The Role of Different TET Proteins in Cytosine Demethylation Revealed by Mathematical Modeling.

In living cells, some reactions can be conducted by more than one enzyme and sometimes it is difficult to establish which enzyme is responsible. Such is the case with proteins from the TET family, capable of converting 5-methyl-2'-deoxycytidine (5-mdC) in DNA to 5-(hydroxymethyl)-2'-deoxycytidine (5-hmdC) and further to 5-formyl-2'-deoxycytidine (5-fdC) and 5-carboxy-2'-deoxycytidine (5-cadC). The estimation of the efficiency of particular TETs in particular oxidative reactions and different cell types is important but experimentally difficult. Here, we propose an approach with mathematical modeling in which methylation and known deoxycytidine modification pathways are presented by 343 possible model versions with assumed different combinations of TET1, 2, and 3 activities in different pathways. Model parameters were calculated on the basis of 5-mdC, 5-hmdC, 5-fdC, 5-cadC, and 5-hmdU levels experimentally assessed in five human cultured cell lines and previously published. Selection of the model versions that give in simulations the best average fit to experimental data suggested that not all TET proteins participate in all modification reactions and that TET3 activity may be especially important in the reaction of 5-fdC removal.

Expression of miRNA-Targeted and Not-Targeted Reporter Genes Shows Mutual Influence and Intercellular Specificity.

The regulation of translation by RNA-induced silencing complexes (RISCs) composed of Argonaute proteins and micro-RNAs is well established; however, the mechanisms underlying specific cellular responses to miRNAs and how specific complexes arise are not completely clear. To explore these questions, we performed experiments with Renilla and firefly luciferase reporter genes transfected in a psiCHECK-2 plasmid into human HCT116 or Me45 cells, where only the Renilla gene contained sequences targeted by microRNAs (miRNAs) in the 3'UTR. The effects of targeting were miRNA-specific; miRNA-21-5p caused strong inhibition of translation, whereas miRNA-24-3p or Let-7 family caused no change or an increase in reporter Renilla luciferase synthesis. The mRNA-protein complexes formed by transcripts regulated by different miRNAs differed from each other and were different in different cell types, as shown by sucrose gradient centrifugation. Unexpectedly, the presence of miRNA targets on Renilla transcripts also affected the expression of the co-transfected but non-targeted firefly luciferase gene in both cell types. Renilla and firefly transcripts were found in the same sucrose gradient fractions and specific anti-miRNA oligoribonucleotides, which influenced the expression of the Renilla gene, and also influenced that of firefly gene. These results suggest that, in addition to targeted transcripts, miRNAs may also modulate the expression of non-targeted transcripts, and using the latter to normalize the results may cause bias. We discuss some hypothetical mechanisms which could explain the observed miRNA-induced effects.

Involvement of miRNAs in cellular responses to radiation.

PURPOSE: Exposure of living cells to ionizing radiation has different consequences, depending on the dose and cell type. Changes in gene expression at the level of transcription and translation, including those regulated by microRNAs (miRNAs), play a role in the intrinsic radiosensitivity of different cells and define their fate, survival or death. The aim of our work was to examine how ionizing radiation may influence the expression of genes regulated by different miRNAs and miRNA biogenesis. MATERIALS AND METHODS: The work was performed on cultured human melanoma Me45 cells, transiently transfected with plasmids containing Renilla luciferase reporter gene targeted by miRNAs Let-7, miR-21 or miR-24. The levels of reporter mRNAs and mRNAs coding for proteins participating in miRNA biogenesis were assayed at different time points in irradiated and non-irradiated cells using RT-qPCR, and reporter protein by luciferase activity assays. MiRNA-targeted motifs in mRNAs coding for proteins engaged in miRNA biogenesis were extracted from the miRTarBase database. RESULTS: Messenger RNA and protein levels of transfected luciferase genes fluctuated in time in patterns that depended on the type of miRNA regulation and changed upon irradiation of the cells. The average levels of reporter mRNAs were higher in irradiated cells, whereas the levels of proteins changed in either direction. Radiation also influenced the levels of miRNAs and the expression of genes engaged in their biogenesis suggesting that the changes in gene expression following ionizing radiation result mainly from these changes in expression of genes regulating miRNA biogenesis and the influence of miRNA on mRNA translation. CONCLUSIONS: Currently, the responses of cells to ionizing radiation are mainly ascribed to changes in their redox conditions and increased intracellular levels of ROS, but the experiments described here suggest that a further important factor is modulation of translation through changes in biogenesis and levels of miRNAs.

