In the last decade, geopolitical instability across the globe has increased the risk of a large-scale radiological event, when radiation biomarkers would be needed for an effective triage of an irradiated population. Ionizing radiation elicits a complex response in the proteome, genome, and metabolome and hence can be leveraged as rapid and sensitive indicators of irradiation-induced damage. We analyzed the plasma of total-body irradiated (TBI) leukemia patients (n = 24) and nonhuman primates (NHPs; n = 10) before and 24 h after irradiation, and we performed a global metabolomic study aiming to provide plasma metabolites as candidate radiation biomarkers for biological dosimetry. Peripheral blood samples were collected according to the appropriate ethical approvals, and metabolites were extracted and analyzed by liquid chromatography mass spectrometry. We identified an array of metabolites significantly altered by irradiation, including bilirubin, cholesterol, and 18-hydroxycorticosterone, which were detected in leukemia patients and NHPs. Pathway analysis showed overlapping perturbations in steroidogenesis, porphyrin metabolism, and steroid hormone biosynthesis and metabolism. Additionally, we observed dysregulation in bile acid biosynthesis and tyrosine metabolism in the TBI patient cohort. This investigation is, to our best knowledge, among the first to provide valuable insights into a comparison between human and NHP irradiation models. The findings from this study could be leveraged for translational biological dosimetry.
- MeSH
- biologické markery krev MeSH
- celotělové ozáření * MeSH
- dospělí MeSH
- ionizující záření MeSH
- leukemie krev metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- Macaca mulatta MeSH
- metabolom * MeSH
- metabolomika metody MeSH
- zvířata MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The increasing risk of acute large-scale radiological/nuclear exposures of population underlines the necessity of developing new, rapid and high throughput biodosimetric tools for estimation of received dose and initial triage. We aimed to compare the induction and persistence of different radiation exposure biomarkers in human peripheral blood in vivo. Blood samples of patients with indicated radiotherapy (RT) undergoing partial body irradiation (PBI) were obtained soon before the first treatment and then after 24 h, 48 h, and 5 weeks; i.e. after 1, 2, and 25 fractionated RT procedures. We collected circulating peripheral blood from ten patients with tumor of endometrium (1.8 Gy per fraction) and eight patients with tumor of head and neck (2.0-2.121 Gy per fraction). Incidence of dicentrics and micronuclei was monitored as well as determination of apoptosis and the transcription level of selected radiation-responsive genes. Since mitochondrial DNA (mtDNA) has been reported to be a potential indicator of radiation damage in vitro, we also assessed mtDNA content and deletions by novel multiplex quantitative PCR. Cytogenetic data confirmed linear dose-dependent increase in dicentrics (p < 0.01) and micronuclei (p < 0.001) in peripheral blood mononuclear cells after PBI. Significant up-regulations of five previously identified transcriptional biomarkers of radiation exposure (PHPT1, CCNG1, CDKN1A, GADD45, and SESN1) were also found (p < 0.01). No statistical change in mtDNA deletion levels was detected; however, our data indicate that the total mtDNA content decreased with increasing number of RT fractions. Interestingly, the number of micronuclei appears to correlate with late radiation toxicity (r2 = 0.9025) in endometrial patients suggesting the possibility of predicting the severity of RT-related toxicity by monitoring this parameter. Overall, these data represent, to our best knowledge, the first study providing a multiparametric comparison of radiation biomarkers in human blood in vivo, which have potential for improving biological dosimetry.
- MeSH
- biologické markery krev MeSH
- celková dávka radioterapie MeSH
- chromozomální aberace MeSH
- genetická transkripce účinky záření MeSH
- leukocyty patologie účinky záření MeSH
- lidé středního věku MeSH
- lidé MeSH
- mikrojádra chromozomálně defektní MeSH
- mitochondriální DNA účinky záření MeSH
- nádory endometria krev radioterapie MeSH
- nádory hlavy a krku krev radioterapie MeSH
- radiační expozice * MeSH
- radiometrie metody MeSH
- radioterapie škodlivé účinky MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- vztah dávky záření a odpovědi MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
This review summarizes recent progress in understanding the role of p53-upregulated mediator of apoptosis (PUMA) in molecular pathways with respect to its potential therapeutic applications. Particular emphasis is given to the PUMA´s role in ionizing radiation-induced signalling as radiotoxicity of normal tissue is mediated mostly via apoptosis. PUMA and its p53-dependent and p53- independent induction are described and potential use as a new target for the development of radioprotective agents is suggested. Further implications, including targeting PUMA to prevent and treat cardiovascular and neurodegenerative diseases, are also discussed together with an overview of other therapeutic applications. Finally, basic chemical structures for the development of novel PUMA modulators such as pifithrine derivatives, kinase inhibitors or modulators of Bcl-2 protein family are described.
