Radiation Signature in Plasma Metabolome of Total-Body Irradiated Nonhuman Primates and Clinical Patients.

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.

Transcriptional Inflammatory Signature in Healthy Donors and Different Radiotherapy Cancer Patients.

Cancer and ionizing radiation exposure are associated with inflammation. To identify a set of radiation-specific signatures of inflammation-associated genes in the blood of partially exposed radiotherapy patients, differential expression of 249 inflammatory genes was analyzed in blood samples from cancer patients and healthy individuals. The gene expression analysis on a cohort of 63 cancer patients (endometrial, head and neck, and prostate cancer) before and during radiotherapy (24 h, 48 h, ~1 week, ~4-8 weeks, and 1 month after the last fraction) identified 31 genes and 15 up- and 16 down-regulated genes. Transcription variability under normal conditions was determined using blood drawn on three separate occasions from four healthy donors. No difference in inflammatory expression between healthy donors and cancer patients could be detected prior to radiotherapy. Remarkably, repeated sampling of healthy donors revealed an individual endogenous inflammatory signature. Next, the potential confounding effect of concomitant inflammation was studied in the blood of seven healthy donors taken before and 24 h after a flu vaccine or ex vivo LPS (lipopolysaccharide) treatment; flu vaccination was not detected at the transcriptional level and LPS did not have any effect on the radiation-induced signature identified. Finally, we identified a radiation-specific signature of 31 genes in the blood of radiotherapy patients that were common for all cancers, regardless of the immune status of patients. Confirmation via MQRT-PCR was obtained for BCL6, MYD88, MYC, IL7, CCR4 and CCR7. This study offers the foundation for future research on biomarkers of radiation exposure, radiation sensitivity, and radiation toxicity for personalized radiotherapy treatment.

Fractionation of Enriched Phosphopeptides Using pH/Acetonitrile-Gradient-Reversed-Phase Microcolumn Separation in Combination with LC-MS/MS Analysis.

Mass spectrometry (MS) is a powerful and sensitive method often used for the identification of phosphoproteins. However, in phosphoproteomics, there is an identified need to compensate for the low abundance, insufficient ionization, and suppression effects of non-phosphorylated peptides. These may hamper the subsequent liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, resulting in incomplete phosphoproteome characterization, even when using high-resolution instruments. To overcome these drawbacks, we present here an effective microgradient chromatographic technique that yields specific fractions of enriched phosphopeptides compatible with LC-MS/MS analysis. The purpose of our study was to increase the number of identified phosphopeptides, and thus, the coverage of the sample phosphoproteome using the reproducible and straightforward fractionation method. This protocol includes a phosphopeptide enrichment step followed by the optimized microgradient fractionation of enriched phosphopeptides and final LC-MS/MS analysis of the obtained fractions. The simple fractionation system consists of a gas-tight microsyringe delivering the optimized gradient mobile phase to reversed-phase microcolumn. Our data indicate that combining the phosphopeptide enrichment with the microgradient separation is a promising technique for in-depth phosphoproteomic analysis due to moderate input material requirements and more than 3-fold enhanced protein identification.

In Vivo Validation of Alternative FDXR Transcripts in Human Blood in Response to Ionizing Radiation.

Following cell stress such as ionising radiation (IR) exposure, multiple cellular pathways are activated. We recently demonstrated that ferredoxin reductase (FDXR) has a remarkable IR-induced transcriptional responsiveness in blood. Here, we provided a first comprehensive FDXR variant profile following DNA damage. First, specific quantitative real-time polymerase chain reaction (qPCR) primers were designed to establish dose-responses for eight curated FDXR variants, all up-regulated after IR in a dose-dependent manner. The potential role of gender on the expression of these variants was tested, and neither the variants response to IR nor the background level of expression was profoundly affected; moreover, in vitro induction of inflammation temporarily counteracted IR response early after exposure. Importantly, transcriptional up-regulation of these variants was further confirmed in vivo in blood of radiotherapy patients. Full-length nanopore sequencing was performed to identify other FDXR variants and revealed the high responsiveness of FDXR-201 and FDXR-208. Moreover, FDXR-218 and FDXR-219 showed no detectable endogenous expression, but a clear detection after IR. Overall, we characterised 14 FDXR transcript variants and identified for the first time their response to DNA damage in vivo. Future studies are required to unravel the function of these splicing variants, but they already represent a new class of radiation exposure biomarkers.

