Radiation-induced Division Delay Research Articles

Spontaneous premature chromosome condensation and mitotic catastrophe following irradiation of HeLa S3 cells

Purpose : To study the mechanisms underlying the loss of G2 /M checkpoint control which leads to mitotic catastrophe in human tumour cells following exposure to ionizing radiation. Materials and methods : Asynchronous HeLa S3 cells were irradiated with doses of 5, 10 and 20 Gy X-rays. Cell-cycle progression and cyclin B1 levels were measured using bivariate flow-cytometric techniques as a function of time after irradiation. As indicators of mitotic catastrophe, the appearance of spontaneous premature chromosome condensation (SPCC) and cells presenting nuclear fragmentation were analysed using microscopy. Cyclin B1- dependent kinase activity was determined in immunoprecipitates and analysed using gel electrophoresis. Results : After X-irradiation of HeLa cells, delays in late S and G2 phases of the cell cycle were followed by SPCC and nuclear fragmentation, both indicative of mitotic catastrophe. The kinetics of appearance of cells that had apparently undergone mitotic catastrophe (i.e. the fraction of cells exhibiting nuclear fragmentation) was independent of the dose-dependent radiation-induced division delay, while the extent of fragmentation (expressed as the number of nuclear fragments per fragmented cell) did increase with dose. Also observed was a 5-fold elevation of cyclin B1 levels in late S/G2 cells, which correlated temporally with the observed delays late in the cell cycle. Following the appearance of elevated cyclin levels, cyclin B1-associated histone H1 kinase activity showed similar increases; these increases in kinase activity occurred prior to increases in the fraction of cells exhibiting nuclear fragmentation. Conclusions : In human cells, cyclin B1 gene expression occurs in late S and G2 phases, and thus the increase observed in this protein may be due to the increased time spent by cells in these phases as a result of cell-cycle delays caused by the radiation exposure. It is possible that, under these conditions, over accumulation of cyclin B1 dilutes the mitosis-inhibitory action of the wee1 or other inhibitory pathways. Thus, this study presents a possible mechanism for G2 /M checkpoint abrogation following ionizing radiation which may depend solely on effects associated with perturbed cell-cycle progression.

Association between the Division Delay Target and DNA Late in the Cell Cycle

The precise cell cycle time of association between labeled DNA (the radiation source) and the non-DNA cell structure whose damage is responsible for radiation-induced division delay was measured. Mitotic cells were selected from a monolayer of Chinese hamster ovary cells for 80 min (nine shakes) to establish the rate of cell progression into mitosis. The cell monolayers were then exposed to 0.1295 MBq/ml 125IUdR for 10 min to label the cells in S phase. After pulse labeling, mitotic cell selection was continued for various times (between 0 and 120 min) before 125I decays were accumulated at 4 degrees C. After 2 h in the cold, the cells were rewarmed and the selection of mitotic cells was continued. (Cooling had a small, transient affect on subsequent cell progression.) As the time between labeling and cooling was increased, the fraction of cells selected in mitosis decreased, indicating that an increasing proportion of 125I-labeled cells had entered a sensitive phase of the cell cycle where 125I decays are particularly effective in producing radiation-induced division delay. It is hypothesized that during this sensitive period (from -25 to +90 min of the S/G2 boundary), the labeled DNA comes into sufficiently close contact with a non-DNA structure to facilitate damage to this structure by overlap irradiation from 125I decays in the DNA.

