Radiation-induced Cell Lethality Research Articles

Targeting NPM1 in irradiated cells inhibits NPM1 binding to RAD51, RAD51 foci formation and radiosensitizes NSCLC.

The ability of chemo-radiation therapy to control locally advanced stage III non-small cell lung cancer (NSCLC) is poor. While addition of consolidation immunotherapy has improved outcomes in subsets of patients there is still an urgent need for new therapeutic targets. Emerging research indicates that nucleophosmin1 (NPM1) is over-expressed in NSCLC, promotes tumor growth and that over-expression correlates with a lower survival probability. NPM1 is critical for APE1 base excision activity and for RAD51-mediated repair of DNA double strand breaks (DSBs). YTR107 is a small molecule radiation sensitizer that has been shown to bind to NPM1, suppressing pentamer formation. Here we show that in irradiated cells YTR107 inhibits SUMOylated NPM1 from associating with RAD51, RAD51 foci formation and repair of DSBs. YTR107 acts synergistically with the PARP1/2 inhibitor ABT 888 to increase replication stress and radiation-induced cell lethality. YTR107 was found to radiosensitize tumor initiating cells. Congruent with this knowledge, adding YTR107 to a fractionated irradiation regimen diminished NSCLC xenograft growth and increased overall survival. These data support the hypothesis that YTR107 represents a therapeutic target for control of NSCLC.

Modulating effects of melatonin, vitamin C and saffron alone or in combination on radiation induced cell lethality; an in vitro study with Hela cells

Introduction: Radiation countermeasures are defined as use of an agent to minimize the deleterious effects of radiation therapy by administering the compound after the radiation exposure has occurred, regardless of toxicity. In spite of extensive study in this field, yet there is no suitable radioprotector introduced. In this study two naturally occurring antioxidants were studied on radiation induced cell lethality. Materials and Methods: Various doses of melatonin, saffron and vitamin C were examined for their antioxidant capacity by using DPPH test. Hela cells were treated with optimum doses of melatonin, saffron and vitamin C; 2 hours prior to gamma irradiation alone or in combination. Treated cells were irradiated with various doses of gamma rays (2, 4, 8 Gy), then cells were plated in 96 well plates for MTT assay. Cells were fixed at 48 and 72 hours after plating and reading was done ELISA reader. Results: Results showed cell viability reduced slightly after 48 h irradiation. Pretreatment of cells with melatonin and vitamin C increased cell lethality considerably for dopses of 2 and 4 Gy, but surprisingly presence of melatonine and vitamin C led to an increase in cell viability when combined with 8 Gy gamma rays. Combination of these agents with saffron showed similar effects. Similar results were observed for 72 h sampling time. However, protective effect of these agents was more pronounced when using 8 Gy radiation dose. Conclusion: Results indicate that despite the antioxidant potential of melatonin, saffron and vitamin C, when combined with low and moderate doses of gamma rays (2 and 4 Gy) led to increased cytotoxic effects of ionizing radiation. However, at high dose of gamma rays (8 Gy) radioprotective effect was seen. The dual action of these agents with low and high doses of ionizing radiation is not known.

68 - Correlation of Particle Traversals with Clonogenic Survival Using Cell-Fluorescent Ion Track Hybrid Detector

Development of novel approaches linking the physical characteristics of particles with biological responses are of high relevance for the field of particle therapy. In radiobiology, the clonogenic survival of cells is considered the gold standard assay for the assessment of cellular sensitivity to ionizing radiation. Toward further development of next generation biodosimeters in particle therapy, cell-fluorescent ion track hybrid detector (Cell-FIT-HD) was recently engineered by our group and successfully employed to study physical particle track information in correlation with irradiation-induced DNA damage in cell nuclei. In this work, we investigated the feasibility of Cell-FIT-HD as a tool to study the effects of clinical beams on cellular clonogenic survival. Tumor cells were grown on the fluorescent nuclear track detector as cell culture, mimicking the standard procedures for clonogenic assay. Cell-FIT-HD was used to detect the spatial distribution of particle tracks within colony-initiating cells. The physical data were associated with radiation-induced foci as surrogates for DNA double-strand breaks, the hallmark of radiation-induced cell lethality. Long-term cell fate was monitored to determine the ability of cells to form colonies. We report the first successful detection of particle traversal within colony-initiating cells at subcellular resolution using Cell-FIT-HD.

