Application of Pulsed Field Gel Electrophoresis to Determine γ-ray-induced Double-strand Breaks in Yeast Chromosomal Molecules

The frequency of DNA double-strand breaks (dsb) was determined in yeast cells exposed to γ-rays under anoxic conditions. Genomic DNA of treated cells was separated by pulsed field gel electrophoresis, and two different approaches for the evaluation of the gels were employed: (1) The DNA mass distribution profile obtained by electrophoresis was compared to computed profiles, and the number of DSB per unit length was then derived in terms of a fitting procedure; (2) hybridization of selected chromosomes was performed, and a comparison of the hybridization signals in treated and untreated samples was then used to derive the frequency of dsb

Repair of potentially lethal damage in unfed plateau phase cultures of ehrlich ascites tumour cells: II. Monolayer cultures.

Monolayer cultures of EAT cells when plated immediately after irradiation show a decrease in survival as they age in the plateau phase of growth. This decrease, which is manifest as a diminution of the shoulder width of the survival curve down to values approaching zero, is reversible if the cells are kept in their growth medium for some hours after irradiation before trypsinization and plating. Survival curves obtained by this holding procedure are similar in shape to those shown by exponentially growing or early plateau phase cells. We interpret this effect in terms of repair of potentially lethal damage which occurs after immediate plating in young cultures but only declared during plating in cultures which have aged in the plateau phase. The kinetics of this repair and the effects caused by the addition of serum after irradiation in the cultures have been studied

The mutagenicity of alpha particles in Ehrlich ascites tumor cells.

Cell killing and the induction of mutation to thioguanine resistance (HGPRT enzyme deficiency) were measured after exposure of Ehrlich ascites tumor cells to 150-kV X rays and 241Am α particles. The curve describing the induction of mutations was almost linear after exposure to α particles (slope: 14.1 x 10-5 Gy-1) but upward bending after exposure to X rays, apparently reaching a final slope similar to that obtained after exposure to α particles. The number of mutants induced per viable cell by α particles at a given level of cell killing was similar to that induced by X rays. The RBE values obtained for cell killing and the induction of mutations are compared with each other, and the possible involvement of repair processes in determining the RBE is discussed

Evidence that repair and expression of potentially lethal damage cause the variations in cell survival after X irratiation observed through the cell cycle in Ehrlich ascites tumor cells.

The survival of synchronously growing Ehrlich ascites tumor cells (EAT cells) was measured after X irradiation in various stages of the cell cycle. Cells at the beginning of S or in G2 + M phase showed a high level of killing, whereas cells irradiated in G1 or in the middle of S phase were more resistant. These changes resulted fom a change in the survival curve shoulder width (D(q)) as cells passed through the cell cycle, and the mean lethal dose (D(0)) remained practically unchanged (0.8 ± 0.05 Gy). When synchronization of the cell population was further sharpened using nocodazole, exponential survival curves were obtained at the beginning of S phase and at mitosis with a D(0) = 0.8 Gy. When cells (in all stages) were incubated in balanced salt solution for 6 hr after irradiation, repair of potentially lethal damage (PLD) was observed, resulting in an increase in D(q), while D(0) remained constant. Treatment of the cells after irradiation with either caffeine (2-6 mM) or β-arabinofuranosyladenine (β-araA) (60-100 μM) or hypertonic medium resulted in an expression of PLD and reduced the D(q) of the survival curve, which approached or reached an exponential line with D(0) = 0.8 ± 0.1 Gy. We measured the rate of the loss of sensitivity of these treatments that we assume reflects the rate of repair of PLD. For caffeine (6 mM) treatment (S cells, 5 hr) we found a repair time constant (t50) or about 1 hr, similar to that observed for repair of PLD in growth medium containing 0.5 μg/ml aphidicolin. With hypertonic treatment we detected two repair components, a fast one that restored the slope of the survival curve, and a slow one with a t50 of about 1 hr that restored the shoulder of the survival curve. PLD induced by irradiating in G1 phase was repaired when cells were arrested for some hours either in G1 phase or in the subsequent mitosis but was not repaired if the cells were arrested in S phase. PLD induced in S or G2 + M phase was repaired only when the cells were arrested in the cell cycle before division. Results indicate that the shoulder width D(q) of the survival curve in cells irradiated at various stages of the cell cycle results from repair of PLD. This repair of PLD probably takes place in the interval between irradiation and the next S phase or mitosis and is therefore minimal for cells irradiated at the G1/S border or in mitosis (D(q) = 0). PLD still unrepaired when the cells reach these phases is assumed to be expressed, as was found for PLD repaired in cells incubated in balanced salt solution, for some hours after irradiation. We therefore suggest that the variations observed in cell survival through the cell cycle might reflect variations in the final amount of PLD either repaired or expressed as the cells progress through the various stages of the cell cycle

