Comparative analysis of chlorambucil-induced DNA lesion formation and repair in a spectrum of different human cell systems.

Chlorambucil (CLB) belongs to the class of nitrogen mustards (NMs), which are highly reactive bifunctional alkylating agents and were the first chemotherapeutic agents developed. They form DNA interstrand crosslinks (ICLs), which cause a blockage of DNA strand separation, inhibiting essential processes in DNA metabolism like replication and transcription. In fast replicating cells, e.g., tumor cells, this can induce cell death. The upregulation of ICL repair is thought to be a key factor for the resistance of tumor cells to ICL-inducing cytostatic agents including NMs. To monitor induction and repair of CLB-induced ICLs, we adjusted the automated reversed fluorometric analysis of alkaline DNA unwinding assay (rFADU) for the detection of ICLs in adherent cells. For the detection of monoalkylated DNA bases we established an LC-MS/MS method. We performed a comparative analysis of adduct formation and removal in five human cell lines and in peripheral blood mononuclear cells (PBMCs) after treatment with CLB. Dose-dependent increases in adduct formation were observed, and suitable treatment concentrations were identified for each cell line, which were then used for monitoring the kinetics of adduct formation. We observed significant differences in the repair kinetics of the cell lines tested. For example, in A2780 cells, hTERT immortalized VH10 cells, and in PBMCs a time-dependent repair of the two main monoalkylated DNA-adducts was confirmed. Regarding ICLs, repair was observed in all cell systems except for PBMCs. In conclusion, LC-MS/MS analyses combined with the rFADU technique are powerful tools to study the molecular mechanisms of NM-induced DNA damage and repair. By applying these methods to a spectrum of human cell systems of different origin and transformation status, we obtained insight into the cell-type specific repair of different CLB-induced DNA lesions, which may help identify novel resistance mechanisms of tumors and define molecular targets for therapeutic interventions.

DNA Damage and Radiosensitivity in Blood Cells from Subjects Undergoing 45 Days of Isolation and Confinement: An Explorative Study.

The effect of confined and isolated experience on astronauts' health is an important factor to consider for future space exploration missions. The more confined and isolated humans are, the more likely they are to develop negative behavioral or cognitive conditions such as a mood decline, sleep disorder, depression, fatigue and/or physiological problems associated with chronic stress. Molecular mediators of chronic stress, such as cytokines, stress hormones or reactive oxygen species (ROS) are known to induce cellular damage including damage to the DNA. In view of the growing evidence of chronic stress-induced DNA damage, we conducted an explorative study and measured DNA strand breaks in 20 healthy adults. The participants were grouped into five teams (missions). Each team was composed of four participants, who spent 45 days in isolation and confinement in NASA's Human Exploration Research Analog (HERA). Endogenous DNA integrity, ex-vivo radiation-induced DNA damage and the rates of DNA repair were assessed every week. Our results show a high inter-individual variability as well as differences between the missions, which cannot be explained by inter-individual variability alone. The ages and sex of the participants did not appear to influence the results.

The Radioprotective Effect of Procaine and Procaine-Derived Product Gerovital H3 in Lymphocytes from Young and Aged Individuals.

Ionizing radiation induces genomic instability in living organisms, and several studies reported an ageing-dependent radiosensitivity. Chemical compounds, such as scavengers, radioprotectors, and modifiers, contribute to reducing the radiation-associated toxicity. These compounds are often antioxidants, and therefore, in order to be effective, they must be present before or during exposure to radiation. However, not all antioxidants provide radioprotection. In this study, we investigated the effects of procaine and of a procaine-based product Gerovital H3 (GH3) on the formation of endogenous and X-ray-induced DNA strand breaks in peripheral blood mononuclear cells (PBMCs) isolated from young and elderly individuals. Interestingly, GH3 showed the strongest radioprotective effects in PBMCs from young subjects, while procaine reduced the endogenous amount of DNA strand breaks more pronounced in aged individuals. Both procaine and GH3 inhibited lipid peroxidation, but procaine was more effective in inhibiting mitochondria free radicals' generation, while GH3 showed a higher antioxidant action on macrophage-induced low-density lipoprotein oxidation. Our findings provide new insights into the mechanisms underlying the distinct effects of procaine and GH3 on DNA damage.

Synergistic Effects of Weightlessness, Isoproterenol, and Radiation on DNA Damage Response and Cytokine Production in Immune Cells.

