Terms Of Cell Killing Research Articles

The G-quadruplex-stabilizing ligand RHPS4 enhances sensitivity of U251MG glioblastoma cells to clinical carbon ion beams.

The pentacyclic acridine RHPS4 is a highly potent and specific G-quadruplex (G4) ligand, which binds and stabilizes telomeric G4 leading to the block of the replication forks at telomeres and consequently to telomere dysfunctionalization. In turn, the cell recognizes unprotected telomeres as DNA double-strand breaks with consequent activation of DNA repair response at telomeres, cellular growth impairment, and death. Data from the literature showed the capability of this compound to sensitize U251MG glioblastoma radioresistant cell line to X-rays sparsely ionizing radiations. In the present paper, it was investigated whether RHPS4 is also able to increase the effect of clinical carbon ion beams (cells irradiated in the middle of a spread-out Bragg peak, in the energy range of 246-312MeV·μm-1 and a dose-averaged linear energy transfer of 46keV·μm-1 ). Interestingly, also for charged particles whose damage inflicted to DNA is more complex than that of sparsely ionizing radiations and results in higher Relative Biological Effectiveness (RBE), RHPS4 significantly potentiated the radiation effect in terms of cell killing, delayed rejoining of DNA double-strand breaks (γ-H2AX and 53BBP1 immunofluorescence staining), chromosome aberrations (pan-centromeric/telomeric FISH and multicolor FISH), and G2 /M-phase accumulation in GBM cells. Overall, the results provide the first evidence that the combined administration of the G4-ligand RHPS4 with charged particles interfere with cellular processes involved in cell survival leading to radiosensitization of highly radioresistant tumor cells.

Heavier Ions with a Different Linear Energy Transfer Spectrum Kill More Cells Due to Similar Interference with the Ku-Dependent DNA Repair Pathway

Ionizing radiation kills cells mainly due to the generation of DNA double-strand breaks (DSBs). High-linear energy transfer (LET) ionizing radiation induces more cell death and generates a higher relative biological effect (RBE) than low-LET ionizing radiation (such as X or γ ray). Although it is known that interference with the Ku-dependent nonhomologous ending-joining (NHEJ) pathway appears to be the major cause of iron-ion- and carbon-ion-induced cell death, it remains unclear whether other ions with a similar or different LET and higher RBE in terms of cell killing are controlled in the same way. In this study, we compared the clonogenic survival frequency of Ku80+/+ (NHEJ-proficient) and Ku80-/- (NHEJ-deficient) cells after exposure to iron (175 keV/μm), silicon (75 keV/μm), oxygen (25 keV/μm) and X ray (low-LET). The results showed that Ku80-/- cells had the same RBE value of 1 for cell killing for all types of ionizing radiation, whereas Ku80+/+ cells had different RBE values for cell killing that depended on the specific type of ionizing radiation. The results indicate that the Ku-dependent NHEJ is the major repair pathway that heavier ions interfere with, resulting in higher RBE for cell killing. These results provide useful information for followup studies that will focus on improving high-LET protection or heavier ion radiotherapy in the near future.

Lung Fibrosis and Lung Cancer Incidence in Beagle Dogs that Inhaled 238PuO2 or 239PuO2

Determination of radiation protection guidelines for persons working with plutonium has been complicated by limited human data on the biological behavior and subsequent health effects from internally deposited plutonium. One solution has been the use of animal models to predict likely health effects in humans. To compare the relationships between plutonium inhalation and lung fibrosis and lung cancer, data from life-span studies of beagle dogs given a single exposure to either plutonium-238 dioxide (238PuO2) or plutonium-239 dioxide(239PuO2) were analyzed. Estimates of the cumulative hazard of lung fibrosis and lung cancer after exposure to either were generated. The hazard of lung fibrosis was not consistent with a linear no-threshold model, although the magnitude of the threshold differed by radionuclide. In dogs given 239PuO2,the best model of lung fibrosis incorporated a linear dose response function; a linear-quadratic dose-response function fit the data better in dogs given 238PuO2. At any given cumulative dose, the lung fibrosis hazard was greater for dogs given 238PuO2. In dogs given 238PuO2, with or without covariates, aquadratic dose-response function for lung cancer hazard fit better than a linear no-threshold model. In dogs given 239PuO2, models of lung cancer with the dose-response function as the sole predictor variable were consistent with a linear no-threshold model; however, a quadratic dose-response function with a cell-killing term fit better. These findings have implications for radiation protection because, while lung cancer hazard was dependent on cumulative dose, regardless of isotope, the lung fibrosis hazard depended on both cumulative dose and isotope.

