低剂量电离辐射诱导的EL-4细胞PARP-1蛋白表达及其意义的研究

目的:研究低剂量电离辐射诱导EL-4细胞适应性反应中PARP-1基因表达,为探讨低剂量电离辐射诱导适应性反应的DNA损伤修复机制提供实验依据.方法:将EL-4细胞分设空白对照组、较大剂量照射组(D2),其照射剂量分别为1、2和3 Gy以及诱导适应性反应照射组(D1+D2),其照射剂量分别为75 mGy+1 Gy、75 mGy+2 Gy和75 mGy+3 Gy,应用流式细胞仪和RT-PCR方法分别检测PARP-1基因蛋白及其转录水平表达.结果:单纯照射组PARP-1蛋白水平较对照组显著升高(P<0.01),预先给予75 mGy照射诱导的适应性反应组其PARP-1蛋白水平较大剂量照射组显著升高,P<0.01.PARP-1 mRNA水平表现出相同的变化.结论:PARP-1蛋白在诱导适应性反应照射组处于高表达,说明其在低剂量电离辐射诱导适应性反应中可能起重要作用.     

Roles of BRCA1 and BRCA2 in homologous recombination,DNA replication fidelity and the cellular response to ionizing radiation

Inheritance of one defective copy of either of the two breast cancer susceptibility genes, BRCA1 and BRCA2, predisposes individuals to breast and ovarian cancers. Current progress in determining the function of these genes suggests that they participate in a common pathway to facilitate orderly homologous recombination and thereby maintain genomic integrity. As a consequence of this defect in homologous recombination, tumors that arise in BRCA carriers are likely to be more sensitive to ionizing radiation. This review summarizes recent investigations about the nature of the defect in DNA repair, and highlights the unanswered questions about the tumor suppressor paradox of BRCA genes. The unsolved mystery is the other genetic changes that must occur to turn a BRCA-deficient cell from a nonviable cell into a tumor cell capable of endless growth.     

What role do glutathione S-transferases play in the cellular response to ionizing radiation?

The glutathione S-transferases (GST's) are cytosolic dimeric proteins that are composed of three family members, alpha, pi, and mu, and a fourth microsomal member. These four family members are primarily involved in cellular detoxification of xenobiotics and hydroperoxides. Recently, a strong correlation has been found between the overexpression of GST's and resistance to chemotherapeutic drugs. In comparison to chemotherapy, little is known about the role of GST's in the cellular response to ionizing radiation. To determine which GST's may be involved in this response, we have identified Chinese hamster ovary cell lines that possess different levels of alpha and pi GST isozyme activity. The survival of these cell lines to ionizing radiation was similar to that of wild-type Chinese hamster ovary-KI cells from which they were derived. Although differences in GST levels did not affect ionizing radiation sensitivity per se, we found that ionizing radiation decreased the amount of cytosolic pi GST without affecting alpha GST levels. Taken together, these data suggest that GST's are involved in the cellular response against oxidative stress generated by ionizing radiation.     

Induction of interleukin-1beta and interleukin-6 mRNA by low doses of ionizing radiation in macrophages

We have previously reported the antimetastatic effects and augmentation of immune responses, which would be a mechanism of the antimetastatic effects, of 0.1 to 0.2 Gy total body irradiation. To elucidate the cellular mechanisms of the augmentation of immune response, we investigated the effects of low-dose irradiation on gene expression of interleukin-1beta (IL-1beta) and IL-6 using mouse peritoneal macrophages in vitro. Absolute mRNA quantification was carried out using competitive polymerase chain reaction. Gene expression of IL-1beta and IL-6 was increased 1 to 2 hr after 2.0 Gy irradiation and then decreased to below the basal expression level 4 hr after irradiation. Irradiation with 0.1 Gy increased IL-6 expression 2 hr after irradiation, but it did not affect IL-1beta expression. Downregulation of IL-1beta and IL-6 observed 4 hr after 2.0 Gy irradiation was not observed with 0.1 Gy irradiation. The protein kinase C (PKC) inhibitor H7 and the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin inhibited induction of IL-1beta and IL-6 expression, which suggests that radiation-induced IL-1beta and IL-6 expression is achieved by PKC- and PI3-kinase-mediated signaling.     