The implementation of rotating-wall vessels (RWVs) for studying the effect of lack of gravity has attracted attention, especially in the fields of stem cells, tissue regeneration, and cancer research. Immune cells incubated in RWVs exhibit several features of immunosuppression including impaired leukocyte proliferation, cytokine responses, and antibody production. Interestingly, stress hormones influence cellular immune pathways affected by microgravity, such as cell proliferation, apoptosis, DNA repair, and T cell activation. These pathways are crucial defense mechanisms that protect the cell from toxins, pathogens, and radiation. Despite the importance of the adrenergic receptor in regulating the immune system, the effect of microgravity on the adrenergic system has been poorly studied. Thus, we elected to investigate the synergistic effects of isoproterenol (a sympathomimetic drug), radiation, and microgravity in nonstimulated immune cells. Peripheral blood mononuclear cells were treated with the sympathomimetic drug isoproterenol, exposed to 0.8 or 2 Gy γ-radiation, and incubated in RWVs. Mixed model regression analyses showed significant synergistic effects on the expression of the ß2-adrenergic receptor gene (ADRB2). Radiation alone increased ADRB2 expression, and cells incubated in microgravity had more DNA strand breaks than cells incubated in normal gravity. We observed radiation-induced cytokine production only in microgravity. Prior treatment with isoproterenol clearly prevents most of the microgravity-mediated effects. RWVs may be a useful tool to provide insight into novel regulatory pathways, providing benefit not only to astronauts but also to patients suffering from immune disorders or undergoing radiotherapy.

The degree of radiation-induced DNA strand breaks is altered by acute sleep deprivation and psychological stress and is associated with cognitive performance in humans.

Study Objectives: Sleep deprivation is associated with impaired immune responses, cancer, and morbidity and mortality, and can degrade cognitive performance, although individual differences exist in such responses. Sleep deprivation induces DNA strand breaks and DNA base oxidation in animals, and psychological stress is associated with increased DNA damage in humans. It remains unknown whether sleep deprivation or psychological stress in humans affects DNA damage response from environmental stressors, and whether these responses predict cognitive performance during sleep deprivation. Methods: Sixteen healthy adults (ages 29-52 years; mean age ± SD, 36.4 ± 7.1 years; seven women) participated in a 5-day experiment involving two 8 hr time-in-bed (TIB) baseline nights, followed by 39 hr total sleep deprivation (TSD), and two 8-10 hr TIB recovery nights. A modified Trier Social Stress Test was conducted on the day after TSD. The Psychomotor Vigilance Test measured behavioral attention. DNA damage was assessed in blood cells collected at 5 time points, and blood cells were irradiated ex vivo. Results: TSD, alone or in combination with psychological stress, did not induce significant increases in DNA damage. By contrast, radiation-induced DNA damage decreased significantly in response to TSD, but increased back to baseline when combined with psychological stress. Cognitively vulnerable individuals had more radiation-induced DNA strand breaks before TSD, indicating their greater sensitivity to DNA damage from environmental stressors. Conclusions: Our results provide novel insights into the molecular consequences of sleep deprivation, psychological stress, and performance vulnerability. They are important for fields involving sleep loss, radiation exposure, and cognitive deficits, including cancer therapy, environmental toxicology, and space medicine.

Impaired PARP activity in response to the ß-adrenergic receptor agonist isoproterenol.

Psychological stress has been associated with DNA damage, thus increasing the risk of numerous diseases including cancer. Here, we investigate the effect of acute and chronic stress on poly(ADP-ribose) polymerase-1 (PARP-1), a sensor of DNA damage and DNA repair initiator. In order to mimic the chronic release of epinephrine, human peripheral blood mononuclear cells (PBMCs) were treated repeatedly with the sympathomimetic drug isoproterenol. We found significant induction of DNA strand breaks that remained unrepaired 24 h after ex vivo incubation. Isoproterenol-induced DNA strand breaks could be partially prevented by pre-treatment with the ß-adrenergic receptor antagonist propranolol. Furthermore, the level of PARP-1 protein and PARP activity decreased and the levels of the PARP substrate nicotinamide adenine dinucleotide (NAD+) and of adenosine triphosphate (ATP), necessary to replenish NAD+ pools, were lowered by isoproterenol treatment. In conclusion our data provide novel insights into the mechanisms of isoproterenol-induced genotoxicity linking ß-adrenergic stimulation and PARP-1.

RNA polymerase III-specific general transcription factor IIIC contains a heterodimer resembling TFIIF Rap30/Rap74.

Transcription of tRNA-encoding genes by RNA polymerase (Pol) III requires the six-subunit general transcription factor IIIC that uses subcomplexes τA and τB to recognize two gene-internal promoter elements named A- and B-box. The Schizosaccharomyces pombe τA subcomplex comprises subunits Sfc1, Sfc4 and Sfc7. The crystal structure of the Sfc1/Sfc7 heterodimer reveals similar domains and overall domain architecture to the Pol II-specific general transcription factor TFIIF Rap30/Rap74. The N-terminal Sfc1/Sfc7 dimerization module consists of a triple ß-barrel similar to the N-terminal TFIIF Rap30/Rap74 dimerization module, whereas the C-terminal Sfc1 DNA-binding domain contains a winged-helix domain most similar to the TFIIF Rap30 C-terminal winged-helix domain. Sfc1 DNA-binding domain recognizes single and double-stranded DNA by an unknown mechanism. Several features observed for A-box recognition by τA resemble the recognition of promoters by bacterial RNA polymerase, where σ factor unfolds double-stranded DNA and stabilizes the non-coding DNA strand in an open conformation. Such a function has also been proposed for TFIIF, suggesting that the observed structural similarity between Sfc1/Sfc7 and TFIIF Rap30/Rap74 might also reflect similar functions.