Proliferation Kinetics of a Murine Fibrosarcoma during Fractionated Irradiation

Accelerated growth of tumor clonogens during the course of fractionated irradiation has been considered one of the major causes of radiation treatment failure. Alterations in clonogen growth rate could occur through three basic mechanisms: changes in cell-loss factor, changes in cell-cycle time, and recruitment of previously quiescent cells into the proliferative pool. This study was designed to assess changes in the cell-cycle time of clonogens of a murine fibrosarcoma during fractionated irradiation using an artificial pulmonary micrometastasis model. Lung colonies of various ages (4 h, 1 day, or 4 days) were exposed to single doses of irradiation ranging from 5-13 Gy; the fraction of surviving colonies was used to determine the preirradiation growth kinetics. The growth kinetics during fractionated irradiation was derived from colony-survival data of 4-day-old micrometastasis exposed to single doses or to 2, 5, 9, and 15 fractions separated by 4, 12, or 24-h intervals. The size of dose fractions used ranged from 1.7 to 14 Gy. The estimated clonogen doubling times before irradiation and during overall treatment periods of up to 14 days were 0.71 and 1.1 days, respectively. This significant (P less than 0.0001) increase in the doubling time was most likely a consequence of lengthening of the overall cell-cycle time of the clonogens by radiation-induced division delay. This observation suggests that accelerated growth, when it occurs in some tumors during fractionated treatment, is the result of a decreased cell-loss factor or recruitment of quiescent cells, but not a shortening of the cell-cycle time of the clonogens.

Radiation-Induced Division Delay in 9L Spheroid versus Monolayer Cells

The technique of percentage labeled mitoses was used to compare radiation-induced division delay in 9L rat gliosarcoma cells growing as spheroids or as exponential monolayers. The length of delay induced by each of five X-ray doses was determined as the difference between control and irradiated cultures in the time required to reach the half-height of the first peak of labeled mitoses. Spheroid cells were delayed significantly longer than monolayer cells; the slopes of the dose responses were 32 and 13 min/Gy, respectively. Cells in small spheroids (150 micron diameter) were delayed to the same extent as cells in large spheroids (800 micron diameter). Like the contact effect previously observed as enhanced radiation survival of cells grown as spheroids, the increased radiation-induced delay may be a consequence of the growth of cells in three-dimensional contact.

G 2 Cells: Progression Delay and Survival

The progression of Chinese hamster ovary (CHO) G2 cells into mitosis and their survival was measured after X-ray doses up to 4.0 Gy. S-phase cells were prevented from reaching mitosis by labeling with 125IUdR for 10 min prior to irradiation of the exponentially growing monolayer of cells. Mitotic cells, located past the radiation-induced division delay transition point, did not suffer a delay and were selected separately prior to the recovery of the G2 cells. The results show that (1) up to 400 min after radiation only 55% of the G2 cells recovered after about 2.5 Gy; (2) the progression delay of the G2 cells that recovered was 52.5 min/Gy; and (3) the survival curve D0 for these cells, 2.45 Gy, indicated a radioresistant population.

Modification of radiation-induced division delay by caffeine analogues and dibutyryl cyclic AMP.

The mitotic selection procedure for cell cycle analysis was utilized to investigate the concentration-dependent modification of radiation-induced division delay in Chinese hamster ovary (CHO) cells by methyl xanthines (caffeine, theophylline, and theobromine) and by dibutyryl cyclic AMP. The methyl xanthines (concentrations from 0.5 to 1000 micrograms/ml) all reduced radiation-induced division delay with the effect being linear between approximately 100 and 1000 micrograms/ml. After doses of 100-300 rad, delay was reduced by 75, 94 or 83 per cent at 1000 micrograms/ml for each drug, respectively. However, the addition of dibutyryl cyclic AMP had an opposite effect: radiation-induced delay was increased by the concentration range of 0.3 to 300 micrograms/ml. These results indicate that in mammalian cells the control of cell cycle progression and the modification of radiation-induced division delay are not simply related to intracellular levels of cyclic AMP. Rather, there appear to be at least two competing mechanisms which are differentially affected by caffeine analogues or by direct addition of dibutyryl cyclic AMP. The direct effect of caffeine and the methyl xanthines on membrane calcium permeability is considered.

