[Keyword] low-dose radiation low-dose radiation ultra-sensitive molecular mechanisms

    Low dose radiation hypersensitivity (hyper  radiosensitivity, HRS) cells are sensitive to very low doses of radiation (about 0.02 ~ 0.50Gy) decreased sensitivity of its post-dose region (0.50 ~ 1.00Gy) phenomenon [1]. When the phenomenon first from corn below 0.5Gy dose irradiation study in 1963 confirmed that it received the low dose (<0.5Gy) showed corn gamma ray irradiation, there is hypersensitive to pollen induced mutation and lethal sensitive than the high dose. Subsequently, in many in vitro and in vivo experiments have confirmed the existence of the phenomenon known more than 40 kinds of cell lines exist in the HRS [2]. The HRS study has profound practical significance, but its mechanism of occurrence is not very clear.

    So far, the HRS mechanisms, mainly from apoptosis, cell cycle regulation and DNA breaks repair of double-stranded (DNA DSBs) three; Some genes, proteins, such as p53, bcl  2 c  myc, ataxia telangiectasia mutated (ataxia the  telangiectasia mutated, ATM) kinase and DNA-dependent protein kinase (DNA  dependent protein kinase, DNA  PK) is considered likely to be related to the occurrence of HRS. In this paper, from the above three aspects to elaborate the possible mechanism.

    1 cells with HRS

    There are many studies have shown that cells in the low-dose radiation, apoptosis is the main form of death [3-]; gene or protein, such as p53, bcl  2, c-myc, p21ras (ras gene family encoding a protein product), etc., the expression levels appeared significant change in [3,6  9]. In these genes, the most studied p53 and bcl  2 gene.

    Mothersill et al [3] observed in 1995 isolated human urothelial cells in different doses of irradiation, the proliferation of cell survival, radiation dose <1 Gy, there HRS, apoptosis; and, p53, bcl  of c  myc, p21ras and epidermal growth factor receptor (EGFR) expression was significantly increased. Subsequently, Enns et al [4] studied the human lung adenocarcinoma A549 glioma T98G and breast cancer MCF7 cell lines A549 and T98G cell lines exist HRS different response characteristics of the γ-ray irradiation (0 ~ 2Gy) , MCF7 cell line does not exist HRS. Then, the cells were observed by detecting the three cell lines in the activation of caspase  3 and Annexin V radiation apoptosis in a 0 ~ 1Gy dose region, A549 and T98G cells apoptosis, apoptosis peaks the 0.1Gy and 0.2Gy Department, MCF7 cells no apoptosis occurred significantly (P <0.005). In addition, after the intervention of the A549 and T98G cell line with the p53 inhibitor pifithrin cells HRS disappear. Observation similar to this model, the p53-deficient cell lines did not (skin fibroblast cell lines 2800T and colorectal cancer cells Department HCT116), the same conditions of exposure, HRS and apoptosis was discovered. The resulting, p53 is a key factor in the regulation of apoptosis and HRS.

    In the low-dose radiation, p53 as a tumor suppressor gene on the growth of tumor cells proliferation regulation, but also by enhancing the low dose of tumor cells to radiation hypersensitivity to enhance the killing of tumor cells. However, it has been reported HRS p53-independent point of view [5,6]. Krueger et al [5] studied the T98G line U373 with MR4 and 3.7 cells HRS phenomenon found the HRS cell apoptosis; T98G and U373 cells expressing mutant p53 MR4 and 3.7 cells expressing wild-type p53 T98G and MR4 the cells exhibit HRS, U373 and 3.7 cells does not appear to HRS, which come to the conclusion the HRS and p53 independent. Tip HRS phenomenon may exist with other p53-independent apoptotic pathways.

    expression of bcl  2 gene is another gene of low dose radiation research more. Many studies have found that low-dose radiation, bcl  2 gene and protein expression decreased. Dey et al [6] of the head and neck squamous cell carcinoma cell line SCC  61 and SQ  20B (both of which exist HRS) given 0.5Gy / time, total dose irradiation 2Gy and bcl  protein immunoblot (Western blot) 2 protein detection, found its expression compared with radiation decreased. Kroger et al [7] lymphoma obtained results of bcl  2 gene and protein expression decreased. However, for low-dose irradiation bcl  2 expression levels of different reports. , Sur [8] close radiation therapy of esophageal cancer, observed before and after treatment bcl  2 expression levels did not change. Szostak et al [9] for prostate cancer brachytherapy treatment of patients of bcl  2 detection, found that after treatment, regardless of group success or failure of the group, bcl  2 were higher than before treatment, but the failure group is higher than the success group ( 30.5% and 13.1%, P <0.05).

    We can draw from the top, in the low-dose radiation, apoptosis is the main mechanism of tumor cell death. HRS occurrence of apoptosis-related genes, such as p53, bcl  2 and other genes related to the HRS p53 and bcl  2 gene expression levels of relationship advice is still controversial, but also requires further study.

