Mykyta V. Sokolov

Ionizing radiation induces DNA double-strand breaks in bystander primary human fibroblasts.

That irradiated cells affect their unirradiated ‘bystander’ neighbors is evidenced by reports of increased clonogenic mortality, genomic instability, and expression of DNA-repair genes in the bystander cell populations. The mechanisms underlying the bystander effect are obscure, but genomic instability suggests DNA double-strand breaks (DSBs) may be involved. Formation of DSBs induces the phosphorylation of the tumor suppressor protein, histone H2AX and this phosphorylated form, named γ-H2AX, forms foci at DSB sites. Here we report that irradiation of target cells induces γ-H2AX focus formation in bystander cell populations. The effect is manifested by increases in the fraction of cells in a population that contains multiple γ-H2AX foci. After 18 h coculture with cells irradiated with 20 α-particles, the fraction of bystander cells with multiple foci increased 3.7-fold. Similar changes occurred in bystander populations mixed and grown with cells irradiated with γ-rays, and in cultures containing media conditioned on γ-irradiated cells. DNA DSB repair proteins accumulated at γ-H2AX foci, indicating that they are sites of DNA DSB repair. Lindane, which blocks gap-junctions, prevented the bystander effect in mixing but not in media transfer protocols, while c-PTIO and aminoguanidine, which lower nitric oxide levels, prevented the bystander effect in both protocols. Thus, multiple mechanisms may be involved in transmitting bystander effects. These studies show that H2AX phosphorylation is an early step in the bystander effect and that the DNA DSBs underlying γ-H2AX focus formation may be responsible for its downstream manifestations.

Intercellular communication of cellular stress monitored by γ-H2AX induction

When cells are exposed to ionizing radiation (IR), unexposed cells that share media with damaged cells exhibit similar effects to irradiated cells including increased levels of DNA double-strand breaks (DSBs). Hypothesizing that this effect, known as the radiation-induced bystander effect, may be a specific instance of communication between damaged and undamaged cells regardless of damage source, we demonstrated that exposure of target cells to non-IR induces bystander damage in non-targeted cells as measured by gamma-H2AX and 53BP1 focal formation. Initially, bystander damage was found primarily in S-phase cells, but at later times, non-S-phase cells were also affected. In addition, media from undamaged malignant and senescent cells also was found to induce DSBs in primary cultures. Media conditioned on cells targeted with either ionizing or non-IR as well as on undamaged malignant and senescent cells contained elevated levels of several cytokines. One of these, transforming growth factor beta (TGF-beta), and nitric oxide (NO) were found to elevate numbers of gamma-H2AX/53BP1 foci in normal cell cultures similar to levels found in bystander cells, and this elevation was abrogated by NO synthase inhibitors, TGF-beta blocking antibody and antioxidants. These findings support the hypothesis that damage in bystander cells results from their exposure to cytokines or reactive compounds released from stressed cells, regardless of damage source. These results have implications for oncogenesis in that they indicate that damaged normal cells or undamaged tumor cells may induce genomic instability, leading to an increased risk of oncogenic transformation in other cells with which they share media or contact directly.

γH2AX in Bystander Cells: Not Just a Radiation-Triggered Event, a Cellular Response to Stress Mediated by Intercellular Communication

The recent years have witnessed a rapid accumulation of experimental data showing that ionizing radiation elicits a plethora of biological effects in unirradiated cells receiving bystander signals from hit cells. This so-called radiation-induced bystander effect (RIBE) manifests in various ways including changes in gene expression, genetic and epigenetic alterations, as well as increases in cell transformation and cell death. Our group and others found that DNA double-stranded breaks (DSBs), directly measured by the γ-H2AX focus formation assay, accumulate in bystander cells in a number of experimental systems such as human cultured cells, human 3-dimensional tissue models and in mice. In addition, we recently found that various other sources of cell stress, including media from cancerous cells resulted in a DNA damage response (DDR) in normal human cells that is reminiscent of RIBE. These results suggest that the RIBE may be part of a more general stress response, however, the molecular mechanism underpinning the formation of DNA DSBs in bystander cells is still unclear. This extra view points to some possibilities that might explain why DDR in human cells can be observed under bystander conditions.

