Effect of exposure to ionizing radiation on competitive proliferation and differentiation of hESC.

PURPOSE: We studied the effects of computed tomography (CT) scan irradiation on proliferation and differentiation of human embryonic stem cells (hESCs). It was reported that hESC is extremely radiosensitive; exposure of hESC in cultures to 1 Gy of ionizing radiation (IR) results in massive apoptosis of the damaged cells and, thus, they are eliminated from the cultures. However, after recovery the surviving cells proliferate and differentiate normally. We hypothesized that IR-exposed hESC may still have growth rate disadvantage when they proliferate or differentiate in the presence of non-irradiated hESC, as has been shown for mouse hematopoietic stem cells in vivo. MATERIALS AND METHODS: To study such competitive proliferation and differentiation, we obtained cells of H9 hESC line that stably express green fluorescent protein (H9GFP). Irradiated with 50 mGy or 500 mGy H9GFP and non-irradiated H9 cells (or vice versa) were mixed and allowed to grow under pluripotency maintaining conditions or under conditions of directed differentiation into neuronal lineage for several passages. The ratio of H9GFP to H9 cells was measured after every passage or approximately every week. RESULTS: We observed competition of H9 and H9GFP cells; we found that the ratio of H9GFP to H9 cells increased with time in both proliferation and differentiation conditions regardless of irradiation, i.e. the H9GFP cells in general grew faster than H9 cells in the mixtures. However, we did not observe any consistent changes in the relative growth rate of irradiated versus non-irradiated hESC. CONCLUSIONS: We conclude that population of pluripotent hESC is very resilient; while damaged cells are eliminated from colonies, the surviving cells retain their pluripotency, ability to differentiate, and compete with non-irradiated isogenic cells. These findings are consistent with the results of our previous studies, and with the concept that early in pregnancy omnipotent cells injured by IR can be replaced by non-damaged cells with no impact on embryo development.

Effect of Ionizing Radiation on Transcriptome during Neural Differentiation of Human Embryonic Stem Cells.

Human embryonic brain development is highly sensitive to ionizing radiation. However, detailed information on the mechanisms of this sensitivity is not available due to limited experimental data. In this study, differentiation of human embryonic stem cells (hESCs) to neural lineages was used as a model for early embryonic brain development to assess the effect of exposure to low (17 mGy) and high (572 mGy) doses of radiation on gene expression. Transcriptomes were assessed using RNA sequencing during neural differentiation at three time points in control and irradiated samples. The first time point was when the cells were still pluripotent (day 0), the second time point was during the stage of embryoid body formation (day 6), and the third and final time point was during the stage of neural rosette formation (day 10). Analysis of the transcriptomes revealed neurodifferentiation in both the control and irradiated cells. Low-dose irradiation did not result in changes in gene expression at any of the time points, whereas high-dose irradiation resulted in downregulation of some major neurodifferentiation markers on days 6 and 10. Gene ontology analysis showed that pathways related to nervous system development, neurogenesis and generation of neurons were among the most affected. Expression of such key regulators of neuronal development as NEUROG1, ARX, ASCL1, RFX4 and INSM1 was reduced more than twofold. In conclusion, exposure to a 17 mGy low dose of radiation was well tolerated by hESCs while exposure to 572 mGy significantly affected their genetic reprogramming into neuronal lineages.

Effect of Ionizing Radiation from Computed Tomography on Differentiation of Human Embryonic Stem Cells into Neural Precursors.

We studied the effect of radiation from computed tomography (CT) scans on differentiation of human embryonic stem cells (hESCs) into neuronal lineage. hESCs were divided into three radiation exposure groups: 0-dose, low-dose, or high-dose exposure. Low dose was accomplished with a single 15 mGy CT dose index (CTDI) CT scan that approximated the dose for abdominal/pelvic CT examinations in adults while the high dose was achieved with several consecutive CT scans yielding a cumulative dose of 500 mGy CTDI. The neural induction was characterized by immunocytochemistry. Quantitative polymerase chain reaction (qPCR) and Western blots were used to measure expression of the neuronal markers PAX6 and NES and pluripotency marker OCT4. We did not find any visible morphological differences between neural precursors from irradiated and non-irradiated cells. However, quantitative analyses of neuronal markers showed that PAX6 expression was reduced following exposure to the high dose compared to 0-dose controls, while no such decrease in PAX6 expression was observed following exposure to the low dose. Similarly, a statistically significant reduction in expression of NES was observed following high-dose exposure, while after low-dose exposure, a modest but statistically significant reduction in NES expression was only observed on Day 8 of differentiation. Further studies are warranted to elucidate how lower or delayed expression of PAX6 and NES can impact human fetal brain development.