Radiation-Induced Division Delay in Chinese Hamster Ovary Fibroblast and Carcinoma Cells: Dose Effect and Ploidy

The mitotic selection procedure for cell cycle analysis was utilized to investigate the G/sub 2/ transition point for and the duration of radiation-induced division delay in diploid and tetraploid Chinese hamster ovary (CHO) fibroblasts and in Chinese hamster ovarian carcinoma cells. The location of the radiation-induced division delay transition point was dose independent at high doses and located approximately 42 min before division. At lower doses only an estimate of the point of blockade was possible; but the G/sub 2/ transition point appeared to be earlier in the cell cycle. The duration of radiation-induced division delay was dose dependent. This response is consistent with a sensitive population of cells in late G/sub 2/ that define the location of the transition point and the length of division delay. There was no difference observed in the dose response for radiation-induced division delay between the pseudotetraploid cell line of CHO and the pseudodiploid parent strain. However, in the cell line derived from a spontaneous Chinese hamster ovarian carcinoma the division delay was 39 +- 4 min/Gy. Therefore, radiation-induced division delay is independent of chromosome ploidy, but can show intraspecies cell line specificity.

The Target for Radiation-Induced Division Delay

In an effort to elucidate the subcellular target responsible for radiation-induced division delay, Chinese hamster ovary (CHO) cells growing in monolayer cultures were pulse-labeled with /sup 125/IUdR and the cell kinetics monitored by counting the mitotic cells selected every 10 min. Our results showed that /sup 125/I had to be incorporated into DNA to cause a perturbation of cell progression; unlabeled G/sub 2/ cells were unperturbed. To evaluate the mechanism of /sup 125/I-induced division delay, /sup 125/IUdR-labeled cells were permitted to accumulate /sup 125/I decays either during the S phase or during both S and G/sub 2/ phases. The results indicated that cells which accumulated /sup 125/I decays only during the S phase did not experience enhanced delay. In contrast, the yield of mitotic cells was reduced in cells which accumulated /sup 125/I decays during S plus G/sub 2/. Analysis of the data suggests that the target for radiation-induced mitotic delay is not the DNA, but a cell structure which comes in contact with the DNA during G/sub 2/ or early M phase.

The effect of bleomycin and irradiation on G2 progression

The interaction of bleomycln and X-irradiation on the induction of G 2 delay in Chinese hamster ovary cab was investigated utilizing the mitotic selection procedure for cell cycle analysis. Following the addition of BLM the number of cells selected in mitosis remained at control level for a refractory period and then decreased. The location of the transition point, I.e. the age in G 2 at which cells become refractory to a progression blockade, was concentration-dependent, ranging from the S G 2 boundary at low concentrations to the G 2 M boundary at high concentrations. Depending upon the concentration of the drug used and the duration of exposure, the mitotic rate either decreased to zero or else leveled off at some intermediate value and then recovered to the control level. The duration of BLM-induced division delay was thus dependent upon the concentration used and the duration of exposure. When cells were treated with pulses of bleomycin (10–500 μg/ml) in addition to X-irradiation, the mitotic rate declined as with exposure to X-ray alone. However, the recovery from radiation-induced division delay and the subsequent reappearance of mitotic cells in the selection window was delayed until the cells had recovered from their BLM-induced division delay. This implies that, in contrast to the synergistic effects observed for cell lethality, BLM and radiation do not interact in the production of a progression blockade and the resultant division delay.