DNA-dependent protein kinase: a major protein involved in the cellular response to ionizing radiation

DNA-dependent protein kinase (DNA-PK) is a DNA-activated nuclear serine/threonine protein kinase. DNA-PK consists of a regulatory sub-unit, the heterodimeric Ku protein (composed of a 70- and a 86-kDa subunit) which binds DNA ends and targets the catalytic sub-unit, DNA-PKcs to DNA strand breaks. DNA-PK plays a major role in the repair of double-strand breaks induced in DNA after exposure to ionizing radiation as shown by the extreme radiosensitivity of cells with mutations in Ku86, Ku70 or DNA-PKcs genes. Cells deficient in DNA-PK activity also exhibit hypersensitivity to genotoxic drugs such as cisplatin and nitrogen mustards. In the first part of this review, the current knowledge on the biochemical characteristics of DNA-PK, its mechanism of action in DNA repair and the phenotype of DNA-PK deficient cells is summarized. These results suggest that DNA-PK might play a role in the acquisition of a resistant phenotype of human tumors to radiotherapy, chemotherapy using genotoxic drugs or to both treatments. In the second part of this review, the studies devoted to inhibition of DNA-PK in order to enhance cancer therapy by DNA-damaging agents are presented.     

Distinct functional domains of Nbs1 modulate the timing and magnitude of ATM activation after low doses of ionizing radiation

The ATM kinase is a tumour suppressor and a key activator of genome integrity checkpoints in mammalian cells exposed to ionizing radiation (IR) and other insults that elicit DNA double-strand breaks (DSBs). In response to IR, autophosphorylation on serine 1981 causes dissociation of ATM dimers and initiates cellular ATM kinase activity. Here, we show that the kinetics and magnitude of ATM Ser1981 phosphorylation after exposure of human fibroblasts to low doses (2 Gy) of IR are altered in cells deficient in Nbs1, a substrate of ATM and a component of the MRN (Mre11-Rad50-Nbs1) complex involved in processing/repair of DSBs and ATM-dependent cell cycle checkpoints. Timely phosphorylation of both ATM Ser1981 and the ATM substrate Smc1 after IR were rescued via retrovirally mediated reconstitution of Nbs1-deficient cells by wild-type Nbs1 or mutants of Nbs1 defective in the FHA domain or nonphosphorylatable by ATM, but not by Nbs1 lacking the Mre11-interaction domain. Our data indicate that apart from its role downstream of ATM in the DNA damage checkpoint network, the MRN complex serves also as a modulator/amplifier of ATM activity. Although not absolutely required for ATM activation, the MRN nuclease complex may help reach the threshold activity of ATM necessary for optimal genome maintenance and prevention of cancer.     

Transient suppression of nuclear Cdc2 activity in response to ionizing radiation

Suppression of Cdc2 activity is essential for DNA damage-induced G2 arrest. In the present study, we elucidated regulatory mechanism of Cdc2 activity during radiation-induced transient G2 arrest. Exposure of the cells to gamma-radiation (4 Gy) led to a transient increase of cells in G2 at 12 h rather than M phase and then the cells resumed cell cycle progression from the G2 arrest. However, the levels of cyclin B1 and Cdc2 activity were increased in the whole cell extracts at 12 h. Despite cyclin B1 induction and increased level of Cdc2 activity after irradiation the activities in the nuclear fractions were transiently decreased at 12 h and returned to control levels by 24-48 h, demonstrating transient inhibition of nuclear translocation of cyclin B1 in response to radiation. Moreover, inhibitory phosphorylation of the Cdc2 on Tyr15 and the Cdc25C on Ser216 were increased concomitant with transient G2 arrest. The level of phosphorylated Wee1 and its activity were also markedly increased at 12 h after irradiation. In addition, radiation caused nuclear accumulation of p21(CIP1/WAF1) at 12 h, resulting in increased-binding of p21(CIP1/WAF1) to Cdc2. Nuclear p21(CIP1/WAF1) protein level and its binding to Cdc2 gradually returned to control level when the cells resumed cell cycle progression. However, total protein level of p21(CIP1/WAF1) continued to increase until 48 h after irradiation. Collectively, these results indicate that the suppression of nuclear import of cyclin B1, the induction of Wee1 kinase activity, and the transient nuclear accumulation of p21(CIP1/WAF1) may play important roles in the transient cell cycle delay in response to ionizing radiation.     

CK2 inhibits apoptosis and changes its cellular localization following ionizing radiation

In this study, we show that CK2 (casein kinase II, CKII) participates in apoptotic responses following ionizing radiation (IR). Using HeLa human cervical carcinoma cells, we find that transfection of small interfering RNA against the CK2 alpha and/or alpha' catalytic subunits results in enhanced apoptosis following IR damage as measured by flow cytometry techniques, compared with a control small interfering RNA. Within 2 to 6 hours of IR, CK2 alpha partially localizes to perinuclear structures, whereas a marked nuclear localization of alpha' occurs. Treatment with a pan-caspase inhibitor or transfection of ARC (apoptosis repressor with caspase recruitment domain) suppresses the apoptotic response to IR in the CK2-reduced cells, indicating involvement of caspases. Additionally, we find that CK2 alpha and/or alpha' reduction affects cell cycle progression independent of IR damage in this human cell line. However, the G2-M checkpoint following IR is not affected in CK2 alpha- and/or alpha'-reduced cells. Thus, our data suggest that CK2 participates in inhibition of apoptosis and negatively regulates caspase activity following IR damage.     