Micro RNAs in Regulation of Cellular Redox Homeostasis.

In living cells Reactive Oxygen Species (ROS) participate in intra- and inter-cellular signaling and all cells contain specific systems that guard redox homeostasis. These systems contain both enzymes which may produce ROS such as NADPH-dependent and other oxidases or nitric oxide synthases, and ROS-neutralizing enzymes such as catalase, peroxiredoxins, thioredoxins, thioredoxin reductases, glutathione reductases, and many others. Most of the genes coding for these enzymes contain sequences targeted by micro RNAs (miRNAs), which are components of RNA-induced silencing complexes and play important roles in inhibiting translation of their targeted messenger RNAs (mRNAs). In this review we describe miRNAs that directly target and can influence enzymes responsible for scavenging of ROS and their possible role in cellular redox homeostasis. Regulation of antioxidant enzymes aims to adjust cells to survive in unstable oxidative environments; however, sometimes seemingly paradoxical phenomena appear where oxidative stress induces an increase in the levels of miRNAs which target genes which are supposed to neutralize ROS and therefore would be expected to decrease antioxidant levels. Here we show examples of such cellular behaviors and discuss the possible roles of miRNAs in redox regulatory circuits and further cell responses to stress.

Circuits Regulating Superoxide and Nitric Oxide Production and Neutralization in Different Cell Types: Expression of Participating Genes and Changes Induced by Ionizing Radiation.

Superoxide radicals, together with nitric oxide (NO), determine the oxidative status of cells, which use different pathways to control their levels in response to stressing conditions. Using gene expression data available in the Cancer Cell Line Encyclopedia and microarray results, we compared the expression of genes engaged in pathways controlling reactive oxygen species and NO production, neutralization, and changes in response to the exposure of cells to ionizing radiation (IR) in human cancer cell lines originating from different tissues. The expression of NADPH oxidases and NO synthases that participate in superoxide radical and NO production was low in all cell types. Superoxide dismutase, glutathione peroxidase, thioredoxin, and peroxiredoxins participating in radical neutralization showed high expression in nearly all cell types. Some enzymes that may indirectly influence superoxide radical and NO levels showed tissue-specific expression and differences in response to IR. Using fluorescence microscopy and specific dyes, we followed the levels and the distribution of superoxide and NO radicals in living melanoma cells at different times after exposure to IR. Directly after irradiation, we observed an increase of superoxide radicals and NO coexistent in the same subcellular locations, suggesting a switch of NO synthase to the production of superoxide radicals.

A mathematical model as a tool to identify microRNAs with highest impact on transcriptome changes.

BACKGROUND: Rapid changes in the expression of many messenger RNA (mRNA) species follow exposure of cells to ionizing radiation. One of the hypothetical mechanisms of this response may include microRNA (miRNA) regulation, since the amounts of miRNAs in cells also vary upon irradiation. To address this possibility, we designed experiments using cancer-derived cell lines transfected with luciferase reporter gene containing sequences targeted by different miRNA species in its 3'- untranslated region. We focus on the early time-course response (1 h past irradiation) to eliminate secondary mRNA expression waves. RESULTS: Experiments revealed that the irradiation-induced changes in the mRNA expression depend on the miRNAs which interact with mRNA. To identify the strongest interactions, we propose a mathematical model which predicts the mRNA fold expression changes, caused by perturbation of microRNA-mRNA interactions. Model was applied to experimental data including various cell lines, irradiation doses and observation times, both ours and literature-based. Comparison of modelled and experimental mRNA expression levels given miRNA level changes allows estimating how many and which miRNAs play a significant role in transcriptome response to stress conditions in different cell types. As an example, in the human melanoma cell line the comparison suggests that, globally, a major part of the irradiation-induced changes of mRNA expression can be explained by perturbed miRNA-mRNA interactions. A subset of about 30 out of a few hundred miRNAs expressed in these cells appears to account for the changes. These miRNAs play crucial roles in regulatory mechanisms observed after irradiation. In addition, these miRNAs have a higher average content of GC and a higher number of targeted transcripts, and many have been reported to play a role in the development of cancer. CONCLUSIONS: Our proposed mathematical modeling approach may be used to identify miRNAs which participate in responses of cells to ionizing radiation, and other stress factors such as extremes of temperature, exposure to toxins, and drugs.

