Radiation Sensitivities Are Not Related to the Sizes of DNA Supercoiled Domains in L5178Y-R and L5178Y-S Cells

Summary

Survival of murine lymphoblasts L5178Y-R and L5178Y-S irradiated with ⁶⁰Co gamma radiation was determined. The parameters of the survival curves were D₀ = 1·18 Gy, n = 1·56 and D₀ = 0·55 Gy, n = 1·00 for L5178Y-R and L5178Y-S cells respectively. The sizes of DNA supercoiled domains were estimated using sedimentation of nucleoids from cells irradiated with doses from 1 to 7 Gy. These sizes were 2·44 × 10⁹ and 5·13 × 10⁸ Da for L5178Y-R cells and 1·30 × 10⁹ and 4·07 × 10⁸ Da for L5178Y-S cells. Hence, higher radiosensitivity of L5178Y-S cells was not compatible with the larger size of the DNA supercoiled domains, as suggested by Filippovich et al. (1982). We have not found any simple relation between the sizes of DNA supercoiled domains and the susceptibility of L5178Y sublines to ionizing radiation.     

The Effect of 238Pu α-particles on the Mouse Fibroblast Cell Line C3H 10T1/2: Characterization of Source and RBE for Cell Survival

Summary

Considerable interest has been aroused in recent years by reports that the transforming and carcinogenic effectiveness of low doses of high LET radiations can be increased by reducing the dose rate, especially for transformation of 10T1/2 cells in vitro by fission-spectrum neutrons. We report on conditions which have been established for irradiation of 10T1/2 cells with high LET monoenergetic α-particles (energy of 3·2 MeV, LET of 124 keV μm^?1) from ²³⁸Pu. The α-particle irradiator allows convenient irradiation of multiple dishes of cells at selectable high or low dose rates and temperatures. The survival curves of irradiated cells showed that the mean lethal dose of α-particles was 0·6 Gy and corresponded to an RBE, at high dose rates, of 7·9 at 80 per cent survival and 4·6 at 5 per cent survival, relative to ⁶⁰Co γ-rays. The mean areas of the 10T1/2 nuclei, perpendicular to the incident α-particles, was measured as 201 μm², from which it follows that, on average, only one in six of the α-particle traversals through a cell nucleus is lethal. Under the well-characterized conditions of these experiments the event frequency of α-particle traversals through cell nuclei is 9·8 Gy^?1.     

Induction of Cytogenetic Adaptive Response of Somatic and Germ Cells in Vivo and in Vitro by Low-dose X-irradiation

Summary

The cytogenetic adaptive response induced by low-level radiation was studied using human and rabbit lymphocytes in vitro and bone marrow cells and germ cells in vivo. The inductive dose of X-rays was 10 mGy for the in vitro studies at a dose rate of 10 mGy/min, and 2, 10, 50, 75 and 100 mGy for the in vivo studies at a dose rate of 50 mGy/min. The challenging dose was 1·5 Gy X-rays for the in vitro experiments and 0·65 or 0·75 Gy for the in vivo experiments at a dose rate of 0·44 Gy/min. The results reported here, in addition to those that have appeared in the literature, show the following characteristics documented for the first time: (1) 10 mGy could induce the adaptive response in human as well as rabbit lymphocytes irradiated not only in G₁, S and G₂ phases, but also in the G₀ state; (2) although the induced adaptive response could only last three cell cycles, it could be revived when the inductive dose was repeated after the third cell cycle; (3) the adaptive response could be induced by low-dose X-rays in somatic cells, both in vitro (lymphocytes) and in vivo (bone marrow cells), and also in germ cells (spermatocytes); (4) the magnitude of the adaptive response induced by whole-body irradiation was found to be dose-dependent–the lower the inductive dose the more the reduction of the frequency of chromatid aberrations following the challenging dose.     

