The effect of repeated small doses of radiation on recovery from sub-lethal damage by Chinese hamster cells irradiated in the plateau phase of growth.

Unfed plateau-phase cells have been irradiated with either single doses or up to ten fractions of X-rays 6 hours apart. The single-dose survival curve had an extrapolation number of 11-4, and the oxygen-enhancement ratio (o.e.r.) was 3-1. Cells were exposed to multiple fractions of 200 rad or 150 rad in air and 600 rad or 450 rad in hypoxia. The resulting survival curves did not fit a multi-target, single-hit model of cell survival, being much steeper than that would predict. The curves were exponential up to five fractions of X-rays, but tended to bend downwards with increasing number of fractions. Cells that had survived five fractions of 200 rad (or 600 rad in hypoxia) 6 hours apart, were less able to absorb damage as sub-lethal than those which had not previously been exposed to radiation. The ratio of the initial slopes of the fractionated survival curves for irradiation in air and hypoxia was 2-1, implying that the o.e.r. "on the shoulder" may be less than that in the exponential region of survival.     

Modification of radiation response in HeLa cells by misonidazole.

The hypoxic radiosensitizer misonidazole decreases survival in dense cultures of HeLa cells irradiated with gamma rays in a non-dose modifying fashion. Survival curves of treated hypoxic cells display a much larger extrapolation number than untreated cells. In oxygenated randomly dividing cells, drug treatment has an effect opposite to that in hypoxic cells, increasing survival. In cultures initiated from mitotic cells and irradiated soon afterwards, a smaller sensitization under hypoxia and no increase in survival of oxygenated cells was observed. It was concluded that metabolic as well as radiochemical events take place in misonidazole-treated and then irradiated HeLa cells which modify survival.     

Dependence of the nucleoside effect on linear energy transfer.

L929 cells were irradiated by cyclotron-produced neutrons and by 14.8 MeV monoenergetic neutrons. For comparison cells were also irradiated by 60Co gamma rays. Following irradiation cells were treated by an equimolar solution of deoxyribonucleosides, and the effect on cell survival measured. Results show that nucleoside treatment was efficient after low-LET irradiation: gamma ray survival curves were altered by deoxyribonucleosides in terms of significantly increased extrapolation numbers only, but without Do change. Cells irradiated by neutrons from either of the two sources did not respond to nucleoside treatment, and consequently their survival curves remained unaltered. These results show that the nucleoside effect does occur after low-LET irradiation, but apparently not following high-LET irradiation. Since deoxyribonucleosides as well as other cell breakdown products are released in irradiated and necrotic tumours due to massive cell destruction, such a nucleoside effect could possibly enhance the cell survival and thus effect the result of radiotherapy. Absence of the nucleoside effect in case of high-LET irradiation may therefore be an additional potential gain from neutrons in radiotherapy.     

The Effect of Freezing on the Radiation Sensitivity of Vegetative Bacteria

S ummary : Five strains of bacteria were irradiated, suspended in heart infusion broth or in phosphate buffer, in aerated or anoxic conditions, at temperatures of 10–13° or -79°. Survivors under the different conditions were enumerated by plate counts on heart infusion agar.
Exponential survivor-dose curves were obtained with a Pseudomonas strain and with Escherichia coli B/r when irradiated at room temperature with aeration, whereas an Alcaligenes strain and 2 strains of Streptococcus faecium gave sigmoid curves. The decreased radiosensitivity in the frozen state was measured by comparing the D₁₀ values for exponential curves, or for the exponential portion of sigmoid curves, with that observed for irradiation at room temperature with aeration. This 'D₁₀ ratio' svaried between 2°5 and 8·5. For the Alcaligenes strain it was about 4, whether the frozen irradiation took place in the presence or absence of oxygen. With the Pseudomonas irradiated in the frozen state in the absence of oxygen the 'D₁₀ ratio' was usually about 1·5 times higher than when oxygen was present. The highest ratio (8·5) was obtained for anoxic irradiation of the Pseudomonas strain.
In general, the shapes of survival curves for frozen irradiation differed from those obtained at room temperature. The sigmoid curves for the Alcaligenes strain irradiated under aerobic conditions when frozen showed a marked decrease in extrapolation numbers. E. coli B/r when frozen in heart infusion broth gave a double exponential curve with a shallow slope initially, followed by a steeper slope. The most radiation resistant strain, Strep. faecium R53, gave sigmoid curves with a D₁₀ value of 300 Krads when frozen, and was then of similar resistance to Clostridium botulinum spores.     

