DBS and diathermy interaction induces severe CNS damage.

Pulse-modulated radiofrequency diathermy treatment to the maxilla produced permanent diencephalic and brainstem lesions and a vegetative state in a patient with PD with implanted subthalamic electrodes for deep brain stimulation.

Increased collagen production in fibroblasts cultured from irradiated skin and effect of TGF beta(1)- clinical study.

Fibrosis in normal tissues is a common and dose-limiting late complication of radiotherapy at many cancer sites, but its pathogenesis is poorly understood. We undertook a controlled study of the effect of irradiation on the collagen production of fibroblasts cultured from skin biopsies taken from patients undergoing radiotherapy treatment. Eight weeks after a single 8 Gy fraction using 300 kV X-rays, five patients treated at the Royal Marsden Hospital underwent biopsy of the irradiated site and of the contralateral, unirradiated body site. Fibroblasts from irradiated and control, unirradiated sites were cultured in vitro, and collagen production rates were measured during a 48-hour incubation under standardized conditions and in the presence and absence of transforming growth factor beta(1)(TGF beta(1)), 1 ng/ml, using HPLC. Collagen production was elevated in cells cultured from irradiated skin; median collagen production rates 61.16 pmoles hydroxyproline/10(5)cells/hour in irradiated cells, 39.78 pmoles hydroxyproline/10(5)cells/hour in unirradiated cells, P = 0.016 (Mann-Whitney U-test). In fibroblasts from unirradiated sites, collagen production rates were increased by the addition of TGF beta(1); however, in three of the cell lines cultured from irradiated sites this effect of TGF beta(1)on collagen production was not observed.

The influence of hypoxia on the relative sensitivity of human tumor cells to 62.5 MeV (p-->Be) fast neutrons and 4 MeV photons.

Fast neutrons have been used in the clinical radiation therapy of tumors largely because of experimental evidence that their cytotoxic effects are much less dependent on oxygen levels than those of low-LET photons. The potential therapeutic advantage of fast neutrons based on hypoxia alone can be calculated as the "hypoxic gain factor", which is the ratio of the OERs for the fast-neutron compared to the photon beams. The hypoxic gain factor that is generally anticipated based on studies with established mammalian cell lines is about 1.6. However, surprisingly few studies have examined the influence of hypoxia on the fast-neutron radiosensitivity of human tumor cells of different histological types. For this reason, we have determined the OERs of five human tumor cell lines exposed to 62.5 MeV (p-->Be) cyclotron-generated fast neutrons or 4 MeV photons from a clinical linear accelerator. The OERs for four chemotherapy-naive cell lines, HT29/5, Hep2, HeLa and RT112, were invariably greater for photons than for neutrons, but all of these values were lower than expected on the basis of the previous literature. Despite their low OERs, these cell lines showed hypoxic gain factors that were within the range of 1.31-1.63, indicating that such effects cannot entirely explain the disappointing clinical results obtained with fast neutrons. In contrast, comparison of the surviving fractions at clinically relevant doses (1.6 Gy of neutrons and 2.0 Gy of photons) for these four tumor cell lines suggested that little benefit should result from neutron treatment. Only the cisplatin-resistant OAW42-CP line showed a significant hypoxic gain factor by this method of analysis. We conclude that, at the dose fractions used in clinical radiation therapy, there may not be a radiobiological precedent for higher local control rates after fast-neutron irradiation of hypoxic tumor cells.

Spinal cord infarction during use of zolmitriptan: a case report.

A 50-year-old woman with a history of migraine without aura, predominantly occurring around her menstrual periods, developed a spinal cord lesion following the use of zolmitriptan. The partial lesion of the cord at T7 predominantly involved the spinothalamic pathways on the left side. Clinical features suggested that the lesion was an ischemic infarct, and this was confirmed by an MRI scan. There were no other known risk factors for vascular disease. There has been mild improvement of her symptoms, but most of the symptoms did not resolve. There are isolated case reports of stroke secondary to the use of triptans, however, this is the first case of spinal cord infarction reported following the use of this group of drugs. The temporal relationship suggests that the spinal cord infarction may be related to the use of zolmitriptan.

