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For many years the linear-quadratic (LQ) model has been widely used to describe the effects of total dose and dose per fraction at low-to-intermediate doses in conventional fractionated radiotherapy. Recent advances in stereotactic radiosurgery (SRS) and stereotactic radiotherapy (SRT) have increased the interest in finding a reliable cell survival model, which will be accurate at high doses, as well. Different models have been proposed for improving descriptions of high dose survival responses, such as the Universal Survival Curve (USC), the Kavanagh-Newman (KN) and several generalizations of the LQ model, e.g. the Linear-Quadratic-Linear (LQL) model and the Padé Linear Quadratic (PLQ) model. The purpose of the present study is to compare a number of models in order to find the best option(s) which could successfully be used as a fractionation correction method in SRT. In this work, six independent experimental data sets were used: CHOAA8 (Chinese hamster fibroblast), H460 (non-small cell lung cancer, NSLC), NCI-H841 (small cell lung cancer, SCLC), CP3 and DU145 (human prostate carcinoma cell lines) and U1690 (SCLC). By detailed comparisons with these measurements, the performance of nine different radiobiological models was examined for the entire dose range, including high doses beyond the shoulder of the survival curves. Using the computed and measured cell surviving fractions, comparison of the goodness-of-fit for all the models was performed by means of the reduced χ2-test with a 95% confidence interval. The obtained results indicate that models with dose-independent final slopes and extrapolation numbers generally represent better choices for SRT. This is especially important at high doses where the final slope and extrapolation numbers are presently found to play a major role. The PLQ, USC and LQL models have the least number of shortcomings at all doses. The extrapolation numbers and final slopes of these models do not depend on dose. Their asymptotes for the cell surviving fractions are exponentials at low as well as high doses, and this is in agreement with the behaviour of the corresponding experimental data. This is an important improvement over the LQ model which predicts a Gaussian at high doses. Overall and for the highlighted reasons, it was concluded that the PLQ, USC and LQL models are theoretically well-founded. They could prove useful compared to the other proposed radiobiological models in clinical applications for obtaining uniformly accurate cell surviving fractions encountered in stereotactic high-dose radiotherapy as well as at medium and low doses.

Imatinib is currently in early clinical trials as targeted therapy for relapsed neuroblastomas and other childhood solid tumors expressing platelet-derived growth factor receptors (PDGFR) or c-Kit. Short-term treatment with imatinib in clinically achievable concentrations is ineffective in neuroblastoma in vitro. However, clinically, imatinib is administered daily over long time periods. The effects of combining imatinib with chemotherapy in neuroblastoma are unknown. Here, a panel of neuroblastoma cell lines (n = 5) were studied, representing tumors with different biological (MYCN-amplification +/-) and clinical (drug resistance) features. Using a protracted low-dose treatment schedule (1-3 weeks; 0.5-5microM) imatinib dose-dependently inhibited proliferation and clonogenic survival for all tested cell lines with IC50

The aim was to investigate and compare the influence of linear energy transfer (LET), dose and time on the induction of apoptosis in a human melanoma cell line exposed to accelerated light boron ((10)B) ions and photons. Cells were exposed in vitro to doses up to 6 Gy accelerated boron ions (40, 80, 125 and 160 eV nm(-1)) and up to 12 Gy photons (0.2 eV nm(-1)). The induction of apoptosis was measured up to 9 days after irradiation using morphological characterization of apoptotic cells and bodies. In parallel, measurements of cell-cycle distribution, monitored by DNA flow cytometry, and cell survival based on the clonogenic cell survival assay, were performed. In addition, the induction and repair of DNA double-strand breaks (DSB), using pulsed-field gel electrophoresis (PFGE) were studied. Accelerated boron ions induced a significant increase in apoptosis as compared with photons at all time points studied. At 1-5 h the percentage of radiation-induced apoptotic cells increased with both dose and LET. At the later time points (24-216 h) the apoptotic response was more complex and did not increase in a strictly LET-dependent manner. The early premitotic apoptotic cells disappeared at 24 h following exposure to the highest LET (160 eV nm(-1)). A postmitotic apoptotic response was seen after release of the dose-, time- and LET-dependent G2/M accumulations. The loss of clonogenic ability was dose- and LET-dependent and the fraction of un-rejoined DSB increased with increasing LET. Despite the LET-dependent clonogenic cell killing, it was not possible to measure quantitatively a LET-dependent apoptotic response. This was due to the different time course of appearance and disappearance of apoptotic cells.