Single nucleotide variations in cultured cancer cells: Effect of mismatch repair.

We assessed single nucleotide variations (SNVs) between individual cells in two cancer cell lines; DU145, from brain metastasis of prostate tumor with deficient mismatch repair; and HT1080, a fibrosarcoma cell line. Clones of individual cells were isolated, and sequenced using Ion Ampliseq comprehensive cancer panel that covered the exomes of 409 oncogenes and tumor suppressor genes. Five clones of DU145 and four clones of HT1080 cells were analyzed. We found from 7 to 12 unique SNVs between DU145 clones, while HT1080 clones showed no more than one unique SNV. We then sub-cloned individual cells from some of these isolated clones of DU145 and HT1080 cells. The sub-clones were expanded from a single cell to approximately one million cells after about 20 cell divisions. The sub-clones of DU145 cells had from one to four new unique SNVs within the sequenced regions. No unique SNVs were found between sub-clones of HT1080 cells. Our data demonstrate that the extent of genetic variation at the single nucleotide level in cultured cancer cells is significantly affected by the status of the DNA mismatch repair system.

Effect of ionizing radiation on the proliferation of human embryonic stem cells.

We studied the effect of ionizing radiation (IR) on continuous growth of seven hESC lines. Cells were exposed to 0, 0.2, or 1 Gy of X-rays, and the growth rates of cell populations were assessed by measuring areas of the same individual colonies versus time. The population doubling times (DT) of sham-irradiated cells varied from 18.9 to 28.7 hours for different cell lines. All cell lines showed similar reaction to IR, i.e. cell populations dropped within 24-48 hours post IR; after that they recovered and grew with the same rate as the sham-irradiated cells. The relative cell survival (RCS), i.e. the ratio of normalized cell population in the irradiated samples to that of the sham-irradiated ones varied from 0.6 to 0.8 after 0.2 Gy, and from 0.1 to 0.2 after 1 Gy IR for different cell lines. We found that the RCS values of hESC lines correlated directly with their DT, i.e. the faster cells grow the more radiosensitive they are. We also found that DT and RCS values of individual colonies varied significantly within all hESC lines. We believe that the method developed herein can be useful for assessing other cytotoxic insults on cultures of hESC.

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.

A Genomic Study of DNA Alteration Events Caused by Ionizing Radiation in Human Embryonic Stem Cells via Next-Generation Sequencing.

Ionizing radiation (IR) is a known mutagen that is widely employed for medical diagnostic and therapeutic purposes. To study the extent of genetic variations in DNA caused by IR, we used IR-sensitive human embryonic stem cells (hESCs). Four hESC cell lines, H1, H7, H9, and H14, were subjected to IR at 0.2 or 1 Gy dose and then maintained in culture for four days before being harvested for DNA isolation. Irradiation with 1 Gy dose resulted in significant cell death, ranging from 60% to 90% reduction in cell population. Since IR is often implicated as a risk for inducing cancer, a primer pool targeting genomic "hotspot" regions that are frequently mutated in human cancer genes was used to generate libraries from irradiated and control samples. Using a semiconductor-based next-generation sequencing approach, we were able to consistently sequence these samples with deep coverage for reliable data analysis. A possible rare nucleotide variant was identified in the KIT gene (chr4:55593481) exclusively in H1 hESCs irradiated with 1 Gy dose. More extensive further studies are warranted to assess the extent and distribution of genetic changes in hESCs after IR exposure.

G-quadruplex formation between G-rich PNA and homologous sequences in oligonucleotides and supercoiled plasmid DNA.

Guanine (G)-rich DNA sequences can adopt four-stranded quadruplex conformations that may play a role in the regulation of genetic processes. To explore the possibility of targeted molecular recognition of DNA sequences with short G-rich peptide nucleic acids (PNA) and to assess the strand arrangement in such complexes, we used PNA and DNA with the Oxytricha nova telomeric sequence d(G4T4G4) as a model. PNA probes were complexed with DNA targets in the following forms: single-stranded oligonucleotides, a loop of DNA in a hairpin conformation, and as supercoiled plasmid with the (G4T4G4)/(C4A4C4) insert. Gel-shift mobility assays demonstrated formation of stable hybrid complexes between the homologous G4T4G4 PNA and DNA with multiple modes of binding. Chemical and enzymatic probing revealed sequence-specific and G-quadruplex dependent binding of G4T4G4 PNA to dsDNA. Spectroscopic and electrophoretic analysis of the complex formed between PNA and the synthetic DNA hairpin containing the G4T4G4 loop showed that the stoichiometry of a prevailing complex is three PNA strands per one DNA strand. We speculate how this new PNA-DNA complex architecture can help to design more selective, quadruplex-specific PNA probes.