    2 cell cycle regulation and HRS

    As we all know, DNA damage repair and cell cycle regulation are two important factors affecting cell radiosensitivity. Known ionizing radiation mammals will result in the cells in G1, S and G2 phase delay, these delay is mediated by a series of cell cycle checkpoint. S phase checkpoint is to prevent the replication of damaged DNA, the G1 / S and G2 / M phase checkpoint is damaged DNA have enough time to repair before entering S or M phase. The entire process including the induction recognition of damaged DNA, and thus initiate a series of signal transduction, and the final effector phase (cell death, DNA fragmentation chain repair, gene mutation) [10]. Which has an important role in the identification of the injury, it has a direct impact on the next series has a decisive role in the process of cell death, repair, and gene mutations. Currently, ATM is the most important factor of the identification of DNA breakage duplexes its activation will cause the detection of cell cycle arrest and DNA repair.

    2.1 ATM Overview

    ATM is the genes that cause ataxia-telangiectasia (ataxia  telangiectasia, AT), AT is an autosomal recessive genetic disease, the clinical features of cerebellar degradation, immunodeficiency, chromosomal instability, cell cycle disorders , is extremely sensitive to radiation, and class-ray cytotoxic drugs. AT patients with this particular genetic radiosensitivity, ATM to become one of the important object of the radiation biologists study radiation sensitivity mechanism.

    ATM gene is located on human chromosome 11q22  23, and its encoded protein ATM contains 3056 amino acids relative molecular mass of 350 KD macromolecular nucleoprotein, belongs to the phosphatidylinositol  3  kinase (PI3K) family members.

    Currently considered to play an important role in cell signaling pathways, ATM, which is mainly involved in the regulation of the cell cycle and DNA damage recognition and repair. The passage: DNA damage ATM kinase is activated, mainly 1981 serine phosphorylated (ATM1981Ser  P), followed by phosphorylation of its downstream of the corresponding protein, the cells in the cell cycle checkpoint arrest splitting, is mainly the G1 / S phase and the G2 / M phase arrest, so the damage could be repaired, the repair failed cells into apoptosis process.

    2.2 ATM-mediated cell cycle arrest signal transduction pathways

    G1 / S phase arrest signal transduction pathways as follows: damage receptors ATM feelings identify DNA fragmentation chain, its 1981 serine autophosphorylation and activation, and subsequently cause elevated levels of p53 protein expression and phosphorylation, the phosphorylation of p53 promote cell cycle dependent phosphorylation inhibitor (CDKI) p21 expression, which is inhibited with cyclin of CyclinE, the expression of CyclinA combination of CDK activity, and thus prevent the cells from G1 to S phase [11].

    G2 / M phase arrest the signal transduction pathways as follows: damage receptors activated ATM experience identifying DNA fragmentation chain, followed by ATM through the activation of cell cycle checkpoint kinase 2 (CHK2) CDC25 phosphorylation and inactivation of the latter inactivation phosphate groups to go after not the cyclin-dependent kinase cdc2 (ie CDK1, the regulation of the cell cycle plays a regulation of G2 to M phase) Thr14/Tyrl5 phosphorylation, making cdc2 can not be activated, and cell cycle arrest in G2 / M at the junction [12].

    2.3 ATM HRS

    HRS simple to understand: the cells in the low-dose radiation, repair, reduce, increase in deaths; the subsequent dose region radiation, repair increased to reduce deaths. So different effects, so researchers invest in damage recognition and signal transduction stage the center of attention. ATM is the most important factor in identifying DNA breakage duplexes.

    Currently, there is no direct relationship between the ATM and the HRS. However, in the low-dose radiation on cell ATM1981Ser  P level changes following studies: Collis [13] of four different tumor cell lines of the same dose of radiation of different dose rates, found that a high dose rate (HDR ) compared to low dose rate (LDR) radiation under strong cell killing effect lower level ATM1981Ser  P; LDR before chloroquine pre-activation of ATM kinase or inhibition of ATM protein expression using siRNA technology HDR before, LDR and HDR considerable cell-killing effect. The study also found that, the LDR radiation cell survival fraction below the predictive value of the dose  response curve, suggesting that only when the number of double-stranded DNA breaks when you reach a certain threshold, to effectively start the monitoring mechanism of the cell. Bakkenist, etc. [14] in ATM activation mechanism study of human fibroblasts to different doses (0 ~ 9 Gy) of radiation detected by Western blot ATM1981Ser  P levels found ATM1981Ser  P 0.1Gy when appear to 0.4Gy peaked, while a 1 ~ 9Gy interval level without further elevated. Step ATM kinase activation has a threshold radiation dose, and the activation threshold dose is similar to the dose region of HRS. Thus speculated that the ATM may be a HRS generating system [15].

    The role of ATM in the HRS may be: low-dose radiation, DNA damage fails to activate receptors in the ATM DNA damage signal can not be transmitted the ATM mediated cell repair pathway can not be activated, resulting in cell killing enhanced role, showing HRS; With the increase of the radiation dose, the DNA damage receptors ATM is activated, the DNA damage signal transduction, the cells were blocked, repair, cell killing worse, showing the radiation resistance.

