Enhancement Of Radiation Sensitivity Research Articles

Proteasome inhibition improves fractionated radiation treatment against non-small cell lung cancer: An antioxidant connection

Non-small cell lung cancer frequently presents as a locally advanced disease. In this setting, radiation has a prominent role in cancer therapy. However, tumor adaptation to oxidative stress may lessen the efficacy of radiation therapy. Recent studies demonstrate that proteasome inhibitors increase the efficacy of radiation against a range of tumors. Although proteasome inhibition impacts on NF-kappaB translocation, the precise mechanism through which proteasome inhibitors induce tumor cell death and promote radiation efficacy remains unclear. The purpose of this study is to evaluate the potential of the proteasome inhibitor, MG-132, to improve the efficacy of radiation therapy and to determine whether its effect is linked to the suppression of the antioxidant enzyme, manganese superoxide dismutase (MnSOD). Human NSCLC (A549) cells were utilized both in vivo and in vitro to evaluate proteasome inhibition on radiation response. In vivo, mice that received combined treatments of 2.5 microg/g body weight MG-132 and 30 Gy demonstrated a delay in tumor regrowth in comparison to the 30 Gy control group. In vitro, clonegenic survival assays confirmed a dose-dependent enhancement of radiation sensitivity in combination with MG-132 and a significant interaction between the two. The levels of IkappaB-alpha, a NF-kappaB target gene and also an inhibitor of NF-kappaB nuclear translocation, decreased in a time-dependent manner following administration of MG-132 confirming the inhibition of the 26S proteasome. The MnSOD protein level was increased consistent with lower levels of IkappaB-alpha, confirming a NF-kappaB-mediated effect. Cells treated with radiation demonstrated an induction of MnSOD; however, the administration of MG-132 suppressed this induction These results support the hypothesis that proteasome inhibitors such as MG-132 can increase the efficacy of radiation therapy, in part, by suppression of cytoprotective NF-kappaB-mediated MnSOD expression.

P53-independent enhancement of radiation sensitivity by siRNA targeted for NBS1 in human tongue cancer cells

p53-independent enhancement of radiation sensitivity by siRNA targeted for NBS1 in human tongue cancer cells

Irradiated homozygous TGF- β 1 knockout fibroblasts show enhanced clonogenic survival as compared with TGF- β 1 wild-type fibroblasts

Purpose : To study the role of transforming growth factor β 1 (TGF- β 1) on cellular radiation sensitivity by analysing mouse lung fibroblasts of different TGF- β 1 genotypes. Materials and methods : Heterozygous TGF- β 1 knock-out mice were mated to produce offspring of different TGF- β 1 genotypes as confirmed by PCR-genotyping. Primary lung fibroblast populations were established from new-born animals of specific genotypes (TGF- β 1 +/+, TGF- β 1 +/-, TGF- β 1 -/-) . Production of TGF- β 1 was tested by ELISA. TGF- β 1 receptor-II mRNA expression was analysed by RT-PCR. Colony formation of untreated, TGF- β 1-treated and/or irradiated primary lung fibroblasts was determined under different medium conditions. Results : Plating efficiencies under different medium conditions were independent of TGF- β 1 genotype. Production of TGF- β 1 correlated with the genotype: heterozygous TGF- β 1 knock-out fibroblasts (TGF- β 1 +/-) produced 60-65% of wild-type (TGF- β 1 +/+ cells). As expected, homozygous TGF- β 1 knock-out fibroblasts (TGF- β 1 -/-) did not produce TGF- β 1. Radiation exposure significantly enhanced TGF- β 1 production in TGF- β 1 +/+ cells by a factor of 2. No such stimulation was observed in TGF- β 1 +/- cells. TGF- β 1 +/- and especially TGF- β 1 -/- cells were significantly more radioresistant than TGF- β 1 +/+ cells. TGF- β 1 treatment significantly reduced clonogenic survival for both TGF- β 1 +/+ and TGF- β 1 -/- cells. TGF- β 1 treatment of TGF- β 1 -/- cells resulted in an enhancement of radiation sensitivity. Conclusion : The data are the first direct evidence that TGF- β 1 is a major autocrine regulator of intrinsic radiation sensitivity of mouse lung fibroblasts.