Changes in human pluripotent stem cell gene expression after genotoxic stress exposures.

Human pluripotent stem cells (hPSCs) represent heterogeneous populations, including induced pluripotent stem cells (iPSCs), endogenous plastic somatic cells, and embryonic stem cells (ESCs). Human ESCs are derived from the inner cell mass of the blastocyst, and they are characterized by the abilities to self-renew indefinitely, and to give rise to all cell types of embryonic lineage (pluripotency) under the guidance of the appropriate chemical, mechanical and environmental cues. The combination of these critical features is unique to hESCs, and set them apart from other human cells. The expectations are high to utilize hESCs for treating injuries and degenerative diseases; for modeling of complex illnesses and development; for screening and testing of pharmacological products; and for examining toxicity, mutagenicity, teratogenicity, and potential carcinogenic effects of a variety of environmental factors, including ionizing radiation (IR). Exposures to genotoxic stresses, such as background IR, are unavoidable; moreover, IR is widely used in diagnostic and therapeutic procedures in medicine on a routine basis. One of the key outcomes of cell exposures to IR is the change in gene expression, which may underlie the ultimate hESCs fate after such a stress. However, gaps in our knowledge about basic biology of hESCs impose a serious limitation to fully realize the potential of hESCs in practice. The purpose of this review is to examine the available evidence of alterations in gene expression in human pluripotent stem cells after genotoxic stress, and to discuss strategies for future research in this important area.

PPG peptide nucleic acids that promote DNA guanine quadruplexes.

Recent studies have shown that guanine-rich (G-rich) sequences with the potential to form quadruplexes might play a role in normal transcription as well as overexpression of oncogenes. Chemical tools that allow examination of the specific roles of G-quadruplex formation in vivo, and their association with gene regulation will be essential to understanding the functions of these quadruplexes and might lead to beneficial therapies. Properly designed peptide nucleic acids (PNAs) can invade G-rich DNA duplexes and induce the formation of a G-quadruplex in the free DNA strand. Replacing guanines in the PNA sequence with pyrazolo[3,4-d]pyrimidine guanine (PPG) nucleobases eliminates G-quadruplex formation with PNA and promotes invasion of the target DNA.

Rational development of radiopharmaceuticals for HIV-1.

The global battle against HIV-1 would benefit from a sensitive and specific radiopharmaceutical to localize HIV-infected cells. Ideally, this probe would be able to identify latently infected host cells containing replication competent HIV sequences. Clinical and research applications would include assessment of reservoirs, informing clinical management by facilitating assessment of burden of infection in different compartments, monitoring disease progression and monitoring response to therapy. A "rational" development approach could facilitate efficient identification of an appropriate targeted radiopharmaceutical. Rational development starts with understanding characteristics of the disease that can be effectively targeted and then engineering radiopharmaceuticals to hone in on an appropriate target, which in the case of HIV-1 (HIV) might be an HIV-specific product on or in the host cell, a differentially expressed gene product, an integrated DNA sequence specific enzymatic activity, part of the inflammatory response, or a combination of these. This is different from the current approach that starts with a radiopharmaceutical for a target associated with a disease, mostly from autopsy studies, without a strong rationale for the potential to impact patient care. At present, no targeted therapies are available for HIV latency, although a number of approaches are under study. Here we discuss requirements for a radiopharmaceutical useful in strategies targeting persistently infected cells. The radiopharmaceutical for HIV should be developed based on HIV biology, studied in an animal model and then in humans, and ultimately used in clinical and research settings.

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.

An in vitro DNA double-strand break repair assay based on end-joining of defined duplex oligonucleotides.