Enhanced Survival of Gamma-irradiated Escherichia Coli Following Pretreatment with Dithiothreitol

Summary

Survival of three strains of Escherichia coli K12 was studied with respect to radiation protection by dithiothreitol (DTT). The three strains compared were AB2462 recA, AB2470 rec21 and their DNA repair-competent prototype, AB1157. The strains were incubated in 10 mmol dm^?3 DTT for 60 min and allowed an expression period for SOS functions to appear which may have been induced by DTT. Following the expression period the DTT-incubated cells and incubated control cells were irradiated. When AB1157 cells were pretreated with chloramphenicol (200 µg cm^?3) for a period of 30 min prior to addition of the induction media no increase in survival was seen. When catalase (0·1 mg cm^?3) was added to the AB1157 cells prior to the induction media a decrease in the degree of induction was noted with an enhancement ratio (ER) of 0·893 (ER^?1 = 1·12). Furthermore, DTT-treated AB2462 and AB2470 demonstrated no increase in survival when compared to control cells. In radiation experiments on either strain of E. coli with or without DTT present during irradiation, the following were observed: (1) survival of AB1157 was enhanced with a dose modification factor (DMF) of 1·7 with DTT present and 1·3 with pretreatment; (2) the rec mutants showed no change in survival at any dose with a DMF of approximately 1·0. Results indicate that, using our protocol, inducible repair is of more importance than free radical scavenging by DTT. Furthermore, DTT-treated AB2462 demonstrated no increase in survival when compared to control cells. In radiation experiments on either strain of E. coli with and without DTT present during irradiation, the following were observed: (1) survival of AB1157 was enhanced with a DMF of 1·7 with DTT present during irradiation and 1·3 with only pretreatment; (2) the recA and recB mutants showed no change in cell survival at any dose with a DMF of approximately 1·0. Results indicate that, using our pretreatment protocol, inducible repair is of more importance in protection than free radical scavenging by DTT.     

Enhancement by Hyperthermia of the ‘Early Delayed’ and ‘Late Delayed’ Radiation Response of the Rat Cervical Spinal Cord

Summary

The cervical spinal cord (C5–T5) of female Wistar WU rats was irradiated with 250 kV X-rays (15–32 Gy). Heat was applied at approximately the same site 7 ± 1 min after X-rays. ‘Early delayed’ paralysis of the forelegs was observed 5–10 months after treatment. The ED₅₀ (± SE) after single-dose irradiation alone was 25·8 ± 0·4 Gy. ‘Late delayed’ paralysis and paresis were observed 11–21 months after irradiation with an ED₅₀ (X-rays alone) of 22·7 ± 0·6 Gy. The data for late paralysis, late paresis and minor neurological symptoms were pooled resulting in an ED₅₀ (± SE) of 20·6 ± 0·7 Gy. Hyperthermia enhanced the radiation response. Thermal enhancement ratios (TER) in the ‘early delayed’ response after a 30 min treatment with 41·1 ± 0·4°C, 42·1 ± 0·4°C and 42·9 ± 0·4°C were 1·07 ± 0·08, 1·17 ± 0·08 and 1·12 ± 0·04 respectively. For the ‘late delayed’ radiation response concerning paralysis and paresis the TER after 30 min at 41·1°C and 42·1°C were 1·25 ± 0·10 and 1·31 ± 0·07, respectively. The latent period for paralysis was not significantly affected. Pathological examination of the spinal cord after combined treatment of X-rays and hyperthermia showed focal demyelination with white matter necrosis and vascular injury in animals as an indication of ‘early delayed’ and ‘late delayed’ paralysis, respectively. This was not different from histopathological changes observed after irradiation alone.     

The Effect of Unilateral Nephrectomy on the Subsequent Radiation Response of the Pig Kidney