Involvement of DNA-PK and ATM in Radiation- and Heat-induced DNA Damage Recognition and Apoptotic Cell Death

Exposure to ionizing radiation and hyperthermia results in important biological consequences, e.g. cell death, chromosomal aberrations, mutations, and DNA strand breaks. There is good evidence that the nucleus, specifically cellular DNA, is the principal target for radiation-induced cell lethality. DNA double-strand breaks (DSBs) are considered to be the most serious type of DNA damage induced by ionizing radiation. On the other hand, verifiable mechanisms which can lead to heat-induced cell death are damage to the plasma membrane and/or inactivation of heat-labile proteins caused by protein denaturation and subsequent aggregation. Recently, several reports have suggested that DSBs can be induced after hyperthermia because heat-induced phosphorylated histone H2AX (γ-H2AX) foci formation can be observed in several mammalian cell lines. In mammalian cells, DSBs are repaired primarily through two distinct and complementary mechanisms: non-homologous end joining (NHEJ), and homologous recombination (HR) or homology-directed repair (HDR). DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) are key players in the initiation of DSB repair and phosphorylate and/or activate many substrates, including themselves. These phosphorylated substrates have important roles in the functioning of cell cycle checkpoints and in cell death, as well as in DSB repair. Apoptotic cell death is a crucial cell suicide mechanism during development and in the defense of homeostasis. If DSBs are unrepaired or misrepaired, apoptosis is a very important system which can protect an organism against carcinogenesis. This paper reviews recently obtained results and current topics concerning the role of DNA-PK and ATM in heat- or radiation-induced apoptotic cell death.

大腸菌及び関連細菌の放射線感受性に及ぼすフリーラジカルと培養基の影響

Effects of gamma-irradiation on some strains of Escherichia coli, Salmonella enteritidis and Staphylococcus aureuswere investigated in the presence of N2, N2O and O2and with the hydroxyl radical (OH) scavengers glycerol, polyethylene glycol and formate. Injured cell membrane of bacteria was detected using with MacConkey agar forE.. coliandS. enteritidisand 7% NaC1 Triptic soy agar forSt. aureusinstead of Tryptic soy agar for recovery medium. From this study, addition of glycerol significantly reduced the sensitivity in all of strains, and cell membrane was not injured significantly except in radiation sensitive strainE.. coliA4-1. When superoxide radicals (O2-) were generated during irradiation in the presence of formate, injured cell membrane increased significantly in all of strains. However, molecular oxygen (O2) and OH radicals also had some effects on the damage of cell membrane. These results suggest that most radiation induced cell lethality was responsible to the cooperative effects of intracellular OH radicals and O2on DNA with lessor effect of damage on cell membrane by O2-radicals, O2and OH radicals. On the radiation sensitive strain of E.. coli, cell lethality occurred significantly by the injury of cell membrane compared with other strains.

The effect of 2-2 difluorodeoxycytidine (dFdC, gemcitabine) on radiation-induced cell lethality in two human head and neck squamous carcinoma cell lines differing in intrinsic radiosensitivity

Purpose: The present study investigated in vitro radio-enhancement by gemcitabine (dFdC) in two head and neck squamous cell carcinomas with different intrinsic cellular radiosensitivity. Materials and methods: Radiosensitive (SCC61, SF2 0.16) and radioresistant (SQD9, SF2 0.49) human head and neck squamous cell carcinomas were used. Confluent cells were incubated with dFdC and irradiated in drug-free medium with a single dose of 250kV X-rays (0-12Gy). Cell survival curves were corrected for the toxicity of the drug alone. Results: In both cell lines, radio-enhancement was observed with 5 mu m dFdC incubated for 3h prior to irradiation. Dose modification factors (DMF) at a surviving fraction level of 0.5 reached 1.3 (95% CI 1.1-1.6) and 1.5 (95% CI 1.4-1.5) for SQD9 and SCC61 cells, respectively. Radio-enhancement was associated with a modest increase in the alpha term of the linear-quadratic model. In SQD9 cells, radio-enhancement increased with dFdC incubation time. At 24h, DMF reached a value of 1.5 (95% CI 0.9-3.2). In SCC61 cells at 24h, DMF reached a value of 1.1 (95%CI 0.9-1.2). In both celllines, radio-enhancement increased with dFdC concentration up to 5-10 mu m from which values it levelled o ff up to 100 mu m. Conclusions: The data indicated that dFdC induced a modest radio-enhancement in both cell lines. For a short incubation time, dFdC did not radio-enhance preferentially the more radioresistant cells, whereas the opposite was observed for a longer time. In both cell lines, radio-enhancement was saturated above a dFdC concentration of 5-10 mu m.