Radiation-Induced Bystander Effects in Cultured Human Stem Cells

Background The radiation-induced “bystander effect” (RIBE) was shown to occur in a number of experimental systems both in vitro and in vivo as a result of exposure to ionizing radiation (IR). RIBE manifests itself by intercellular communication from irradiated cells to non-irradiated cells which may cause DNA damage and eventual death in these bystander cells. It is known that human stem cells (hSC) are ultimately involved in numerous crucial biological processes such as embryologic development; maintenance of normal homeostasis; aging; and aging-related pathologies such as cancerogenesis and other diseases. However, very little is known about radiation-induced bystander effect in hSC. To mechanistically interrogate RIBE responses and to gain novel insights into RIBE specifically in hSC compartment, both medium transfer and cell co-culture bystander protocols were employed. Methodology/Principal Findings Human bone-marrow mesenchymal stem cells (hMSC) and embryonic stem cells (hESC) were irradiated with doses 0.2 Gy, 2 Gy and 10 Gy of X-rays, allowed to recover either for 1 hr or 24 hr. Then conditioned medium was collected and transferred to non-irradiated hSC for time course studies. In addition, irradiated hMSC were labeled with a vital CMRA dye and co-cultured with non-irradiated bystander hMSC. The medium transfer data showed no evidence for RIBE either in hMSC and hESC by the criteria of induction of DNA damage and for apoptotic cell death compared to non-irradiated cells (p>0.05). A lack of robust RIBE was also demonstrated in hMSC co-cultured with irradiated cells (p>0.05). Conclusions/Significance These data indicate that hSC might not be susceptible to damaging effects of RIBE signaling compared to differentiated adult human somatic cells as shown previously. This finding could have profound implications in a field of radiation biology/oncology, in evaluating radiation risk of IR exposures, and for the safety and efficacy of hSC regenerative-based therapies.

Expression of pluripotency-associated genes in the surviving fraction of cultured human embryonic stem cells is not significantly affected by ionizing radiation.

Human embryonic stem cells (hESC) are capable to give rise to all cell types in the human body during the normal course of development. Therefore, these cells hold a great promise in regenerative cell replacement based therapeutical approaches. However, some controversy exists in literature concerning the ultimate fate of hESC after exposure to genotoxic agents, in particular, regarding the effect of DNA damaging insults on pluripotency of hESC. To comprehensively address this issue, we performed an analysis of the expression of marker genes, associated with pluripotent state of hESC, such as Oct-4, Nanog, Sox-2, SSEA-4, TERT, TRA-1-60 and TRA-1-81 up to 65h after exposure to ionizing radiation (IR) using flow cytometry, immunocytochemistry and quantitative real-time polymerase chain reaction techniques. We show that irradiation with relatively low doses of gamma-radiation (0.2Gy and 1Gy) does not lead to loss of expression of the pluripotency-associated markers in the surviving hESC. While changes in the levels of expression of some of the pluripotency markers were observed at different time points after IR exposure, these alterations were not persistent, and, in most cases, the expression of the pluripotency-associated markers remained significantly higher than that observed in fully differentiated human fibroblasts, and in hESCs differentiated into definitive endodermal lineage. Our data suggest that exposure of hESC to relatively low doses of IR as a model genotoxic agent does not significantly affect pluripotency of the surviving fraction of hESC.

Dynamics of the transcriptome response of cultured human embryonic stem cells to ionizing radiation exposure.

One of the key consequences of exposure of human cells to genotoxic agents is the activation of DNA damage responses (DDR). While the mechanisms underpinning DDR in fully differentiated somatic human cells have been studied extensively, molecular signaling events and pathways involved in DDR in pluripotent human embryonic stem cells (hESC) remain largely unexplored. We studied changes in the human genome-wide transcriptome of H9 hESC line following exposures to 1Gy of gamma-radiation at 2h and 16h post-irradiation. Quantitative real-time PCR was performed to verify the expression data for a subset of genes. In parallel, the cell growth, DDR kinetics, and expression of pluripotency markers in irradiated hESC were monitored. The changes in gene expression in hESC after exposure to ionizing radiation (IR) are substantially different from those observed in somatic human cell lines. Gene expression patterns at 2h post-IR showed almost an exclusively p53-dependent, predominantly pro-apoptotic, signature with a total of only 30 up-regulated genes. In contrast, the gene expression patterns at 16h post-IR showed 354 differentially expressed genes, mostly involved in pro-survival pathways, such as increased expression of metallothioneins, ubiquitin cycle, and general metabolism signaling. Cell growth data paralleled trends in gene expression changes. DDR in hESC followed the kinetics reported for human somatic differentiated cells. The expression of pluripotency markers characteristic of undifferentiated hESC was not affected by exposure to IR during the time course of our analysis. Our data on dynamics of transcriptome response of irradiated hESCs may provide a valuable tool to screen for markers of IR exposure of human cells in their most naive state; thus unmasking the key elements of DDR; at the same time, avoiding the complexity of interpreting distinct cell type-dependent genotoxic stress responses of terminally differentiated cells.