Substituted Piperazines as Novel Potential Radioprotective Agents.

The increasing risk of radiation exposure underlines the need for novel radioprotective agents. Hence, a series of novel 1-(2-hydroxyethyl)piperazine derivatives were designed and synthesized. Some of the compounds protected human cells against radiation-induced apoptosis and exhibited low cytotoxicity. Compared to the previous series of piperazine derivatives, compound 8 exhibited a radioprotective effect on cell survival in vitro and low toxicity in vivo. It also enhanced the survival of mice 30 days after whole-body irradiation (although this increase was not statistically significant). Taken together, our in vitro and in vivo data indicate that some of our compounds are valuable for further research as potential radioprotectors.

Biological dosimetry and modern (-omic) methods.

The increased risk of acute large-scale radiation exposure of the population underlies the necessity to develop new methods that could provide a rapid assessment of the doses received while using modern high-throughput technologies. At the same time, there is a growing interest in discovering new biomarkers enabling the categorization of irradiated individuals that could be used in epidemiological studies to correlate the estimated absorbed doses with the consequent impact on patients health. The aim of this study was to summarize the current literature on biological dosimetry, specifically ionizing radiation-responsive biomarkers. We briefly describe current knowledge in the field of radiation genomics, metabolomics, and proteomics. Although the majority of studies that provided a plethora of useful information were conducted in animal models, oncological patients remain the crucial experimental model. The authors describe various biological materials that could be potentially used to predict the effect of ionizing radiation. Plasma proteins appear to be ideal for this purpose. Out of many candidate markers, the ferredoxin reductase (FDXR) seems to be promising, as it has been confirmed in several biodosimetric studies at the level of both human gene and protein.

Novel quinazolin-4-one derivatives as potentiating agents of doxorubicin cytotoxicity.

We report the design, synthesis and biological evaluation of 17 novel 8-aryl-2-morpholino-3,4-dihydroquinazoline derivatives based on the standard model of DNA-PK and PI3K inhibitors. Novel compounds are sub-divided into two series where the second series of five derivatives was designed to have a better solubility profile over the first one. A combination of in vitro and in silico techniques suggested a plausible synergistic effect with doxorubicin of the most potent compound 14d on cell proliferation via DNA-PK and poly(ADP-ribose) polymerase-1 (PARP-1) inhibition, while alone having a negligible effect on cell proliferation.

A Potent Autophagy Inhibitor (Lys05) Enhances the Impact of Ionizing Radiation on Human Lung Cancer Cells H1299.

Autophagy inhibition through small-molecule inhibitors is one of the approaches to increase the efficiency of radiotherapy in oncological patients. A new inhibitor-Lys05-with the potential to accumulate within lysosomes and to block autophagy was discovered a few years ago. Several studies have addressed its chemosensitizing effects but nothing is known about its impact in the context of ionizing radiation (IR). To describe its role in radiosensitization, we employed radioresistant human non-small cell lung carcinoma cells (H1299, p53-negative). Combined treatment of H1299 cells by Lys05 together with IR decreased cell survival in the clonogenic assay and real-time monitoring of cell growth more than either Lys05 or IR alone. Immunodetection of LC3 and p62/SQSTM1 indicated that autophagy was inhibited, which correlated with increased SQSTM1 and decreased BNIP3 gene expression determined by qRT-PCR. Fluorescence microscopy and flow cytometry uncovered an accumulation of lysosomes. Similarly, transmission electron microscopy demonstrated the accumulation of autophagosomes confirming the ability of Lys05 to potentiate autophagy inhibition in H1299 cells. We report here for the first time that Lys05 could be utilized in combination with IR as a promising future strategy in the eradication of lung cancer cells.

Radio-sensitization of human leukaemic MOLT-4 cells by DNA-dependent protein kinase inhibitor, NU7441.

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.

Radiosensitization of human leukemic HL-60 cells by ATR kinase inhibitor (VE-821): phosphoproteomic analysis.

DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR)-triggered by radiation-induced double strand breaks-is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs): Ataxia teleangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK) and ATM and Rad3-related kinase (ATR). Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide), has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient) without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative). Hydrophilic interaction liquid chromatography (HILIC)-prefractionation with TiO2-enrichment and nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells.

Second-generation piperazine derivatives as promising radiation countermeasures.