Protective Effects of Melatonin Against Low- and High-LET Irradiation

To investigate the protective effects of melatonin against high-LET ionizing radiation, V79 Chinese hamster cells were irradiated with 100 keV/microm carbon beam. Parallel experiments were performed with 200 kV X-rays. To avoid the impact from extra solvents, melatonin was dissolved directly in culture medium. Cells were cultured in melatonin medium for 1 hr before irradiation. Cell inactivation was measured with conventional colony forming assay, medium containing 6-thioguanine was used for the selection of mutants at hprt locus, and the cell cycle was monitored by flow cytometry. Both carbon beam and X-rays induced cell inactivation, hprt gene mutation and cell cycle G2 block dose-dependently. But carbon beam showed stronger effects as indicated by all three endpoints and the relative biological effectiveness (RBE) was 3.5 for cell killing (at 10% survival level) and 2.9 for mutation induction (at 5 x 10(-5) mutants/cell level). Melatonin showed protective effects against ionizing radiation in a dose-dependent manner. In terms of cell killing, melatonin only increased the survival level of those samples exposed to 8Gy or larger of X-rays or 6 Gy or larger of carbon beam. In the induction of hprt mutation and G2 block, melatonin reduced such effects induced by carbon beam but not by X-rays. The results suggest that melatonin reduces the direct interaction of particles with cells rather than an indirect interaction. Further studies are required to disclose the underlying mechanisms.

Cytotoxicity of PM 2.5 and PM 2.5–10 ambient air pollutants assessed by the MTT and the Comet assays

Ambient air particulate matters are classified into two distinct modes in size distribution, namely the coarse and fine particles. Correlation between high particulate concentration and adverse effects on human populations has long been recognized, however, the toxicology of these adverse effects has not been clarified. In the current report, the cytotoxic effects of the solvent-extractable organic compounds (SEOC) from fine particles smaller than 2.5 μm (PM 2.5) and from coarse particles between 2.5–10 μm (PM 2.5–10) were studied. Nine 24 h consecutive monthly samples were tested to determine the correlation between cytotoxicity and total SEOC in two size fractions of particulate air pollution. Cytotoxicity of SEOC was measured by two micro-scale mammalian cells-based bioassays: the MTT cell proliferation assay, and the Comet assay for the detection of DNA damage. A well-defined mammalian cell line — Rat 6 rodent fibroblast was employed in the study. The SEOC extracts of air particulate matters were sub divided into two equal parts. One part was dissolved in DMSO, the other in KOH/hexane and then conjugated with bovine serum albumin to produce a lipid-soluble fraction for testing. The DMSO fraction would contain mainly the polycyclic aromatic hydrocarbons (PAH), alkanes and alkanols, while the lipid-soluble fraction would be enriched with fatty acids. The results from MTT assay showed that cytotoxicity of the PM 2.5 was much more severe than the PM 2.5–10, suggesting that toxic SEOC were confined to the fine particles. By and large, the DMSO solubles were much more toxic than the lipid solubles. The degree of cytotoxicity of the DMSO soluble samples is positively correlated to the amount of particulates present in the ambient air. For the PM 2.5, the winter samples were significantly more toxic than the summer samples in terms of cell killing, which seemed to be a direct reflection of the total loading of organic matter in the samples. Results from Comet assays showed that SEOC samples of PM 2.5 derived from winter months induced DNA damage at dosages resulting in no obvious cell killing in the MTT assay. Thus, long-term exposure to non-killing dosage of air pollutants may lead to the accumulation of DNA lesions, which may be one of the mechanisms responsible for the chronic adverse health effects of particulate air pollution.

Biologically-based risk estimation for radiation-induced chronic myeloid leukemia.