The effect of adriamycin and radiation on G 2 cell survival

The effect of Adriamycin (ADR) and irradiation on the ultimate fate of Chinese hamster ovary cells treated in G 2, phase was investigated utilizing the mitotic selection procedure for cell cycle analysis coupled with a plating efficiency assay. As cells were selected in mitosis, they were inoculated at appropriate numbers and incubated for analysis of colony-forming ability. The parameters obtained, expressed as a function of time after initiation of treatment, were the number of mitotic cells selected per shake (the mitotic yield), the plating efficiency (surviving fraction), and the number of surviving cells per shake (the product of the first two parameters). Cells treated with a 20 min pulse of 1.0 μ/ml ADR exhibited a transient division delay with a decrease in plating efficiency at the nadir of the mitotic yield curve. Cells treated with 150 rad of X-ray displayed a similar response except that surviving fraction decreased immediately after treatment. As cells recovered from the radiation-induced division delay, the surviving fraction increased to a plateau level close to that of the control. When cells were treated with both ADR and X-ray, the result was an increase in the duration of the division delay, a decreased surviving fraction, and consequently, a decreased number of surviving cells per shake. These results suggest the possibility of predicting optimum schedules for sequential treatment with these two agents based on a knowledge of the perturbatory effects on cell cycle progression for those cells most likely to retain clonogenic ability.

Alterations in the Radiosensitivity of CHO Cells by Anisotonic Treatments: Correlations between Cell Lethality and Chromosomal Aberrations

The frequency of chromosomal aberrations and cell lethality induced in synchronous CHO cells by X irradiation alone or X irradiation combined with a treatment of 0.05) or 1.5 M NaCl has been examined. Treatment with hypotonic 0.05 M NaCl increased the radiosensitivity, i.e., decreased the D/sub 0/ in M (D/sub 0/ from 87 to 61 rad), in G/sub 1/ (D/sub 0/ from 156 to 122 rad) and in S (D/sub 0/ from 165 to 128 rad). Exposure to hypertonic (1.5 M) NaCl decreased the radiosensitivity in G/sub 1/ and S; i.e., increased the D/sub 0/ in G/sub 1/ (D/sub 0/ from 156 to 182 rad) and in S (D/sub 0/ from 165 to 232 rad), while exposure to 1.5 M NaCl increased the radiosensitivity in mitosis (D/sub 0/ from 87 to 78 rad). The 0.05 or 1.5 M salt treatments had no observable effect on the radiation-induced division delay. The appearance of tetraploid cells following treatment of mitotic cells with 1.5 M NaCl indicates that the hypertonic treatment probably had a deleterious effect on the mitotic apparatus. Except for a decrease in the frequency of radiation-induced chromosomal aberrations in mitotic cells treated with 1.5 M NaCl, the changes in themore » frequency of chromosomal aberrations caused by the salt treatments correlated well with the changes in lethality delineated above. These positive correlations can be illustrated best by a plot of the logarithm of survival versus aberration frequency where the data points fell on a straight line havinga D tilde/sub 0/ of 0.8 aberration per cell. The salt-induced changes in cellular radiosensitivity are hypothesized to be caused by changes in the indirect effect of radiation, associated with free radicals produced in water and interacting in chromatin.« less

X-Irradiation-Induced Nuclear Lesions in Cultured Mammalian Cells: An Ultrastructural Analysis

Electron-dense chromatin aggregates, hereafter referred to as lesions, have been characterized morphologically within interphase nuclei of Chinese hamster cells (line CHO) after a single acute exposure to 400, 800, 1200, or 2000 rad of x irradiation. At all doses studied, lesions were observed only after termination of radiation-induced division delay. Cell profiles were scored by electron microscopy for the presence or absence of nuclear lesions at various times after irradiation. The mitotic fraction from each irradiated population was also scored for each sample by light and electron microscopy. From these data and from simultaneous cell-density counts for each sample, it is apparent that postirradiation cell division is a prerequisite to formation of interphase nuclear lesions. Irradiated cell populations blocked in mitosis by Colcemid beyond the normal period of postirradiation division-delay failed to display nuclear lesions until after Colcemid was removed and cell division was completed. Enzyme digestions of isolated nuclei from irradiated cells with DNase I, RNase A, and Pronase suggest that the nuclear lesions are comprised primarily of chromatin. Nucleolar lesions, as well as various aberrant morphological forms of nucleoli, were also observed in cell populations after the onset of postirradiation cell division during the first 72 hr followingmore » exposure to irradiation. Delayed radiation-induced ultrastructural alterations of the nucleus included the formation of cytoplasmic invaginations into the nuclear space and inclusions of membranes within nuclei.« less