Response of lymphosarcoma LS/BL cells to continuous irradiation

Mouse lymphosarcoma LS/BL cells growing as an ascites tumor in the peritoneal cavity of C57BL mice were continuously irradiated in vivo at a low exposure rate of 1.2 Gy per day (5 rad/hr). The growth of the ascites tumor evaluated by direct counting of the cells in the peritoneal cavity and their capacity to form colonies in livers declined with increasing time of continuous irradiation. The radiosensitivity and repair ability of LS/BL cells were studied by a serial dilution method using host survival time as the end point and by the liver colony assay. The radiosensitivity of continuously irradiated LS/BL-CI cells showed no remarkable change as measured by the Do values, but from the 150th week of irradiation the initial shoulder on the survival curves appeared and its width increased with time of exposure. The extrapolation number (n) increased from 1.0 to 8.4 after 350 weeks of irradiation. The reappearance of the initial shoulder was proved with the split-dose technique.     

X-Ray and Ultraviolet Sensitivity of Synchronized Chinese Hamster Cells at Various Stages of the Cell Cycle

Populations of Chinese hamster cells, synchronized by selecting for cells at or close to division, were exposed to 250 kvp x-rays and to ultraviolet light at different stages of the cell cycle and colony-forming ability examined thereafter. These cells were found to be most resistant to x-rays during the latter part of the DNA synthetic period (S) and to be about equally sensitive before (G₁) and after (G₂) this period. Multitarget type curves of the same slope (D_o ~ 200 rad) only approximately fitted the survival data at different stages in the cycle. The changes in response were primarily due to variations in the shoulders (or extrapolation numbers) of the curves however. The response to ultraviolet light differed from that to x-rays. Resistance was greatest in G₂ and changes in both shoulder and slope of the survival curves occurred throughout the cell cycle. The x-ray and ultraviolet responses for component stages of the cell cycle were respectively compounded into expected survival data for a log phase asynchronous population of hamster cells and found to agree well with direct experiment.     

Radiosensitivity and recovery of mouse L cells during the cell cycle

Summary Mouse fibroblasts, subline L-929 F were synchronized by mitotic detachment. The synchronized cell cultures were irradiated with 200 kVp X-rays at different time after mitosis, and age reponse functions and dose effect curves were determined using the colony test. The cell age in the mitotic cycle was obtained from a computer analysis of flow cytometric DNA histograms. Both intrinsic radiosensitivity 1/D ₀ and extrapolation numbern were found to vary during the cell cycle. TheD ₀ has a maximum value of 176 ± 1 rad in the middle ofG ₁ phase and a minimum of 71 ± 1 rad at theS/G ₂ transition, while the extrapolation number is rather constant from the beginning ofG ₁ phase (1.9 ± 0.1) to the middle ofS phase (2.3 ± 0.1) and reaches a steep maximum of 9.3 ± 1.1 atS/G ₂ transition. The values ofn in the various phases of cell cycle are compared with the respective values of the recovery factor determined after fractionated irradiation. - Cell survival after a single dose of 616 rad has minima for irradiation atG ₁/S transition and in earlyG ₂ phase; the survival in earlyG ₂ being about 40 times smaller than in earlyG ₁ phase. Implications for a cell cycle specific therapy are discussed.Supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg     

Evidence for reduced capacity for damage accumulation and repair in plateau-phase C3H 10T1/2 cells following multiple-dose irradiation with gamma rays

The effects of multiple-dose gamma irradiation on the shape of survival curves were studied with mouse C3H 10T1/2 cells maintained in contact-inhibited plateau phase. The dose-fractionation intervals included 3, 6, and 24 h. Following three fractionated doses (5 Gy per dose) of exposures, cells responded to further irradiation by displaying a survival curve with a much reduced shoulder width (Dq) compared to that of the survival curve measured in cells irradiated with single-graded doses alone. The effect on the mean lethal dose (D0) was small and appeared to be significant. The effect on reduction of Dq could not be completely overcome by lengthening the fractionation intervals from 3 to 6 h or 24 h, times in which repair of sublethal damage (SLD) measured by simple split-dose scheme and potentially lethal damage (PLD) measured by postirradiation incubation was completed. Other experiments showed that pretreatments of cells with fractionated irradiation appeared to slow down the cellular repair processes of SLD and PLD. Therefore, the observed change in the shape of survival curves after fractionation treatments may be attributed to a reduction of the cells' capacity for damage accumulation by an enhancement of the lethal expression of SLD and PLD. Although the molecular mechanism(s) is not known, the results of this study indicate that the acute graded dose-survival curve cannot be used a priori to extrapolate and reliably predict results of hyperfractionation. It is probable that for a nondividing or slowly dividing cell population, such an extrapolation may lead to an underestimation of cell killing. Furthermore, the findings of this investigation appear to support an interpretation, alternative to the high-linear energy transfer (LET) track-end postulate, for the effects on cell survival seen at low doses or low dose rates.     