Cell type-specific differences in redox regulation and proliferation after low UVA doses.

Ultraviolet A (UVA) radiation is harmful for living organisms but in low doses may stimulate cell proliferation. Our aim was to examine the relationships between exposure to different low UVA doses, cellular proliferation, and changes in cellular reactive oxygen species levels. In human colon cancer (HCT116) and melanoma (Me45) cells exposed to UVA doses comparable to environmental, the highest doses (30-50 kJ/m2) reduced clonogenic potential but some lower doses (1 and 10 kJ/m2) induced proliferation. This effect was cell type and dose specific. In both cell lines the levels of reactive oxygen species and nitric oxide fluctuated with dynamics which were influenced differently by UVA; in Me45 cells decreased proliferation accompanied the changes in the dynamics of H2O2 while in HCT116 cells those of superoxide. Genes coding for proteins engaged in redox systems were expressed differently in each cell line; transcripts for thioredoxin, peroxiredoxin and glutathione peroxidase showed higher expression in HCT116 cells whereas those for glutathione transferases and copper chaperone were more abundant in Me45 cells. We conclude that these two cell types utilize different pathways for regulating their redox status. Many mechanisms engaged in maintaining cellular redox balance have been described. Here we show that the different cellular responses to a stimulus such as a specific dose of UVA may be consequences of the use of different redox control pathways. Assays of superoxide and hydrogen peroxide level changes after exposure to UVA may clarify mechanisms of cellular redox regulation and help in understanding responses to stressing factors.

Correction: Profiles of a broad spectrum of epigenetic DNA modifications in normal and malignant human cell lines: Proliferation rate is not the major factor responsible for the 5-hydroxymethyl-2'-deoxycytidine level in cultured cancerous cell lines.

[This corrects the article DOI: 10.1371/journal.pone.0188856.].

Modulation by neighboring cells of the responses and fate of melanoma cells irradiated with UVA.

UVA radiation, which accounts for about 95% of the solar spectrum, contributes to and may be the etiological factor of skin cancers of which malignant melanoma is the most aggressive. UVA causes oxidative stress in various types of cells in the skin, keratinocyte, melanocytes, and fibroblasts, which is responsible for its cytotoxic effect. Here we used a transwell system to explore how the responses of melanoma cells to a low dose of UVA (20kJ/m2, ~10% of the minimal erythema dose) are influenced by neighboring co-cultured melanoma cells or fibroblasts. This dose had a low toxicity for melanoma cells, but after irradiation, co-culture with non-irradiated melanoma cells caused a strong decline in their viability and an increased frequency of apoptosis, whereas co-culture with fibroblast exerted a protective effect on irradiated melanoma cells. At the same time, the presence of non-irradiated cells, especially fibroblasts, decreased the level of UVA-induced reactive oxygen and nitrogen species. Interleukins efficiently produced by fibroblasts seem to be main players in these effects. Our studies reveal that coexistence of fibroblasts with melanoma cells may strongly modulate the direct action and may change bystander effects exerted by UVA light. Similar modulation of the effect of UVA on melanoma cells in vivo by bystander-like signaling from neighboring cells would have consequences for the development of malignant melanoma.

Profiles of a broad spectrum of epigenetic DNA modifications in normal and malignant human cell lines: Proliferation rate is not the major factor responsible for the 5-hydroxymethyl-2'-deoxycytidine level in cultured cancerous cell lines.