Effect of Hydroxyurea on Radiation-Induced Division Delay in CHO Cells

The effects of the DNA synthesis-inhibitor hydroxyurea (HU) on radiation-induced division delay in CHO cells were studied using the mitotic selection procedure for cell cycle analysis. After the addition of 1.0 mM HU, the mitotic yield (number of mitotic cells selected from an asynchronous population) remained unaltered for a time and then decreased eventually to zero with a point of action at the S/G/sub 2/ boundary, i.e., cells in S were halted while cells in G/sub 2/ and M proceeded through mitosis and were selected, but at a slower rate than normal. Pulsed treatment with HU for up to 2 hr caused a decline in mitotic yield, but after the pulse-length-dependent S delay, S-phase cells entered G/sub 2/ and progressed to mitosis. Treatment with HU pulses (less than or equal to 2 hr) either before or after X irradiation did not alter the number of cells refractory to radiation-induced division delay nor the duration of the delay. This indicates that DNA synthesis was not necessary for recovery from radiation-induced division delay, and that the first cells to recover from radiation-induced delay were in G/sub 2/ at the time of irradiation. Finally, utilizing HU pulses to partially synchronize cells in G/submore » 2/, no differential division delay was observed for cells irradiated within the G/sub 2/ phase. However, if a cell in G/sub 2/ was exposed to HU for prolonged periods (i.e., treatment before irradiation and continuing during division delay and after mitotic recovery), considerable retardation of progression occurred.« less

Division Delay after Low X-Ray Doses and Treatment with Cycloheximide

SCHNEIDERMAN, M. H., BRABY, L. A., AND ROESCH, W. C. Division Delay after Low X-Ray Doses and Treatment with Cycloheximide. Radiat. Res. 70, 130-140 (1977). Radiation-induced division delay of Chinese hamster ovary cells located in G2, and in G2 between the cycloheximide and X-ray transition points, was measured by the mitotic cell selection technique. The mitotic yield (number of mitotic cells after treatment expressed as a fraction of the control) decreased with increasing radiation dose (4.5 to 34 rad). However, either because some cells were not delayed or because delayed cells recovered rapidly, the mitotic yield did not fall to zero. When cycloheximide was combined with radiation to prevent repair of the radiation damage, only cells which were past the cycloheximide transition point and not delayed by the radiation were selected. The location of the transition points determined from the combined drug plus low-dose radiation (4.5 to 34 rad) experiments indicate a dose-dependent relationship, with more cells delayed as the dose was increased. In addition, the transition point for cells treated with cycloheximide plus 150 rad of X rays was closer to division than the 150 rad of X rays alone. These results are discussed in light of a recent model for radiation-induced division delay proposed by Dewey and Highfield [Radiat. Res. 65, 511-528 (1976)].

Radionuclide Toxicity in Cultured Mammalian Cells: Elucidation of the Primary Site for Radiation-Induced Division Delay

Chinese hamster ovary cells, arrested in late S or early G1 phase, were exposed to various doses of membrane-associated125 I (administered as125 I-labeled, monovalent concanavalin A) and DNA-associated125 I (administered as ${}^{125}{\rm I}\text{-iododeoxyuridine}$ . Cell division was evaluated by monitoring the formation of duplet cells in monolayer. On the basis of radionuclide incorporation data, cellular dimensions, and subcellular radionuclide distributions, the cumulative dose to various subcellular regions was calculated from the known decay properties of125 I. In terms of125 I decays/cell, plasma membrane-associated125 I was very inefficient in producing division delay (0.01 min/decay), whereas DNA-associated125 I was extremely effective (1.45 min/decay). In terms of radiation dose to the cell nucleus, membrane-associated125 I caused exactly the same division delay as external X irradiation (0.45 min/rad), but nuclear125 I remained much more toxic (1.80 min/rad). These findings...