Effects of the multidrug transporter P-glycoprotein on cellular responses to ionizing radiation

Ionizing radiation induces apoptosis, mitotic catastrophe, and senescence-like terminal proliferation arrest in tumor cells. We investigated the effect of the MDR1 P-glycoprotein (Pgp), recently shown to inhibit caspase-mediated apoptosis, on cellular responses to radiation. Pgp strongly inhibited radiation-induced apoptosis in a HeLa-derived cell line with inducible MDR1 expression and in NIH 3T3 cells transduced with a MDR1-expressing retroviral vector. The inhibition of apoptosis by Pgp was associated, however, with increases in radiation-induced mitotic catastrophe and senescence and produced only a marginal change in the survival of irradiated cells. Pgp had no effect on radiation responses in apoptosis-resistant HT1080 cells. These results indicate that Pgp inhibits radiation-induced apoptosis, but this effect of Pgp provides no substantial increase in radiation resistance of the tested cell lines because apoptosis-resistant cells die from mitotic catastrophe or undergo senescence-like terminal proliferation arrest.     

Effects of p53 mutations on cellular sensitivity to ionizing radiation

Mutations in the p53 tumor suppressor gene have been found in more than 50% of human tumors including those in breast, colon, lung, and oral cavity. However, the significance of p53 mutation in radiation sensitivity and its underlying mechanisms still remains unclear. In this study, we have measured the effects of p53 mutation on cell cycle delay, apoptosis, and radiation sensitivity using mouse cells transfected with different forms of p53 mutations. Wild-type p53 and p53-Null mouse embryo fibroblast cells were used as positive and negative controls, respectively. Exponentially growing cells were irradiated with 0- to 9-Gy gamma rays and then assayed for cell survival, p53 expression, cell cycle checkpoint, and apoptosis. Cell survivals determined by clonogenic assay show that p53 mutant cells are generally more sensitive to ionizing radiation than cells with wild-type p53. Western blot analysis indicates that exposure to 6-Gy gamma rays increases the p53 expression levels by two- to threefold in wild-type p53 cells. However, the p53 level remains unchanged in cells with mutant p53 during the same postirradiation period. Irradiation with 6-Gy gamma rays produces G2/M arrest in all cell lines, indicating that p53 is probably not involved in the G2/M checkpoint. However, all mutant cells fail to show any significant G1/S arrest after irradiation, suggesting that G1/S arrest may be implicated in radiation sensitivity. Finally, there is very little apoptosis (<3% by Tat-mediated dUTP nick-end labeling [TUNNEL] and morphologic assays) detected in wild-type and p53 mutant cell lines after 6-Gy gamma rays. Our results suggest that mutant forms of p53 represent a phenotype that affects the radiation sensitivity and is not dependent on the apoptotic pathway.     

AMPK regulates metabolism and survival in response to ionizing radiation

Background and purpose

AMPK is a metabolic sensor and an upstream inhibitor of mTOR activity. AMPK is phosphorylated by ionizing radiation (IR) in an ATM dependent manner, but the cellular consequences of this phosphorylation event have remained unclear. The objective of this study was to assess whether AMPK plays a functional role in regulating cellular responses to IR.

Methods

The importance of AMPK expression for radiation responses was investigated using both MEFs (mouse embryo fibroblasts) double knockout for AMPK α1/α2 subunits and human colorectal carcinoma cells (HCT 116) with AMPK α1/α2 shRNA mediated knockdown.

Results

We demonstrate here that IR results in phosphorylation of both AMPK and its substrate, ACC. IR moderately stimulated mTOR activity, and this was substantially exacerbated in the absence of AMPK. AMPK was required for IR induced expression of the mTOR inhibitor REDD1, indicating that AMPK restrains mTOR activity through multiple mechanisms. Likewise, cellular metabolism was deregulated following irradiation in the absence of AMPK, as evidenced by a substantial increase in oxygen consumption rates and lactate production. AMPK deficient cells showed impairment of the G1/S cell cycle checkpoint, and were unable to support long-term proliferation during starvation following radiation. Lastly, we show that AMPK proficiency is important for clonogenic survival after radiation during starvation.

Conclusions

These data reveal novel functional roles for AMPK in regulating mTOR signaling, cell cycle, survival and metabolic responses to IR.     

Redox cycling by motexafin gadolinium enhances cellular response to ionizing radiation by forming reactive oxygen species.