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 D(0), D(q), n and alpha, beta values to facilitate comparison with classical cell survival models.

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.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 gamma-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.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.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.

The use of ion radiation therapy is growing due to the continuously increasing positive clinical experience obtained. Therefore, there is a high interest in radio-biological experiments comparing the relative efficiency in cell killing of ions and photons as photons are currently the main radiation modality used for cancer treatment. This comparison is particularly important since the treatment planning systems (TPSs) used at the main ion therapy Centers make use of parameters describing the cellular response to photons, respectively ions, determined in vitro. It was, therefore, the aim of this article to compare the effects of high linear energy transfer (LET) ion radiation with low LET photons and determine whether the cellular response to low LET could predict the response to high LET irradiation.Clonogenic cell survival data of five tumor cell lines irradiated with different ion beams of similar, clinically-relevant, LET were studied in relation to response to low LET photons. Two mathematical models were used to fit the data, the repairable-conditionally repairable damage (RCR) model and the linear quadratic (LQ) model.The results indicate that the relative biological efficiency of the high LET radiation assessed with the RCR model could be predicted based only on the response to the low LET irradiation.The particular features of the RCR model indicate that tumor cells showing a large capacity for repairing the damage will have the larger benefit from radiation therapy with ion beams.

The purpose of this study was to quantify and model various types of cell death for a small-cell lung cancer (SCLC) cell line (U1690) after exposure to a 137Cs source and as well as to compare the linear-quadratic (LQ) and repairable-conditionally repairable model (RCR). This study is based on four different experiments that were taken place at Cancer Centrum Karolinska (CCK). A human small-cell lung cancer (SCLC) cell line after the exposure to a 137Cs source was used for the extraction of the clonogenic cell survival curve. Additionally, for the determination and quantification of various modes of cell death the method of fluorescence staining was implemented, where the cell deaths were categorized based on morphological characteristics. The percentage of cells in each phase of the cell cycle was investigated with flow cytometry analysis. The quantification of senescent cells was performed by staining the samples with senescence-associated β-galactosidase (SA-β-Gal) solution and then scoring as senescent cells those that had incorporated the substance. These data were introduced into a maximum likelihood fitting to calculate the best estimates of the parameters used by the examined model. In this model, the modes of cell death are divided into three categories: apoptotic, senescent and other types of cell death (necrotic/apoptotic, necrotic, micronuclei and giant). In the clonogenic cell survival assay, the fitting of the RCR model gives a χ(2)-value of 6.10 whereas for the LQ model became 9.61. In the fluorescence microscopy and senescence assay, the probability of the three different modes of cell death on day 2 seems to increases with a dose up to about 10 Gy where there is saturation. On day 7 a significant induction of apoptosis in a dose- and time-dependent manner was evident, whereas senescence was slightly increased in response to dose but not to time. As for the 'other types of cell death' mode on day 7 showed a higher probability than the one on day 2 and as well as a prominent dose-dependence. The RCR model fits better to the experimental data than the LQ model. On day 2 there is a slight increase of the apoptotic and senescent probability with dose. On the other hand, on day 7 the shape of the curve of apoptosis differs and a sigmoidal increase with dose is observed. At both time-points, the present model fits the data reasonably well. Due to the fact that the clonogenic survival does not coincide with the one extracted from the fluorescence microscopy, a more accurate way to quantify cell death needs to be used, e.g. computerized video time-lapse (CVTL).

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.

Using treatment with cytochalasin-B (Cyt-B) for the induction of a cytokinetic block, the significance of the proportion of binucleate cells (BNC) in the micronucleus (MN) assay was investigated in a methodological study. A Chinese hamster cell line V79 was used in which MN were induced by radiation. In complementary tests the radiation effect in inducing MN was enhanced by depletion of the cellular glutathione content with buthionine sulfoximine (BSO). The data indicated that the concentration of Cyt-B is the major factor which determines the proportion of BNC. This proportion was shown to be independent of radiation dose and of BSO. Furthermore, the MN frequency was not related to the percentage of BNC. Therefore, a high proportion of BNC may be practical for the MN assay, but may not make the technique more accurate.