- MeSH
- apoptóza účinky léků genetika MeSH
- cílená molekulární terapie metody MeSH
- kardiovaskulární látky farmakologie terapeutické užití MeSH
- kardiovaskulární nemoci farmakoterapie patologie MeSH
- lidé MeSH
- nádorový supresorový protein p53 antagonisté a inhibitory genetika metabolismus MeSH
- nádory genetika radioterapie MeSH
- neurodegenerativní nemoci farmakoterapie patologie MeSH
- neuroprotektivní látky farmakologie terapeutické užití MeSH
- poškození DNA účinky léků účinky záření MeSH
- proteiny regulující apoptózu antagonisté a inhibitory metabolismus MeSH
- protoonkogenní proteiny antagonisté a inhibitory metabolismus MeSH
- radiační poranění prevence a kontrola MeSH
- radioprotektivní látky farmakologie terapeutické užití MeSH
- signální transdukce účinky léků genetika MeSH
- tolerance záření účinky léků MeSH
- vazba proteinů účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
We studied the effect of pre-incubation with NU7441, a specific inhibitor of DNA-dependent protein kinase (DNA-PK), on molecular mechanisms triggered by ionizing radiation (IR). The experimental design involved four groups of human T-lymphocyte leukaemic MOLT-4 cells: control, NU7441-treated (1 μM), IR-treated (1 Gy), and combination of NU7441 and IR. We used flow cytometry for apoptosis assessment, Western blotting and ELISA for detection of proteins involved in DNA repair signalling and epifluorescence microscopy for detection of IR-induced phosphorylation of histone H2A.X. We did not observe any major changes in the amount of DNA-PK subunits Ku70/80 caused by the combination of NU7441 and radiation. Their combination led to an increased phosphorylation of H2A.X, a hallmark of DNA damage. However, it did not prevent up-regulation of neither p53 (and its phosphorylation at Ser 15 and 392) nor p21. We observed a decrease in the levels of anti-apoptotic Mcl-1, cdc25A phosphatase, cleavage of PARP and a significant increase in apoptosis in the group treated with combination. In conclusion, the combination of NU7441 with IR caused increased phosphorylation of H2A.X early after irradiation and subsequent induction of apoptosis. It was efficient in MOLT-4 cells in 10× lower concentration than the inhibitor NU7026. NU7441 proved as a potent radio-sensitizing agent, and it might provide a platform for development of new radio-sensitizers in radiotherapy.
- MeSH
- apoptóza účinky léků účinky záření MeSH
- časové faktory MeSH
- chromony farmakologie MeSH
- fosforylace účinky léků účinky záření MeSH
- histony metabolismus MeSH
- inhibitory proteinkinas farmakologie MeSH
- leukemie patologie MeSH
- lidé MeSH
- morfoliny farmakologie MeSH
- nádorové buněčné linie MeSH
- oprava DNA účinky léků účinky záření MeSH
- poškození DNA MeSH
- proliferace buněk účinky léků účinky záření MeSH
- proteinkinasa aktivovaná DNA antagonisté a inhibitory MeSH
- radiosenzibilizující látky farmakologie MeSH
- signální transdukce účinky léků účinky záření MeSH
- tolerance záření účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In this paper we describe the influence of NU7026, a specific inhibitor of DNA -dependent protein kinase, phosphoinositide 3-kinase, and AT M-kinase on molecular and cellular mechanisms triggered by ionising irradiation in human T-lymphocyte leukaemic MOLT -4 cells. We studied the effect of this inhibitor (10 μM) combined with gammaradiation (1 Gy) leading to DNA damage response and induction of apoptosis. We used methods for apoptosis assessment (cell viability count and flow-cytometric analysis) and cell cycle analysis (DNA content measurement) and we detected expression and post-translational modifications (Western blotting) of proteins involved in DNA repair signalling pathways. Pre-treatment with NU7026 resulted into decreased activation of checkpoint kinase-2 (Thr68), p53 (Ser15 and Ser392), and histone H2A.X (Ser139) 2 hours after irradiation. Subsequently, combination of radiation and inhibitor led to decreased amount of cells in G2-phase arrest and into increased apoptosis after 72 hours. Our results indicate that in leukaemic cells the pre-incubation with inhibitor NU7026 followed by low doses of ionising radiation results in radio-sensitising of MOLT -4 cells via diminished DNA repair and delayed but pronounced apoptosis. This novel approach might offer new strategies in combined treatment of leukaemia diseases.
- MeSH
- apoptóza účinky záření MeSH
- buněčný cyklus účinky záření MeSH
- chromony farmakologie MeSH
- leukemie T-buněčná radioterapie MeSH
- lidé MeSH
- morfoliny farmakologie MeSH
- nádorové buněčné linie účinky záření MeSH
- oprava DNA účinky záření MeSH
- poškození DNA účinky záření MeSH
- proliferace buněk účinky záření MeSH
- proteinkinasa aktivovaná DNA antagonisté a inhibitory MeSH
- radiosenzibilizující látky farmakologie MeSH
- tolerance záření účinky léků MeSH
- záření gama MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
This review describes a drug target for cancer therapy, family of phosphatidylinositol-3 kinase related kinases (PIKKs), and it gives a comprehensive review of recent information. Besides general information about phosphatidylinositol-3 kinase superfamily, it characterizes a DNA-damage response pathway since it is monitored by PIKKs.