Radiation cancer risks are typically determined by the use of simple statistical descriptions of epidemiological data. It is important in risk assessment in general, however, to attempt to incorporate as much biological information into the risk models as possible. We illustrate this by presenting a biologically-based linear-quadratic-exponential (LQE) incidence rate model for radiation-induced chronic myeloid leukemia (CML). The model consists of a linear-quadratic dose-response for the induction of BCR-ABL, a waiting time distribution between BCR-ABL formation and detection of CML, and an exponential cell-killing term that multiplies both the background and induced incidence rates. Using data exclusive of the A-bomb survivor cohort, Bayesian priors are defined for each of the nine parameters in this LQE model. The priors are based on chromosomal translocations in lymphocytes, hematopoietic stem cell survival experiments, CML waiting times in women irradiated for benign disease, the background CML incidence rate in the U.S. population, and genomic DNA target sizes of BCR and ABL. Fixing three of the LQE model parameters to the means of their priors, maximum likelihood estimates of the remaining six parameters were obtained using A-bomb survivor incidence data for Hiroshima males. The likelihood estimates and the corresponding six prior distributions, both approximated as multivariate normal, were then used to form Bayesian posteriors for the six parameters not fixed. With these posteriors the LQE model yields Qgamma*=0.0042 Gy(-1) where Qgamma* is the upper 95% confidence bound of the lifetime CML risk per person-gray in the limit of low doses of gamma-rays. This value is slightly less than Qgamma*=0.0049 Gy(-1) obtained from likelihood estimates of the LQE parameters, and substantially less than Qgamma*=0.0158 Gy(-1) obtained for a simple statistical model linear in dose for kermas less than 4 Gy.

Lack of differential G2 chromosomal radiosensitivity between non-tumorigenic and tumorigenic human hybrid cells (HeLa x skin fibroblasts).

Previous studies have shown, that for log phase cultures, tumorigenic segregants of HeLa x skin fibroblast human hybrid cells are slightly more radiosensitive in terms of cell killing than their nontumorigenic parents (Redpath et al. 1985, Colman et al. 1988). Other studies have shown that these same tumorigenic segregants exhibit a markedly enhanced G2 chromosomal radiosensitivity (Sanford et al. 1986) thus offering a possible explanation for the cell killing data. The present study set out to examine the G2-phase radiosensitivity of these cells in terms of cell killing with the expectation that an enhanced sensitivity in the tumorigenic cells would be seen. No enhanced sensitivity was observed. The G2 chromosomal radiosensitivity was then examined and no differential was seen between the non-tumorigenic and tumorigenic cells. This lack of confirmation of previously reported studies may be due to some technical differences in the experimental protocols.

Reparability of DNA double-strand breaks and radiation sensitivity in five mammalian cell lines.

We examined the relationship between gamma-ray-induced DNA double-strand breaks (dsb) and cell lethality in five mammalian cell lines differing in radiosensitivity; HA-1, HMV-I, L5178Y, M10 and LX830. These cells were derived from Chinese hamster, human melanoma, and mouse lymphoma (parent and its radiosensitive mutants), respectively. HA-1 cells were the most radioresistant and LX830 cells were the most radiosensitive among these five lines. Although the induction of dsb by gamma-rays for HA-1 was significantly different from other curves (p less than 0.05 for HMV-I and p less than 0.01 for L5178Y and M10), those for the other four lines were similar to one another. In addition, the most radioresistant cell line, HA-1, showed the highest dsb induction among five cell lines. Therefore, there is no correlation between radiosensitivity and the induction of dsb in these five lines. On the other hand, residual dsb after repair incubation (non-reparable dsb) do differ from each other. When the relative number of non-reparable dsb was plotted against the radiation dose, the dose-response curves for all the cell lines were concave, and the slopes of curves for M10 and LX830 were steeper than those for other cell lines. These curves are a mirror-image of the survival curves. The results suggest that there is a correlation between the radio-resistance in terms of cell killing and the capacity of cells to repair dsb.