The increasing threat of nuclear incidents and the widespread use of ionizing radiation (IR) in medical treatments underscore the urgent need for effective radiation countermeasures. Despite the availability of compounds such as amifostine, their clinical utility is significantly limited by adverse side effects and logistical challenges in administration. This study focuses on the synthesis and evaluation of novel piperazine derivatives as potential radioprotective agents, with the aim of overcoming the limitations associated with current countermeasures. We designed, synthesized, and evaluated a series of 1-(2-hydroxyethyl)piperazine derivatives. The compounds were assessed for cytotoxicity across a panel of human cell lines, and for their radioprotective effects in the MOLT-4 lymphoblastic leukemia cell line and in peripheral blood mononuclear cells (PBMCs) exposed to gamma radiation. The radioprotective efficacy was further quantified using the dicentric chromosome assay (DCA) to measure DNA damage mitigation. Among the synthesized derivatives, compound 6 demonstrated the most significant radioprotective effects in vitro, with minimal cytotoxicity across the tested cell lines. Compound 3 also showed notable efficacy, particularly in reducing dicentric chromosomes, thus indicating its potential to mitigate DNA damage from IR. Both compounds exhibited superior safety profiles and effectiveness compared to amifostine, suggesting their potential as more viable radioprotective agents. This study highlights the development of novel piperazine derivatives with promising radioprotective properties. Compound 6 emerged as the leading candidate, offering an optimal balance between efficacy and safety, with compound 3 also displaying significant potential. These findings support the further development and clinical evaluation of these compounds as safer, and more effective radiation countermeasures.

Inhibition of ATR kinase with the selective inhibitor VE-821 results in radiosensitization of cells of promyelocytic leukaemia (HL-60).

We compared the effects of inhibitors of kinases ATM (KU55933) and ATR (VE-821) (incubated for 30 min before irradiation) on the radiosensitization of human promyelocyte leukaemia cells (HL-60), lacking functional protein p53. VE-821 reduces phosphorylation of check-point kinase 1 at serine 345, and KU55933 reduces phosphorylation of check-point kinase 2 on threonine 68 as assayed 4 h after irradiation by the dose of 6 Gy. Within 24 h after gamma-irradiation with a dose of 3 Gy, the cells accumulated in the G2 phase (67 %) and the number of cells in S phase decreased. KU55933 (10 µM) did not affect the accumulation of cells in G2 phase and did not affect the decrease in the number of cells in S phase after irradiation. VE-821 (2 and 10 µM) reduced the number of irradiated cells in the G2 phase to the level of non-irradiated cells and increased the number of irradiated cells in S phase, compared to irradiated cells not treated with inhibitors. In the 144 h interval after irradiation with 3 Gy, there was a considerable induction of apoptosis in the VE-821 group (10 µM). The repair of the radiation damage, as observed 72 h after irradiation, was more rapid in the group exposed solely to irradiation and in the group treated with KU55933 (80 and 77 % of cells, respectively, were free of DSBs), whereas in the group incubated with 10 µM VE-821, there were only 61 % of cells free of DSBs. The inhibition of kinase ATR with its specific inhibitor VE-821 resulted in a more pronounced radiosensitizing effect in HL-60 cells as compared to the inhibition of kinase ATM with the inhibitor KU55933. In contrast to KU55933, the VE-821 treatment prevented HL-60 cells from undergoing G2 cell cycle arrest. Taken together, we conclude that the ATR kinase inhibition offers a new possibility of radiosensitization of tumour cells lacking functional protein p53.

Looking for the phoenix: the current research on radiation countermeasures.

PURPOSE: Ionizing radiation (IR) is widely applied in radiotherapy for the treatment of over 50% of cancer patients. IR is also intensively used in medical diagnostics on a daily basis in imaging. Moreover, recent geopolitical events have re-ignited the real threat of the use of nuclear weapons. Medical radiation countermeasures represent one of the effective protection strategies against the effects of IR. The aim of this review was to summarize the most commonly used strategies and procedures in the development of radiation countermeasures and to evaluate the current state of their research, with a focus on those in the clinical trial phase. METHODS: Clinical trials for this review were selected in accordance with the preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement. The search was performed in the clinicaltrials.gov database as of May 2022. RESULTS: Our search returned 263 studies, which were screened and of which 25 were included in the review. 10 of these studies had been completed, 3 with promising results: KMRC011 increased G-CSF, IL-6, and neutrophil counts suggesting potential for the treatment of hematopoietic acute radiation syndrome (H-ARS); GC4419 reduced the number of patients with severe oral mucositis and its duration; the combination of enoxaparin, pentoxifylline, and ursodeoxycholic acid reduced the incidence of focal radiation-induced liver injury. CONCLUSION: The agents discovered so far show significant side effects or low efficacy, and hence most of the tested agents terminate in the early stages of development. In addition, the low profitability of this type of drug demotivates the private sector to invest in such research. To overcome this problem, there is a need to involve more public resources in funding. Among the technological opportunities, a deeper use of in silico approaches seems to be prospective.

