Chromosome aberrations and radiation-induced cell death. I. Transmission and survival parameters of aberrations

Synchronous cultures of V₇₉ Chinese hamster cells were irradiated in G₁ with 300 rad of X-rays. Cells were collected for 2-h intervals after synchronization to include the first three post-irradiation divisions and were scored for chromosome aberrations. After the first post-irradiation division, asymmetrical exchanges were distributed according to the Poisson formula and both the asymmetrical exchange frequency and the acentric fragment frequency exhibited significant variations with collection time. Formulae derived from a previous mathematical analysis were used in conjunction with the aberration frequencies observed at the first, second, and third post-irradiation divisions to predict transmission and survival parameters for specific chromosomal aberrations.The probability, 2T, that an acentric fragment will be transmitted to a daughter cell at anaphase was found to be 0.57. The probability, W, that a two-break aberration (asymmetrical exchange) will be transmitted and observed at the next division was 0.56. Finally, the probability, P, that a cell will survive to a subsequent mitosis after losing a single acentric fragment was about 1.0 for one post-irradiation generation but somewhat less for two generations.     

The stage of chromosome duplication in the cell cycle as revealed by X-ray breakage and 3H-thymidine labeling

By means of combined experiments of X-irradiation and ³H-thymidine labeling of the chromosomes which are in the phase of synthesis, and the subsequent analysis at metaphase on the autoradiographs of the chromosomal damage induced during interphase, it was shown that in somatic cells from a quasi-diploid Chinese hamster line cultured in vitro the chromosomes change their response to radiation from single (chromosome type aberrations) to double (chromatid type aberrations) in late G₁. These results are interpreted to indicate that the chromosome splits into two chromatids in G₁, before DNA replication. — By extending the observations at the second metaphase after irradiation, it was also seen that cells irradiated while in G₂ or late S when they reach the second post-irradiation mitosis still exhibit, beside chromosome type aberrations, many chromatid exchanges, some of which are labeled. Two hypotheses are suggested to account for this unexpected reappearance of chromatid aberrations at the second post-irradiation division. The first hypothesis is that they arise from half-chromatid aberrations. The second hypothesis, which derives from a new interpretation of the mechanisms of production of chromosome aberrations recently forwarded by Evans, is that they arise from gaps or achromatic lesions which undergo, as the cells go through the next cycle, a two-step repair process culminating in the production of aberrations.This work was supported in part by grant No. RH-00304 from the Division of Radiological Health, Bureau of State Services, Public Health Service, U.S.A.     

Post-irradiation DNA synthesis inhibition and G2 phase delay in radiosensitive body cells from non-Hodgkin's lymphoma patients: an indication of cell cycle defects

In the present study, both post-irradiation DNA synthesis and G₁ phase accumulation were analyzed in lymphoblastoid cell lines (LCLs) and fibroblast cell strains derived from (Saudi) patients with non-Hodgkin's lymphoma (NHL), ataxia telangiectasia (AT), AT heterozygotes and normal subjects. A comparison of the percent DNA synthesis inhibition (assayed by ³H-thymidine uptake 30 min after irradiation), and a 24 h post-irradiation G₂ phase accumulation determined by flow cytometry placed the AT heterozygotes and the NHL patients in an intermediate position between the normal subjects (with maximum DNA synthesis inhibition and minimum G₂ phase accumulation) and the AT homozygotes (with minimum DNA synthesis inhibition and maximum G₂ accumulation). The similarity between AT heterozygotes and the NHL patients with respect to the two parameters studied after irradiation was statistically significant. The data indicating a moderate abnormality in the control of cell cycle progression after irradiation in the LCLs and fibroblasts from NHL patients may explain the enhanced cellular and chromosomal radiosensitivity in these patients reported by us earlier. In addition to demonstrating a link between cell cyle abnormality and radiosensitivity as a possible basis for cancer susceptibility, particularly in the NHL patients, the present studies emphasized the usefulness of the assay for 24 h post-irradiation G₂ phase accumulation developed by Lavin et al. (1992) in characterizing AT heterozygote-like cell cycle anomally in cancer patients irrespective of whether they carried the AT gene or any other affecting the cell cycle.     

Cell-cycle dependency of radiosensitivity and mutagenesis in fertilized egg cells of rice,Oryza sativa L.

Summary In order to examine changes in survival and mutation rates during a cell cycle in higher plant, fertilized egg cells of rice were irradiated with X-rays at 2 h intervals for the first 36 h after pollination, i.e., at different phases of the first and second cell cycles. The most sensitive phase in lethality was late G₁ to early S, followed by late G₂ to M, which were more sensitive than the other phases. In both M₁ and M₂ generations, sterile plants appeared most frequently when fertilized egg cells were irradiated at G₂ and M phases. Different kinds of mutated characters gave rise to the respective maximum mutation rates at different phases of a cell cycle: namely, albino and viridis were efficiently induced at early G₁, xantha at early S, short-culm mutant at mid G₂, heading-date mutant at M to early G₁. The present study suggests the possibility that the differential mutation spectrums concerning agronomic traits are obtained by selecting the time of irradiation after pollination.     