Summary

The left kidney of 14 Large White female pigs, approximately 14 weeks of age, was surgically removed. Thirty weeks after unilateral nephrectomy (UN) the remaining kidney was irradiated with a single dose of between 11·9 and 15·6 Gy of ⁶⁰Co γ-rays; three pigs received sham irradiation. Following irradiation glomerular filtration rate (GFR), effective renal plasma flow (ERPF) and haematocrit (Hct) were determined for up to 48 weeks after irradiation. Irradiation resulted in a dose-dependent decline in GFR, evident 8 weeks after irradiation. This was followed by a gradual improvement in GFR, although after doses of 14·0 Gy GFR remained below control values throughout the study. A similar pattern of response was seen in terms of ERPF, but this was not dose-related. Doses of ? 14·0 Gy also caused a significant reduction (p < 0·001) in Hct within 4–8 weeks. Minimal levels were evident 16 weeks after irradiation; Hct then increased, but remained below preirradiation values. Dose–effect curves were obtained by determining the percentage of irradiated kidneys which showed a ? 50% reduction in GFR and ERPF, fitted by probit analysis, and ED₅₀ values (± SE) were calculated for each parameter. The ED₅₀ values for GFR and ERPF were 14·49 ± 0·27 Gy and 12·56 ± 0·98 Gy, respectively. These values were not significantly different from those obtained from intact agematched pigs in which the right kidney alone was irradiated; UN did not compromise or alter the radiation response of the kidney to irradiation. However, the ED₅₀ values obtained for the UN pigs were significantly greater (p < 0·001) than the values of 9·76 ± 0·17 Gy and 6·19 ± 0·93 Gy, seen for GFR and ERPF in intact age-matched pigs in which both kidneys were irradiated. Thus although both experimental situations involved irradiating the entire renal tissue, the relative radiosensitivity of the kidneys varied considerably. These findings show that the radiosensitivity of the pig kidney can vary markedly, depending on the physiological status of the kidney at the time of irradiation.     

Response of Human Bone Marrow Progenitor Cells to X-rays in Vitro

Summary

In 25 out of 33 cases the survival response of myeloid progenitor cells from fresh human bone marrows, X-irradiated in vitro in phosphate-buffered saline at 23°C, was characterized by a two-term exponential relationship, with D₀ values of 0·42 ± 0·19 Gy and 1·38 ± 0·37 Gy, respectively. In the remaining eight cases for which the colony-forming efficiency was 10 times lower, survival followed a single exponential function with D₀ = 1·18 ± 0·15 Gy. The biphasic response at 23°C became a single exponential response when the temperature at irradiation was 30 or 37°C.     

Rescue of Marker Phenotypes: Effects of BUdR Sensitization,Hypoxia and High LET

Summary

The survival curve of colony-forming ability of Chinese hamster wg3h cells has been compared with the dose—response curve for the expression of an active thymidine kinase (TK) gene from these cells. The TK⁺ phenotype was measured by hybrid colony formation after fusion of wg3h (TK⁺) cells with Chinese hamster A23 (TK^?) cells. The TK⁺ survival data fitted a multi-target curve up to 3 krad of ¹³⁷Cs irradiation, when a highly resistant fraction of hybrid colonies was seen at about 1 per cent survival. The D₀ of TK⁺ survival for the multi-target region was 3·1–4·0 times greater than that of wg3h survival, even when the D₀ for cell survival varied between 136 and 545 rad by 14 MeV neutrons and hypoxia respectively. This parallel modification of cell and TK⁺ sensitivities suggests that the lesions causing cell inactivation are of the same type as those that cause marker inactivation. Using 14 MeV neutron data the approximate target size for TK inactivation was calculated to be 0·54–0·91 per cent of the DNA content of the cell (or about one-fifth to one-tenth of a chromosome). The data support the idea that marker inactivation results primarily from damage occurring outside the marker gene. BUdR labelling of wg3h cells before irradiation caused slight toxicity (30 per cent reduction in plating efficiency) and a twofold increase in cell sensitivity. However, the sensitivity of the TK⁺ phenotype increases by only 30 per cent. The increased cell sensitivity thus appeared to result from synergism between increased sensitivity of DNA to strand breakage and metabolic toxicity, the latter being largely overcome by fusion with normal cells.     