Levels of p21(WAF1/CIP1) do not affect radiation-induced cell death in human breast epithelial cells

Loss of the wild-type p53 activity and/or overexpression of the proto-oncogene bcl-2 are frequently detected in breast cancer and suggested to be related to chemotherapy and radiation therapy resistance. To identify the downstream signaling molecules for anti-proliferative and apoptotic activities of p53 and to investigate the interaction of bcl-2 with p53 in human breast epithelial cells, we have used the MCF10A cell line. We previously showed that overexpression of bcl-2 downregulates expression of p21(WAF1/CIP1) (a cyclin dependent kinase inhibitor which mediates p53 dependent G(1) arrest) and suppresses DNA damage-induced apoptosis in MCF10A cells. In the present study, we constitutively overexpressed p21(WAF1/CIP1) in bcl-2 overexpressing MCF10A cells to determine whether downregulation of p21(WAF1/CIP1) is necessary for the anti apoptotic activity of bcl-2, and to investigate the roles of p21(WAF1/CIP1) in p53-mediated cell death upon irradiation. Overexpression of p21(WAF1/CIP1) resulted in growth inhibition, but had no effect on bcl-2 inhibition of apoptosis following irradiation. Also, overexpression of p21(WAF1/CIP1) did not affect the dose-dependent radiation-induced cell lethality as determined by a clonogenic survival assay. These results suggest that bcl-2 downregulation of p21(WAF1/CIP1) is independent of the anti-apoptotic activity of bcl-2, and that p21(WAF/CIP1) is not involved in the p53-mediated cell death pathway.

Radiation-Induced Cell Lethality of Salmonella typhimurium ATCC 14028: Cooperative Effect of Hydroxyl Radical and Oxygen

The lethality of gamma-radiation doses of 0.2 to 1.0 kGy for Salmonella typhimurium ATCC 14028 was measured in the presence of air, N2 and N2O and with the hydroxyl radical scavengers formate and polyethylene glycol (PEG), M(r) 8,000. Saturation of cell suspensions with either N2O or N2/N2O (1:1, v/v) gas was expected to double the number of hydroxyl radicals (OH.) and to produce an equivalent increase in lethality, but this did not occur. Adding 10% (v/v) O2 to either N2 or N2O gas produced approximately the same gamma-irradiation lethality for S. typhimurium as did air. Addition of hydroxyl radical scavengers, 40 mM formate and 1.5% (w/v) PEG, significantly reduced the lethality of gamma radiation for S. typhimurium in the presence of air but not in the presence of N2 or N2O gases. Membrane-permeable formate provided slightly better protection than nonpermeable PEG. Cells of S. typhimurium grown under anaerobic conditions were more sensitive to radiation, and were less protected by hydroxyl radical scavengers, especially formate, than when cells grown under aerobic conditions were irradiated in the presence of oxygen. Hydroxyl radical scavengers provided no further protection during irradiation in the absence of oxygen. These results indicated that the increased radiation sensitivity of cells grown under anaerobic conditions may be related to superoxide radicals which could increase intercellular damage during irradiation in the presence of oxygen. However, endogenous superoxide dismutase and catalase activities did not protect cells from the radiation-induced lethality of S. typhimurium. Cytoplasmic extracts protected bacterial DNA in vitro in either the presence or absence of oxygen, and no radiation-induced lipid peroxidation of the cellular components was identified by measuring the levels of 2-thiobarbituric acid. These results suggest that most radiation-induced cell lethality was related to the cooperative effects of extracellular OH. and O2 on the cell surface as the radiation dose increased.

Inhibition of Topoisomerase IIα Activity in CHO K1 Cells by 2-[(Aminopropyl)Amino]Ethanethiol (WR-1065)

The aminothiol 2-[(aminopropyl)amino]ethanethiol (WR-1065) is the active thiol of the clinically studied radioprotective agent S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721). WR-1065 is an effective radiation protector when it is administered 30 min prior to exposure of Chinese hamster ovary K1 cells to radiation (i.e., a dose modification factor of 1.4) at a concentration of 4 mM. Under these exposure conditions, topoisomerase (Topo) I and II alpha activities and associated protein contents were measured in cells of the K1 cell line using the DNA relaxation assay, the P4 unknotting assay and immunoblotting, respectively. WR-1065 was ineffective in modifying Topo I activity, but it did reduce Topo II alpha activity by an average of 50%. The magnitude of Topo II alpha protein content, however, was not affected by these exposure conditions. The effects on the cell cycle were monitored by the method of flow cytometry. Exposure of cells to 4 mM WR-1065 for up to 6 h resulted in a build-up of cells in the G2/M-phase compartment. However, under these conditions and in contrast to Topo II inhibitors used in chemotherapy, WR-1065 is an effective radioprotective agent capable of protecting against both radiation-induced cell lethality and mutagenesis. One of several mechanisms of action attributed to aminothiol compounds such as WR-1065 has been their ability to affect endogenous enzymatic reactions involved in DNA synthesis and repair and progression of cells through the phases of the cell cycle. These results are consistent with such a proposed mechanism and demonstrate in particular a modifying effect by WR-1065 on Topo II, which is involved in DNA synthesis.