Active demethylation of 5-methylcytosine moiety in DNA occurs by its sequential oxidation to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine, catalysed by enzymes of the Ten-Eleven Translocation family proteins (TETs 1, 2 and 3). Here we analyzed for the first time all the intermediate products of DNA demethylation pathway in the form of deoxynucleosides (5-methyl-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine and 5-carboxy-2'-deoxycytidine as well as 5-(hydroxymethyl)-2'-deoxyuridine) using automated isotope-dilution online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. DNA was isolated from human malignant cell lines of colon adenocarcinoma (HCT 116), melanoma (Me45), myelogenous leukemia bone marrow blasts (K562), EBV-positive Burkitt's lymphoma lymphoblasts (Raji), EBV-negative Burkitt's lymphoma lymphoblasts (male-CA46 and female-ST486), as well as normal neonatal dermal fibroblasts (NHDF-Neo). The expression levels of TET1, TET2, TET3, SMUG1, and TDG genes were also assayed by RT-qPCR. Our results show a global erasure of 5-hydroxymethyl-2'-deoxycytidine and 5-carboxy-2'-deoxycytidine in DNA of cultured cells compared with DNA from primary malignant tissue. Moreover, malignant cells in culture have a quite different DNA epigenetic profile than cultured normal cells, and different types of malignant cells display different and characteristic profiles of DNA epigenetic marks. Similar analyses of a broader spectrum of epigenetic modifications, not restricted to 5-methyl-2'-deoxycytidine, could lead to better understanding of the mechanism(s) responsible for emergence of different types of cancer cells.

The Impact of DIDS-Induced Inhibition of Voltage-Dependent Anion Channels (VDAC) on Cellular Response of Lymphoblastoid Cells to Ionizing Radiation.

BACKGROUND: The voltage-dependent anion channels (VDAC) play an essential role in the cross talk between mitochondria and the rest of the cell. Their implication in cell life and cell death has been studied extensively in recent years. In this work we studied the impact of mitochondrial membrane (VDACs) on cell survival and response to X-ionizing radiation (IR) of human lymphoblastoid K562 cells. METHODS: The inhibition of VDACs was achieved by 4,4`-diisothiocyanostilbene-2,2`-disulfonic acid (DIDS) inhibitor and in vitro experiments including clonogenity assay, UV-visible spectrophotometry, comet assay and FACS analysis were implemented. RESULTS: Inhibition of VDAC led to augmentation of IR-induced apoptosis and ROS production. Additionally, DIDS affected repair of IR-induced DNA strand breaks and was in line with both induction of apoptosis and caspase activity. The IR-induced NO production was potently reduced by inhibition of VDAC. CONCLUSION: Our results suggest that VDAC control cellular response to ionizing radiation through modulation of the ROS- and NO-dependent signaling pathways. Inhibition of VDAC with DIDS induced apoptosis in irradiated K562 lymphoblastoid cells points at DIDS, as a promising agent to enhance the effectiveness of radiotherapy.

Vitamin C enhances substantially formation of 5-hydroxymethyluracil in cellular DNA.

The most plausible mechanism behind active demethylation of 5-methylcytosine involves TET proteins which participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine; the latter is further oxidized to 5-formylcytosine and 5-carboxycytosine. 5-Hydroxymethyluracil can be also generated from thymine in a TET-catalyzed process. Ascorbate was previously demonstrated to enhance generation of 5-hydroxymethylcytosine in cultured cells. The aim of this study was to determine the levels of the abovementioned TET-mediated oxidation products of 5-methylcytosine and thymine after addition of ascorbate, using an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with electrospray ionization tandem mass spectrometry. Intracellular concentration of ascorbate was determined by means of ultra-performance liquid chromatography with UV detection. Irrespective of its concentration in culture medium (10-100µM) and inside the cell, ascorbate stimulated a moderate (2- to 3-fold) albeit persistent (up to 96-h) increase in the level of 5-hydroxymethylcytosine. However, exposure of cells to higher concentrations of ascorbate (100µM or 1mM) stimulated a substantial increase in 5-formylcytosine and 5-carboxycytosine levels. Moreover, for the first time we demonstrated a spectacular (up to 18.5-fold) increase in 5-hydroxymethyluracil content what, in turn, suggests that TET enzymes contributed to the presence of the modification in cellular DNA. These findings suggest that physiological concentrations of ascorbate in human serum (10-100µM) are sufficient to maintain a stable level of 5-hydroxymethylcytosine in cellular DNA. However, markedly higher concentrations of ascorbate (ca. 100µM in the cell milieu or ca. 1mM inside the cell) were needed to obtain a sustained increase in 5-formylcytosine, 5-carboxycytosine and 5-hydroxymethyluracil levels. Such feedback to elevated concentrations of ascorbate may reflect adaptation of the cell to environmental conditions.

