genome-wide association studies (GWAS), variants showing consistent effect directions across populations are considered as true discoveries. We model this information in an Effect Direction MEta-analysis (EDME) to quantify pleiotropy using GWAS of 34 Cholesky-decorrelated traits in 44,000+ cattle with sequence variants. The effect-direction agreement between independent bull and cow datasets was used to quantify the false discovery rate by effect direction (FDRed) and the number of affected traits for prioritised variants. Variants with multi-trait p < 1e-6 affected 1∼22 traits with an average of 10 traits. EDME assigns pleiotropic variants to each trait which informs the biology behind complex traits. New pleiotropic loci are identified, including signals from the cattle FTO locus mirroring its bystander effects on human obesity. When validated in the 1000-Bull Genome database, the prioritized pleiotropic variants consistently predicted expected phenotypic differences between dairy and beef cattle. EDME provides robust approaches to control GWAS FDR and quantify pleiotropy.

RADIATION-INDUCED BYSTANDER EFFECT - MODELING, MANIFESTATION, MECHANISMS, PERSISTENCE, CANCER RISKS (literature review). / RADIATsIY̆NO-INDUKOVANYY̆ EFEKT SVIDKA ­ MODELIuVANNIa, PROIaVY, MEKhANIZMY ROZVYTKU, PERSYSTENTsIIa, ONKOLOGIChNI RYZYKY (ogliad literatury).

The review summarizes and analyzes the data of world scientific literature and the results of the own research con- cerning one of the main non-targeted effects of ionizing radiation - the radiation induced bystander effect (RIBE) - the ability of irradiated target cells to induce secondary biological changes in non-irradiated receptor cells. The his- tory of studies of this phenomenon is presented - it described under various names since 1905, began to study from the end of the twentieth century when named as RIBE and caused particular interest in the scientific community during recent decades. It is shown that the development of biological science and the improvement of research methods allowed to get new in-depth data on the development of RIBE not only at the level of the whole organism, but even at the genome level. The review highlights the key points of numerous RIBE investigations including mod- eling; methodological approaches to studying; classification; features of interaction between irradiated and intact cells; the role of the immune system, oxidative stress, cytogenetic disorders, changes in gene expression in the mechanism of development of RIBE; rescue effect, abscopal effect, persistence, modification, medical effects. It is emphasized that despite the considerable amount of research concerning the bystander response as the universal phenomenon and RIBE as one of its manifestations, there are still enough «white spots¼ in determining the mech- anisms of the RIBE formation and assessing the possible consequences of its development for human health.

Suicide Gene Therapy Against Malignant Gliomas by the Local Delivery of Genetically Engineered Umbilical Cord Mesenchymal Stem Cells as Cellular Vehicles.

BACKGROUND: Glioblastoma (GBM) is a malignant tumor that is difficult to eliminate, and new therapies are thus strongly desired. Mesenchymal stem cells (MSCs) have the ability to locate to injured tissues, inflammation sites and tumors and are thus good candidates for carrying antitumor genes for the treatment of tumors. Treating GBM with MSCs that have been transduced with the herpes simplex virus thymidine kinase (HSV-TK) gene has brought significant advances because MSCs can exert a bystander effect on tumor cells upon treatment with the prodrug ganciclovir (GCV). OBJECTIVE: In this study, we aimed to determine whether HSV-TK-expressing umbilical cord mesenchymal stem cells (MSCTKs) together with prodrug GCV treatment could exert a bystander killing effect on GBM. METHODS AND RESULTS: Compared with MSCTK: U87 ratio at 1:10,1:100 and 1:100, GCV concentration at 2.5µM or 250µM, when MSCTKs were cocultured with U87 cells at a ratio of 1:1, 25 µM GCV exerted a more stable killing effect. Higher amounts of MSCTKs cocultured with U87 cells were correlated with a better bystander effect exerted by the MSCTK/GCV system. We built U87-driven subcutaneous tumor models and brain intracranial tumor models to evaluate the efficiency of the MSCTK/GCV system on subcutaneous and intracranial tumors and found that MSCTK/GCV was effective in both models. The ratio of MSCTKs and tumor cells played a critical role in this therapeutic effect, with a higher MSCTK/U87 ratio exerting a better effect. CONCLUSION: This research suggested that the MSCTK/GCV system exerts a strong bystander effect on GBM tumor cells, and this system may be a promising assistant method for GBM postoperative therapy.

Radiation-Induced Bystander Effect is Mediated by Mitochondrial DNA in Exosome-Like Vesicles.