A comparison of the chemical repair rates of free radical precursors of DNA damage and cell killing in Chinese hamster V79 cells.

One of the important temporal stages of radiation action in cellular systems is the chemical phase, where oxygen fixation reactions compete with chemical repair reactions involving reducing agents such as GSH. Using the gas explosion technique it is possible to follow the kinetics of these fast (greater than 1 ms) reactions in intact cells. We have compared the chemical repair kinetics of the oxygen-dependent free radical precursors leading to DNA single-strand and double-strand breaks, measured using filter elution techniques, with those leading to cell killing in V79 cells. The chemical repair rates for DNA dsb (670s-1 at pH 7.2 and 380s-1 at pH 9.6) and cell killing (530s-1) were similar. This is in agreement with the important role of DNA dsb in radiation induced cell lethality. The rate for DNA ssb precursors was significantly slower (210s-1). The difference in rate between DNA ssb and dsb precursors may be explained on the basis of a dsb free radical precursor consisting of a paired radical, one radical on each strand. The instantaneous probability of one or other of these radicals being chemically repaired and not proceeding to form a dsb will be twice that of ssb radical precursor. This agrees well with the concept of locally multiply damaged sites (LMDS) produced from clusters of ionizations in DNA (Ward 1985).

A potential pitfall in the use of electroporation: cellular radiosensitization by pulsed high-voltage electric fields.

Chinese hamster ovary cells have been exposed to high-voltage electric fields causing electroporation (EP) and the interaction between EP and radiation-induced cell lethality investigated. There was a voltage-dependent decrease in plating efficiency, assessed immediately following EP, and cell viability, assessed at 24 h. A linear decrease was seen for both. These decreases were accompanied by a voltage-dependent increase in cell volume, assessed immediately following EP. A good correlation between increases in cell volume and decreases in plating efficiency was seen (r = -0.91). The application of electric fields immediately prior to, or following, irradiation led to a radiosensitization of the cells. This radiosensitization still occurred when a 6 h interval was left between radiation and EP but was lost when cells were irradiated 24 h prior to EP. When cells were irradiated following EP, the radiosensitization was lost with a 1 h interval between the two treatments. These results suggest that, when studying the combined cellular effects of EP of macromolecules and radiation, care should be taken that sufficient time has elapsed between the two modalities to prevent the radiosensitization of cells.

The Irradiation of V79 Mammalian Cells by Protons with Energies below 2 MeV. Part II. Measurement of Oxygen Enhancement Ratios and DNA Damage

The effectiveness of low-energy (below 2 MeV) protons at inducing DNA damage in the form of single- and double-strand breaks has been determined. Protons with mean energies of 1.90 MeV, 1.15 MeV and 0.76 MeV corresponding to track average LETs of 17 keV/microns, 24 keV/microns and 32 keV/microns, respectively, have been used and compared with 250 kVp X-rays and 3.8 MeV 238Pu alpha-particles. Although there was variation in the RBE for DNA ssb induction with LET, the RBEs for dsb induction at all three proton energies and for 3.8 MeV alpha-particles were all around 1.0. This suggests that, if DNA dsb are important in radiation-induced cell lethality, the probability of an induced dsb leading to a lethal event increases with increasing LET of radiation. Oxygen enhancement ratios were measured for both cell survival and DNA dsb induction, and in both cases a decrease in OER with increasing LET was observed.

The radioprotector WR1065 reduces radiation-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in V79 cells.

N-(2-mercaptoethyl)-1,3-diaminopropane (WR1065) protects against radiation-induced cell killing and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 Chinese hamster lung fibroblast cells. At a concentration of 4 mM, WR1065 was found to be effective in protecting against radiation-induced cell lethality only if present during irradiation, e.g., a dose modification factor (DMF) of 1.9. No protective effect was observed if the protector was added within 5 min after irradiation or 3 h later, e.g., DMFs of 1.0 and 1.1, respectively. The effect of WR1065 on radiation-induced mutation, expressed as resistance to the cytotoxic purine analogue 6-thioguanine (HGPRT), was also investigated. In contrast to the treatment-schedule dependence for protection by WR1065 against cell killing, this agent was effective in reducing radiation-induced mutations regardless of when it was administered. Following a dose of 10 Gy of 60Co gamma-rays, the mutation frequencies observed per 10(6) survivors were 77 +/- 8, 27 +/- 6, 42 +/- 7, and 42 +/- 7 for radiation only, and WR1065 present during, immediately after, or 3 h after irradiation. These data suggest that although a segment of radiation-induced damage leading to reproductive death cannot be modulated through the postirradiation action of WR1065, processes leading to the fixation of gross genetic damage and mutation induction in surviving cells can be effectively altered and interfered with leading to a marked reduction in mutation frequency.