DNA double-strand breaks (DSBs) are caused by endogenous cellular processes such as oxidative metabolism, or by exogenous events like exposure to ionizing radiation or other genotoxic agents. Repair of these DSBs is essential for the maintenance of cellular genomic integrity. In human cells, and cells of other higher eukaryotes, DSBs are primarily repaired by the nonhomologous end-joining (NHEJ) DSB repair pathway. Most in vitro assays that have been designed to measure NHEJ activity employ linear plasmid DNA as end-joining substrates, and such assays have made significant contributions to our understanding of the biochemical mechanisms of NHEJ. Here we describe an in vitro end-joining assay employing linear oligonucleotides that has distinct advantages over plasmid-based assays for the study of structure-function relationships between the proteins of the NHEJ pathway and synthetic DNA end-joining substrates possessing predetermined DSB configurations and chemistries.

Whole-genome gene expression profiling reveals the major role of nitric oxide in mediating the cellular transcriptional response to ionizing radiation in normal human fibroblasts.

The indirect biological effects of ionizing radiation (IR) are thought to be mediated largely by reactive oxygen and nitrogen species (ROS and RNS). However, no data are available on how nitric oxide (NO) modulates the response of normal human cells to IR exposures at the level of the whole transcriptome. Here, we examined the effects of NO and ROS scavengers, carboxy-PTIO and DMSO, on changes in global gene expression in cultured normal human fibroblasts after exposures to gamma-rays, aiming to elucidate the involvement of ROS and RNS in transcriptional response to IR. We found that NO depletion dramatically affects the gene expression in normal human cells following irradiation with gamma-rays. We observed striking (more than seven-fold) reduction of the number of upregulated genes upon NO scavenging compared to reference irradiated cell cultures. NO scavenging in irradiated IMR-90 cells results in induction of p53 signaling, DNA damage and DNA repair pathways.

Effect of 5-[(125)I]iodo-2'-deoxyuridine uptake on the proliferation and pluripotency of human embryonic stem cells.

PURPOSE: Human embryonic stem cells (hESC) hold a great potential for regenerative medicine because, in principle, they can differentiate into any cell type found in the human body. In addition, studying the effect of ionizing radiation (IR) on hESC may provide valuable information about the response of human cells to IR exposure in their most naive state, as well as the consequences of IR exposure on the development of organisms. However, the effect of IR, in particular radionuclide uptake, on the pluripotency, proliferation and survival of hESC has not been extensively studied. METHODS: In this study we treated cultured hESC with 5-[(125)I]iodo-2'-deoxyuridine ((125)IdU), a precursor of DNA synthesis. Then we measured the expansion of colonies and expression of pluripotency markers in hESC. RESULTS: We found that uptake of (125)IdU was similar in both hESC and HT1080 human fibrosarcoma cells. However, treatment with 0.1 µCi/ml (125)IdU for 24 hours resulted in complete death of the hESC population; whereas HT1080 cancer cells continued to grow. Treatment with a 10-fold lower dose (125)IdU (0.01 µCi/ml) resulted in colonies of hESC becoming less defined with numerous cells growing in monolayer outside of the colonies showing signs of differentiation. Then we analyzed the expression of pluripotency markers (octamer-binding transcription factor 4 [Oct-4] and stage-specific embryonic antigen-4 [SSEA4]) in the surviving hESC. We found that hESC in the surviving colonies expressed pluripotency markers at levels comparable with those in the non-treated controls. CONCLUSIONS: Our results provide important initial insights into the sensitivity of hESC to IR, and especially that produced by the decay of an internalized radionuclide.

Radioprobing the conformation of DNA in a p53-DNA complex.

PURPOSE: The frequency of DNA strand breaks produced by the decay of Auger electron-emitting radionuclides is inversely proportional to the distance of DNA nucleotides from the decay site; and thus is very sensitive to changes in the local conformation of the DNA. Analysis of the frequency of DNA breaks, or radioprobing, gives valuable information about the local DNA structure. More than 10 years ago, we demonstrated the feasibility of radioprobing using a DNA-repressor complex with a known structure. Herein, we used radioprobing to study the conformation of DNA in complex with the tumor suppressor protein 53 (p53). Several structures of p53-DNA complexes have been solved by X-ray crystallography. These structures, obtained with the p53 DNA binding domain, a truncated form, laid the groundwork for understanding p53-DNA interactions and their relation to p53 functions. However, whether all observed stereochemical details are relevant to the native p53-DNA complex remains unclear. A common theme of the crystallographic structures is the lack of significant bending in the central part of the DNA response element. In contrast, gel electrophoresis and electron microscopy data showed strong DNA bending and overtwisting upon binding to the native p53 tetramer. METHODS: To analyze DNA in complex with p53, we incorporated (125)I-dCTP in two different positions of synthetic duplexes containing the consensus p53-binding site. RESULTS: The most significant changes in the break frequency distributions were detected close to the center of the binding site, which is consistent with an increase in DNA twisting in this region and local DNA bending and sliding. CONCLUSIONS: Our data confirm the main results of the studies made in solution and lay a foundation for systematic examination of interactions between DNA and native p53 using (125)I radioprobing.

