Margareta Edgren

Induction and Repair of Single-strand DNA Breaks after X-irradiation of Human Fibroblasts Deficient in Glutathione

Using the unwinding technique in weak alkali, the induction and repair of DNA single-strand breaks was determined after aerobic and anerobic X-irradiation of human fibroblasts, obtained from a patient suffering from 5-oxoprolinuria, and from a clinically healthy control. The metabolic disorder associated with 5-oxprolinuria is a deficiency in glutathione synthetase activity resulting in a greatly reduced glutathione content in the cells. A small dose-modifying effect of oxygen (o.e.r. = 1.1) was found for these cells in comparison to an o.e.r. of 2.5 for control cells with normal glutathione content. No significant difference was found between the repair capacity of cells with normal and deficient glutathione content, and repair was nearly completed within 60 min of anoxic irradiation in each case. In contrast, after aerobic irradiation of glutathione-deficient cells repaired less than 70 per cent of the breaks during the same period. When the glutathione-deficient cells were incubated with either dithiothreitol or mercaptopropionylglycine directly after aerobic irradiation, almost complete repair was obtained within 60 Min. The data are interpreted as indicating that the repair mechanism for oxically and anoxically induced single-strand breaks is qualitatively different, and requires glutathione in the former case.

Repairable–Conditionally Repairable Damage Model Based on Dual Poisson Processes

Abstract Lind, B. K., Persson, L. M., Edgren, M. R., Hedlöf, I. and Brahme, A. Repairable–Conditionally Repairable Damage Model Based on Dual Poisson Processes. Radiat. Res. 160, 366–375 (2003). The advent of intensity-modulated radiation therapy makes it increasingly important to model the response accurately when large volumes of normal tissues are irradiated by controlled graded dose distributions aimed at maximizing tumor cure and minimizing normal tissue toxicity. The cell survival model proposed here is very useful and flexible for accurate description of the response of healthy tissues as well as tumors in classical and truly radiobiologically optimized radiation therapy. The repairable–conditionally repairable (RCR) model distinguishes between two different types of damage, namely the potentially repairable, which may also be lethal, i.e. if unrepaired or misrepaired, and the conditionally repairable, which may be repaired or may lead to apoptosis if it has not been repaired correctly. When potentially repairable damage is being repaired, for example by nonhomologous end joining, conditionally repairable damage may require in addition a high-fidelity correction by homologous repair. The induction of both types of damage is assumed to be described by Poisson statistics. The resultant cell survival expression has the unique ability to fit most experimental data well at low doses (the initial hypersensitive range), intermediate doses (on the shoulder of the survival curve), and high doses (on the quasi-exponential region of the survival curve). The complete Poisson expression can be approximated well by a simple bi-exponential cell survival expression, S(D) = e–aD + bDe–cD, where the first term describes the survival of undamaged cells and the last term represents survival after complete repair of sublethal damage. The bi-exponential expression makes it easy to derive D0, Dq, n and α, β values to facilitate comparison with classical cell survival models.

Lack of oxygen effect in glutathione-deficient human cells in culture

The frequency of X-ray-induced DNA breaks was determined in human cell lines which are deficient in glutathione synthetase and have a greatly reduced glutathione content. Hydroxyapatite chromatography was used for the estimation of the DNA breaks in cell cultures, which were derived either from lymphoblasts transformed by infection with EB virus or from fibroblasts. The dose-effect relationship for the induction of breaks when radiation exposure was made in argon, was similar to that found when exposure was made in air. In control cultures with normal glutathione content, the induction of breaks was enhanced when irradiation was made under aerobic, instead of anaerobic, conditions. Treatment of the glutathione-deficient cells with the hypoxic radiosensitizer misonidazole did not enhance the induction of breaks by radiation delivered either in air or in argon. In control cultures, radiation induction of breaks was enhanced by misonidazole under anaerobic but not under aerobic conditions. When the glutathione-deficient cells were pretreated with cysteamine however, irradiation in the absence of oxygen resulted in a decreased frequency of DNA breaks.

Nuclear Glutathione and Oxygen Enhancement of Radiosensitivity

Treatment with buthionine sulphoximine depletes glutathione from whole cells and nuclei to different extents resulting in different relationships between o.e.r. and the glutathione content in the two cases.

Release of Thiols from Cellular Mixed Disulphides and Its Possible Role in Radiation Protection

SummaryThe non-protein-bound part of cellular mixed disulphides between proteins and thiols was analysed by gel-filtration after reduction of the disulphide bonds. Glutathione (GSH) was found to be the only detectable low-molecular thiol bound to cellular proteins. Reduction of disulphide bonds resulted, in addition to the release of GSH, in the liberation of sulphydryl-containing protein fragments of various sizes.Using cells in which the sulphur was labelled with 35S, it was demonstrated that about 15 per cent of the incorporated cysteamine was bound to the proteins, and that GSH was released in comparable amounts. The protein-cysteamine bound was stable for at least 95 min.It is concluded that GSH is released from proteins by cysteamine treatment in an exchange reaction. The data suggest, furthermore, that protein-bound GSH may be regarded as a reservoir of a radioprotective substance which can be released under certain conditions and, when released, can contribute to enhance the radioresistance of cells.