Comparison of DNA repair protein expression and activities between human fibroblast cell lines with different radiosensitivities.

In order to investigate the molecular basis of variation in response to ionising radiation (IR) in radiotherapy patients, we have studied the expression of several genes involved in DNA double-strand break repair pathways in fibroblast cell lines. Ten lines were established from skin biopsies of cancer patients with different normal-tissue reactions to IR, and 3 from a control individual. For all 10 test cell lines, the cellular radiosensitivity was also known. Using Western blots we measured, in non-irradiated cells, the basal expression levels of ATM, Rad1 and Hus1, involved in the control of cellular DNA damage checkpoints, together with DNA-PKcs, Ku70, Ku80; XRCC4, ligaseIV and Rad51, involved in radiation- induced DSB repair. We also analysed the in vitro enzymatic activities, under non-irradiated conditions, of the DNA-PK and XRCC4/ligaseIV complexes. The levels of expression of the different proteins were similar in all the cell lines, but the activities of the DNA-PK and XRCC4/ligaseIV complexes showed some differences. These differences did not correlate with either the normal tissue response of the patient in vivo or with cellular radiation sensitivity in vitro. The activity differences of these enzyme complexes, therefore, do not account for the variation of responses seen between patients.

Describing patients' normal tissue reactions: concerning the possibility of individualising radiotherapy dose prescriptions based on potential predictive assays of normal tissue radiosensitivity. Steering Committee of the BioMed2 European Union Concerted Action Programme on the Development of Predictive Tests of Normal Tissue Response to Radiation Therapy.

Clinical radiotherapeutic doses are limited by the tolerance of normal tissues. Patients given a standard treatment exhibit a range of normal tissue reactions, and a better understanding of this individual variation might allow for individualisation of radiotherapeutic prescriptions, with consequent improvement in the therapeutic ratio. At present, there is no simple way to describe normal tissue reactions, which hampers communication between clinic and laboratory and between groups from different centres. There is also no method for comparing the severity of reactions in different normal tissues. This arises largely because there is no definition of a "normal" reaction, an "extreme" reaction or the particular term "over-reactor" (OR). This report proposes definitions for these terms, as well as a simple terminology for describing normal tissue reactions in patients having radiotherapy. The "normal" range represents the individual variation in normal tissue reactions amongst large numbers of patients treated in the same way which is within clinically acceptable limits. The term "OR" is applied to an individual whose reaction is more severe than the normal range but also implies that this forced a major change in the radiotherapeutic prescription or that the reactions were very severe or fatal. A "severe OR" would develop serious problems with a typical radical dose, while an "extreme OR" would have such difficulties at a much lower dose. To describe the normal range, a numerical scale is suggested, from 1 to 5, resistant to sensitive. The term "highly radiosensitive" (HR) is suggested for category 5. An "informal" relative scale, as suggested here, is quick and simple. It should allow comparison between different hospitals, compensate for differences in radiotherapeutic dose and technique and allow comparison of reactions between different anatomical sites. It should be adequate for discriminating patients at the extremes of the normal range from those at the centre. It is hoped that the definitions and terminology proposed here will aid communication in the field of predictive testing of normal tissue radiosensitivity.

Comparison between pulsed-field gel electrophoresis and the comet assay as predictive assays for radiosensitivity in fibroblasts.