Lack of Dose Rate Modification (0·0049 vs. 0·12 Gy/min) of Fission-neutron-induced Neoplastic Transformation in C3H/10T½ Cells

Summary

Clonogenic survival and neoplastic transformation of asynchronous cultures of C3H/10T½ cells were used to assay the effect of dose protraction of reactor-produced fission neutrons. Cells were exposed to eight neutron doses ranging from 0·05 to 0·9 Gy delivered at 11·7 or at 0·49 cGy/min. For each dose level, high and low dose rate irradiations were performed on the same day. At each dose a similar effectiveness of fission neutron irradiation at high or low dose rates was measured for both cell survival and transformation. The combined high and low dose-rate data were analysed by two- or three-parameter models. Depending on the model used, values of the effectiveness per unit dose derived as parameters of linear terms of the respective dose-response curves were 0·9–1·2 Gy^?1 for clonogenic survival and 5–8 × 10^?4 Gy^?1 for neoplastic transformation. It is concluded that the modification of fission neutron dose-response curves by dose rate is negligible or absent in the range of doses and dose rates examined, in contrast to results with other sources of fission or fast neutrons.     

Differences in Heat-induced Cell Killing as Determined in Three Mammalian Cell Lines Do Not Correspond with the Extent of Heat Radiosensitization

Summary

Three different cell lines, Ehrlich ascites tumour (EAT) cells, HeLa S₃ cells and LM mouse fibroblasts, were used to investigate whether or not the extent of heat killing (44°C) and heat radio-sensitization (44°C before 0–6 Gy X-irradiation) are related. Although HeLa cells were the most heat-resistant cell line and showed the least heat radiosensitization, we found that the most heat-sensitive EAT cells (D₀, EAT = 8·0 min; D₀, LM = 10·0 min; D₀ HeLa = 12·5 min) showed less radiosensitization than the more heat-resistant LM fibroblasts (TER_HeLa < TER_EAT < TER_LM). Therefore, it is concluded that the routes leading to heat-induced cell death are not identical to those determining heat radiosensitization. Furthermore the inactivation of DNA polymerase α and β activities by heat seemed not to correlate with heat survival alone but showed a positive relationship to heat radiosensitization. The possibility of these enzymes being a determinant in heat radiosensitization is discussed.     

Hyperthermia Promotes the Incidence of Tumours Following X-irradiation of the Rat Cervical Cord Region

Summary

The cervical region of the rat, including the spinal cord (cervical 5-thoracic 2) was irradiated with single doses of 15–32 Gy 250 kV X-rays. Hyperthermia, at temperatures of 42-, 43- and 44 ± 0·1°C for 30 min was applied to the cervical vertebral column and immediate adjacent tissues for 5–10 min or 7 h after X-irradiation. Over a period of 18–21 months, animals were followed up to monitor neurological complications occurring as a result of damage to the spinal cord (Sminia et al. 1991). We also noted the development of neoplasms either inside or outside the cervical region. The data on tumour incidence were analysed retrospectively using the actuarial method. Although hyperthermia alone was not carcinogenic, it led to a significant increase of radiation-induced tumours. This increase of radiation carcinogenesis was observed both with hyperthermia applied 5–10 min after X-rays and with an interval of 7 h between X-rays and heat. Cancer induction was highest after the lower radiation doses (16 Gy) combined with high heat doses (30 min 44°C). The latent period for induction of tumours by X-rays was 472 ± 19 days (mean ± SEM; n = 24). Latency was significantly shortened by hyperthermia to 404 ± 34 days (n = 22) if applied 5–10 min after X-rays and to 348 ± 6 days (n = 33) with an interval of 7 h. Histology revealed that 86% (38/44) of the examined tumours found inside the volume treated with hyperthermia and irradiation were sarcomas. The percentage of animals with a tumour outside the treated volume was almost the same for all treatment groups. Most of these tumours were of the mammary gland type.     