Exosome-like vesicles (ELV) are involved in mediating radiation-induced bystander effect (RIBE). Here, we used ELV from control cell conditioned medium (CCCM) and from 4 Gy of X-ray irradiated cell conditioned medium (ICCM), which has been used to culture normal human fibroblast cells to examine the possibility of ELV mediating RIBE signals. We investigated whether ELV from 4 Gy irradiated mouse serum mediate RIBE signals. Induction of DNA damage was observed in cells that were treated with ICCM ELV and ELV from 4 Gy irradiated mouse serum. In addition, we treated CCCM ELV and ICCM ELV with RNases, DNases, and proteinases to determine which component of ELV is responsible for RIBE. Induction of DNA damage by ICCM ELV was not observed after treatment with DNases. After treatment, DNA damages were not induced in CCCM ELV or ICCM ELV from mitochondria depleted (ρ0) normal human fibroblast cells. Further, we found significant increase in mitochondrial DNA (mtDNA) in ICCM ELV and ELV from 4 Gy irradiated mouse serum. ELV carrying amplified mtDNA (ND1, ND5) induced DNA damage in treated cells. These data suggest that the secretion of mtDNA through exosomes is involved in mediating RIBE signals.

Hydroxyurea-induced senescent peripheral blood mesenchymal stromal cells inhibit bystander cell proliferation of JAK2V617F-positive human erythroleukemia cells.

Hydroxyurea (HU) is a nonalkylating antineoplastic agent used in the treatment of hematological malignancies. HU is a DNA replication stress inducer, and as such, it may induce a premature senescence-like cell phenotype; however, its repercussion on bystander cell proliferation has not been revealed so far. Our results indicate that HU strongly inhibited peripheral blood mesenchymal stromal cells (PBMSC) proliferation by cell cycle arrest in S phase, and that, consequently, PBMSC acquire senescence-related phenotypical changes. HU-treated PBMSC display increased senescence-associated ß-galactosidase levels and p16INK4 expression, as well as DNA damage response and genotoxic effects, evidenced by expression of γH2A.X and micronuclei. Moreover, HU-induced PBMSC senescence is mediated by increased reactive oxygen species (ROS) levels, as demonstrated by the inhibition of senescence markers in the presence of ROS scavenger N-acetylcysteine and NADPH oxidase inhibitor Apocynin. To determine the HU-induced bystander effect, we used the JAK2V617F-positive human erythroleukemia 92.1.7 (HEL) cells. Co-culture with HU-induced senescent PBMSC (HU-S-PBMSC) strongly inhibited bystander HEL cell proliferation, and this effect is mediated by both ROS and transforming growth factor (TGF)-ß expression. Besides induction of premature senescence, HU educates PBMSC toward an inhibitory phenotype of HEL cell proliferation. Finally, our study contributes to the understanding of the role of HU-induced PBMSC senescence as a potential adjuvant in hematological malignancy therapies.

DNA Damage and Cytokine Production in Non-Target Irradiated Lymphocytes.

In advanced radiotherapy, treatment of the tumor with high-intensity modulated fields is balanced with normal tissue sparing. However, the non-target dose delivered to surrounding healthy tissue within the irradiated volume is a potential cause for concern. Whether the effects observed are caused after exposure to out-of-field radiation or bystander effects through neighboring irradiated cells is not fully understood. The goal of this study was to determine the effect of exposure to out-of-field radiation in lymphocyte cell lines and primary blood cells. The role of cellular radiosensitivity in altering bystander responses in out-of-field exposed cells was also investigated. Target cells were positioned in a phantom in the center of the radiation field (in-field dose) and exposed to 2 Gy irradiation. Lymphocyte cell lines (C1, AT3ABR, Jurkat, THP-1, AT2Bi and AT3Bi) and peripheral blood were placed 1 cm away from the radiation field edge (out-of-field dose) and received an average dose of 10.8 ± 4.2 cGy. Double-stranded DNA damage, cell growth and gene expression were measured in the out-of-field cells. Radiosensitive AT3ABR and primary blood cells demonstrated the largest increase in γ-H2AX foci after irradiation. Exposure of normal cells to bystander factors from irradiated radiosensitive cell lines also increased DNA damage. Expression of IL-1, IL-6, TNFα and TGFß after addition of bystander factors from radiosensitive cells showed differential effects in normally responding cells, with some evidence of an adaptive response observed. Exposure to out-of-field radiation induces DNA damage and reduces growth in radiosensitive cells. Bystander factors produced by directly irradiated cells in combination with out-of-field exposure may upregulate pro- and anti-inflammatory genes in responding cells of different radiosensitivities, with the potential of affecting the tumor microenvironment. A greater understanding of the radio-biological response in normal cells outside the primary treatment field would assist in radiation treatment planning and in reducing early and late toxicities.