The classification, structure and functioning of Ago proteins in Eukaryotes.

Ago proteins are members of the highly specialized and conserved Argonaute family, primarily responsible for regulation of gene expression. As a part of RNA-induced silencing complexes (RISCs) Ago proteins are responsible for binding a short RNA and cleavage/inhibition of translation of target mRNAs. Phosphorylation may work as the switch between those two functions, but the role of magnesium ion concentration is also taken into consideration. Recent reports indicate that Ago proteins can interact with an mRNA and cause inhibition of translation without the participation of a short RNA. As key elements in RNA interference processes, Ago proteins are an important and intensively exploited area of research. Furthermore, these proteins are involved in the repair of DNA double-strand breaks by homologous recombination, modifications of chromatin, and alternative splicing. Their role in the cell cycle and senescence is also being studied. In addition, Ago expression is tissue-specific, which potentially may be used for diagnostic purposes. Understanding the mechanisms of Ago functioning is therefore crucial for understanding many cellular processes. The following article presents a detailed description of the Ago proteins including their post-translational modifications, recent data and hypotheses concerning their interactions with short RNAs and mRNAs as well as the mechanisms of siRNA/miRNA sorting into individual members of the Ago subfamily, and their role in eukaryotic cells. The latest classification of Ago proteins within the Argonaute family based on evolutionary studies and their possible interactions with DNA are also described.

Development of a population of cancer cells: Observation and modeling by a Mixed Spatial Evolutionary Games approach.

Living cells, like whole living organisms during evolution, communicate with their neighbors, interact with the environment, divide, change their phenotypes, and eventually die. The development of specific ways of communication (through signaling molecules and receptors) allows some cellular subpopulations to survive better, to coordinate their physiological status, and during embryonal development to create tissues and organs or in some conditions to become tumors. Populations of cells cultured in vitro interact similarly, also competing for space and nutrients and stimulating each other to better survive or to die. The results of these intercellular interactions of different types seem to be good examples of biological evolutionary games, and have been the subjects of simulations by the methods of evolutionary game theory where individual cells are treated as players. Here we present examples of intercellular contacts in a population of living human cancer HeLa cells cultured in vitro and propose an evolutionary game theory approach to model the development of such populations. We propose a new technique termed Mixed Spatial Evolutionary Games (MSEG) which are played on multiple lattices corresponding to the possible cellular phenotypes which gives the possibility of simulating and investigating the effects of heterogeneity at the cellular level in addition to the population level. Analyses performed with MSEG suggested different ways in which cellular populations develop in the case of cells communicating directly and through factors released to the environment.

The expression pattern of PFKFB3 enzyme distinguishes between induced-pluripotent stem cells and cancer stem cells.

Induced pluripotent stem cells (iPS) have become crucial in medicine and biology. Several studies indicate their phenotypic similarities with cancer stem cells (CSCs) and a propensity to form tumors. Thus it is desirable to identify a trait which differentiates iPS populations and CSCs. Searching for such a feature, in this work we compare the restriction (R) point-governed regulation of cell cycle progression in different cell types (iPS, cancer, CSC and normal cells) based on the expression profile of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase3 (PFKFB3) and phosphofructokinase (PFK1). Our study reveals that PFKFB3 and PFK1 expression allows discrimination between iPS and CSCs. Moreover, cancer and iPS cells, when cultured under hypoxic conditions, alter their expression level of PFKFB3 and PFK1 to resemble those in CSCs. We also observed cell type-related differences in response to inhibition of PFKFB3. This possibility to distinguish CSC from iPS cells or non-stem cancer cells by PFKB3 and PFK1 expression improves the outlook for clinical application of stem cell-based therapies and for more precise detection of CSCs.

