Irina V. Panyutin

Phosphorylation of Histone H2AX in Radiation-Induced Micronuclei

Abstract Medvedeva, N. G., Panyutin, I. V., Panyutin, I. G. and Neumann, R. D. Phosphorylation of Histone H2AX in Radiation-Induced Micronuclei. Radiat. Res. 168, 493–498 (2007). DNA double-strand breaks are thought to precede the formation of most radiation-induced micronuclei. Phosphorylation of the histone H2AX is an early indicator of DNA double-strand breaks. Here we studied the phosphorylation status of the histone H2AX in micronuclei after exposure of cultured cells to ionizing radiation or treatment with colchicine. In human astrocytoma SF268 cells, after exposure to γ radiation, the proportion of γ-H2AX-positive to γ-H2AX-negative micronuclei increases. The majority of the γ-H2AX-positive micronuclei are centromere-negative. The number of γ-H2AX-positive micronuclei continues to increase even 24 h postirradiation when most γ-H2AX foci in the main nucleus have disappeared. In contrast, in normal human fibroblasts (BJ), the proportion of γ-H2AX-positive to γ-H2AX-negative micronuclei remains constant, and the majority of the centromere-negative cells are γ-H2AX-negative. Treatment of both cell lines with colchicine results in mostly centromere-positive, γ-H2AX-negative micronuclei. Immunostaining revealed co-localization of MDC1 and ATM with γ-H2AX foci in both main nuclei and micronuclei; however, other repair proteins, such as Rad50, 53BP1 and Rad17, that co-localized with γ-H2AX foci in the main nuclei were not found in the micronuclei. Combination of the micronucleus assay with γ-H2AX immunostaining provides new insights into the mechanisms of the formation and fate of micronuclei.

Repair of Sequence-specific 125I-induced Double-strand Breaks by Nonhomologous DNA End Joining in Mammalian Cell-free Extracts

In mammalian cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair. Rejoining of DSB produced by decay of 125I positioned against a specific target site in plasmid DNA via a triplex-forming oligonucleotide (TFO) was investigated in cell-free extracts from Chinese hamster ovary cells. The efficiency and quality of NHEJ of the “complex” DSB induced by the 125I-TFO was compared with that of “simple” DSB induced by restriction enzymes. We demonstrate that the extracts are indeed able to rejoin 125I-TFO-induced DSB, although at approximately 10-fold decreased efficiency compared with restriction enzyme-induced DSB. The resulting spectrum of junctions is highly heterogeneous exhibiting deletions (1–30 bp), base pair substitutions, and insertions and reflects the heterogeneity of DSB induced by the125I-TFO within its target site. We show that NHEJ of125I-TFO-induced DSB is not a random process that solely depends on the position of the DSB but is driven by the availability of microhomology patches in the target sequence. The similarity of the junctions obtained with the ones found in vivo after125I-TFO-mediated radiodamage indicates that our in vitro system may be a useful tool to elucidate the mechanisms of ionizing radiation-induced mutagenesis and repair.

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.

Strand breaks in whole plasmid DNA produced by the decay of 125I in a triplex-forming oligonucleotide

Abstract Panyutin, I. V., Luu, A. N., Panyutin, I. G. and Neumann, R. D. Strand Breaks in Whole Plasmid DNA Produced by the Decay of 125I in a Triplex-Forming Oligonucleotide. Radiat. Res. 156, 158–166 (2001). DNA strand breaks produced by the decay of 125I positioned against a specific site in plasmid DNA via a triplex-forming oligonucleotide were studied both in the immediate vicinity of the site of the decay with a single nucleotide resolution and in the whole plasmid by measuring the percentages of supercoiled, open-circular and linear forms. The localized breaks are distributed within 10 bp in each direction from the decay site with maxima in both strands just opposite the 125I-dC residue in the triplex-forming oligonucleotide. The distributions of breaks in the two DNA strands are almost symmetrical, in agreement with the geometry of the pyrimidine motif triplex. We found that about 25% of the double-strand breaks were located outside the 90-bp fragment containing the triplex-forming oligonucleotide binding sequence. The ratio of single- to double-strand breaks in the whole plasmid was 11 for bound triplex-forming oligonucleotide compared to 26 when the triplex-forming oligonucleotide was free in solution. The number of double-strand breaks per decay of 125I was 0.46 for bound triplex-forming oligonucleotide and 0.17 for free triplex-forming oligonucleotide. Comparing the data on the localized damage and those for the whole plasmid, we concluded that, in addition to DNA breaks that are confined to a helical turn around the 125I atom, the decay can produce breaks hundreds of base pairs away in the plasmid molecule. This linear plasmid molecule containing radiation-induced damage at a specific DNA site should be useful in studies of the molecular mechanisms of DNA repair.

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.

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.

Assessment of DNA damage produced by 125I‐triplex‐forming oligonucleotides in cells

Purpose: Triplex‐forming oligodeoxyribonucleotides (TFOs) bind specifically to their target sequences by forming hydrogen bonds within the major groove of the target duplex. When labeled with Auger‐electron‐emitting radioisotopes, TFOs are able to damage the target gene in a process named antigene radiotherapy. We compared radiotoxicity and the amount of DNA damage produced within cultured cells by two 125I‐labeled TFOs, one with a single target in the genome and another with multiple targets. Materials and methods: Radiotoxicity was measured by clonogenic assay while DNA damage was assessed by the number of histone γ‐H2AX foci formed at the sites of DNA double strand breaks (DSBs). Results: The TFO with multiple nuclear targets was 1.7 fold more radiotoxic and produced on average 1.9 fold more γ‐H2AX foci per cell than the TFO with a single target. Conclusion: Since the two methods gave comparable results, measuring the number of γ‐H2AX foci per decay may be a useful procedure for the assessment of cytotoxic effects and the intranuclear localization of radionuclides when they produce DSBs.