Method for probing cells in radiation-induced G2 arrest: demonstration of potentially lethal damage repair

Chinese hamster ovary cells were arrested in the G2 phase of the cell cycle by X-irradiation. When subsequently treated with 5 mM caffeine the arrested population progressed into mitosis as a synchronous cohort where it was harvested by mitotic cell selection. This procedure provides a means to isolate cell populations treated in G2, for the investigation of G2 arrest. Comparisons were made of the number of cells retrieved from G2 arrest with the number suffering arrest, as determined by flow cytometry and by matrix algebraic simulations of irradiated cell progression. The retrieved population was not significantly less than expected for doses up to 3.5 Gy, indicating that the retrieval process does not favour the isolation of any population subset below this dose. Cell populations retrieved from arrest at varying intervals (0-3 h) after irradiation (0-3.5 Gy) showed an increase in survival with increase in interval, consistent with repair of potentially lethal damage. The repair curves (surviving fraction vs time) were each described by a single exponential. G2 cells that were brought to mitosis without a period of arrest exhibited the same radiation response as cells irradiated in mitosis.     

Sensitivity to ultraviolet radiation as a function of DNA content in Escherichia coli B/r.

Populations of Escherichia coli B/r A were grown to log phase at various growth rates determined by the richness of the medium. The genome content, G, was calculated from log phase doubling times by means of the Cooper-Helmstetter formula. Cell volumes were measured and found to vary linearly with this genome content. Cells with various DNA contents were prepared for ultraviolet irradiation and plated for dark repair under similar conditions. The resulting logarithmic survival curves were all similar in shape: convex up, with straight line portions having approximately the same slope (D0 = 11.4 +/- 0.2 J/m2). The shoulders however increase in width with calculated DNA content giving an extrapolation number which varies roughly as exp(G) or exp (0.6 Gmax).     

Independent effect of a mixed-beam regimen of fast neutrons and gamma rays on a murine fibrosarcoma

Biological effectiveness of a mixed-beam regimen of fast neutrons and photons was studied in an animal tumor system. NFSa , a spontaneous fibrosarcoma in a C3H mouse, was transplanted in the right hind legs of syngeneic male mice and locally irradiated with a single dose or five daily doses. Tumor control experiments showed that five gamma-ray doses increased TCD50 values by 20 Gy and produced a shallower slope on the dose-response curve compared to that after a single fraction. Fractionated neutron doses also increased the TCD50 value by 9 Gy without changing the slope of the dose-response curve. A mixed-beam regimen of N-gamma-gamma-gamma-N resulted in an independent effect on the tumor. Second, tumor cell survival was examined by the lung colony assay. Nembutal anesthesia reduced the tumor oxic cell fraction, resulting in a single component dose-response curve after a single gamma ray. Five fractionated doses of gamma rays increased both D0 and extrapolation number while those of fast neutrons increased only extrapolation number. The D0 and extrapolation number of the mixed-beam regimen were again identical to those values assuming that the mixed-beam effect was independent. RBEs obtained from cell survival were fairly close to those from TCD50 assays except single-dose experiments.     

The initial part of the survival curve: does it predict the outcome of fractionated radiotherapy?

The publication of a number of single-cell survival curves in vitro has stimulated radiobiologists and radiotherapists to analyze the survival characteristics of these curves for their ability to be predictive of the radioresponses of the tumors from which they were derived. Parameters of interest have been the steepness of the initial slope, the single-dose survival at 2 Gy, the mean inactivation dose, and the extrapolation number along with the D0 dose. An assessment of these correlations shows considerable overlap between the values of particular survival parameters even when tumors thought to be the most responsive are compared to those thought to be the least responsive. The importance of the full repair of sublethal damage in the analysis is noted, and a number of factors which may limit effective correlations between cell survival parameters and tumor response are discussed.     