Microarray experiments and factors which affect their reliability.

Oligonucleotide microarrays belong to the basic tools of molecular biology and allow for simultaneous assessment of the expression level of thousands of genes. Analysis of microarray data is however very complex, requiring sophisticated methods to control for various factors that are inherent to the procedures used. In this article we describe the individual steps of a microarray experiment, highlighting important elements and factors that may affect the processes involved and that influence the interpretation of the results. Additionally, we describe methods that can be used to estimate the influence of these factors, and to control the way in which they affect the expression estimates. A comprehensive understanding of the experimental protocol used in a microarray experiment aids the interpretation of the obtained results. By describing known factors which affect expression estimates this article provides guidelines for appropriate quality control and pre-processing of the data, additionally applicable to other transcriptome analysis methods that utilize similar sample handling protocols.

The different radiation response and radiation-induced bystander effects in colorectal carcinoma cells differing in p53 status.

Radiation-induced bystander effect, appearing as different biological changes in cells that are not directly exposed to ionizing radiation but are under the influence of molecular signals secreted by irradiated neighbors, have recently attracted considerable interest due to their possible implication for radiotherapy. However, various cells present diverse radiosensitivity and bystander responses that depend, inter alia, on genetic status including TP53, the gene controlling the cell cycle, DNA repair and apoptosis. Here we compared the ionizing radiation and bystander responses of human colorectal carcinoma HCT116 cells with wild type or knockout TP53 using a transwell co-culture system. The viability of exposed to X-rays (0-8 Gy) and bystander cells of both lines showed a roughly comparable decline with increasing dose. The frequency of micronuclei was also comparable at lower doses but at higher increased considerably, especially in bystander TP53-/- cells. Moreover, the TP53-/- cells showed a significantly elevated frequency of apoptosis, while TP53+/+ counterparts expressed high level of senescence. The cross-matched experiments where irradiated cells of one line were co-cultured with non-irradiated cells of opposite line show that both cell lines were also able to induce bystander effects in their counterparts, however different endpoints revealed with different strength. Potential mediators of bystander effects, IL-6 and IL-8, were also generated differently in both lines. The knockout cells secreted IL-6 at lower doses whereas wild type cells only at higher doses. Secretion of IL-8 by TP53-/- control cells was many times lower than that by TP53+/+ but increased significantly after irradiation. Transcription of the NFκBIA was induced in irradiated TP53+/+ mainly, but in bystanders a higher level was observed in TP53-/- cells, suggesting that TP53 is required for induction of NFκB pathway after irradiation but another mechanism of activation must operate in bystander cells.

Human-gyrovirus-Apoptin triggers mitochondrial death pathway--Nur77 is required for apoptosis triggering.

The human gyrovirus derived protein Apoptin (HGV-Apoptin) a homologue of the chicken anemia virus Apoptin (CAV-Apoptin), a protein with high cancer cells selective toxicity, triggers apoptosis selectively in cancer cells. In this paper, we show that HGV-Apoptin acts independently from the death receptor pathway as it induces apoptosis in similar rates in Jurkat cells deficient in either FADD (fas-associated death domain) function or caspase-8 (key players of the extrinsic pathway) and their parental clones. HGV-Apoptin induces apoptosis via the activation of the mitochondrial intrinsic pathway. It induces both mitochondrial inner and outer membrane permebilization, characterized by the loss of the mitochondrial potential and the release into cytoplasm of the pro-apoptotic molecules including apoptosis inducing factor and cytochrome c. HGV-Apoptin acts via the apoptosome, as lack of expression of apoptotic protease-activating factor 1 in murine embryonic fibroblast strongly protected the cells from HGV-Apoptin-induced apoptosis. Moreover, QVD-oph a broad-spectrum caspase inhibitor delayed HGV-Apoptin-induced death. On the other hand, overexpression of the anti-apoptotic BCL-XL confers resistance to HGV-Apoptin-induced cell death. In contrast, cells that lack the expression of the pro-apoptotic BAX and BAK are protected from HGV-Apoptin induced apoptosis. Furthermore, HGV-Apoptin acts independently from p53 signal but triggers the cytoplasmic translocation of Nur77. Taking together these data indicate that HGV-Apoptin acts through the mitochondrial pathway, in a caspase-dependent manner but independently from the death receptor pathway.