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. Original Publication:Carsten Berndt, Tino Kurz, Markus Selenius, Aristi P Fernandes, Margareta R Edgren and Ulf T Brunk, Chelation of lysosomal iron protects against ionizing radiation., 2010, Biochemical Journal, (432), 2, 295-301.http://dx.doi.org/10.1042/BJ20100996Copyright: Portland Press -- Londonhttp://www.portlandpress.com/

Purpose To investigate and compare the cell cycle progression in relation to cell death in the human glioma cell lines, M059J and M059K, after exposure to DNA double-strand break–inducing agents. Methods and materials The M059J and M059K cells, deficient and proficient in the catalytic subunit of the DNA-dependent protein kinase, respectively, were exposed to 1 and 4 Gy of photons or accelerated nitrogen ions. In addition, M059J and M059K cells were treated with 10 and 40 μg/mL of bleomycin for 30 min, respectively. Cell cycle progression, monitored by DNA flow cytometry, was measured up to 72 h after treatment. Results M059J, but not M059K, cells displayed G 2 /M accumulation after low linear energy transfer irradiation. High linear energy transfer radiation exposure however, resulted in a substantial increase of M059K cells in the G 2 /M phase detected at 48 h. At 72 h, the number of cells in the G 2 /M phase was equivalent to its control. M059J cells accumulated mainly in S phase after high linear energy transfer irradiation. In contrast to M059K, M059J cells were still blocked at 72 h. Bleomycin induced G 2 /M accumulation for both M059J and M059K cells detected 24 h after treatment. At 48 h, the percentage of bleomycin-treated M059J cells in G 2 /M phase remained high, and the number of M059K cells had decreased to control levels. Neither cell line showed cell cycle arrest (≤10 h) after exposure to these agents. Conclusion Distinct cell cycle block and release is dependent on the complexity of the induced DNA damage and the presence of the DNA-dependent protein kinase catalytic subunit.

Purpose: To compare the repair efficiency of X‐ray (low linear energy transfer [LET]) and nitrogen ion (high LET)‐induced single‐strand breaks (SSB) in a human cell‐free end‐joining system.Materials and methods: SSB were introduced into a bacterial plasmid, pBR322, by X‐rays (4 MeV photons) and nitrogen ions with an LET=125 keV µm−1. Repair efficiency was studied under incubation with the protein extracts from human squamous carcinoma cells, UT‐SCC‐5.Results: A several fold higher dose of nitrogen ion radiation compared with X‐ray radiation was needed to induce a similar loss of supercoiled plasmid DNA. There was no difference in the repair efficiency of SSB induced by these two types of radiation.Conclusion: The data indicate that X‐rays at 25 Gy and nitroging ions at 100 Gy radiation doses, under condition of low scavenging capacity (10 mM Tris), induce SSB of similar complexity or, alternatively, differences in SSB complexity do not alter the repair rate.

The aim of this article is to present a method for studying the shape of the dose and repair responses for X-ray-induced double-strand breaks (DSBs) as measured by neutral filter elution (NFE). The approach is closely related to a method we developed for the use of specific molecular size markers and used for determination of the absolute number of randomly distributed radiation-induced DSBs by pulsed-field gel electrophoresis (PFGE). Mouse leukemia L1210 cells were X-irradiated with 0-50 Gy. Samples were then evaluated both with PFGE and with NFE. Assuming that with both migration (PFGE) and elution (NFE), a heterogeneous population of double-stranded DNA fragments will start with the smallest fragments and proceed with increasingly larger fragments, it is possible to match the migration behavior of fractions of fragments smaller than a certain size to the fraction eluted at a specific time. This assumption does not exclude the possibility of DNA being sheared in the NFE filter. The yield, as determined by the size markers in PFGE, was used to find the corresponding elution times in the NFE experiment. These experimentally used elution times could then reversely be interpreted as size markers which finally were used to calculate DSBs/Mbp as a function of X-ray dose. The resulting lines were almost straight. The data were also plotted as relative elution and showed that, as expected, the dose response then appears with a more pronounced sigmoid shape.