The radiosensitivity of skin fibroblasts derived from patients as measured in vitro by a clonogenic survival assay appears to correlate with the risk of developing severe late reactions to radiation. Unfortunately, these assays are clinically impractical as a predictive test for radiosensitivity. The purpose of this study was to assess the utility of two possible surrogate assays for radiosensitivity, pulsed-field gel electrophoresis (PFGE) and single-cell gel electrophoresis (comet assay), both of which can be used to measure DNA double-strand breaks. Twenty-three nontransformed human fibroblast cell lines exhibiting a range of radiosensitivities were studied with both of these assays. The results were correlated with measurements of radiosensitivity obtained as part of a larger study examining the correlation between cellular radiosensitivity and clinical response. [2-(14)C]Thymidine-labeled confluent cultures were irradiated at 1.0 Gy/min with doses of 0 to 150 Gy. After allowing 4 h for repair at 37 degrees C, cells were trypsinized and aliquots were used for preparing slides for the comet assay. After neutral lysis and electrophoresis, the slides were stained with ethidium bromide and 50 comet moments were measured for each dose. The remainder of the cells were formed into agarose plugs and, after neutral lysis, were subjected to PFGE. The fraction of activity released (FAR) from the well was measured by scintillation counting of appropriate segments of each gel lane. Cellular radiosensitivity was measured with a standard clonogenic assay at a low dose rate of 1.2 cGy/min, and the dose that resulted in a surviving fraction of 0.01 (D0.01) was calculated. The slope of the plot of comet moment as a function of dose for each cell line did not correlate with D0.01 (R = 0.36, P > 0.1). In contrast, the slope of the FAR as a function of dose had a weak inverse correlation with D0.01 (R = 0.43 and P = 0.05) such that the more radiosensitive cell lines exhibited a steeper dose response for FAR. Although the correlation between the slope of the dose response for FAR and D0.01 was weak, refinement of the PFGE technique may provide a potentially useful predictive assay for radiosensitivity.

DNA damage and prediction of radiation response in lymphocytes and epidermal skin human cells.

The success of radiotherapy in eradicating tumours depends on the total radiation dose, but what limits this dose is the tolerance of the normal tissues within the treatment volume. Studies involving fibroblast survival have demonstrated the theoretical feasibility of a predictive assay of radiation sensitivity, but such an assay is still far from clinical application. Using pulsed-field gel electrophoresis (PFGE), we have quantified the initial "apparent" number of DNA double-strand breaks (dsb) induced by the radiation as an alternative measure of sensitivity in 2 different normal cell types from the same patients, epidermal skin cells and lymphocytes. We found significant inter-individual variation in the measured dsb (1-5 dsb/Gy/DNA unit). We also found a linear correlation between molecular damage in lymphocytes and skin samples from the same patient (slope = 0.83; r = 0.694; p = 0.0001). These results suggest that the initial number of dsb could be used as an indicator of the in vivo response to radiation.

Chromatin structure and cellular radiosensitivity: a comparison of two human tumour cell lines.

The role of variation in susceptibility to DNA damage induction was studied as a determinant for cellular radiosensitivity. Comparison of the radiosensitive HX142 and radioresistant RT112 cell lines previously revealed higher susceptibility to X-ray-induced DNA damage in the sensitive cell line using non-denaturing elution, but not when using alkaline unwinding. The present data also show that no difference in the amount of initial damage is seen when pulsed-field gel electrophoresis (PFGE) or comet analysis are used for DNA damage assessment. However, using the halo assay or a modified version of PFGE in which the higher DNA architecture remained partially intact, the radiosensitive cells showed steeper dose-response curves for initial DNA damage than the radioresistant cells. Analysis of the protein composition, of DNA-nucleoid structures revealed substantial differences when isolated from HX142 or RT112 cells. From our data, it is concluded that HX142 and RT112 differ in their structural organization of chromatin. As no differences in the kinetics of DNA damage rejoining were found, it is hypothesized that the same amount of lesions have a different impact in the two cell lines in that the 'presentation' of DNA damage alters the ratio of repairable to non-repairable DNA damage.

Low dose-rate fibroblast radiosensitivity and the prediction of patient response to radiotherapy.