Irradiated stem cells and ageing of the haematopoietic system.

In the work presented here, changes in haematopoiesis of mice (B6129SF2/J) were studied 1 year after their whole-body exposure to a dose of 7 Gy (72% of mice survived). The irradiated mice were compared with non-irradiated younger (4 months of age) and older (16 months of age) mice. There was a significant increase in the relative abundance of primitive stem cells with long-term capability of the haematopoiesis recovery lin(-)/Sca-1(+)/CD117(+)/CD34(-) in the bone marrow of mice aged 16 months (irradiated and non-irradiated) compared with those aged 4 months. In terms of the ability to respond to further whole-body irradiation at a dose of 1 Gy, the presence of γH2A.X foci was studied in lin(-) bone marrow cells. There was a considerable number of persisting foci in lin(-) stem cells isolated from the bone marrow of the older irradiated mice. In the blood count from the peripheral blood of the older mice (both non-irradiated and irradiated at 7 Gy), there was a significant increase in granulocytes. In the group exposed to 7 Gy, the numbers of thrombocytes significantly increased, and on the contrary, the numbers of erythrocytes, the amount of haemoglobin, and haematocrit significantly decreased.

Radio-sensitization of human leukaemic molt-4 cells by DNA-dependent protein kinase inhibitor, NU7026.

In this paper we describe the influence of NU7026, a specific inhibitor of DNA-dependent protein kinase, phosphoinositide 3-kinase, and ATM-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 1microM) combined with gamma-radiation (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.

Mitigation of Ionizing Radiation-Induced Gastrointestinal Damage by Insulin-Like Growth Factor-1 in Mice.

Purpose: Insulin-like growth factor-1 (IGF-1) stimulates epithelial regeneration but may also induce life-threatening hypoglycemia. In our study, we first assessed its safety. Subsequently, we examined the effect of IGF-1 administered in different dose regimens on gastrointestinal damage induced by high doses of gamma radiation. Material and methods: First, fasting C57BL/6 mice were injected subcutaneously with IGF-1 at a single dose of 0, 0.2, 1, and 2 mg/kg to determine the maximum tolerated dose (MTD). The glycemic effect of MTD (1 mg/kg) was additionally tested in non-fasting animals. Subsequently, a survival experiment was performed. Animals were irradiated (60Co; 14, 14.5, or 15 Gy; shielded head), and IGF-1 was administered subcutaneously at 1 mg/kg 1, 24, and 48 h after irradiation. Simultaneously, mice were irradiated (60Co; 12, 14, or 15 Gy; shielded head), and IGF-1 was administered subcutaneously under the same regimen. Jejunum and lung damage were assessed 84 h after irradiation. Finally, we evaluated the effect of six different IGF-1 dosage regimens administered subcutaneously on gastrointestinal damage and peripheral blood changes in mice 6 days after irradiation (60Co; 12 and 14 Gy; shielded head). The regimens differed in the number of doses (one to five doses) and the onset of administration (starting at 1 [five regimens] or 24 h [one regimen] after irradiation). Results: MTD was established at 1 mg/kg. MTD mitigated lethality induced by 14 Gy and reduced jejunum and lung damage caused by 12 and 14 Gy. However, different dosing regimens showed different efficacy, with three and four doses (administered 1, 24, and 48 h and 1, 24, 48, and 72 h after irradiation, respectively) being the most effective. The three-dose regimens supported intestinal regeneration even if the administration started at 24 h after irradiation, but its potency decreased. Conclusion: IGF-1 seems promising in the mitigation of high-dose irradiation damage. However, the selected dosage regimen affects its efficacy.