Sigma-2 Receptor as Potential Indicator of Stem Cell Differentiation

PurposeThe sigma-2 (σ2) receptor is a potential biomarker of proliferative status of solid tumors. Specific synthetic probes using N-substituted-9-azabicyclo [3.3.1]nonan-3α-yl carbamate analogs have been designed and implemented for experimental cancer diagnosis and therapy.ProceduresWe employed the fluorescently labeled σ2 receptor probe, SW120, to evaluate σ2 receptor expression in human stem cells (SC), including: bone marrow stromal, neural progenitor, amniotic fluid, hematopoetic, and embryonic stem cells. We concurrently evaluated the intensity of SW120 and 5-ethynyl-2′-deoxyuridine (EdU) relative to passage number and multi-potency.ResultsWe substantiated significantly higher σ2 receptor density among proliferating SC relative to lineage-restricted cell types. Additionally, cellular internalization of the σ2 receptor in SC was consistent with receptor-mediated endocytosis and confocal microscopy indicated SW120 specific co-localization with a fluorescent marker of lysosomes in all SC imaged.ConclusionThese results suggest that σ2 receptors may serve to monitor stem cell differentiation in future experimental studies.

Effect of Chromatin Structure on the Extent and Distribution of DNA Double Strand Breaks Produced by Ionizing Radiation; Comparative Study of hESC and Differentiated Cells Lines

Chromatin structure affects the extent of DNA damage and repair. Thus, it has been shown that heterochromatin is more protective against DNA double strand breaks (DSB) formation by ionizing radiation (IR); and that DNA DSB repair may proceed differently in hetero- and euchromatin regions. Human embryonic stem cells (hESC) have a more open chromatin structure than differentiated cells. Here, we study the effect of chromatin structure in hESC on initial DSB formation and subsequent DSB repair. DSB were scored by comet assay; and DSB repair was assessed by repair foci formation via 53BP1 antibody staining. We found that in hESC, heterochromatin is confined to distinct regions, while in differentiated cells it is distributed more evenly within the nuclei. The same dose of ionizing radiation produced considerably more DSB in hESC than in differentiated derivatives, normal human fibroblasts; and one cancer cell line. At the same time, the number of DNA repair foci were not statistically different among these cells. We showed that in hESC, DNA repair foci localized almost exclusively outside the heterochromatin regions. We also noticed that exposure to ionizing radiation resulted in an increase in heterochromatin marker H3K9me3 in cancer HT1080 cells, and to a lesser extent in IMR90 normal fibroblasts, but not in hESCs. These results demonstrate the importance of chromatin conformation for DNA protection and DNA damage repair; and indicate the difference of these processes in hESC.

DNA Damage Produced by 125I-Triplex-Forming Oligonucleotides as a Measure of Their Succesful Delivery into Cell Nuclei

Decay of an Auger‐electron‐emitting radioisotope can knock out a targeted gene by producing DNA strand breaks within its sequence. For delivery of Auger emitters to genomic targets we used triplex‐forming oligonucleotides (TFOs) that bind specifically to their target sequences by forming hydrogen bonds within the major groove of the target duplex. We named this approach antigene radiotherapy. In our previous studies, we demonstrated that 125I‐labeled TFOs targeted against the human MDR1 gene produced sequence‐specific double strand breaks (DSBs) within this gene in live cultured cells. We also found that conjugation of TFO with nuclear localization signal peptide significantly increased the efficiency of targeting. To screen the wide variety of possible TFO modifications a sensitive and robust assay of DNA damage produced by such 125I‐TFOs would be highly desirable. Recently we showed a direct correspondence between the number of decays of 125I incorporated into DNA as 125I‐UdR and the number of histone γ‐H2AX foci per cell revealed by staining with γ‐H2AX antibodies. The technique is 100‐fold more sensitive than other DSB‐detection methods, thus it is possible to detect as few as an average of 0.5 DSBs per cell in a population of cultured cells. Here we applied this method to evaluate the intracellular DNA damage produced by two 125I‐TFOs, the first targeted to the single‐copy HPRT gene (125I‐TFO‐HPRT) and second to a multicopy repeated sequence (GA)n that occurs almost 7000 times in the human genome (125I‐TFO‐GA). DNA damage produced by 125I‐TFO was assessed by staining the cells with γ‐H2AX antibody followed by either direct counting γ‐H2AX foci or by measuring the γ‐H2AX signal using flow cytometry. Both methods produced quantitatively close results; 125I‐TFO‐GA with multiple nuclear targets produced on average 1.93 times more γ‐H2AX foci per cell and generated 1.96 times increase in γ‐H2AX antibody staining signal than 125I‐TFO‐HPRT with a single target. The γ‐H2AX‐based assay requires considerably less time and effort than the direct measurement of DSB by Southern hybridization applied previously. Therefore, we believe that γ‐H2AX‐based measurement of DNA damage could be useful for evaluation and cellular DNA accessibility by 125I‐labeled DNA targeting agents.