The Effect of Caffeine on Radiation-induced Division Delay

The Effect of Caffeine on Radiation-induced Division Delay

Effects of ionizing radiation upon intracellular levels of soluble microtubule protein in cultured mammalian cells

Intracellular soluble microtubule protein levels were determined in cultured mammalian cells following X-irradiation. Microtubule protein levels rose along with corresponding RNA and total soluble protein levels as cells accumulated in G 2 and declined as cells resumed division. These results suggest that radiation-induced division delay of mammalian cells does not arise from a radiation-induced defect in synthesis and accumulation of microtubule protein.

Radiobiological properties of Acholeplasma laidlawii B.

Survival data were obtained on Acholeplasma laidlawii B after exposure to ionizing radiation at various ages, pH, temperature, and supplements to the medium. The D0 value varied slightly with pH in air but much more after irradiations in N2 or N2O. Replacement of the growth medium with fresh broth resulted in an increase in D0 while dilution of the growth medium with Tris-NaCl 1:100 caused a decrease in D0 both in air and N2. The D0 varied with the age of the culture and the temperature at the time of irradiation. In air the ratio of increment in D0 (ΔD0) over the increment in temperature (ΔT) had positive values as a function of age of the cells while under N2 this ratio was always negative. In N2 the D0 decreased in a smoothly non-linear fashion with an increase in temperature while in air it remained constant below 12° and increased linearly above that temperature at a rate equivalent to 1.65 kcal/mol. ATP was found to radioprotect the cells in N2 at pH 6.3 but radiosensitize them both in air and N2 at pH 7.5. The adenine nucleotides ATP, ADP, and AMP eliminated radiation-induced division delay in the cells in air. Cyclic AMP had no effect on the division delay in air but was found to synchronize cell growth.

Radiation-Induced Division Delay in Synchronized Chinese Hamster Ovary Cells in Monolayer Culture

Chinese hamster ovary cells in monolayer culture, synchronized by mechanically selecting mitotic cells in the absence of drugs, were x-irradiated with 150, 300, or 600 rads at frequent intervals during the cell cycle and examined visually until completion of the first wave of cell division. The synchronized populations consisted of greater than 90% mitotic cells and their subsequent distribution throughout the cycle was carefully documented. At each time-dose combination, growth curves were analyzed for division delay (first, mean, and last cells to divide), the fraction of cells completing the first division, and the duration of the division wave. Results showed that radiation-induced division delay increased exponentially with cell age between early-mid G1 (0.29 minute/rad) and late S-early G2, (0.80 minute/rad) and linearly with dose at any given position in the cell cycle. Mitotic cells were more sensitive than mid- G1 cells (0.43 vs 0.29 minute/rad) and the increase in sensitivity leveled off in G2 a...

Radiation-induced division delay in Dictyostelium discoideum: relation to survival in resistant and sensitive strains.

SummaryVegetative amoeboid cells of the cellular slime mould, Dictyostelium discoideum NC—4, are very resistant to gamma-radiation. Mutant strains have been isolated which are significantly more radiosensitive. Studies were conducted to measure division delays and other growth responses after irradiation of strain NC—4 and two mutant strains. Some division of non-surviving (non-colonyforming) cells was observed in all strains. Total cell populations (surviving plus non-surviving cells) in all three strains showed essentially equivalent division-delay responses. However, surviving cells of the mutant strains revealed division delays that were much longer than those of the total cell populations. A model is proposed to interpret these results. A basic premise of the model is that, if more time is allowed for repair of potentially lethal damage incurred by the sensitive strains, cellular survival will be greater. Support for this model was obtained by producing artificially long delays in populations consisting of irradiated mutant cells. Increases in survival were observed for the sensitive strains but not for strain NC—4.