Stimulation of cultured h9 human embryonic stem cells with thyroid stimulating hormone does not lead to formation of thyroid-like cells.

The sodium-iodine symporter (NIS) is expressed on the cell membrane of many thyroid cancer cells, and is responsible for the radioactive iodine accumulation. However, treatment of anaplastic thyroid cancer is ineffective due to the low expression of NIS on cell membranes of these tumor cells. Human embryonic stem cells (ESCs) provide a potential vehicle to study the mechanisms of NIS expression regulation during differentiation. Human ESCs were maintained on feeder-independent culture conditions. RT-qPCR and immunocytochemistry were used to study differentiation marker expression, (125)I uptake to study NIS function. We designed a two-step protocol for human ESC differentiation into thyroid-like cells, as was previously done for mouse embryonic stem cells. First, we obtained definitive endoderm from human ESCs. Second, we directed differentiation of definitive endoderm cells into thyroid-like cells using various factors, with thyroid stimulating hormone (TSH) as the main differentiating factor. Expression of pluripotency, endoderm and thyroid markers and (125)I uptake were monitored throughout the differentiation steps. These approaches did not result in efficient induction of thyroid-like cells. We conclude that differentiation of human ESCs into thyroid cells cannot be induced by TSH media supplementation alone and most likely involves complicated developmental patterns that are yet to be understood.

Study of charge transport mechanisms in (125)I-induced DNA damage at various temperatures.

PURPOSE: Iodine-125 decay induces localized DNA damage by three major mechanisms: (1) Direct damage by the emitted Auger electrons, (2) indirect damage by diffusible free radicals, and (3) charge neutralization of the residual, highly positively charged, tellurium daughter atom by stripping electrons from neighboring residues. The charge neutralization mechanism of (125)I-induced DNA damage is poorly understood. Charge transport along a DNA molecules can occur by either a hopping mechanism initiated by charge injection into DNA and propagated by charge migration through DNA bases along the DNA length, or by a tunneling mechanism in which charge transfers directly from a donor to an acceptor residue. In the first case additional damage in DNA nucleotides can be inflicted by the traveling charge; therefore, it is important to learn if charge hopping plays a role in (125)I-decay-induced DNA damage. In our previous work, we determined that at 193K the charge hopping mechanism was not an appreciable component of the mechanism of (125)I-induced DNA damage. However, the question whether this is also the case at higher temperatures remained open. METHODS: In the current study we used a well-known chemical barrier for charge hopping, 8-oxo-7, 8,-dihydroguanine (8-oxo-G), to assess the role of this mechanism in (125)I-decay-induced DNA damage at the following temperatures: 198, 253, 277 and 298 K. RESULTS: We found that varying the temperature had little effect on the distribution of (125)I-induced DNA breaks, as well as on the breaks found at the 8-oxo-G probe both with and without piperidine treatment. CONCLUSIONS: We thus conclude that charge transport by the hopping mechanism is not a major factor in (125)I-decay-induced DNA damage at biologically relevant temperatures.

Targeting DNA G-quadruplex structures with peptide nucleic acids.

Regulation of genetic functions based on targeting DNA or RNA sequences with complementary oligonucleotides is especially attractive in the post-genome era. Oligonucleotides can be rationally designed to bind their targets based on simple nucleic acid base pairing rules. However, the use of natural DNA and RNA oligonucleotides as targeting probes can cause numerous off-target effects. In addition, natural nucleic acids are prone to degradation in vivo by various nucleases. To address these problems, nucleic acid mimics such as peptide nucleic acids (PNA) have been developed. They are more stable, show less off-target effects, and, in general, have better binding affinity to their targets. However, their high affinity to DNA can reduce their sequence-specificity. The formation of alternative DNA secondary structures, such as the G-quadruplex, provides an extra level of specificity as targets for PNA oligomers. PNA probes can target the loops of G-quadruplex, invade the core by forming PNA-DNA guanine-tetrads, or bind to the open bases on the complementary cytosine-rich strand. Not only could the development of such G-quadruplex-specific probes allow regulation of gene expression, but it will also provide a means to clarify the biological roles G-quadruplex structures may possess.