Detection of adaptive response at chromosome level.

We have just started the basic study to detect the genetic alterations at chromosome level as a result of radioadaptive response. The assay system is based upon the analysis of loss of heterozygosity (LOH) induced in human lymphoblastoid cell TK6, which were pre-irradiated with low-doses of ionizing radiation (IR) before the challenging irradiation. In our previous study, this analysis was shown to be very sensitive to IR because the radiation-specific hemizygous LOHs (interstitial deletions) were observed after 10 cGy of IR (X-rays or accelerated carbon-ion beam). Here, we would like to introduce our plan how to detect the changes in such radiation-specific LOH patterns by the pre-irradiation of TK6. If we succeed the detection, the radioadaptation assay system can be used for elucidating the biological effects of low-doses of space ionizing radiation. In addition, we are also considering the modification of assay system by introducing the site-specific chromosome breakage (DNA double-strand break) instead of challenging IR. Furthermore, the preliminary results of the experiments using frozen TK6 cells for the preparation of ISS experiments.     

The role of regulatory networks of the cell response to damages in radiation effects

In view of modern knowledge and concepts about components, function and mechanisms of response of cell molecular structures to damaging effects, response which is generating specialized modules of reactions, it is shown that main components of the mechanism of maintenance of genome constancy at ionizing radiation exposure are checkpoints of cell cycle, DNA repair and apoptosis. They operate under the control of a genetic system at participation of Tp53 gene, corresponding protein and of regulatory networks formed by cascades of mitogen-activated protein kinases (MAPK). At ionizing radiation exposure the MAPK special modules participate in formation of radiation effect: ERK 1/2 (extracellular signal-regulated kinase 1 and 2), JNK/SAPK (c-Jun N-terminal kinase/stress activated protein kinase) and p38 MAPK. Executing physiological functions of maintenance of normal life activity of cells, they do not lose this capacity after exposure to ionizing radiation, participating in formation of radiation effect in a wide range of doses, and are inactivated only by exposure to very high doses. It is concluded that in light of the modern data the main problem is not a problem of mechanisms of biological effect of ionizing radiation but a problem of biological mechanisms of radiation exposure.     

Phosphorylation and nuclear accumulation are distinct events contributing to the activation of p53

It has been recently shown that ionizing radiation (IR) and the mRNA synthesis inhibitor 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) act in synergy to induce p53-mediated transactivation of reporter plasmids in human cells [Oncogene 19 (2000) 3829]. We have extended these studies and show that ionizing radiation and DRB also act in synergy to induce ATM-mediated phosphorylation of the ser15 site of p53 and enhance the expression of endogenous p21 protein. Examination of the localization of p53 revealed that while DRB did not induce phosphorylation of the ser15 site of p53 but efficiently accumulated p53 in the nucleus, ionizing radiation induced phosphorylation of the ser15 site of p53 without prolonged nuclear accumulation. Importantly, the combination of DRB and IR resulted in a strong accumulation of phosphorylated p53 in the nucleus that was more persistent then p53 accumulation after IR alone. Furthermore, the nuclear export inhibitor leptomycin B showed a similar synergy with IR as did DRB regarding ser15 phosphorylation of p53 and p21 induction. These results suggest that the synergistic activation of the p53 response by the combination treatment is due to the activation of two distinct pathways where DRB causes the prolonged nuclear accumulation of p53 while ionizing radiation activates p53 by ATM-mediated phosphorylation.     

JNK/p53 mediated cell death response in K562 exposed to etoposide-ionizing radiation combined treatment

The study of the ability of chemotherapeutic agents and/or ionizing radiation (IR) to induce cell death in tumor cells is essential for setting up new and more efficient therapies against human cancer. Since drug and ionizing radiation resistance is an impediment to successful chemotherapy against cancer, we wanted to check if etoposide/ionizing radiation combined treatment could have a synergic effect to improve cell death in K562, a well-known human erythroleukemia ionizing radiation resistant cell line. In this study, we examined the role played by JNK/SAPK, p53, and mitochondrial pathways in cell death response of K562 cells to etoposide and IR treatment. Our results let us suppose that the induction of cell death, already evident in 15 Gy exposed cells, mainly in 15 Gy plus etoposide, may be mediated by JNK/SAPK pathway. Moreover, p53 is a potential substrate for JNK and may act as a JNK target for etoposide and ionizing radiation. Thus further investigation on these and other molecular mechanisms underlying the cell death response following etoposide and ionizing radiation exposure could be useful to overcome resistance mechanisms in tumor cells.     