In order to compare the biological effectiveness of a 50 MV scanned bremsstrahlung beam to (60)Co and 6 MV photons, the survival of Chinese hamster cells (V79-379A), human normal fibroblasts cells (GSH(+/+)) and human small cell lung cancer cells (U-1690) were analysed.Cells were irradiated in vitro under aerobic conditions in a plastic phantom. Dose to the cells was determined using ferrous sulphate and ionization chamber dosimetry. A number of cell survival models were fitted to the experimental data, including the standard LQ model with and without the induced repair. In particular, a new model treating damage and repair separately was used in combination with a new technique for accurate RBE determination.The measured RBE for the three cell lines were 0.988 (0.984-0.992), 0.999 (0.996-1.002) and 1.013 (1.009-1.016) for V79-379A, GSH(+/+) and U-1690 respectively and thus 50 MV scanned beams did not differ more than a fraction of a per cent from conventional therapy beams.The present study gives RBE consistent with previously calculated RBEs based on photonuclear reaction cross-sections of high-energy photons.

To investigate the cellular effects of 1.1 MHz high-intensity focused continuous wave (CW) ultrasound alone and in combination with X-rays.V79 cells were exposed to ultrasound of different intensities for 30s (non-hyperthermic conditions). In the combined treatments, cells were exposed to ultrasound (63 W/cm2) either before or after 2 Gy X-irradiation. Cellular effects studied were clonogenic survival, DNA strand breaks (pulsed-field gel electrophoresis, DNA precipitation assay) and membrane integrity (morphological analysis).With increasing ultrasound intensity, cell survival decreased in a dose-dependent manner as the induction of DNA strand breaks, the fraction of cells with lost membrane integrity and cell lysis increased. In the treatments with combined exposures, the regimen with X-rays before ultrasound had a nearly additive effect on cell kill, whereas the reverse regimen with ultrasound exposure before X-irradiation resulted in a synergistic effect (p0.012).High-intensity focused CW ultrasound induces an intensity-dependent reduction in clonogenic survival in V79 cells, which seems to depend on both DNA and membrane damage. Combined exposures of ultrasound and X-rays resulted in a synergistic reduction in cell survival when cells were exposed to ultrasound before X-rays but not for the reverse regimen. Thus, a larger fraction of the repairable sublethal cell damage induced by an initial ultrasound exposure was rendered non-repairable by a subsequent X-ray exposure than if the reverse treatment order was used.

O 6 -methylguanine-DNA methyltransferase (MGMT), glutathione transferase (GST) M3-3 and glutathione (GSH) have all been implicated in the resistance of cells to the cytostatic drug carmustine. U1810, a human non-small cell lung cancer cell line, expresses all of these putative resistance factors. The U1810 cells show a 4.4-fold lower sensitivity to carmustine compared with the U1690 cell line, a human small cell lung cancer cell line lacking detectable levels of both MGMT and GST M3-3. We investigated the effect of the MGMT inhibitor O 6 -benzylguanine, the GST inhibitor ethacrynic acid and the GSH synthesis inhibitor d,l-buthionine- S,R -sulfoximine (BSO) on the cytotoxicity of carmustine to U1810 cells. No potentiation to carmustine was observed after treatment with ethacrynic acid, while a 2-fold potentiation was found after exposure to O 6 -benzylguanine. Depletion of GSH with BSO showed a similar sensitising effect as that obtained with O 6 -benzylguanine. Thus, MGMT and GSH are the predominant resistance factors to carmustine in the U1810 cell line, whereas it is unclear whether GST M3-3 plays any role.

Fibroblasts from an individual with 5-oxoprolinuria are deficient in glutathione (GSH) as compared to cells from a healthy sibling. Glutathione transferase (GST) levels also differ for some isoenzymes: GSH deficient cells have more GST A1-1, but lack GST M1-1. However, the contents of the quantitatively dominating isoenzyme GST P1-1 show no significant difference. While there is equal sensitivity to ultaviolet irradiation (UV), the GSH deficient cells are more sensitive to cisplatin, carboplatin and melphalan. Depletion of GSH with buthionine sulfoximine sensitizes the normal fibroblasts to these drugs. Only slightly more cisplatin-induced DNA cross-links are detected in the GSH deficient cells, and there is no difference in the removal of DNA cross-links. Similar amounts of DNA strand breaks are seen in normal and GSH deficient cells after UV exposure, but there is a reduced rates of resealing of strand breaks in GSH deficient cells. The main conclusion from the studies of these fibroblasts is that GSH plays a significant role in the cellular resistance to cytostatic drugs such as platinum compounds and melphalan.