The relationship between cellular radiosensitivity and normal-tissue response to radiotherapy in individual cancer patients has attracted increasing attention over the last few years. Recent work has suggested that a correlation exists between fibroblast sensitivity and normal-tissue reactions. We have examined the radiosensitivity of fibroblasts grown from skin biopsies of four normal individuals and three patients identified as having suffered unexpectedly severe reactions to clinical radiotherapy, called here 'over-reactor' (OR) patients. Clonogenic survival was measured after high (HDR) and low dose-rate (LDR) irradiation. By comparing the two, and LDR Recovery Factor was derived. Potentially-lethal damage repair was examined in 4 cell strains. After HDR the OR strains were indistinguishable from the normals. At LDR the range of sensitivity was expanded. The OR strains fell at the sensitive end of the range and were characterized by a lack of LDR recovery, which clearly distinguished them from the normal strains. Experimental errors were estimated by considering all the data sets together rather than viewing each experiment individually. Duplicate strains from several patients were tested, and the differences between them were found to be within the estimated experimental errors, suggesting that these differences were not biologically significant. The data are consistent with the hypothesis that normal-tissue response is linked to individual cellular radiosensitivity. Our data confirm the importance of using LDR irradiation in clinical investigations of cellular sensitivity.

Application of a bromodeoxyuridine-Hoechst/ethidium bromide technique for the analysis of radiation-induced cell cycle delays in asynchronous cell populations.

A flow cytometric technique utilizing the continuous incorporation of bromodeoxyuridine (BrdU) into asynchronous cells to measure radiation-induced cell cycle delay is described. Following the incorporation of the BrdU label the cells are stained with ethidium bromide and the bis-benzimidazole Hoechst 33258. These fluorochromes have differential staining patterns. Hoechst 33258 fluoresces blue and is quenched by BrdU incorporated into cellular DNA during S phase. Ethidium bromide fluoresces red and is not quenched by BrdU. Therefore in cells that are cycling and synthesizing DNA new G1 and G2 compartments are created and this can be used to measure cell cycle delays following ionizing radiation to asynchronous cells. We have used this technique to evaluate two cell lines: a normal diploid human embryo fibroblast cell line MRC 5, which has inducible p53 and shows delays at both G1 and G2 checkpoints, and the human cervix carcinoma cell line HX 156. This cell line has been infected with human papilloma virus (HPV) 16, and therefore has inactivated p53 function and is blocked only at the G2 checkpoint. Using this method, cell cycle-dependent effects relating to the G2 block can be observed. The radiation-induced G2 block differs from that induced by drugs or heating in that cells are blocked in G2 irrespective of the phase of the cell cycle they are treated in. This method allows these different types of G2 block to be quantified.

Normal tissue radiosensitivity--how important is it?

The success of radiotherapy in eradicating tumours depends on the total radiation dose, but what limits this dose is the tolerance of the normal tissues within the treatment volume. Selection of the appropriate dose for all patients is based on a balance between minimising the incidence of severe normal tissue complications and maximizing the probability of local control. In patients treated to the same radical dose, a wide range of reactions is seen; in many clinical situations, radical doses are limited by the minority of patients whose normal tissues are particularly sensitive. Clinical studies of radiotherapy reactions have demonstrated that a large part of the spectrum of normal tissue reactions, perhaps as much as 80%, is due to differences in individual normal tissue sensitivity. This suggests that it might be possible to measure this sensitivity and to change treatment accordingly. The main objective of normal tissue sensitivity testing is to permit dose escalation without increased normal tissue complication rates in patients with more resistant normal tissues. Calculations suggest that the most "resistant' 40% of patients could be dose escalated by 17%-18%, which is likely to be associated with significant gains in local control, perhaps by as much as 34%-36%; this should translate into an increase in overall survival. It should also be possible to identify those relatively few patients who suffer serious normal tissue morbidity with conventional doses. Thus, if successful, predictive testing of normal tissue response should improve the therapeutic ratio of radiotherapy.

Glutathione determination by the Tietze enzymatic recycling assay and its relationship to cellular radiation response.

Large fluctuations in glutathione content were observed on a daily basis using the Tietze enzyme recycling assay in a panel of six human cell lines of varying radiosensitivity. Glutathione content tended to increase to a maximum during exponential cell proliferation, and then decreased at different rates as the cells approached plateau phase. By reference to high-performance liquid chromatography and flow cytometry of the fluorescent bimane derivative we were able to verify that these changes were real. However, the Tietze assay was occasionally unable to detect glutathione in two of our cell lines (MGH-U1 and AT5BIVA), although the other methods indicated its presence. The existence of an inhibitory activity responsible for these anomalies was confirmed through spiking our samples with known amounts of glutathione. We were unable to detect a direct relationship between cellular glutathione concentration and aerobic radiosensitivity in our panel of cell lines.