Protein oxidation implicated as the primary determinant of bacterial radioresistance

In the hierarchy of cellular targets damaged by ionizing radiation (IR), classical models of radiation toxicity place DNA at the top. Yet, many prokaryotes are killed by doses of IR that cause little DNA damage. Here we have probed the nature of Mn-facilitated IR resistance in Deinococcus radiodurans, which together with other extremely IR-resistant bacteria have high intracellular Mn/Fe concentration ratios compared to IR-sensitive bacteria. For in vitro and in vivo irradiation, we demonstrate a mechanistic link between Mn(II) ions and protection of proteins from oxidative modifications that introduce carbonyl groups. Conditions that inhibited Mn accumulation or Mn redox cycling rendered D. radiodurans radiation sensitive and highly susceptible to protein oxidation. X-ray fluorescence microprobe analysis showed that Mn is globally distributed in D. radiodurans, but Fe is sequestered in a region between dividing cells. For a group of phylogenetically diverse IR-resistant and IR-sensitive wild-type bacteria, our findings support the idea that the degree of resistance is determined by the level of oxidative protein damage caused during irradiation. We present the case that protein, rather than DNA, is the principal target of the biological action of IR in sensitive bacteria, and extreme resistance in Mn-accumulating bacteria is based on protein protection.     

The effects of low doses of low-intensity ionizing radiation on DNA structure and function

Chronic effects of low doses of low-intensity ionizing radiation (IR) on biological objects have gained great social significance. This has given a considerable impetus to research into the biological effects and mechanisms of such exposures, both in Russia and abroad. In this paper, an overview of the physicochemical and molecular basis of IR influence at low doses is provided. Means of cell protection from radiation damage are studied and an analysis of the typical features and differences in the radiation effects at low and high doses is carried out. We considered DNA radiation damage, both in cell cultures and in vivo, as well as the processes and results of their repair. Particular attention is paid to changes in the basic paradigms of biological radiation effects at low doses.     

Mutagenic adaptive response to high-LET radiation in human lymphoblastoid cells exposed to low doses of heavy-ion radiation

Adaptive response (AR) and bystander effect are two important phenomena involved in biological responses to low doses of ionizing radiation (IR). Furthermore, there is a strong interest in better understanding the biological effects of high-LET radiation. We previously demonstrated the ability of low doses of X-rays to induce an AR to challenging heavy-ion radiation [8]. In this study, we assessed in vitro the ability of priming low doses (0.01Gy) of heavy-ion radiation to induce a similar AR to a subsequent challenging dose (1-4Gy) of high-LET IR (carbon-ion: 20 and 40keV/μm, neon-ion: 150keV/μm) in TK6, AHH-1 and NH32 cells. Our results showed that low doses of high-LET radiation can induce an AR characterized by lower mutation frequencies at hypoxanthine-guanine phosphoribosyl transferase locus and faster DNA repair kinetics, in cells expressing p53.     

Down-regulation of PERK enhances resistance to ionizing radiation

Although, ionizing radiation (IR) has been implicated to cause stress in endoplasmic reticulum (ER), how ER stress signaling and major ER stress sensors modulate cellular response to IR is unclear. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is an ER transmembrane protein which initiates unfolded protein response (UPR) or ER stress signaling when ER homeostasis is disturbed. Here, we report that down-regulation of PERK resulted in increased clonogenic survival, enhanced DNA repair and reduced apoptosis in irradiated cancer cells. Our study demonstrated that PERK has a role in sensitizing cancer cells to IR.     

Rapid activation of ATR by ionizing radiation requires ATM and Mre11

The ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases are crucial regulatory proteins in genotoxic stress response pathways that pause the cell cycle to permit DNA repair. Here we show that Chk1 phosphorylation in response to hydroxyurea and ultraviolet radiation is ATR-dependent and ATM- and Mre11-independent. In contrast, Chk1 phosphorylation in response to ionizing radiation (IR) is dependent on ATR, ATM, and Mre11. The ATR and ATM/Mre11 pathways are generally thought to be separate with ATM activation occurring early and ATR activation occurring as a late response to double strand breaks. However, we demonstrate that ATR is activated rapidly by IR, and ATM and Mre11 enhance ATR signaling. ATR-ATR-interacting protein recruitment to double strand breaks is less efficient in the absence of ATM and Mre11. Furthermore, IR-induced replication protein A foci formation is defective in ATM- and Mre11-deficient cells. Thus, ATM and Mre11 may stimulate the ATR signaling pathway by converting DNA damage generated by IR into structures that recruit and activate ATR.     

Vaccinia-related kinase 1 (VRK1) is an upstream nucleosomal kinase required for the assembly of 53BP1 foci in response to ionizing radiation-induced DNA damage

Cellular responses to DNA damage require the formation of protein complexes in a highly organized fashion. The complete molecular components that participate in the sequential signaling response to DNA damage remain unknown. Here we demonstrate that vaccinia-related kinase 1 (VRK1) in resting cells plays an important role in the formation of ionizing radiation-induced foci that assemble on the 53BP1 scaffold protein during the DNA damage response. The kinase VRK1 is activated by DNA double strand breaks induced by ionizing radiation (IR) and specifically phosphorylates 53BP1 in serum-starved cells. VRK1 knockdown resulted in the defective formation of 53BP1 foci in response to IR both in number and size. This observed effect on 53BP1 foci is p53- and ataxia-telangiectasia mutated (ATM)-independent and can be rescued with VRK1 mutants resistant to siRNA. VRK1 knockdown also prevented the activating phosphorylation of ATM, CHK2, and DNA-dependent protein kinase in response to IR. VRK1 activation in response to DNA damage is a novel and early step in the signaling of mammalian DNA damage responses.     