Cisplatin resistance was developed in the human melanoma cell line RPMI8322 by repeated short-term exposures to cisplatin. The most resistant daughter cell line, RPMI8322/CDDP-300, was 4-fold resistant to cisplatin, and partially cross-resistant to carboplatin, melphalan and UV, but not to BCNU. RPMI8322/CDDP-300 cells showed less apoptosis after cisplatin than the parental cells. The cisplatin resistance was not paralleled by a similar reduction in cellular cisplatin accumulation or DNA cross-links in RPMI8322/CDDP-300 cells, and these cells exhibited no increase in cellular glutathione or in mRNA encoding the DNA excision repair protein ERCC1 and XPB. Induction of c-jun mRNA by cisplatin was considerably lower in RPMI8322/CDDP-300 cells than in RPMI8322 cells, consistent with the possibility that c-jun induction may be involved in a pathway that triggers apoptosis after exposure to DNA damaging agents. However, c-jun induction is not necessary for apoptosis, since cisplatin also induced apoptosis in A14 rat embryo fibroblasts, cells in which the c-jun gene is deleted.

In 1985, Edgren et al. proposed a modified version of the competition theory to explain the interaction of sensitizers and protectors with target molecules damaged by radiation, which was designated the 'X' model. This model incorporates concepts which have been considered previously, namely that a type of radiation damage exists which cannot be chemically repaired, and that cells may contain a naturally occurring sensitizer. The model leads to testable predictions, such as, e.g. the crossing of 'K curves' when the level of protection is varied. It can only be applied to the immediate effects of radiation, i.e. before enzymatic reactions play a part. The present paper is a summary of work carried out since 1985 to test the predictions of the 'X' model and an exposition of the related algebra.

Contradictory obervations have been reported that, on the one hand, the radical competition processes which determine the oxygen effect, are dependent on the cellular level of non-protein-bound sulphydryls (NPSH) and, on the other hand, oxygen enhancement ratios (OER) can be close to unity allthough sulphydryls are present in a considerable amount in the cells (Edgren 1987a). The controversy can be resolved by assuming a compartmentalization of GSH in the cells, and that the effect of buthionine sulphoximine (BSO), used to deplete GSH from the cell is different in the different compartments (Edgren 1987b). The present experiments were conducted to test this possibility.

The cytotoxic effects of the bifunctional DNA-reactive drugs cisplatin, mustine and melphalan were studied in two human melanoma cell lines, RPMI 8322 and A 375. A 375 cells were 3.0 times more sensitive to cisplatin and 1.5 times more sensitive to mustine than RPMI 8322 cells. In contrast, A 375 cells were less sensitive to melphalan than RPMI 8322 cells. The increased sensitivity of A 375 cells was parallelled by an increased induction of DNA interstrand crosslinks following exposure to cisplatin and mustine. After cisplatin exposure A 375 cells also showed higher levels of platinum-DNA intrastrand adducts than RPMI 8322 cells. The increased effect of cisplatin in A 375 cells was not due to an increased drug accumulation in these cells. The higher sensitivity of A 375 cells to cisplatin may be related to lower intranuclear levels of glutathione, compared to RPMI 8322 cell nuclei, while the sensitivity to mustine may depend on lower overall levels of glutathione than in RPMI 8322 cells.

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.

SummaryTreatment 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.

The effect of cysteamine and 2-mercapto-propionyl-glycine on the induction and repair of single-strand breaks in the DNA of Chinese hamster cells by irradiation in vitro in the presence of oxygen or in anoxia was investigated. The substances protected against the formation of breaks by irradiation both under oxic and anoxic conditions. The substances also exhibited a toxic effect which resulted in degradation of DNA. Cysteamine inhibited the rejoining of breaks after radiation exposure.

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.

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.

Evidence obtained in different types of experiments has indicated that glutathione (GSH) can function as an inherent protector of cells against treatment with radiation and many chemotherapeutic agents (Arrick & Nathan, 1984). In an attempt to enhance the effect of such treatments, efforts have been made to control the cellular GSH content artificially. In many of the recent studies, buthionine sulphoximine (BSO) has been used to deplete cellular GSH (Chemical Modifiers of Cancer Treatment, 1986). This substance inhibits the synthesis of gamma-glutamyl-cysteine, a precursor of GSH, by specifically blocking the synthesising enzyme. Edgren (1987) reported observations which suggest that depletion of GSH by BSO occurs to different extents in the nucleus and the cytoplasm. The experiments reported here confirm the earlier observations either when treatment is made with BSO at a single concentration for varying time periods, or at varying concentrations for a fixed period.