Chelation of lysosomal iron protects against ionizing radiation

Ionizing radiation causes DNA damage and consequent apoptosis, mainly due to the production of hydroxyl radicals (HO•) that follows radiolytic splitting of water. However, superoxide (O2•-) and H2O2 also form and induce oxidative stress with resulting LMP (lysosomal membrane permeabilization) arising from iron-catalysed oxidative events. The latter will contribute significantly to radiation-induced cell death and its degree largely depends on the quantities of lysosomal redox-active iron present as a consequence of autophagy and endocytosis of iron-rich compounds. Therefore radiation sensitivity might be depressed by lysosome-targeted iron chelators. In the present study, we have shown that cells in culture are significantly protected from ionizing radiation damage if initially exposed to the lipophilic iron chelator SIH (salicylaldehyde isonicotinoyl hydrazone), and that this effect is based on SIH-dependent lysosomal stabilization against oxidative stress. According to its dose-response-modifying effect, SIH is a most powerful radioprotector and a promising candidate for clinical application, mainly to reduce the radiation sensitivity of normal tissue. We propose, as an example, that inhalation of SIH before each irradiation session by patients undergoing treatment for lung malignancies would protect normally aerated lung tissue against life-threatening pulmonary fibrosis, whereas the sensitivity of malignant lung tumours, which usually are non-aerated, will not be affected by inhaled SIH.

Low and high LET radiation‐induced apoptosis in M059J and M059K cells

Purpose: To investigate and compare the ability of DNA‐dependent protein kinase (DNA‐PK)‐deficient and ‐proficient cells to undergo apoptosis after exposure to low and high linear energy transfer (LET) radiation. Materials and methods: A human glioma cell line M059J lacking the catalytic subunit of DNA‐PK (DNA‐PKcs) and its DNA‐PKcs‐proficient counterpart, M059K, were exposed to 1 and 4 Gy of accelerated nitrogen ions (14N, 140 eV nm−1, 8–12 Gy min−1) or 60Co γ‐rays (0.2 eV nm−1, 0.7 Gy min−1). The induction of apoptosis was studied up to 144 h post‐irradiation using two different methods: morphological characterization of apoptotic cells after fluorescent staining and cell size distribution analysis to detect apoptotic bodies. In parallel, protein expression of DNA‐PKcs and poly(ADP‐ribose) polymerase (PARP) as well as DNA‐PK and caspase‐3 activity were investigated. Results: Low and high LET radiations (4 Gy) induced a time‐dependent apoptotic response in both cell lines. Low LET radiation induced a significantly elevated apoptotic response in M059J as compared with M059K cells at 144 h post‐irradiation. Following high LET radiation exposure, there was no difference between the cell lines at this time. PARP cleavage was detected in M059J cells following both low and high LET irradiation, while only high LET radiation induced PARP cleavage in M059K cells. These cleavages occurred in the absence of caspase‐3 activation. Conclusions: M059J and M059K cells both display radiation‐induced apoptosis, which occur independently of caspase‐3 activation. The apoptotic course differs between the two cell lines and is dependent on the quality of radiation.

Dual Effect of Oxygen on the Induction and Repair of Single-strand Breaks in the DNA of X-irradiated Mammalian Cells

SummaryThe induction and rejoining of single-strand DNA breaks was studied in Chinese hamster cells exposed to radiation under oxic or anoxic conditions. The number of breaks was determined by the alkaline sucrose gradient technique either immediately after irradiation or after a period of oxic or extremely hypoxic incubation. A linear relationship between the dose and the inverse of the molecular weight of irradiated DNA was noted when oxygen was present or absent during radiation exposure. A dose-modifying effect of oxygen by a factor 2·3 was calculated. When quinacrine was used to inhibit rejoining of breaks during irradiation, the dose-modifying factor increased to 2·7. The energy required to induce one single-strand break was estimated to be 29 eV in oxygen, and 77 eV in anoxia.When the cells were incubated after irradiation under oxic conditions for 30 min, about 80 per cent of the oxically-induced breaks, and about 50 per cent of the hypoxically-induced breaks were repaired. The rejoining process w...

Cellular glutathione content and K values

The K values for two cell strains with differing intrinsic GSH concentrations have been measured with the yield of DNA breaks as end-point of the radiation effect. The K value of the strain with reduced GSH content was decreased (1.59 microM O2) in comparison to the K value (3.01 microM O2) for the strain with a normal GSH content. The significance of the observation is discussed in relation to competition models. All variants of the competition model agree in predicting a reduction of K, if GSH is reduced.

Selective toxicity of buthionine sulfoximine (BSO) to melanoma cells in vitro and in vivo

PURPOSE Glutathione (GSH) was found to occur in relatively high concentrations in melanoma cells. The purpose of this study was to test the possible cytotoxic effects of an artificial decrease of the elevated GSH level. METHODS AND MATERIALS The tests were made in vitro and in vivo. In the former case, a total of 11 rodent and human cell lines were studied of which seven were derived from melanomas. After treatment with buthionine sulfoximine (BSO), the decrease of GSH content of the cells and their clonogenic survival was determined. In the in vivo system, single cell suspensions of a subline of the B16 mouse melanoma were injected intravenously into immunocompetent and preirradiated recipients which were subsequently treated with BSO intraperitoneally. Survival time, formation of lung colonies and the weight of metastatic tumor mass in the lungs were the criteria of the BSO effect on the tumor cells. RESULTS The decrease of the GSH level by BSO was associated with impaired clonogenic survival of the melanoma cells in vitro. Nonmelanoma cells were less affected. BSO treatment of mice inoculated intravenously with melanoma cells resulted in prolonged survival of the animals and impaired metastatic spread of the tumor cells. CONCLUSION Melanoma cells are particularly sensitive to disturbance of GSH metabolism by treatment with BSO. In view of this selective cytotoxicity of BSO, treatment with this substance may afford a promising therapeutic potential for melanoma.