Murine Haemopoietic Stem Cells with Long-term Engraftment and Marrow Repopulating Ability are More Resistant to Gamma-radiation than are Spleen Colony Forming Cells

The radiation sensitivity of various subsets in the haemopoietic stem cell hierarchy was defined using a limiting dilution type long-term bone marrow culture technique that was previously shown to allow quantification of cells with spleen colony-forming potential (day-12 CFU-S) and in vivo marrow repopulating ability (MRA). Primitive stem cells that generate new in vitro clonable colony-forming cells (CFU-C) in the irradiated marrow (MRA) and have long-term repopulation ability (LTRA) in vitro (cobblestone area forming cell, CAFC day-28) had D₀ values of 1·25 and 1·38 Gy, respectively. A lower D₀ was found for the less primitive CFU-S day-12, CAFC day-12 and cells with erythroid repopulating ability (0·91, 1·08 and 0·97 Gy, respectively). CFU-S day-7 were the most radiosensitive (D₀ equalling 0·79 Gy), while CFU-C and CAFC day-5 were relatively resistant to irradiation (D₀ 1·33 and 1·77 Gy). Split-dose irradiation with a 6 h interval gave dose sparing for stem cells with MRA and even more with in vitro LTRA, less for CFU-S day-12 and CAFC day-10 and none for CFU-S day-7. The cell survival data of the specified stem cell populations were compared with the ability of a fixed number of B6-Gpi-1^a donor bone marrow cells to provide for short- and long-term engraftment in single- and split-dose irradiated cognenic B6-Gpi-1^b mice. Serial blood glucose phosphate isomerase (Gpi) phenotyping showed less chimerism in the split as compared to the single radiation dose groups beyond 4 weeks after transplant. Radiation dose-response curves corresponding to stable chimerism at 12 weeks for single and fractionated doses revealed appreciable split-dose recovery (D₂–D₁) in the order of 2 Gy. This was comparable to D₂–D₁ estimates for MRA and late-developing CAFC (1·27 and 1·43 Gy, respectively), but differed from the poor dose recovery in cells corresponding to the committed CFU-S day-7/12 and CAFC day-10 population (0·14–0·33 Gy). These data are together consistent with differential radiosensitivity and repair in the haemopoietic stem cell hierarchy, and provide a cellular basis for explaining the dose-sparing effect of fractionated total-body irradiation conditioning on long-term host marrow repopulation.     

Induction and Repair of DNA Strand Breaks in X-irradiated Proliferating and Quiescent CHO Cells

Summary

Induction and repair of DNA strand breaks was studied in X-irradiated proliferating and quiescent CHO cells using the alkaline unwinding technique. The results showed that induction of strand breaks is identical for both states of proliferation, whereas repair is different. The decrease in the number of DNA strand breaks with incubation time at 37°C is best described by a sum of three exponential components I, II and III. The half-times of component I were similar (τ_Ip = 1·73 min versus τ_Iq = 1·66 min) whereas strand breaks comprising component II were repaired slightly faster (τ_IIp = 17·0 min versus τ_IIq = 14·2 min) and those comprising component III were repaired significantly faster (τ_IIIp = 218 min versus τ_IIIq = 113 min) in quiescent as compared with proliferating cells. In contrast, the initial fractions, f, of the three components were closely similar for both states of growth (proliferating cells: f_I = 0·69, f_II = 0·25, f_III = 0·06; quiescent cells: f_I = 0·65, f_II = 0·29, f_III = 0·06). Radiosensitivity as assayed by colony formation was found to be lower for quiescent cells than for proliferating cells. By fitting the survival data to the linear-quadratic equation, ?ln(S/S₀) = αD + βD², the ratios α_p/α_q = 1·7 and β_p/β_q = 1·2 were obtained, which are similar to the ratios of the half-times of components III and II (τ_IIIp/τ_IIIq = 1·9; τ_IIp/τ_IIq = 1·2). This result indicates that the DNA damage represented by components II and III might be involved in cell killing.     