Oxygen enhancement ratios (OER) determined with single-strand DNA breaks as end-point of radiation effect were lower for cells with genetically determined glutathione (GSH) deficiency, than for cells depleted of GSH artificially to a corresponding degree by treatment with buthionine sulfoximine (BSO). In view of a considerable non-protein thiol (NPSH) accumulation found in both cases, the difference can be attributed to different NPSH components. Those in the BSO-treated cells having the capacity to substitute for GSH in the processes which define OER. As an alternative explanation, the particular location of GSH in GSH-proficient cells in close contact with the critical radiation target can be considered. At these sites, as indicated by measurements of NPSH in isolated nuclei, BSO may not deplete thiols as efficiently as at other sites. Observations on a persisting difference found between the OER of genetically GSH-deficient and proficient cells after treatment with dithiothreitol (DTT) can also be given a spatial explanation, assuming that exogenous thiols may not reach the critical sites in the nucleus in efficient concentrations.

The role of glutathione (GSH) in the rejoining of radiation-induced single-strand DNA breaks (ssb) was studied in human fibroblast cultures sensitized to radiation by a 30 min treatment with 1 mM misonidazole (MISO). Hypoxically irradiated cells, deficient in GSH, either inherently, or due to a 16 h incubation with 1 mM buthionine sulphoximine (BSO), rejoined the breaks after MISO treatment at a lower rate and to a lesser extent than did GSH-proficient cells. Without MISO treatment, the hypoxically induced ssb were rejoined in the GSH-deficient cells as effectively as in the proficient cells. It is concluded that a large proportion of the breaks which arise after hypoxic irradiation in the presence of MISO are of a different type to those which arise in the absence of the drug, and require a particular GSH-dependent, enzymatic repair system. This requirement for rejoining in hypoxically irradiated, MISO-treated cells is similar to that seen earlier in MISO-untreated, oxically irradiated cells, and suggests that the ssb induced by radiation in the presence of MISO or oxygen are of a similar nature.

An intracellular radiation-chemical reaction scheme is tested in which solute and solvent radicals R. react with non-target molecules Sa (scavengers) or with target molecules (presumed to be DNA) to produce target radicals T., which may also be produced by direct ionization of DNA. The rate of target radical decomposition to become 'uncommitted damage' that the cell may repair is affected by the concentration of oxygen (O2), thiols (S) and electronaffinic sensitizers (F), which compete with one another to form, respectively, target products TO2, TS and TF. This uncommitted damage is then subject to biochemical modification, including molecular repair, by the cell. The rate equations for this competing reaction scheme were written and programmed for computer simulations of changes in oxygen, thiol and electronaffinic sensitizer concentrations. A reaction scheme that also includes some non-radical target damage was also simulated. Simulations were made using available experimental data concerning intranuclear concentrations and reaction rate constants, respectively, ko, ks and k1 for the reactions T. + O2----TO2, T. + S----TS and T. + F----TF, which produce uncommitted chemical damage. Experimental data on strand-break induction in glutathione-proficient and glutathione-deficient cells, in cells treated with thiol active agents, and in cells treated with hypoxic sensitizers, along with the computer simulations, generally agree that thiol molecules can react with target radicals to reverse T. in competition with O2 and/or electronaffinic sensitizers. Forward reaction rate constants ko, ks (dithiothreitol), ks (glutathione) and k1 (misonidazole) in the approximate ratio 10:0.3:0.02:0.4 satisfied the above reaction scheme, and approximately 5 per cent non-radical target molecule damage could be included with satisfactory agreement with experimental data.

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...

Recent observations in experiments with glutathione-deficient (GSH-) human fibroblasts indicate that endogenous glutathione (GSH) is involved in the development of radiation damage which leads to single-strand DNA breaks (SSB) and cellular survival or lethality (Edgren et al, 1980, 1981; Deschavanne et al, 1981a, b). It has been suggested that GSH may repair radiation-induced radicals by hydrogen donation, and may participate also in some enzymatic repair processes, specifically those concerned with the rejoining of radiation induced SSB.