MITOCHONDRIA IN BIODOSIMETRY: FLOW CYTOMETRY ASSESMENT IN VITRO.

The JC-1 dye is widely used in apoptosis studies to monitor mitochondrial health. The probe was tested in vitro on two established cell lines and peripheral porcine blood lymphocytes after gamma irradiation (IR) to assess its potential in biodosimetric evaluation. In brief, we stained irradiated and non-irradiated cells with the JC-1 dye to determine the existing changes in mitochondrial membrane potential and monitor cell health through flow cytometry. The stage of injury in these cells was evaluated through an irradiated versus non-irradiated ratio (IVNIR), comparing the relative proportion of polarised cells containing red JC-1 aggregates. We observed a decreasing IVNIR as the radiation dose increased (i.e. 0.5; 1; 2; 4; 6; 8 and 10 Gy), performing the analysis at 4, 8 and 24 h after IR in all the tested cells. The results from the JC1-dye test showed that CD4 T lymphocytes were more sensitive to irradiation than other subpopulations.

The Effect of Lactobacillus casei on Experimental Porcine Inflammatory Bowel Disease Induced by Dextran Sodium Sulphate.

BACKGROUND: Gastrointestinal injury caused by dextran sodium sulphate (DSS) is a reliable porcine experimental model of inflammatory bowel disease (IBD). The purpose of this study was to evaluate the effect of probiotic Lactobacillus casei DN 114001 (LC) on DSS-induced experimental IBD. RESULTS: Eighteen female pigs (Sus scrofa f. domestica, weight 33-36 kg, age 4-5 months) were divided into 3 groups (6 animals per group): controls with no treatment, DSS, and DSS + LC. LC was administered to overnight fasting animals in a dietary bolus in the morning on days 1-7 (4.5 × 1010 live bacteria/day). DSS was applied simultaneously on days 3-7 (0.25 g/kg/day). On day 8, the pigs were sacrificed. Histopathological score and length of crypts/glands (stomach, jejunum, ileum, transverse colon), length and width of villi (jejunum, ileum), and mitotic and apoptotic indices (jejunum, ileum, transverse colon) were assessed. DSS increased the length of glands in the stomach, length of crypts and villi in the jejunum and ileum, and the histopathological score of gastrointestinal damage, length of crypts and mitotic activity in the transverse colon. Other changes did not achieve any statistical significance. Administration of LC reduced the length of villi in the jejunum and ileum to control levels and decreased the length of crypts in the jejunum. CONCLUSIONS: Treatment with a probiotic strain of LC significantly accelerated regeneration of the small intestine in a DSS-induced experimental porcine model of IBD.

Exposure to fractionated dose of 60 Gy affects molecular response of HL-60 cells to irradiation.

In this work we evaluated changes in molecular response of human promyelocyte leukemia cells HL-60 and HL-60-IR cells (HL-60 irradiated by 10 cycles of radiation with total dose of 60 Gy, given over a period of 3 months) to irradiation by the dose of 2 and 8 Gy. Analysis of CD11b and apoptosis by flow-cytometry revealed that on 3rd day after irradiation by 8 Gy the HL-60-IR are more resistant to radiation-induced apoptosis and more differentiated (increase in CD11b in non-apoptotic cells) than regular HL-60. We found that both types of cells have high basal level of phosphorylated extracellular signal-regulated kinases Erk1/2 . Irradiation induces decrease in Erk1/2 phosphorylation after 4 and 8 h in both cell types. However, in HL-60-IR cells Erk1/2 phosphorylation is restored faster than in HL-60. Also it was found that in contrary to HL-60 cells, the HL-60-IR cells react to 2 Gy irradiation by p53 independent increase in p21(WAF1/Cip1), and not by activation of checkpoint kinase Chk-2. Therefore we concluded that relatively high dose of radiation (6 Gy) does not lead after 10 repetitive irradiations to eradication of HL-60 cells, but instead increases their radioresistance, increases the ability to differentiate, alters MAPK response, increases amount of p21(WAF1/Cip1), and decreases induction of apoptosis by ionizing radiation. p21(WAF1/Cip1) might prevent apoptosis induction and trigger permanent cell-cycle arrest, which can also contribute to regression of this leukaemia after therapy.

Ataxia-telangiectasia mutated kinase (ATM) as a central regulator of radiation-induced DNA damage response.

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 radiation-induced DNA damage.