Computer programs for the analysis of cellular survival data

Four programs have been written to enable radiobiologists to build a computer data base of cellular dose-survival data, calculate cell survival with a correction for cell multiplicity at the time of irradiation, fit various survival models to the data by iteratively weighted least squares, and calculate the ratio of survival levels corresponding to specified doses or the ratio of doses that produce specified survival levels (e.g., oxygen enhancement ratio or relative biological effectiveness). The programs make plots of survival curves and data, and they calculate standard errors and confidence intervals of the fitted survival curve parameters and ratios. The programs calculate survival curves for the linear-quadratic, repair-saturation, single-hit multitarget, linear-multitarget, and repair-misrepair models of cell survival and have been designed to accommodate the addition of other survival models in the future. The programs can be used to compare the accuracy with which different models fit the data, determine if a difference in fit is statistically significant, and show how the estimated value of a survival curve parameter, such as the extrapolation number or the final slope, varies with the survival model. The repair of radiation-induced damage is analyzed in a novel way using these programs.     

Radiobiology and stathmokinetics of intestinal crypts associated with patches of Peyer

The stathmokinetics and radiobiology of intestinal crypts directly adjoining the lymphoid patches of Peyer, have been compared with those of non-patch-associated crypts. Patch crypts contain an additional one to two rings of cells, the Mitotic Index for the whole crypt is higher than in non-patch crypts, and the apparent cell cycle time is insignificantly lower. Using single and split doses of gamma-rays, dose-survival curves were obtained for whole intestinal crypts, from which single-cell survival curves were derived for the clonogenic cells of the crypt. For a single-hit, multitarget, model, the extrapolation numbers of the cell survival curves for patch and non-patch crypts were the same (approximately 35) but the final D0 for cells of the patch crypts was significantly higher (2.1 versus 1.7 Gy). A linear-quadratic fit gave a similar ratio of alpha/beta (approximately 10) for the two curves. For a given level of crypt depletion, the number of clonogenic cells per crypt derived by the use of equal split doses of radiation, was the same for patch and non-patch crypts. This number is a function of the dose regime employed: the higher the level of crypt depletion, the higher the derived number of cells (range 10 to 45, for non-patch crypts).     

Method For Probing Cells In Radiation-Induced G2Arrest: Demonstration of Potentially Lethal Damage Repair

Abstract. Chinese hamster ovary cells were arrested in the G₂ phase of the cell cycle by X-irradiation. When subsequently treated with 5 mM caffeine the arrested population progressed into mitosis as a synchronous cohort where it was harvested by mitotic cell selection. This procedure provides a means to isolate cell populations treated in G₂, for the investigation of G₂ arrest. Comparisons were made of the number of cells retrieved from G₂ arrest with the number suffering arrest, as determined by flow cytometry and by matrix algebraic simulations of irradiated cell progression. the retrieved population was not significantly less than expected for doses up to 3.5 Gy, indicating that the retrieval process does not favour the isolation of any population subset below this dose. Cell populations retrieved from arrest at varying intervals (0-3 h) after irradiation (0-3.5 Gy) showed an increase in survival with increase in interval, consistent with repair of potentially lethal damage. the repair curves (surviving fraction us time) were each described by a single exponential. G₂ cells that were brought to mitosis without a period of arrest exhibited the same radiation response as cells irradiated in mitosis.     

Patterns of bacterial destruction in solutions by microwave irradiation

The patterns of destruction of several kinds of bacterial cells suspended in solutions by microwave irradiation were studied. The survival curves of Escherichia coli and Staphylococcus aureus were similar and approximated a set of three linear phases. The curves of Pseudomonas fluorescens and Bacillus cereus spores shifted to the shorter and longer irradiation periods, respectively. The rate constant and initial time of destruction for each linear phase of the survival curve were compared among these organisms. When irradiated E. coli cells were incubated in an agar with a high salt level, fewer cells were recovered. The curve of E. coli cells in the logarithmic phase of growth shifted to shorter exposure periods. There were no significant differences in the survival curves of E. coli cells grown at temperatures of 22–36°C, whereas the curve of cells grown at 44°C shifted to longer periods.     

Protection of Normal, Lysogenic, and Pyocinogenic Strains Against Ultraviolet Radiation by Bound Acriflavine

The presence of bound acriflavine protects bacteria against the lethal effects of ultraviolet (UV) light, presumably because pyrimidine dimer formation is inhibited. Although acriflavine present in plating medium usually results in reduced viable counts from irradiated bacteria, no enhancement of lethal effects is observed when acriflavine is added to irradiated bacteria left in suspending buffer for 45 min before plating. Acriflavine remaining bound to the deoxyribonucleic acid of irradiated bacteria at the time they are plated likewise does not affect their survival. Protection is precisely dose-modifying unless some killing of bacteria by UV results from induction of prophage, against which bound acriflavine is less protective, or from induction of pyocin, against which there is no protection at all. It is inferred that prophage induction proceeds in part, and pyocin induction wholly, by virtue of effects of UV other than pyrimidine dimerization. The response of Escherichia coli strain B to radiation has been postulated to be attributable in part to induction of a prophage or a lethal protein; but exact dose modification was observed for this strain, to about the same extent, whether or not the irradiated organisms were grown in conditions thought to enhance the expected contribution to killing if such a mechanism were involved. Our results support the hypothesis that the inhibition by acriflavine of dimer formation is attributable to energy transfer mechanisms. They fail to support the hypothesis that shapes of survival curves (in particular the manifestation of "shoulders") can be attributed to inactivation by radiation of repair enzymes.     