MicroRNAs and reactive oxygen species: are they in the same regulatory circuit?

Interactions of living organisms with their environment mainly involve modulation of gene expression by stimulation or silencing. One of the epigenetic mechanisms that regulate translation and the levels of transcripts is RNA interference, in which microRNAs (miRNAs) address RNA-induced silencing complexes (RISCs) to degrade specific mRNAs or to silence their translation. In this mechanism, double stranded RNA structure is crucial for miRNA biogenesis and the action of RISCs. RNA molecules can be modified structurally by reactive oxygen and nitrogen species (ROS and RNS) that are produced in cells of all aerobic organisms and may be induced by environmental factors. Here we describe experiments in which changes of ROS and transcript levels are induced by X-irradiation and measured using flow cytometer and the fluorescent dye 2',7'-dichlorofluorescein diacetate and microarray methods in cultured human K562, Me45 and HCT116 cells. Analysis of the nucleotide sequences of mRNAs which are up- or down-regulated after irradiation shows significant differences in the distributions of miRNA-targeted motives between these two groups. Immediately after irradiation most miRNAs behave as "up-regulators", showing more targets in up-than in down-regulated transcripts, and this changes about 12h later when we also observe changes in ROS and miRNA levels. Our results suggest that the changes in the transcriptome could result from changes in RNA interference and that these effects could be related to the changed ROS levels in irradiated cells. We propose that such modulation of gene expression at the mRNA level may be implicated more generally in cellular responses to stresses where ROS levels change.

Induction of bystander effects by UVA, UVB, and UVC radiation in human fibroblasts and the implication of reactive oxygen species.

Radiation-induced bystander effects are various types of responses displayed by nonirradiated cells induced by signals transmitted from neighboring irradiated cells. This phenomenon has been well studied after ionizing radiation, but data on bystander effects after UV radiation are limited and so far have been reported mainly after UVA and UVB radiation. The studies described here were aimed at comparing the responses of human dermal fibroblasts exposed directly to UV (A, B, or C wavelength range) and searching for bystander effects induced in unexposed cells using a transwell co-incubation system. Cell survival and apoptosis were used as a measure of radiation effects. Additionally, induction of senescence in UV-exposed and bystander cells was evaluated. Reactive oxygen species (ROS), superoxide radical anions, and nitric oxide inside the cells and secretion of interleukins 6 and 8 (IL-6 and IL-8) into the medium were assayed and evaluated as potential mediators of bystander effects. All three regions of ultraviolet radiation induced bystander effects in unexposed cells, as shown by a diminution of survival and an increase in apoptosis, but the pattern of response to direct exposure and the bystander effects differed depending on the UV spectrum. Although UVA and UVB were more effective than UVC in generation of apoptosis in bystander cells, UVC induced senescence both in irradiated cells and in neighbors. The level of cellular ROS increased significantly shortly after UVA and UVB exposure, suggesting that the bystander effects may be mediated by ROS generated in cells by UV radiation. Interestingly, UVC was more effective at generation of ROS in bystanders than in directly exposed cells and induced a high yield of superoxide in exposed and bystander cells, which, however, was only weakly associated with impairment of mitochondrial membrane potential. Increasing concentration of IL-6 but not IL-8 after exposure to each of the three bands of UV points to its role as a mediator in the bystander effect. Nitric oxide appeared to play a minor role as a mediator of bystander effects in our experiments. The results demonstrating an increase in intracellular oxidation, not only in directly UV-exposed but also in neighboring cells, and generation of proinflammatory cytokines, processes entailing cell damage (decreased viability, apoptosis, senescence), suggest that all bands of UV radiation carry a potential hazard for human health, not only due to direct mechanisms, but also due to bystander effects.