- MeSH
- buněčný cyklus MeSH
- DNA vazebné proteiny MeSH
- fosfatidylinositol-3-kinasy třídy I MeSH
- fosfatidylinositol-3-kinasy třídy II MeSH
- fosfatidylinositol-3-kinasy třídy III MeSH
- fosfatidylinositol-3-kinasy * fyziologie genetika MeSH
- fosfatidylinositol-4-fosfát-3-kinasa MeSH
- ionizující záření * MeSH
- lidé MeSH
- nádorové supresorové proteiny fyziologie genetika MeSH
- nádorový supresorový protein p53 fyziologie genetika MeSH
- nádory genetika radioterapie MeSH
- oprava DNA genetika MeSH
- poškození DNA * účinky záření MeSH
- protein-serin-threoninkinasy fyziologie genetika MeSH
- proteiny buněčného cyklu fyziologie genetika MeSH
- radioterapie škodlivé účinky MeSH
- teleangiektatická ataxie genetika MeSH
- TOR serin-threoninkinasy fyziologie genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
Most of the cellular processes are regulated by reversible phosphorylation of proteins, which in turn plays a critical role in the regulation of gene expression, cell division, signal transduction, metabolism, differentiation, and apoptosis. Mass spectrometry of phosphopeptides obtained from tryptic protein digests has become a powerful tool for characterization of phosphoproteins involved in these processes. However, there is a general need to significantly enrich the phosphopeptide content to compensate their low abundance, insufficient ionization, and suppression effects of non-phosphorylated peptides. This paper aims to give a comprehensive overview on the methods involved in recent phosphoproteomics. It presents a description of contemporary enrichment techniques with references to particular studies and compares different approaches to characterization of phosphoproteome by mass spectrometry.
- MeSH
- apoptóza fyziologie MeSH
- buněčná diferenciace fyziologie MeSH
- buněčné dělení fyziologie MeSH
- fosfoproteiny analýza metabolismus MeSH
- fosforylace fyziologie MeSH
- hmotnostní spektrometrie metody MeSH
- lidé MeSH
- peptidy analýza metabolismus MeSH
- proteomika metody MeSH
- regulace genové exprese fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- srovnávací studie MeSH
Ionising radiation (IR) is one of the main treatment modalities in oncology. However, we still search for substances which can radio-sensitize tumour cells. In this study we used caffeine, a non-specific ataxia-telangiectasia mutated kinase (ATM) inhibitor, and studied its effect on the activation of the proteins involved in cell cycle control and the induction of apoptosis in human T-lymphocyte leukaemic MOLT-4 cells (p53 wt). We evaluated the expression of the tumour-suppressor p53 (itself and phosphorylated on Ser15 and Ser392), the cell cycle regulator p21, and the anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1). After treatment with 2 mM caffeine, the cells were irradiated by 1 or 3 Gy, lysed and the proteins detected by Western-blotting. Apoptosis was determined by flow-cytometric annexin V/propidium iodine detection. Irradiation by 1 or 3 Gy induced p53 phosphorylation at Ser15 and Ser392 after 2 h with maximum after 4 h. Adding caffeine significantly inhibited Ser15 phosphorylation, which is ATM-dependent but surprisingly also Ser392 phosphorylation, which is ATM-independent, suggesting that caffeine might have another cellular target (protein kinase). Similarly, caffeine caused a substantial decrease in p21 in combination with both doses of IR and also Mcl-1 was down-regulated. Three days after irradiation, caffeine significantly increased induction of apoptosis. The ATM/p53 pathway was suppressed by caffeine, which led to increased apoptosis accompanied by a p53-independent decrease in Mcl-1. It also caused down-regulation of p21, which possibly contributed to the shortened cell cycle arrest necessary for effective DNA repair and thus impeded radio-resistance. Caffeine promotes the cytotoxic effect of ionising radiation and provides a possible platform for the development of new anti-cancer therapeutics known as radio-sensitizers.
- MeSH
- apoptóza genetika imunologie účinky léků MeSH
- buněčný cyklus MeSH
- elektroforéza MeSH
- financování organizované MeSH
- geny p53 genetika účinky léků MeSH
- ionizující záření MeSH
- kofein chemie metabolismus MeSH
- kultivační techniky využití MeSH
- leukemie T-buněčná genetika MeSH
- lidé MeSH
- onkogenní protein p21(ras) genetika MeSH
- proteinkinasy genetika imunologie MeSH
- průtoková cytometrie MeSH
- teleangiektatická ataxie genetika MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
Ataxia-telangiectasia mutated kinase (ATM) is a DNA damage-inducible protein kinase, which phosphorylates plethora of substrates participating in DNA damage response. ATM significance for the cell faith is undeniable, since it regulates DNA repair, cell-cycle progress, and apoptosis. Here we describe its main signalling targets and discuss its importance in DNA repair as well as novel findings linked to this key regulatory enzyme in the terms of ionizing radiationinduced DNA damage.