Inhibitors of Repair of Potentially Lethal Damage and DNA Polymerases Also Influence the Recovery of Potentially Neoplastic Transforming Damage in C3H10T-1/2 Cells

The effects of aphidicolin and beta Ara A on radiation sensitivity were evaluated in terms of cell killing, recovery, and neoplastic transformation in the C3H10T-1/2 cell system. When cells were held in plateau phase, recovery of potentially lethal damage (PLD) and potentially transforming damage (PTD) occurred. The addition of beta Ara A resulted in reduced PLD recovery for both the survival and neoplastic transformation end points. The addition of aphidicolin did not affect recovery of PLD or PTD. These data show that the inhibition of polymerase alpha by aphidicolin does not affect recovery of damage leading to cell death or neoplastic transformation. However, the inhibition of both polymerase alpha and beta by beta Ara A resulted in inhibition of recovery of damage leading to both cell death and neoplastic transformation. These data indicated that polymerase beta may be involved in both PLD and PTD recovery.

Fission-neutron-induced Expression of a Tumour-associated Antigen in Human Cell Hybrids (HeLa × Skin Fibroblast): Evidence for Increased Expression at Low Dose Rate

The induction of a tumour-associated antigen in a human cell hybrid line (HeLa x skin fibroblast) following exposure to fission neutrons of average energy 0.85 MeV (Janus reactor, Argonne National Laboratory) at two dose rates, 0.086 and 10.3 cGy/min, has been examined. The dose-response data obtained indicate the lower dose rate to be 2.9-fold more effective than the higher in inducing expression of the tumour-associated antigen, while there was no significant dose-rate effect in terms of cell killing. These results are qualitatively in agreement with previous observations using neutrons from the Janus reactor for the neoplastic transformation of C3H10T1/2 cells and Syrian hamster embryo cells.

Mutation Induction by Very Low Dose Rate γ Rays in Cultured Mouse Leukemia Cells L5178Y

Induction of cell killing and mutation to 6-thioguanine resistance was studied in growing mouse leukemia cells in culture following gamma rays at dose rates of 30 Gy/h, 20 cGy/h, and 6.3 mGy/h, i.e., acute, low dose rate, and very low dose rate irradiation. A marked increase was observed in the cell survival with decreasing dose rate; no reduction in the surviving fraction was detected after irradiation at 6.3 mGy/h until a total dose of 4 Gy. Similarly, the induced mutation frequency decreased after low dose rate irradiation compared to acute irradiation. However, the frequency after irradiation at 6.3 mGy/h was unexpectedly high and remained at a level which was intermediate between acute and low dose rate irradiation. No appreciable changes were observed in the responses to acute gamma rays (in terms of cell killing and mutation induction) in the cells which had experienced very low dose rate irradiation.

Modeling for Comparison of Leukemia Incidence Risk between Nuclear and Coal Power Industries

The leukemia incidence risk for a single coal plant, a single nuclear plant, and a single nuclear accident is used to compute the total industry leukemia incidence risk. In the absence of a nuclear power plant accident, the leukemia incidence risk is normally lower for a nuclear industry than for a coal industry of equivalent size. The nuclear industry risk with accidents was compared to the coal industry risk for six proposed dose response curves. Simplifying assumptions about the negligible effect of the cell-killing term and the linear nature of the linear quadratic curve allowed derivation of risk models for the assumption of both linear and quadratic dose response. These derived models, representing leukemia incidence risk bounds, are used to estimate the total industry risk comparison. Evaluation of an accident's impact on the leukemia incidence risk comparison is done with the risk bounds and compared to the risk evaluations calculated during all six dose response curves. The overlapping plot of the number of nuclear accidents required for equivalent industry environment risks versus the accident fraction allows the conservative function to be defined.

Lung cancer risk at low doses of alpha particles.

A survey of inhabitant exposures arising from the inhalation of 222Rn and 220Rn progeny, and lung cancer mortality has been carried out in two adjacent areas in Guangdong Province, People's Republic of China, designated as the "high background" and the "control" area. Annual exposure rates are 0.38 working level months (WLM) per year in the high background, and 0.16 WLM/yr in the control area. In 14 yr of continuous study, from 1970 to 1983, age-adjusted mortality rates were found to be 2.7 per 10(5) living persons of all ages in the high background area, and 2.9 per 10(5) living persons in the control area. From this data, we conclude that we are unable to determine excess lung cancers over the normal fluctuations below a cumulative exposure of 15 WLM. This conclusion is supported by lung cancer mortality data from Austrian and Finnish high-background areas. A theoretical analysis of epidemiological data on human lung cancer incidence from inhaled 222Rn and 220Rn progeny, which takes into account cell killing as competitive with malignant transformation, leads to the evaluation of a risk factor which is either a linear-exponential or a quadratic-exponential function of the alpha-particle dose. Animal lung cancer data and theoretical considerations can be supplied to support either hypothesis. Thus we conclude that at our current stage of knowledge both the linear-exponential and the quadratic-exponential extrapolation to low doses seem to be equally acceptable for Rn-induced lung cancer risk, possibly suggesting a linear-quadratic transformation function with an exponential cell-killing term, or the influence of risk-modifying factors such as repair or proliferation stimuli.