Aphidicolin promotes repair of potentially lethal damage in irradiated mammalian cells synchronized in S-phase.

Aphidicolin, an inhibitor of the α-polymerase in mammalian cells, at a concentration of 0.5 μg/ml, is shown to enable cells which are growing exponentially and synchronized in the S-phase of the cell cycle, to repair potentially lethal damage caused by exposure to either x-rays or UV light. The drug holds cells up in the S-phase which may serve to allow time for repair and could prevent fixation of damage which may occur when the cells progress through the cell cycle. The possible involvement of α- and β-polymerase in repair of potentially lethal damage is discussed

Effects of the nucleoside analogues α-ara A, β-ara A and β-ara C on cell growth and repair of both potentially lethal damage and DNA double strand breaks in mammalian cells in culture.

The effects of the DNA polymerase inhibitors a-ara A and beta-ara C have been compared with those of beta-ara A on the endpoints of cell growth, repair of x-ray induced potentially lethal damage (PLD) and repair of x-ray induced DNA double strand breaks, a-ara A was found to have no effects on any of the endpoints studied. beta-ara C inhibited cell growth and DNA double strand breaks repair more strongly than beta-ara A but it was less effective in inhibiting repair of PLD. The inhibition of PLD repair by beta-ara C resulted in a diminution of the shoulder width of the x-ray survival curve, a result similar to that previously found for beta-ara A. The effectiveness of beta-ara C was enhanced when cells were treated with the drug in fresh medium rather than under plateau phase conditions. The lower effectiveness of beta-ara C, when compared with beta-ara A in causing expression of PLD, is interpreted in terms of a difference in the ability of the two drugs to cause fixation or misrepair of the DNA double strand breaks during or after treatment with the drugs

Clinical significance of optic disc progression by topographic change analysis maps in glaucoma: An 8-year follow-up study

Aim. To investigate the ability of Heidelberg Retina Tomograph (HRT3) Topographic Change Analysis (TCA) map to predict the subsequent development of clinical change, in patients with glaucoma. Materials. 61 eyes of 61 patients, which, from a retrospective review were defined as stable on optic nerve head (ONH) stereophotographs and visual field (VF), were enrolled in a prospective study. Eyes were classified as TCA-stable or TCA-progressed based on the TCA map. All patients underwent HRT3, VF, and ONH stereophotography at 9-12 months intervals. Clinical glaucoma progression was determined by masked assessment of ONH stereophotographs and VF Guided Progression Analysis. Results. The median (IQR) total HRT follow-up period was 8.1 (7.3, 9.1) years, which included a median retrospective and prospective follow-up time of 3.9 (3.1, 5.0) and 4.0 (3.5, 4.7) years, respectively. In the TCA-stable eyes, VF and/or photographic progression occurred in 5/13 (38.4%) eyes compared to 11/48 (22.9%) of the TCA-progressed eyes. There was no statistically significant association between TCA progression and clinically relevant (photographic and/or VF) progression (hazard ratio, 1.18; P=0.762). The observed median time to clinical progression from enrollment was significantly shorter in the TCA-progressed group compared to the TCA-stable group (P=0.04). Conclusion. Our results indicate that the commercially available TCA progression criteria do not adequately predict subsequent photographic and/or VF progression. © 2014 D. Kourkoutas et al