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.     

Caffeine induces a second wave of apoptosis after low dose-rate gamma radiation of HL-60 cells

Most cell lines that lack functional p53 protein are arrested in the G₂ phase of the cell cycle due to DNA damage. It was previously found that the human promyelocyte leukemia cells HL-60 (TP53 negative) that had been exposed to ionizing radiation at doses up to 10 Gy were arrested in the G₂ phase for a period of 24 h. The radioresistance of HL-60 cells that were exposed to low dose-rate gamma irradiation of 3.9 mGy/min, which resulted in a pronounced accumulation of the cells in the G₂ phase during the exposure period, increased compared with the radioresistance of cells that were exposed to a high dose-rate gamma irradiation of 0.6 Gy/min. The D₀ value (i.e. the radiation dose leading to 37% cell survival) for low dose-rate radiation was 3.7 Gy and for high dose-rate radiation 2.2 Gy. In this study, prevention of G₂ phase arrest by caffeine (2 mM) and irradiation of cells with low dose-rate irradiation in all phases of the cell cycle proved to cause radiosensitization (D₀=2.2 Gy). The irradiation in the presence of caffeine resulted in a second wave of apoptosis on days 5–7post-irradiation. Caffeine-induced apoptosis occurring later than day 7 post-irradiation is postulated to be a result of unscheduled DNA replication and cell cycle progress.     

DNA synthesis and cell cycle progression of synchronized L-cells after irradiation in various phases of the cell cycle

Summary The varying sensitivity to radiation in the different phases of the cell cycle was investigated using L-929 cells of the mouse. The cells were synchronized by mechanical selection of mitotic cells. The synchronous populations were X-irradiated with a single dose of 10 Gy in the middle of the G₁-phase, at the G₁/S-transition or in the middle of the S-phase, respectively. The radiation effect was determined in 2 h intervals a) by¹⁴C-TdR incorporation (IT) into the DNA, b) by autoradiography (AR), c) by flow cytometry (FCM). The incorporation rate decreased in all three cases, but the reasons appeared to be different, as can be derived from FCM and AR data: After irradiation in G₁, a fraction of cells was prevented from entering S-phase, after irradiation at G₁/S a proportion of cells was blocked in the S-phase, and after irradiation in S, DNA synthesis rate was reduced. As a consequence of these effects, the mean transition time through S-phase increased. The G₂ blocks, obtained after irradiation at the three stages of the cycle were also different: Cells irradiated in G₁ are partly released from the block after 10 h. Irradiation at G₁/S caused a persisting accumulation of 50% of the cells in G₂, and for irradiation in S more than 80% of the cells were arrested in G₂.     

A correlation between radiation sensitivity and initial chromatid breaks in cancer cell lines revealed by Calyculin A-induced premature condensation

Three human malignancy cell lines were irradiated with ⁶⁰Co γ-rays. Initial chromatid breaks were measured by using the chemically induced premature chromosome condensation technique. Survival curves of cells exposed to gamma rays was linear-quadratic while the efficiency of Calyculin A in inducing PCC of G₂ PCC was about five times more than G₁ PCC. A dose-dependent increase in radiation-induced chromatid/isochromatid breaks was observed in G₁ and G₂ phase PCC and a nearly positive linear correlation was found between cell survival and chromatin breaks. This study implies that low LET radiation-induced chromatid/isochromatid breaks can potentially be used to predict the radiosensitivity of tumor cells either in in vitro experimentation or in in vivo clinical radiotherapy.     

BRCA1 Is Required for Maintenance of Phospho-Chk1 and G2/M Arrest during DNA Cross-Link Repair in DT40 Cells

The Fanconi anemia DNA repair pathway is pivotal for the efficient repair of DNA interstrand cross-links. Here, we show that FA-defective (Fancc⁻) DT40 cells arrest in G₂ phase following cross-link damage and trigger apoptosis. Strikingly, cell death was reduced in Fancc⁻ cells by additional deletion of the BRCA1 tumor suppressor, resulting in elevated clonogenic survival. Increased resistance to cross-link damage was not due to loss of toxic BRCA1-mediated homologous recombination but rather through the loss of a G₂ checkpoint. This proapoptotic role also required the BRCA1-A complex member ABRAXAS (FAM175A). Finally, we show that BRCA1 promotes G₂ arrest and cell death by prolonging phosphorylation of Chk1 on serine 345 after DNA damage to sustain arrest. Our data imply that DNA-induced cross-link death in cells defective in the FA pathway is dependent on the ability of BRCA1 to prolong cell cycle arrest in G₂ phase.     