The Absence of Late Effects of Radiation on the Cellularity of the Mouse Thymus

Summary

The in vitro cell survival of a human cervix carcinoma cell line (HX156c) has been assessed using ⁶⁰Co γ-rays administered at either high (150 cGy/min) or low (3·2 cGy/min) dose rate. Recovery during low dose-rate irradiation was observed; the dose reduction factor at 10^?2 cell kill for 150 versus 3·2 cGy/min was around 1·3. An insight into the possible underlying mechanisms of this recovery process has been investigated by addition of non-toxic concentrations of various agents thought to inhibit eukaryotic DNA repair. Agents were added 2 h prior to irradiation and removed after 24 h exposure. Differential effects among the inhibitors were observed; aphidicolin had no effect on cell survival, novobiocin, hydroxyurea and 3-aminobenzamide reduced survival by a similar extent at both dose rates, β-ara A and caffeine reduced survival to a greater extent during low dose-rate irradiation. β-ara A and caffeine seemed to exert their effects mainly by increasing the alpha component of the acute survival curve. Since survival curves obtained at dose rates of around 3 cGy/min help define a dominant component of the initial slope of the acute curve we have demonstrated that β-ara A and caffeine modify the initial slope, probably by inhibiting DNA repair processes involved in the sparing of tumour cells during protracted irradiation.     

Sensitivity and Protection of Mouse Bone-marrow Cells X-irradiated in Vitro

Summary

The 30-day survival of mice exposed to a lethal dose (798 r) of x-rays was a function of the number of isologous bone-marrow cells injected. From this calibration curve it was possible to determine the surviving fraction in a suspension of marrow cells irradiated in vitro. The in vitro radiation-response of such suspensions could be represented by a typical exponential survival curve the linear part of which indicated a D₃₇ of 84 r and an extra-polation number of 2·5.

Cysteine, S,2-aminoethylisothiuronium chloride, and anoxia protected marrow cells irradiated in vitro and gave dose reduction factors ranging from 1·7–2·1. Noradrenaline did not protect the cells.     

Interactions of Drugs and Radiation in Haemopoietic Tissue Assessed by Lethality of Mice after Whole-body Irradiation

Summary

Drug-radiation interactions in haemopoietic tissue were assessed as the lethality of mice within 7–28 days after whole-body irradiation. The investigated drugs were adriamycin (ADM), bleomycin (BLM), cyclophosphamide (CTX), 5-fluorouracil (5-FU), methotrexate (MTX), mitomycin C (MM-C) and cis-diamminedichloroplatinum II (cis-DDP). The drugs were administered as single doses 15 min before graded doses of whole-body irradiation or at different intervals from 7 days before to 7 days after fixed radiation doses. ADM, CTX, 5-FU, MM-C and cis-DDP enhanced the radiation response when administered 15 min before irradiation. The dose effect factor (DEF) was 9·11 for 5-FU and in the range 1·25–1·59 for the other drugs. MTX administration 15 min before irradiation had no effect (DEF 1·00). However, MTX increased lethality if given 1–3 days after irradiation (DEF 1·21–1·76) and protected against lethality if given 1–3 days before irradiation (DEF 0·83). A similar time dependence was observed for ADM, CTX, 5-FU, MM-C and cis-DDP. Protection against lethality was not observed but in all these cases the lethality was significantly lower at administration 1–3 days before than 1–3 days after irradiation. A proper investigation of the effect of BLM was not possible as the combination of this drug and whole-body irradiation caused a high rate of gastrointestinal deaths.     

Dose-rate effect was observed in T98G glioma cells following BNCT

BackgroundIt is generally said that low LET radiation produce high dose-rate effect, on the other hand, no significant dose rate effect is observed in high LET radiation. Although high LET radiations are produced in BNCT, little is known about dose-rate effect of BNCT.Materials and methodsT98G cells, which were tumor cells, were irradiated by neutron mixed beam with BPA. As normal tissue derived cells, Chinese hamster ovary (CHO-K1) cells and DNA double strand breaks (DNA-DSBs) repair deficient cells, xrs5 cells were irradiated by the neutrons (not including BPA). To DNA-DSBs analysis, T98G cells were stained immunochemically with 53BP1 antibody. The number of DNA-DSBs was determined by counting 53BP1 foci.ResultsThere was no dose-rate effect in xrs5 cells. D₀ difference between 4 cGy/min and 20 cGy/min irradiation were 0.5 and 5.9 at the neutron and gamma-ray irradiation for CHO-K1, and 0.3 at the neutron for T98G cells. D₀ difference between 20 cGy/min and 80 cGy/min irradiation for T98G cells were 1.2 and 0.6 at neutron irradiation plus BPA and gamma-ray. The differences between neutron irradiations at the dose rate in T98G cells were supported by not only the cell viability but also 53BP1 foci assay at 24 h following irradiation to monitor DNA-DSBs.ConclusionDose-rate effect of BNCT when T98G cells include 20 ppm BPA was greater than that of gamma-ray irradiation. Moreover, Dose-rate effect of the neutron beam when CHO-K1 cells did not include BPA was less than that of gamma-ray irradiation These present results may suggest the importance of dose-rate effect for more efficient BNCT and the side effect reduction.     