Modification of Cell Survival and Transformation by Exposure to Anisotonic Solutions during Irradiation

C3H 10T 1/2 cells were exposed to a wide range of anisotonic NaCl solutions and were irradiated during the last few minutes of the salt treatment. Radiosensitization in terms of cell killing and transformation was observed for hypotonic NaCl treatment. Hypertonic treatment with 0.5 mol/liter NaCl also caused radiosensitization for cell killing and transformation, while extreme hypertonic treatment (1.5 mol/liter) resulted in radioprotection for both end points. Radiosensitization for both end points declined as the hypotonic treatment (0.05 mol/liter) was prolonged. To a lesser extent, a decline in radioprotection by 1.5 mol/liter treatment occurred as exposure time increased.

Estimates of lifetime lung cancer risks resulting from Rn progeny exposure.

Data on five mining populations exposed to Rn progeny have been used to estimate the lifetime risk of lung cancer resulting from occupational and environmental exposure under current standards. Slopes of dose-response relations for lung cancer show a tendency to decrease with increasing dose. Our best estimate of curvilinearity is given by raising dose to the power 0.92 +/- 0.07, but the improvement in fit beyond simple linearity is not significant. On the other hand, the addition of a cell-killing term significantly improves the fit of the linear model. In any event, linear extrapolation is unlikely to underestimate the excess risk at low doses by more than a factor of 1.5. However, these inferences about curvilinearity are highly subject to error from the choice of reference populations, dosimetry, and latency. Under the linear-cell-killing model, our best estimate of excess relative risk is 2.28 +/- 0.35 per 100 working level month (WLM) (a doubling dose of 44 WLM). Attributable risks in these five studies range from 3.4-17.8 per 10(6) person-yr WLM-1. Risks from Rn progeny appear to interact with age and smoking in a form intermediate between additive and multiplicative. The "relative risk" model is therefore preferable for projecting lifetime risks, but life-table projections are described for a wide variety of assumptions. Our best estimate of the effect of a 50-yr occupational exposure to 4 WLM yr-1 is 130 excess lung cancer deaths per 1000 persons (0.65 per 1000 person-WLM), with a range from 60-250 per 1000. Similar calculations for lifetime exposure to an additional 0.02 working level (WL) beyond normal background produces an estimate of 20 excess lung cancers per 1000 persons.

Thermal enhancement of dna strand breakage in mammalian cells treated with bleomycin

Chinese hamster ovary cells were treated with various doses of Bleomycin (BLM) for 1 hr at 37° or 43°C and the effects of these treatments in terms of cell killing and DNA strand breakage were determined. The technique of alkaline elution was utilized for the sensitive detection of the DNA strand breaks thus allowing the use of doses of BLM which also permit the measurement of cell survival. The results of these experiments showed that both the cytotoxic effects and DNA strand breakage were enhanced when the treatments were conducted at 43°C compared to 37°C. However, when the data were plotted as the degree of strand breakage vs the fraction of cells surviving, it appeared that the strand breaks produced under hypertbermic conditions were more effective for cell killing. While most of the breaks were rapidly repaired following treatment at 37°C, the rate of red was reduced and the amount of residual unrepalred breaks was greater when the drug treatment was at 43°C. Thus, it would appear that the repair of BLM induced DNA strand breaks is important for cell survival.