Glutathione manipulation and the radiosensitivity of human tumour and fibroblast cell lines.

We have studied the role of glutathione (GSH) in determining radiation response in five human tumour and one human fibroblast cell line. GSH concentration was measured using the Tietze assay and compared with clonogenic survival following gamma-irradiation. No relationship between GSH concentration and aerobic radiosensitivity was observed. The addition of 10 mM extracellular cysteamine produced protection factors in all cell lines, ranging from 1.6 to 2.1, but had little influence on cellular GSH concentration. Depletion of GSH by buthionine sulphoximine (0.1 mM for 18 h) had negligible effect on cell survival, though moderate radiosensitization resulted from extreme GSH depletion after 30-min treatment with 1 mM dimethylfumarate. The degree of aerobic sensitization did not correlate with GSH levels. Irradiation under hypoxia produced oxygen enhancement ratios varying from 1.6 to 2.6, with no relationship to GSH content.

The relationship between cellular radiation sensitivity and tissue response may provide the basis for individualising radiotherapy schedules.

There is a wide variation in normal tissue reactions to radiotherapy and in many situations the severity of these reactions limits radiotherapy dose. Clinical fractionation studies carried out in Gothenburg have demonstrated that a large part of the spectrum of normal tissue reactions is due to differences in individual normal tissue sensitivity. If this variation in normal tissue reactions is due to differences in intrinsic cellular radiosensitivity, it should be possible to predict tissue response based on measurement of cellular sensitivity. Here we report the initial results of a study aimed at establishing whether a direct relationship exists between cellular radiosensitivity and tissue response. Ten fibroblasts strains, including four duplicates, were established from a group of patients in the Gothenburg fractionation trials who had received radiotherapy following mastectomy. Skin doses were measured and both acute and late skin changes were observed following radiotherapy. Right and left parasternal areas were treated with different dose fractionation schedules. Clonogenic assays were used to assess intrinsic cellular radiosensitivity, and all experiments were carried out without prior knowledge of the clinical response, or which strains were duplicates. Irradiation was carried out using 60Co gamma-rays at high dose-rate (HDR) of 1-2 Gy/min and low dose-rate (LDR) of 1 cGy/min. A spectrum of sensitivity was seen, with SF2 values of 0.17-0.28 at HDR and 0.25-0.34 at LDR, and values of D0.01 of 5.07-6.38 Gy at HDR and 6.43-8.12 Gy at LDR. Comparison of the in vitro results with the clinical normal tissue effects shows a correlation between cellular sensitivity and late tissue reactions, which is highly significant with p = 0.02. A correlation between cellular sensitivity and acute effects was noted in the left-sided parasternal fields, but not the right. This is thought to be coincidental, and without biological significance. Our results suggest that cellular sensitivity might form the basis for the development of an assay system capable of predicting late normal tissue effects to curative radiotherapy, which might allow dose escalation in some patients. Increased local control and cure, with unchanged or improved normal tissue complications, could result from such individualised radiotherapy prescriptions.

Relationship between chromosome aberrations, micronuclei and cell kill in two human tumour cell lines of widely differing radiosensitivity.

The relationship between chromosome aberration frequency, micronucleus (MN) formation and cell kill was investigated in two human tumour cell lines, RT112, a radioresistant bladder carcinoma cell line, and HX142, a radiosensitive neuroblastoma cell line. As MN are generally accepted to result from acentric chromosome fragments that are lost at nuclear division and all unstable aberrations result in an acentric fragment, a 1:1 correlation between aberrations and MN might be expected. There was a good correlation between cell kill and aberration frequency in the two cell lines, however the relationship between cell kill and MN formation in the lines was very different. RT112 showed 0.22 aberrations and 0.21 MN per Gy whilst HX142 showed 0.61 aberrations and 0.078 MN per Gy. Possible reasons for the discrepancy observed in HX142 are discussed.