Mechlorethamine-induced enhancement of radiation sensitivity of guanine.

This study describes and characterizes the interactions of nitrogen mustard mechlorethamine (HN2) with guanine and the radiation sensitivity of guanine in the presence of HN2. Briefly, in an equimolar solution (0.5 mmol dm-3) the pH-dependence (pH 3.0-12.0) and time-dependence (0-36 h) of alkylation of guanine at room temperature were determined using a reverse-phase high-performance liquid chromatography (hplc) column. Based on the hplc peak areas of the product and intact guanine, the optimal pH for alkylation was determined to be 8.0. Similarly, the optimal time required for alkylation was 10 h. Two products, i.e. alkylated guanines, were detected (10:1, peak areas measured at 260 nm) and purified. Structural studies of the products were performed by direct insertion probe-electron impact mass spectrometry. These products were identified as N-(2-chloroethyl)-N-[2-(7-guanyl)ethyl]-methylamine (product 2). At optimal conditions, samples of either guanine or an equimolar solution of guanine and HN2 were 60Co irradiated (gamma-ray) at 25 Gy min-1 at doses up to 400 Gy. Both sets of samples were analysed by hplc. In each case, the sole radiation product observed and characterized was 8-hydroxy-guanine. Dose-yield plots were linear and showed that HN2 enhanced the radiation sensitivity of guanine. This increase in radiation sensitivity is attributed to the differences in electrophilic properties between nitrogen mustard and guanine.

Modification of Radiation Sensitivity by Salts of the Metals Beryllium and Indium and the Rare Earths Cerium, Lanthanum and Scandium

The LD50 of 46 salts of metals and rare earths (lanthanoids) was determined in mice. Half the LD50 of the compounds was then combined with lethal radiation (10.5 Gy) and the modification of survival time was scored. Only the metals beryllium and indium and the rare earths cerium, lanthanum and scandium displayed activity in our assay. They were then tested at a wider range of lower doses and reduced survival time in a dose-dependent fashion. This appears to be compatible with enhancement of radiation sensitivity. The interaction of these metals and rare earths with radiation adds a new facet to their toxicological spectrum and, by enhancing radiation effects, may influence estimates of risk. On the other hand, the radiosensitizing properties of the metals may be useful for further development of compounds to be used as adjuncts in specific situations of cancer radiotherapy.

A Comparison of the Enhancement of Radiation Sensitivity and DNA Polymerase Inactivation by Hyperthermia in Human Glioma Cells

Two human glioma cell lines (U87MG and U373MG) were evaluated for their thermal enhancement of radiation sensitivity and its correlation to the degree of inactivation of DNA polymerase alpha and beta. The data showed that hyperthermia increased radiation sensitivity in a time- and temperature-dependent manner. The differential heat sensitivity of the two cell lines was reflected in the degree of polymerase inactivation. Polymerase inactivation was also dependent on time and temperature and was greater for polymerase beta than alpha. The degree of polymerase inactivation correlated well with the thermal enhancement ratio (TER) calculated at the 1.0% survival level. This correlation was poor for the TER at the 50% survival level. The correlations were better for polymerase beta than alpha. The small differences in thermal sensitivity between the two cell lines primarily at 41 and 42 degrees C could not be explained by correlation between polymerase inactivation and TER. Incubation between hyperthermia and irradiation resulted in recovery of polymerase activity and loss of radiosensitization. Levels of polymerase beta after hyperthermia may be used to predict thermal enhancement of radiosensitivity for low survival levels, but possibly not in the shoulder region of the radiation survival curve. Small cell line-dependent differences in thermal sensitivity may not be resolved in these comparisons.