PURPOSE: To examine the mechanism of radiation enhancement by motexafin gadolinium (Gd-Tex) in vitro. METHODS AND MATERIALS: Oxidation of ascorbate and NADPH by Gd-Tex was evaluated in a neutral buffer. Growth inhibition of human uterine cancer cell line MES-SA was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye. Clonogenic assays were used to measure radiation response in MES-SA, A549 human lung carcinoma, E89, a CHO cell line variant deficient in glucose-6-phosphate dehydrogenase activity, and murine lymphoma cell lines LYAR and LYAS. RESULTS: Gd-Tex catalyzed the oxidation of NADPH and ascorbate under aerobic conditions, forming hydrogen peroxide. Decreased viability was observed in MES-SA cells incubated with Gd-Tex in media containing NADPH or ascorbate. Gd-Tex and ascorbate increased fluorescence in dichlorofluorescin acetate-treated cultures. Synergistic effects on the aerobic radiation response in MES-SA and A549 were seen using Gd-Tex in combination with L-buthionine-(S,R)-sulfoximine (BSO). Incubation with Gd-Tex in the presence of ascorbate increased the aerobic radiation response of E89 and the apoptosis-sensitive B-cell line (LYAS). CONCLUSIONS: Gd-Tex sensitizes cells to ionizing radiation by increasing oxidative stress as a consequence of futile redox cycling. Optimization of the concentration of ascorbate (or other reducing species) may be required when evaluating Gd-Tex activity in vitro.     

Transcriptional response to ionizing radiation in human radiation sensitive cell lines.

BACKGROUND AND PURPOSE: Radiation is a common treatment of cancer, but some patients show severe side effects when exposed to small doses of radiation. The aim of this study was to explore the underlying cause of radiation sensitivity in a group of radiation sensitive patients. MATERIALS AND METHODS: Lymphoblastoid cell lines from 5 normal individuals, 4 Ataxia Telangiectasia (AT), and 12 non-AT radiation sensitive (RS) patients were irradiated. RNA was isolated before and after radiation and hybridized to 15k cDNA microarrays and gene expression was recorded. RESULTS AND CONCLUSION: The RS cell lines showed an expression phenotype different from both the AT and normal cell lines. Six of the RS cell lines had a distinct expression profile before radiation. This implies that the RS patients are a heterogeneous group, but that six of the patients may have a common cause of radiation sensitivity.     

Disruption of ATM in p53-null cells causes multiple functional abnormalities in cellular response to ionizing radiation

ATM is a member of the large phosphatidylinositol-3 kinase family and plays an important role in cellular response to DNA damage. To further define the physiological roles of ATM at the cellular level, we created an isogenic set of stable cell lines differing only in their ATM status from the chicken B cell line DT40 by targeted integration. These stable DT40 cell lines, as most of transformed chicken cell lines, do not express p53. However, ATM-/- DT40 cells displayed retarded cellular proliferation, defective G2/M checkpoint control and radio-resistant DNA synthesis. Furthermore, ATM-/- DT40 cells were sensitive to ionizing radiation and showed highly elevated frequencies of both spontaneous and radiation-induced chromosomal aberrations. In addition, a slight but significant reduction in targeted integration frequency was observed in ATM-/- DT40 cells. These results suggest that ATM has multiple p53-independent functions in cell cycle checkpoint control and in maintenance of chromosomal DNA. These ATM deficient DT40 clones therefore provide a useful model system for analysing p53-independent ATM functions.     

Distinct response to ionizing radiation of human prostate cell lines

The purpose of this study was to determine in vitro the relationship between ionizing radiation (IR) treatment, reactive oxygen species (ROS) production, lipid peroxidation, glutathione (GSH) levels, and DNA damage of the human benign prostate hyperplasia BPH-1 cell line, and two prostate cancer cell lines, LNCaP, which is androgen-sensitive, and DU-145, which is androgen non-responsive. The cells were analysed after exposure to 1.0 or 2.0 Gy of X-ray radiations. The response to IR treatment was evaluated by examining: ROS production by quantitative analysis with fluorescent probe 5 and 6-carboxy-2'7'-dichlorodihydrofluorescein diacetate bis acetomethyl ester (DCFH-DA), GSH levels by 2,2'-dinitro-5,5'-dithio-benzoic acid (DTNB), and lipoperoxidation by thiobarbituric acid reactive substances (TBARS) analysis. To study IR-induced DNA damage, Single Cell Gel Electrophoresis or comet assay was performed. DU-145 cells were characterized by higher DNA damage, more evident extent of lipid peroxidation, and slighter levels of ROS and GSH compared to BPH-1 or LNCaP. Human benign BPH-1 and cancer LNCaP and DU-145 cell lines are not equal regarding their capability of IR resistance in terms of ROS production, antioxidant potential, IR-induced lipid peroxidation and DNA damage.