John J. Eady

Cellular radiosensitivity and complication risk after curative radiotherapy

PURPOSE To test for an association between in vitro fibroblast radiosensitivity and complication risk in a case-control study of breast cancer patients treated under standard conditions in a clinical trial of radiotherapy dose fractionation. PATIENTS AND METHODS A cohort of patients participating in a randomised clinical trial of radiotherapy dose fractionation was selected on the basis of treatment-induced changes in the breast several years later. Thirty-nine cases with marked normal tissue changes were matched on several variables with 65 controls with no changes attributable to radiotherapy. Dermal fibroblast strains were established from duplicate skin biopsies, and clonogenic cell survival assays performed in triplicate after both high ( approximately 1.6 Gy/min) and low ( approximately 1 cGy/min) dose-rate irradiation. Laboratory studies were blind to patient identity, treatment outcome and radiotherapy schedule. RESULTS Analysis of 1128 clonogenic survival curves confirmed significant inter-patient variation in fibroblast radiosensitivity as measured by clonogenic survival. However, no association between fibroblast radiosensitivity and the development of late radiotherapy normal tissue effects was detected. CONCLUSIONS Inter-individual variation in cellular radiosensitivity may not be the main determinant of complication risk in patients undergoing radiotherapy for breast cancer. Other biological and technical factors may be more important in explaining the marked inter-patient differences in normal tissue damage evident several years after curative radiotherapy.

The Intrinsic α/β Ratio for Human Tumour Cells: Is It a Constant?

The radiation response of 15 mammalian cell lines comprising 11 human tumour, two human fibroblast and two murine lymphoma cell lines, has been analysed using the linear-quadratic equation. As well as using conventional analysis of acute dose-survival curves to derive values for α and β (termed αac and βac), low dose-rate and split-dose experiments have been used to derive independent values of α and β (αldr and βRR), respectively. αldr provides a measure of irrecoverable damage, the magnitude of which agreed well with the initial slope of the acute survival curve for most cell lines. βRR derived from split-dose experiments represents a unique measure of recovery for each cell line. Large differences were found between individual values of βac and βRR, especially in the radiosensitive cell lines. Since βRR is a functional measure of recovery we suggest that this is the more relevant parameter in studies of dose sparing. The most striking result of this analysis was found in considering the α/β ratios. No ...

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.

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.

Split-dose and Low Dose-rate Recovery in Four Experimental Tumour Systems

The radiation dose-rate effect has been investigated in three murine tumour cell systems (MT carcinoma, Lewis lung tumour, B16 melanoma) and in the HX34 human melanoma xenograft taken directly from mice and irradiated in vitro. The four tumour types were remarkably similar in their radiation response characteristics, especially at low dose rate; the Lewis lung tumour tended to be the most radiosensitive at high dose rate. The data have been analysed using the Lethal-Potentially Lethal (LPL) model of Curtis and the Incomplete Repair (IR) model of Thames. The data are equally well fitted by both models. The most remarkable feature of these analyses is that both models lead to estimates for the half-time for recovery that are in the region of 0.1 h, considerably shorter than other published values. Split-dose experiments were also performed, taking care to keep the cells at 37 degrees C between exposures. In all cases the split-dose half times were longer than the values derived from dose-rate analysis and in the case of the Lewis lung and HX34 tumour lines the difference was by almost a factor of ten. The discrepancy between these estimates could be the result of biphasic cellular recovery.

Cellular Recovery in Two Sub-lines of the L5178Y Murine Leukaemic Lymphoblast Cell Line Differing in Their Sensitivity to Ionizing Radiation

Cellular recovery was assessed in two sublines of L5178Y murine lymphoma cells of differing radiosensitivity (LY-S and LY-A4) using low dose-rate irradiation and split-dose experiments. No increase in cell survival was observed in the LY-S cell line until the dose-rate was reduced to 2 cGy/min, whereas in the LY-A4 cell line 20 cGy/min was low enough to detect changes in survival. The extent of this change, as assessed by dose reduction factors at 2 logs of cell kill, was greater in the LY-A4 cell line. Fitting these data with the incomplete repair model of Thames led to anomalous values for the half-time of repair. In split-dose experiments the maximum observed recovery ratio increased as a function of dose in a manner that is consistent with the linear-quadratic equation. As was found previously with radiosensitive human tumour cells, the LY-S cell line showed more split-dose recovery at any given dose than the LY-A4 cell line.