Jejunal Crypt Stem-cell Survival after Fractionated γ-irradiation Performed at Different Dose Rates

Summary

Jejunal crypt survival after fractionated total body irradiation of C3H mice given at dose rates between 1·2 and 0·08 Gy/min was studied and the results analysed according to the linear quadratic model. Whereas α was independent of dose rate β decreased with dose rate to approach zero at about 0·01 Gy/min.

During the period of recovery, sublethal damage from doses given at high dose rate interact with low dose rate irradiation given immediately after, and increases its effectiveness.     

In Vitro Studies of the Sensitivity of Canine Bone-marrow Erythroid Burst-forming Units (BFU-E) and Fibroblast Colony-forming Units (CFU-F) to X-irradiation

Summary

The radiosensitivity of the early erythroid progenitor cells (BFU-E) and the progenitor cells of the stroma (CFU-F) in canine bone marrow was studied under steady-state conditions by in vitro irradiation with 280 kV X-rays. The dose—effect relationship for colony formation was determined for BFU-E obtained from the iliac crest marrow, and for CFU-F in bone marrow collected from the iliac crest and the humerus of adult beagles. The BFU-E were adequately stimulated with serum from lethally irradiated dogs to obtain a source of BPA (burst-promoting activity). The BFU-E proved to be extremely radiosensitive, and the survival curve was exponential (D₀ = 15·3 ± 1·8 cGy). We showed that buffy-coat leukocytes separated from bone marrow leukocytes obtained by aspiration were an optimum source of CFU-F. A curve was fitted to the data obtained for CFU-F obtained from the iliac crest or the humerus, resulting in D₀ = 241 ± 38 cGy and an extrapolation number n = 1·38 ± 0·62 or D₀ = 261 ± 40 cGy and n = 1·04 ± 0·42, respectively. According to these findings, and other published data, we conclude that the canine bone marrow BFU-E are presently the most radiosensitive hemopoietic cells detected among all hemopoietic cells of different mammals.     

Differences in the DNA Supercoiling Response of Irradiated Cell Lines from Ataxia-telangiectasia Versus Unaffected Individuals

Summary

In this study the manifestation of DNA damage at the nucleoid level was examined in several AT cell lines using an image analysis system to directly visualize and measure the changes in DNA loop size which occur when increasing concentrations of propidium iodide (PI) are used to titrate the DNA supercoiling response (the ‘fluorescent halo assay’). This response consists of a relaxation (0·5 – 7·5 μg/ml PI) and rewinding phase (10–50 μg/ml PI), the latter of which is impaired by the presence of DNA strand breaks in irradiated cells. In addition to the inhibition of DNA rewinding seen immediately after irradiation at 0°C, the supercoiling response of AT diploid fibroblasts indicated an increased amount of DNA unwinding compared to fibroblasts from unaffected individuals. This difference appeared to saturate, since the excess in DNA loop size over that seen in irradiated fibroblasts from unaffected individuals remained constant after 5 Gy. These results may reflect a greater instability of the DNA-nuclear matrix attachment points in irradiated AT fibroblasts. The DNA supercoiling response in irradiated transformed AT fibroblasts and AT lymphoblasts did not differ from that observed in unaffected cells of the same type. However, all of the immortalized cell lines (AT and unaffected) had inherently larger DNA loop sizes than diploid fibroblasts and exhibited excess unwinding after irradiation.