Modification of radiation and dihydroxyanthraquinone-induced cell lethality by cysteamine and N-ethylmaleimide

The effect of alteration of sulfhydryl levels on the cell lethality induced by ionizing radiation and dihydroxyanthraquinone (DHAQ was investigated in cultured Chinese hamster V79 cells. DHAQ produces a potentiation of radiation-induced cell lethality, both by increasing the slope and decreasing the shoulder of the survival curve. It has been suggested that DHAQ functions through the production of free radicals which then produce DNA strand breaks and crosslinks, resulting in cytotoxicity. If this mode of action predominates, then one would expect to be able to change the degree of cell kill by modifying conditions such that free radical processes were altered. This was accomplished by the addition of N-ethylmaleimide (NEM) or Cysteamine (CYS) to the culture medium during treatment with DHAQ. It was observed that the combination of DHAQ and NEM did not produce more cytotoxicity than would be expected from an additive interaction. Likewise, CYS did not reduce the cytotoxicity induced by DHAQ. When cells were treated with DHAQ and radiation plus either NEM or CYS, the resultant survival was consistent with an additive interaction between the potentiation of DHAQ for radiation-induced cell kill and the extra effect of NEM or CYS. These results indicate that alterations of sulfhydryl levels do not produce changes in the cytotoxicity induced by DHAQ, nor in the enhancement by DHAQ of radiation-induced lethality. More investigation is required before definite conclusions can be reached as to the mechanisms of action by which DHAQ, alone or in combination with ionizing radiation, induces mammalian cell lethality.

Effect of Dihydroxyanthraquinone (DHAQ) and Radiation on the Survival of Cultured Chinese Hamster Ovary Cells

Dihydroxyanthraquinone (DHAQ) is currently being tested as a cancer chemotherapeutic agent because of its structural similarity to Adriamycin (ADR) and other DNA-intercalating antibiotics. The interaction of DHAQ and ionizing radiation on the induction of cell lethality was investigated in Chinese hamster ovary cells in culture. In asynchronous populations of cells, DHAQ produced a slight enhancement of radiation-induced cell lethality as evidenced by changes in both shoulder and slope of the radiation dose-survival curves. However, DHAQ had no effect on either the extent or time course of recovery from sublethal radiation damage. In synchronous populations of cells treated at various times before or after selection in mitosis, the combination of DHAQ and radiation produced greater cell killing than that predicted based on simple additivity of effect, with a decided enhancement for cells treated during S phase. These results indicate that DHAQ is similar to other DNA-intercalating antibiotics in regard to the interaction with ionizing radiation to produce cell lethality.

Cyclic Responses of Cultured 9L Cells to Radiation

Several responses of cultured 9L rat brain tumor cells to ionizing radiation were investigated as a function of their position in the cell cycle. The mitotic selection procedure for cell cycle analysis was utilized to study the blockade of progression of cells through G/sub 2/ and the resultant division delay. The transition point for this blockade (i.e., the last point in the cell cycle at which cells are blocked, and after which they are refractory to delay) was located approximately at the G/sub 2//M boundary 35 min prior to selection. The duration of division delay was a linear function of the x-ray dose, 33 min of delay/Gy. The survival of cells following exposure to a constant dose of x rays at varous times after incubation of mitotically selected cells demonstrated a definite resistance during the G/sub 1/ phase and slight resistance during the S phase, relative to the level of survival at the G/sub 1//S boundary. As shown with other cell lines there was maximum radiosensitivity during G/sub 2/ and mitosis. Thus the response of 9L rat brain tumor cells in culture to ionizing radiation is similar to that of other cultured mammalian cell lines in regard to induction andmore » duration of division delay, and the sensitivity of M and G/sub 2/ cells to radiation-induced cell lethality. However, the radioresistance exhibited over the remainder of the cell cycle results in a relatively flat age response for radiation-induced lethality. This may in part explain the radiation resistance observed for the 9L gliosarcoma in situ.« less