    2.4 cell cycle with HRS

    Studies have shown that, compared with the G1 and S phase, the G2 phase cells HRS more significant. When dose <1Gy cells HRS, HRS occurs mainly in the G2 phase, G1 phase and S phase cells did not appear Marple [10] 0.1 ~ 10Gy the dose of 60Coγ ray irradiation of the Chinese hamster V79 cell lines. Hamster V79 cells, human glioma T98G cell line also exists in the HRS, use of flow cytometry T98G cells in the low-dose radiation (<0.3Gy) intracellular phosphorylation level of histone H3 (histone H3 phosphorylation usually of representatives cells into M phase), found damaged cells in G2 / M phase arrest did not occur, and direct access to the M phase. Therefore come to the HRS may be damaged G2 phase cells directly into the M phase, the case has not been repaired.

    2.5 cell cycle checkpoint HRS

    Checkpoint signaling pathway plays the role of the signal sensor. G1 / S phase arrest is highly dependent on the p53 protein has been found that more than 50% of the malignant cells mutated p53 gene, and that the expression of mutant p53 gene, only through is not dependent on p53 of the G2 / M checkpoint to detect damaged DNA. G2 / M checkpoint including General checkpoint and specific checkpoint two, the former is not dependent on ATM kinase, and radiation dose dependent checkpoint, its role is to accept radiation G1 or S phase cells prevents the G2 phase; In contrast, the latter depends on the ATM kinase and ATM activation similar, and is activated only when the radiation dose of 0.4 Gy only, and does not depend on the dose of radiation in the 1 ~ 10Gy interval activity unchanged, its role is to prevent the G2 phase of the The loss of cells in the case of not repaired into the M phase [16]. Is enable researchers are interested in, specificity checkpoint activation radiation dose threshold value and generating a radiation dose threshold resist Similar. Therefore, it is speculated that the mechanism of HRS may also contain the G2 / M specific checkpoint [17].

    The 3 DNA DSBs repair with HRS

    Currently, there are a large number of research data indicate that DNA is the most important target intracellular radiation lethal effect, in a variety of DNA damage, DNA double-strand breaks and cell death most closely related one. Therefore, cell radiation sensitivity study people first take into account to determine the radiation sensitivity of the cells is the formation and repair of DNA DSBs than cells. Become more consistent view of ionizing radiation-induced DNA double-strand breaks, if linking to each other through some kind of repair mechanisms, cell survival, resist showed Radiation; lack of repair of radiation-induced DNA DSBs repair or error cells a key factor of death.

    DNA DSBs can be repaired through a variety of channels, there are two main ways in eukaryotic cells, that is, non-homologous end joining repair (NHEJ) and homologous recombination repair (HR) [18]. Mediated NHEJ by DNA-dependent protein kinase (DNA  PK) completed or completed by the coordinating role of the protein complex MRE11  RAD50  NBS1. HR in the rad52 gene group and homologues consisting of complexes under the action is complete, it requires the sequence homology between the sister chromosome chain, and DNA replication or in the S  G2 of role. Known in mammals are mainly NHEJ pathway, DNA  PK (by Ku- 70 Ku- 80 and DNA  PKcs three subunits, respectively, by the XRCC5, XRCC6 and XRCC7 genes encoding molecular mass were 70ku 86ku 350ku) play an important role in the repair of DNA DSBs. XR  V15B cells due to lack of Ku-DNA  PK complex  80 subunit, rather than demonstrated radiation resistance responses. The mutant cell lines xrs5 also lack Ku  80 subunit are extremely sensitive to radiation performance, but the cells by transfection Ku  80 gene radiation reduced susceptibility. Human malignant glioma cell M059J not express DNA  PKcs protein, the lack of DNA DSBs repair capacity, and therefore very sensitive to radiation; human malignant glioma cells M059K, expressing normal DNA  PK DNA DSBs repair, its radiation sensitivity is far less than the former. Of detecting DNA <0.5Gy low dose radiation  PK activity, found the DNA  PK activity was significantly decreased in 6 HRS’s tumor cell lines, while in the four cell lines without the HRS DNA  PK did not reduce the expression [19]. Prompted HRS is closely related with the expression of the DNA  PK cells.

    4 Conclusion and Outlook

    , HRS has recently attracted more and more attention, it is traditional radiobiology a useful complement and development of important guiding significance for clinical radiotherapy. Some 2Gy performance of conventional fractionated dose of radiation resistance cells extremely sensitive to low doses of radiation they exhibit, and thus it is possible to take advantage of the low-dose hypersensitivity phenomenon to design a new way of dividing, and thus improve the therapeutic gain ratio, but a prerequisite for radio-resistant tumors than in normal tissue hypersensitivity stronger. If we can identify the molecular decided to low-dose hypersensitivity, we will be able to detect cells in the presence of these molecules selected individuals and cells that are sensitive to low doses of radiation. HRS study only at a beginning, a large number of findings from research on human HRS cells in vitro and animal studies, is still relatively small, in many ways, such as human tissues existence of HRS and its characteristics sensitivity differences between tumor and normal tissue to low doses of radiation, and low-dose radiation and carcinogenic and other, yet to be further research.