Effect of low doses of low-intensity ionizing radiation on the DNA structure and functions

Chronic effects of low doses of low intensity ionizing radiation (IR) on biological objects have now become of great social significance. This has given a considerable impetus to research into biological effects and mechanisms of such exposures both in Russia and abroad. This paper provides an overview of physicochemical and molecular bases of the IR influence at small doses and the ways of cell protection from the radiation damage, as well as the analysis of characteristic features and differences in the effects of radiation at small and high doses. We consider the DNA radiation damage both in cell cultures and in vivo, as well as processes and results of their repair. Particular attention is paid to the changes in the basic paradigms of radiation biological effects of small doses.     

Enhancement of radiosensitivity in H1299 cancer cells by actin-associated protein cofilin

Cofilin is an actin-associated protein that belongs to the actin depolymerization factor/cofilin family and is important for regulation of actin dynamics. Cofilin can import actin monomers into the nucleus under certain stress conditions, however the biological effects of nuclear transport are unclear. In this study, we found that over-expression of cofilin led to increased radiation sensitivity in human non-small lung cancer H1299 cells. Cell survival as determined by colony forming assay showed that cells over-expressing cofilin were more sensitive to ionizing radiation (IR) than normal cells. To determine whether the DNA repair capacity was altered in cofilin over-expressing cells, comet assays were performed on irradiated cells. Repair of DNA damage caused by ionizing radiation was detected in cofilin over-expressing cells after 24 h of recovery. Consistent with this observation, the key components for repair of DNA double-strand breaks, including Rad51, Rad52, and Ku70/Ku80, were down-regulated in cofilin over-expressing cells after IR exposure. These findings suggest that cofilin can influence radiosensitivity by altering DNA repair capacity.     

Cellular response to DNA damage. Link between p53 and DNA-PK

Cells which lack DNA-activated protein kinase (DNA-PK) are very susceptible to ionizing radiation and display an inability to repair double strand DNA breaks. DNA-PK is a member of a protein kinase family that includes ATR and ATM which have strong homology in their carboxy-terminal kinase domain with PL-3 kinase. ATM has been proposed to act upstream of p53 in cellular response to ionizing radiation. DNA-PK may similarly interact with p53 in cellular growth control and in mediation of the response to ionizing radiation.     

Differential DNA methylation alterations in radiation-sensitive and -resistant cells

An understanding of cellular processes that determine the response to ionizing radiation (IR) exposure is essential to improve radiotherapy and to assess risks to human health after accidental radiation exposure. Exposure to IR induces a multitude of biological effects. Recent studies have indicated the involvement of epigenetic events in regulating the responses of irradiated cells. DNA methylation, where the cytosine bases in CpG dimers are converted to 5-methyl cytosine, is an epigenetic event that has been shown to regulate a variety of biological processes. We investigated the DNA methylation changes in irradiated TK6 and WTK1 human cells that differ in sensitivity to IR. The global DNA methylation alterations as measured by an enzyme-linked immunosorbent assay-based assay showed hypomethylation in both type of cells. Using an arbitrarily primed polymerase chain reaction (AP-PCR) approach, we observed time-dependent dynamic changes in the regional genomic DNA methylation patterns in both cell lines. The AP-PCR DNA methylation profiles were different between TK6 and WTK1 cells, indicating the involvement of differential genomic DNA responses to radiation treatment. The analysis of the components of the DNA methylation machinery showed the modulation of maintenance and de novo methyltransferases in irradiated cells. DNMT1 mRNA levels were increased in TK6 cells after irradiation but were repressed in WTK1 cells. DNMT3A and DNMT3B were induced in both cells after radiation treatment. TET1, involved in the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), was induced in both cells. This study demonstrates that irradiated cells acquire epigenetic changes in the DNA methylation patterns, and the associated cellular machinery are involved in the response to radiation exposure. This study also shows that DNA methylation patterns change at different genomic regions and are dependent on time after irradiation and the genetic background of the cell.     

Endogenous hSNM1B/Apollo interacts with TRF2 and stimulates ATM in response to ionizing radiation

Human SNM1B/Apollo is involved in the cellular response to DNA-damage, however, its precise role is unknown. Recent reports have implicated hSNM1B in the protection of telomeres. We have found hSNM1B to interact with TRF2, a protein which functions in telomere protection and in an early response to ionizing radiation. Here we show that endogenous hSNM1B forms foci which colocalize at telomeres with TRF1 and TRF2. However, we observed that additional hSNM1B foci could be induced upon exposure to ionizing radiation (IR). In live-cell-imaging experiments, hSNM1B localized to photo-induced double-strand breaks (DSBs) within 10s post-induction. Further supporting a role for hSNM1B in the early stages of the cellular response to DSBs, we observed that autophosphorylation of ATM, as well as the phosphorylation of ATM target proteins in response to IR, was attenuated in cells depleted of hSNM1B. These observations suggest an important role for hSNM1B in the response to IR damage, a role that may be, in part, upstream of the central player in maintenance of genome integrity, ATM.