Thermal radiosensitization in two pairs of CHO wild-type and radiation-sensitive mutant cell lines.

Two pairs of Chinese hamster ovary cell lines, each comprising a parental normal-responding and a radiation-sensitive mutant, were tested for their X-radiation sensitivity, hyperthermia sensitivity and thermal radiosensitization. The relative radiation responses were similar to those already published in the literature, indicating that the mutant cell lines, V-3 and 5-11, were extremely sensitive to radiation while their parental strains (AA8-4 and K1) exhibited normal sensitivity. The hyperthermia response of V3 was the same as that of the parental cell line AA8-4; the thermal sensitivity of 5-11 was greater than that of its parental line K1 but this difference decreased at higher temperatures (greater than 43 degrees C). These data indicate that the hyperthermia response and its targets are probably not related to the mutation-causing radiation sensitivity in the two pairs of parent-mutant cell lines. When heat and radiation were combined, the thermal enhancement of radiation sensitivity for different heat treatments was the same or larger in the mutant than in the parental cell lines. The mutants are known to be deficient in potentially lethal damage repair (PLDR), sublethal damage repair (SLDR) and repair of double-stranded DNA breaks; however, these deficiencies did not impair thermal radiosensitization, indicating that thermal radiosensitization may not be through these pathways in these particular mutants. In addition, the parental cell lines exhibited a significant sequence dependence of thermal radiosensitization (i.e. whether heat was applied before or after irradiation) at low survival levels (10(-4); this sequence dependence was very small to absent in the mutant cell lines at these survival levels.

Enhancement of Radiation Sensitivity in Iododeoxyuridine Labelled Cells Exposed to Low Energy X-Rays

In order to investigate if low-energy x-rays induce Auger cascades by photoelectric absorption in iodine present in DNA, CHO cells were labelled with iododeoxyuridine (IUdR) for 72 hours. Following labelling, the cells were either irradiated with low-energy x-rays (75 kV, 4 mm Al) or 137Cs-gamma-rays. The radiation response was measured using clonogenic survival, and the survival parameters were analyzed according to the linear quadratic model. The dose modifying factors were determined as the ratios of the alpha-coefficients. The IUdR labelled cells were found to be about 3.2 times as sensitive as the control cells when irradiated with low-energy x-rays. For 137Cs-gamma the ratio was about 1.5. The standard deviations were estimated by Gauss' approximation to be about 0.5 for both irradiation conditions.

Enhancement of Radiation Sensitivity by Postirradiation Hypoxia: Time Course and Oxygen Concentration Dependency

We have recently demonstrated that postirradiation hypoxia during colony formation in vitro enhanced the radiation sensitivity of murine tumor cells irradiated and maintained at 0.1% O2. The effect of postirradiation hypoxia was expressed by a significant reduction in the oxygen enhancement ratio. We now demonstrate that this enhancement of radiation sensitivity by postirradiation hypoxia is also observed in a human tumor cell line. The effect was observed at [O2] less than or equal to 0.1%, but was not present at [O2] greater than or equal to 0.5%. Time-course experiments suggested that this enhancement of cell killing by X rays required prolonged exposure to hypoxic conditions.

Effect of Dimethyl Fumarate on the Radiation Sensitivity of Mammalian Cells in Vitro

Dimethylfumarate (DMF) depletes intracellular glutathione (GSH) by covalent bond formation in a reaction which may be mediated by GSH-S-transferase. In Chinese hamster ovary cells this depletion is rapid; e.g., 0.5 mM DMF depletes GSH to less than 10% of control in 5 min at room temperature. DMF is a very effective hypoxic cell radiosensitizer, with an enhancement ratio (ER) of about 3 obtained by a 5-min exposure of cells at room temperature to 5 mM DMF, without significant toxicity. At this same concentration of drug, there is a small enhancement of aerobic cells (ER = 1.3), but the 5 mM DMF in hypoxia results in nearly a complete collapse of the hypoxic dose-response curve to the same level as seen in air with DMF. It has been suggested previously that DMF sensitizes cells via electron affinic mechanisms. However, this appears not to be the case in this study, as shown by the fact that cells pretreated with DMF and then washed free of the drug remained equally radiosensitive as cells irradiated in the presence of the drug. This large enhancement of radiation sensitivity appears to be related to the drug's ability to deplete thiols; i.e., thiols appear to be a major factor responsible for radioresistance of hypoxic cells.