Lessons Learned about Human Stem Cell Responses to Ionizing Radiation Exposures: A Long Road Still Ahead of Us

Human stem cells (hSC) possess several distinct characteristics that set them apart from other cell types. First, hSC are self-renewing, capable of undergoing both asymmetric and symmetric cell divisions. Second, these cells can be coaxed to differentiate into various specialized cell types and, as such, hold great promise for regenerative medicine. Recent progresses in hSC biology fostered the characterization of the responses of hSC to genotoxic stresses, including ionizing radiation (IR). Here, we examine how different types of hSC respond to IR, with a special emphasis on their radiosensitivity, cell cycle, signaling networks, DNA damage response (DDR) and DNA repair. We show that human embryonic stem cells (hESCs) possess unique characteristics in how they react to IR that clearly distinguish these cells from all adult hSC studied thus far. On the other hand, a manifestation of radiation injuries/toxicity in human bodies may depend to a large extent on hSC populating corresponding tissues, such as human mesenchymal stem cells (hMSC), human hematopoietic stem cells (hHSC), neural hSC, intestine hSC, etc. We discuss here that hSC responses to IR differ notably across many types of hSC which may represent the distinct roles these cells play in development, regeneration and/or maintenance of homeostasis.

Unraveling the Global microRNAome Responses to Ionizing Radiation in Human Embryonic Stem Cells

MicroRNAs (miRNA) comprise a group of short ribonucleic acid molecules implicated in regulation of key biological processes and functions at the post-transcriptional level. Ionizing radiation (IR) causes DNA damage and generally triggers cellular stress response. However, the role of miRNAs in IR-induced response in human embryonic stem cells (hESC) has not been defined yet. Here, by using system biology approaches, we show for the first time, that miRNAome undergoes global alterations in hESC (H1 and H9 lines) after IR. Interrogation of expression levels of 1,090 miRNA species in irradiated hESC showed statistically significant changes in 54 genes following 1 Gy of X-ray exposures; global miRNAome alterations were found to be highly temporally and cell line - dependent in hESC. Time-course studies showed that the 16 hr miRNAome radiation response of hESC is much more robust compared to 2 hr-response signature (only eight genes), and may be involved in regulating the cell cycle. Quantitative real-time PCR performed on some miRNA species confirms the robustness of our miRNA microarray platform. Positive regulation of differentiation-, cell cycle-, ion transport- and endomembrane system-related processes were predicted to be negatively affected by miRNAome changes in irradiated hESC. Our findings reveal a fundamental role of miRNAome in modulating the radiation response, and identify novel molecular targets of radiation in hESC.

Effects of Low Doses of Ionizing Radiation Exposures on Stress-Responsive Gene Expression in Human Embryonic Stem Cells

There is a great deal of uncertainty on how low (≤0.1 Gy) doses of ionizing radiation (IR) affect human cells, partly due to a lack of suitable experimental model systems for such studies. The uncertainties arising from low-dose IR human data undermine practical societal needs to predict health risks emerging from diagnostic medical tests’ radiation, natural background radiation, and environmental radiological accidents. To eliminate a variability associated with remarkable differences in radioresponses of hundreds of differentiated cell types, we established a novel, human embryonic stem cell (hESC)-based model to examine the radiobiological effects in human cells. Our aim is to comprehensively elucidate the gene expression changes in a panel of various hESC lines following low IR doses of 0.01; 0.05; 0.1 Gy; and, as a reference, relatively high dose of 1 Gy of IR. Here, we examined the dynamics of transcriptional changes of well-established IR-responsive set of genes, including CDKN1A, GADD45A, etc. at 2 and 16 h post-IR, representing “early” and “late” radioresponses of hESCs. Our findings suggest the temporal- and hESC line-dependence of stress gene radioresponses with no statistically significant evidence for a linear dose-response relationship within the lowest doses of IR exposures.

Human Embryonic Stem Cell Responses to Ionizing Radiation Exposures: Current State of Knowledge and Future Challenges

Human embryonic stem cells, which are derived from the inner cell mass of the blastocyst, have become an object of intense study over the last decade. They possess two unique properties that distinguish them from many other cell types: (i) the ability to self-renew indefinitely in culture under permissive conditions, and (ii) the pluripotency, defined as the capability of giving rise to all cell types of embryonic lineage under the guidance of the appropriate developmental cues. The focus of many recent efforts has been on the elucidating the signaling pathways and molecular networks operating in human embryonic stem cells. These cells hold great promise in cell-based regenerative therapies, disease modeling, drug screening and testing, assessing genotoxic and mutagenic risks associated with exposures to a variety of environmental factors, and so forth. Ionizing radiation is ubiquitous in nature, and it is widely used in diagnostic and therapeutic procedures in medicine. In this paper, our goal is to summarize the recent progress in understanding how human embryonic stem cells respond to ionizing radiation exposures, using novel methodologies based on “omics” approaches, and to provide a critical discussion of what remains unknown; thus proposing a roadmap for the future research in this area.