The Radiotoxicity of Iodine-125 in Mammalian Cells: II. A Comparative Study on Cell Survival and Cytogenetic Responses to 125 IUdR, 131 IUdR, and 3 HTdR

Hydrogen-3, iodine-125, or iodine-131 was incorporated into cellular DNA in the form of thymidine or its analog 5-iodo-2-deoxyuridine. The effects of these radionuclides on cell survival and chromosomes were examined in the V79 Chinese hamster cell line. It was found that in terms of cell killing, iodine-125 was considerably more effective than iodine-131 and hydrogen-3. The mean radioactivity contents at 37 percent survival were 0.10, 1.16, and 1.64 pCi per cell for iodine-125, iodine-131, and hydrogen-3, respectively. Similarly, iodine-125 was much more effective in inducing chromosome aberrations than iodine-131 or hydrogen-3. For example, 0.1 pCi per cell of iodine-125 gave rise to three chromosome breaks per cell; it took 1.0 pCi per cell of iodine-131 or 1.7 pCi per cell of hydrogen-3 to produce the same effect. The cytogenetic consequences of irradiation by these radionuclides, which decay by disparate processes, were directly related to their lethal effects and strengthens the theory that radiation-induced cell death results from damage in chromatin structures.

Effect of Heat and Radiation on Synchronous Chinese Hamster Cells: Killing and Repair

GERWECK, L. E., GILLETTE, E. L., AND DEWEY, W. C. Effect of Heat and Radiation on Synchronous Chinese Hamster Cells: Killing and Repair. Radiat. Res. 64, 611-623 (1975). Synchronous Chinese hamster cells were heated to 45.500C before, during, or after Xirradiation. The lethal effects of heat and radiation treatment were determined by the capacity of single cells to form colonies. Heat treatment prior to irradiation during G, or S was more effective, in terms of cell killing than heat treatment during or after irradiation. The time interval between irradiation and subsequent heat treatment influenced survival. The half-time for maximal survival when heat followed irradiation was 20-30 min in both G1 and S phase cells. Survival of cells which were heated, and then irradiated after heat treatment, indicated an independent, additive or synergistic interaction between heat and radiation, depending on the time of heat treatment and the phase of the cell cycle treated. Nine minutes of heat treatment during G, or 7 min during S decreased the shoulder region and increased the slope of the radiation survival curves. But most important, 7 min of heat prior to irradiation radiosensitized the relatively radioresistant S phase cells more than the relatively radiosensitive G, cells, and thus virtually eliminated the differences in radiosensitivity normally observed between G1 and S. Repair of heat damage as determined by survival to subsequent irradiation, began approximately 6 hr after 9 min heat treatment during Gx, and approximately 12 hr after 7 min heat treatment during S. In both cases, heat damage was repaired in the absence of cell division; for heating in Gi, repair occurred during Gi, but for heating during S, repair may have been associated with movement into G2. Cells exposed to split heat treatments during S repaired heat damage while they were still in S phase, and maximum survival was attained when 6 hr elapsed between the heat treatments.

In vivo effects of methotrexate. Inhibition of DNA synthesis and cell death in sensitive and resistant L1210 ascites populations.

The effect of methotrexate on 125I-iododeoxy-uridine (125IUDR) incorporation into the DNA of L1210 and L1210/MTX ascites cells was determined at different stages of tumor development. Methotrexate administration to drug sensitive L1210 cells during the early phases of tumor growth invariably produced a dramatic decline in precursor utilization. In advanced L1210 ascites populations small drug doses (1 mg/kg) increased 125IUDR incorporation to about twice the control value, while larger doses (10 mg/kg or more) decreased uptake to less than half the original value. Log phase populations of drug resistant L1210/MTX cells resembled terminal L1210 ascites cells in that precursor uptake was enhanced by small amounts of drug and decreased by larger amounts. During the terminal phase of L1210/MTX development all drug levels up to 100 mg/kg produced an increase in 125IUDR incorporation. In parallel studies an attempt was made to correlate the in vivo perturbations of the DNA precursor metabolism to the lethal action of the drug. In terms of cell killing, L1210/MTX cells proved completely resistant to the action of methotrexate, while peritoneal L1210 cells remained fully sensitive throughout the course of ascites growth. Thus, although the results indicated some overall correlation between drug induced inhibition of DNA synthesis and subsequent cell death, the relationship between these 2 parameters would appear to be too complex and ambiguous to be used as a technique for predicting the chemotherapeutic effectiveness of the drug.