Structural and functional characterization of a phosphatase domain within yeast general transcription factor IIIC.

Saccharomyces cerevisiae τ55, a subunit of the RNA polymerase III-specific general transcription factor TFIIIC, comprises an N-terminal histidine phosphatase domain (τ55-HPD) whose catalytic activity and cellular function is poorly understood. We solved the crystal structures of τ55-HPD and its closely related paralogue Huf and used in silico docking methods to identify phosphoserine- and phosphotyrosine-containing peptides as possible substrates that were subsequently validated using in vitro phosphatase assays. A comparative phosphoproteomic study identified additional phosphopeptides as possible targets that show the involvement of these two phosphatases in the regulation of a variety of cellular functions. Our results identify τ55-HPD and Huf as bona fide protein phosphatases, characterize their substrate specificities, and provide a small set of regulated phosphosite targets in vivo.

The automated FADU-assay, a potential high-throughput in vitro method for early screening of DNA breakage.

Genotoxicity tests are essential to identify compounds that have a potential to compromise not only the environment but also human and animal health, including compounds that increase the risk of cancer. At present, no single test is capable of detecting all types of genotoxic effects; therefore a battery of in vitro and, if positive, in vivo tests is necessary to determine the genotoxicity of a substance. However, the respective specificities of current assays have been criticized for their high percentage of false positive results. We recently published an automated version of the "Fluorimetric detection of Alkaline DNA Unwinding" (FADU) assay for measuring DNA strand breaks in human peripheral blood mononuclear cells or in cell lines. Using this new technology we show detection of DNA strand breaks in cells treated with several compounds known to induce DNA strand breaks by various mechanisms. We also tested toxic compounds that were not expected to induce DNA strand breaks; these were negative in the assay as expected. Finally, we included zinc oxide nanoparticles of high production volume to explore further fields of potential FADU applications. The main advantages of this assay are high reproducibility, easy handling, lack of operator bias, high-throughput, speed, and low cost.

Extrinsic versus intrinsic cues in avian paraxial mesoderm patterning and differentiation.

Somitic and head mesoderm contribute to cartilage and bone and deliver the entire skeletal musculature. Studies on avian somite patterning and cell differentiation led to the view that these processes depend solely on cues from surrounding tissues. However, evidence is accumulating that some developmental decisions depend on information within the somitic tissue itself. Moreover, recent studies established that head and somitic mesoderm, though delivering the same tissue types, are set up to follow their own, distinct developmental programmes. With a particular focus on the chicken embryo, we review the current understanding of how extrinsic signalling, operating in a framework of intrinsically regulated constraints, controls paraxial mesoderm patterning and cell differentiation.

Neural tube derived signals and Fgf8 act antagonistically to specify eye versus mandibular arch muscles.

Recent knockout experiments in the mouse generated amazing craniofacial skeletal muscle phenotypes. Yet none of the genes could be placed into a molecular network, because the programme to control the development of muscles in the head is not known. Here we show that antagonistic signals from the neural tube and the branchial arches specify extraocular versus branchiomeric muscles. Moreover, we identified Fgf8 as the branchial arch derived signal. However, this molecule has an additional function in supporting the proliferative state of myoblasts, suppressing their differentiation, while a further branchial arch derived signal, namely Bmp7, is an overall negative regulator of head myogenesis.

Protein and genomic organisation of vertebrate MyoR and Capsulin genes and their expression during avian development.

The related bHLH transcription factors MyoR and Capsulin control craniofacial myogenesis and the development of a number of mesoderm-derived organs in the mouse. However, their molecular function as regulators of differentiation processes is conversely debated. One approach to clarify the roles of these genes is to comparatively analyse their biological and molecular function in various vertebrate models. For this, a prerequisite is the determination of their similarity and their expression patterns. Here we show that vertebrate MyoR and Capsulin are paralogous genes with a high level of conservation regarding their protein sequence, their cDNA sequence and their chromosomal organisation. In the chick, both genes are co-expressed in the developing branchiomeric muscles, the anterior heart field and the splanchnopleura lining the foregut. However, both genes show unique expression domains in trunk skeletal muscle precursors, in the lateral and intermediate mesoderm.

Structure of a bifunctional DNA primase-polymerase.

Genome replication generally requires primases, which synthesize an initial oligonucleotide primer, and DNA polymerases, which elongate the primer. Primase and DNA polymerase activities are combined, however, in newly identified replicases from archaeal plasmids, such as pRN1 from Sulfolobus islandicus. Here we present a structure-function analysis of the pRN1 primase-polymerase (prim-pol) domain. The crystal structure shows a central depression lined by conserved residues. Mutations on one side of the depression reduce DNA affinity. On the opposite side of the depression cluster three acidic residues and a histidine, which are required for primase and DNA polymerase activity. One acidic residue binds a manganese ion, suggestive of a metal-dependent catalytic mechanism. The structure does not show any similarity to DNA polymerases, but is distantly related to archaeal and eukaryotic primases, with corresponding active-site residues. We propose that archaeal and eukaryotic primases and the prim-pol domain have a common evolutionary ancestor, a bifunctional replicase for small DNA genomes.