Survival of human lymphoblastoid cells after DNA damage measured by growth in microtiter wells

Survival of cells in suspension culture after treatment with damaging agents is usually measured by extrapolation from growth curves or by growth of colonies in soft agar. We have developed a survival assay which measures the ability of small numbers of cells to initiate microscopic cultures in wells of microtiter plates without agar or feeder layers. Suitable human lymphoblastoid lines were obtained by selection of rapidly growing cultures from microtiter wells in which <200 cells were inoculated in 0.2 ml RPMI 1640 medium and incubated at 37° with 5% CO₂ at 95% relative humidity. Survival after damage was measured by inoculating groups of 24 microtiter wells with appropriate serial dilutions of cells. The wells were examined microscopically at intervals and scored for evidence of cell proliferation. Survival was calculated with the Poisson formula on the basis of the fraction of wells in which cells were not proliferating. Survival did not change appeciably after 2–3 weeks incubation. Survival measured by the microtiter-well assay was found to be similar to survival measured by extrapolation from growth curves after damaging the cells with bleomycin or with 8-methoxypsoralen plus long-wavelength ultraviolet radiation. The microtiter-well assay affords a simple, accurate measure of cell survival in human lymphoblastoid cells with suitable growth ability.     

Inactivation of Oriented Bacteria with Polarized Ultraviolet Light

Aligned deoxyribonucleic acid (DNA) molecules exhibit a large absorption anisotropy in the ultraviolet (UV) region of the spectrum (11). Also, the UV action spectra of most bacteria resemble the absorption spectrum of DNA (23), implying that inactivation is directly proportional to the UV absorbed by the bacterial DNA. Hence, the UV sensitivity of aligned uniaxial bacteria might be anisotropic with respect to polarization of the incident UV (17, 19). Any inactivation anisotropy would depend upon the orientation of DNA within the bacteria, as well as upon the alignment of bacteria, and could provide a more sensitive indication of in vivo DNA orientation than is presently available using optical methods (5, 12-16). Using an electric field of 3.5 × 10⁶ cycles/second, samples of bacteria of strain LS-301 were aligned in a quartz cell and were irradiated with UV (λ = 2652 A) polarized perpendicular and parallel to the alignment direction. The resultant survival curves resolved no inactivation anisotropy. This result is interpreted to mean that there was insufficient bacterial DNA alignment to give a detectable anisotropy. The minimum average DNA alignment necessary to have resolved an anisotropy is calculated to be 15 per cent in an axial direction (bases perpendicular to the bacterial axis) or 30 per cent in a radial direction (bases parallel to the bacterial axis).     

Heterogeneous cell-cycle behavior in response to UVB irradiation by a population of single cancer cells visualized by time-lapse FUCCI imaging

The present study analyzed the heterogeneous cell-cycle dependence and fate of single cancer cells in a population treated with UVB using a fluorescence ubiquitination-based cell-cycle (FUCCI) imaging system. HeLa cells expressing FUCCI were irradiated by 100 or 200 J/m² UVB. Modulation of the cell-cycle and apoptosis were observed by time-lapse confocal microscopy imaging every 30 min for 72 h. Correlation between cell survival and factors including cell-cycle phase at the time of the irradiation of UVB, mitosis and the G₁/S transition were analyzed using the Kaplan–Meier method along with the log rank test. Time-lapse FUCCI imaging of HeLa cells demonstrated that UVB irradiation induced cell-cycle arrest in S/G₂/M phase in the majority of the cells. The cells irradiated by 100 or 200 J/m² UVB during G₀/G₁ phase had a higher survival rate than the cells irradiated during S/G₂/M phase. A minority of cells could escape S/G₂/M arrest and undergo mitosis which significantly correlated with decreased survival of the cells. In contrast, G₁/S transition significantly correlated with increased survival of the cells after UVB irradiation. UVB at 200 J/m² resulted in a greater number of apoptotic cells.