Enhancement of Radiation Sensitivity by Postradiation Hypoxia

We demonstrate that postradiation hypoxia during colony formation in vitro enhances radiation sensitivity when cells are irradiated in severely hypoxic states. The presence and magnitude of this phenomenon, if it occurs in vivo, raise questions about the importance of the oxygen effect in the radiation response of tumor cells in vivo and suggest that hypoxia may not be an important factor in the probability of tumor control following radiation therapy.

Effects of Hyperthermia on Repair of Radiation-Induced DNA Strand Breaks

Previous reports have suggested a relationship between the heat-induced changes in nucleoprotein and the hyperthermic enhancement of radiation sensitivity. In an effort to further understand these relationships, we measured the level of initial DNA strand break damage and the DNA strand break rejoining kinetics in Chinese hamster ovary cells following combined hyperthermia and ionizing radiation treatments. The amount of protein associated with DNA measured as the ratio of [3H]leucine to ['4C]thymidine was also compared in chromatin isolated from both heated and unheated cells. The results of these experiments show that the initial level of radiation-induced DNA strand breaks (as measured by the sensitive technique of alkaline elution) is significantly enhanced by a prior hyperthermia treatment of 43?C for 30 min. Treatments at higher temperatures and longer treatments at the same temperature magnified this effect. Hyperthermia was also shown to cause a substantial inhibition of the DNA strand break rejoining after irradiation. Both the initial level of DNA damage and the rejoining kinetics recovered to normal levels with incubation at 37?C between the hyperthermia and radiation treatments. Recovery of these parameters coincided with the return of the amount of protein associated with DNA to normal values, further suggesting a relationship between the changes in nucleoprotein and the hyperthermic enhancement of radiation sensitivity.

Radiation and Temper Embrittlement Processes in Advanced Reactor Weld Metals

Experimental weld filler metals having high resistance to radiation embrittlement at ≃550 F (288 C) have been developed for quenched and tempered A543 and A542 steel. The filler metals are from a special 2-1/4 Cr-1 Mo-0.40Si-0.10C composition series formulated to study the effects of variable copper, nickel, and manganese contents on weld performance. This report presents an advanced evaluation of weld deposit performance based on new Charpy-V (Cv) and tension data and an analysis of temper embrittlement and radiation embrittlement processes. High fluence assessments confirm the high resistance to radiation embrittlement of the low copper content filler metal group. A 530 F (277 C) irradiation of one typical submerged arc weld deposit to a fluence of 3.8 × 1020 n/cm2 > 1 MeV did not elevate its Cv 30 ft-lb transition temperature to above 275 F (135 C) or reduce its Cv shelf energy level to below 50 ft-lb. Radiation embrittlement saturation was not evident. Temper embrittlement and radiation embrittlement development and the probable mechanisms of copper and phosphorus influences on radiation embrittlement sensitivity are analyzed with the aid of experimental data for the weld metals and A543 plate. Temper embrittlement and radiation embrittlement are shown to be additive effects which can occur simultaneously or sequentially. A separate component of irradiation effects, manifested as a strength increase without embrittlement, is revealed. The enhancement of radiation sensitivity by high copper content (≧0.16–0.27 percent Cu) is related to a copper influence on the radiation elevation of yield strength; the enhancement of radiation sensitivity of phosphorus is ascribed to a detrimental effect similar to that of temper embrittlement. It is proposed that copper acts to pin radiation-induced defect aggregates and dislocation arrays in the matrix and that phosphorus segregates during irradiation to weaken the interface of ferrite platelets and carbides.