Investigations on radiosensitive and radioresistant populations of Drosophila melanogaster. IV. The genetics of the relative radioresistance in stage-7 oocytes of the irradiated population RO I

The genetic system that controls the relative radioresistance in an irradiated laboratory population of Drosophila melanogaster (RÖ I) was studied. Comparisons were made between an unirradiated control population (+60, +K), the population RÖ I (after 227–333 generations of irradiation at 2100 R per generation), the sub-population RÖ I₀ (derived from RÖ I after 260 generations of irradiation and kept without irradiation for up to 74 generations), the F₁ hybrids +60/RÖ I, various homo- and heterozygous carriers of the 3 major chromosomes of RÖ I and +60, respectively, in combination with suitable balancers, and several chromosome substitution stocks of +K and RÖ I. The criteria used to assess the magnitude of radiosensitivity were dominant lethals, X-chromosome loss, and sex-linked recessive lethals induced in stage-7 oocytes at various exposure levels of X-irradiation.The data show that the radioresistance in RÖ I is controlled by a stable and homozygous genetic system. The system is semidominant. With respect to the induction of dominant lethals and sex-linked recessive lethals, the relative resistance is mainly contributed by chromosomes I and II. The effects of the two chromosomes are additive, each contributing about half the relative resistance. Resistance to the X-ray induction of X-chromosome loss is solely contributed by chromosome II.The findings suggest that at least 2 different and independent mechanisms are involved in determining the resistance of the RÖ I population.     

Mitotic recombination in the absence of synaptonemal complexes in Saccharomyces cerevisiae.

Summary Mitotic cells of a diploid strain of Saccharomyces cerevisiae with appropriate markers for the detection of mitotic crossing-over and mitotic gene conversion were irradiated with X-rays. Induction of these recombinational events was strong. After irradiation, cells were incubated in a rich growth medium and samples were removed for studying the possible formation of synaptonemal complexes up to a time when most cells had completed the first post-irradiation cell division. No complexes were found during the entire period of sampling, during which mitotic recombination in G₁ (mitotic gene conversion), DNA replication and G₂ (mitotic crossing-over) had occurred. These results are interpreted to mean that synaptonemal complexes are not required for mitotic recombination.     

Extent of excision repair before DNA synthesis determines the mutagenic but not the lethal effect of UV radiation

Excision repair-proficient diploid fibroblasts from normal persons (NF) and repair-deficient cells from a xeroderma pigmentosum patient (XP12BE, group A) were grown to confluence and allowed to enter the G₀ state. Autoradiography studies of cells released from G₀ after 72 h and replated at lower densities (3?9 × 10³ cells/cm²) in fresh medium containing 15% fetal bovine serum showed that semiconservative DNA synthesis (S phase) began ～24 h after the replating. To determine whether the time available for DNA excision repair between ultraviolet irradiation (254 nm) and the onset of DNA synthesis was critical in determining the cytotoxic and/or mutagenic effect of UV in human fibroblasts, we released cultures of NF or XP12BE cells from G₀, allowed them to reattach at lower densities, irradiated them in early G₁ (～18 h prior to the onset of S) or just prior to S phase, and assayed the frequency of mutations to 6-thioguanine resistance and the survival of colony-forming ability. The XP12BE cells, which are virtually incapable of excising UV-induced DNA lesions, showed approximately the same frequency of mutations and survival regardless of the time of UV irradiation. In NF cells, the slope of the dose response for mutations induced in cells irradiated just prior to S was about 7-fold steeper than that of cells irradiated 18 h earlier. However, the two sets of NF cells showed no significant difference in survival. Neither were there significant differences in the survival of NF cells released from G₀, plated at cloning densities and irradiated as soon as they had attached and flattened out (～20 h prior to S) or 4, 8, 12, 16, 20 or 24 h later. We conclude that the frequency of mutations induced by UV is dependent upon the number of unexcised lesions remaining at the time of semi-conservative DNA replication. However, the amount of time available for excision of potentially cytotoxic lesions is not determined primarily by the period between irradiation and the onset of S phase.     

Involvement of RAD9-dependent damage checkpoint control in arrest of cell cycle,induction of cell death,and chromosome instability caused by defects in origin recognition complex in Saccharomyces cerevisiae

Perturbation of origin firing in chromosome replication is a possible cause of spontaneous chromosome instability in multireplicon organisms. Here, we show that chromosomal abnormalities, including aneuploidy and chromosome rearrangement, were significantly increased in yeast diploid cells with defects in the origin recognition complex. The cell cycle of orc1-4/orc1-4 temperature-sensitive mutant was arrested at the G₂/M boundary, after several rounds of cell division at the restrictive temperature. However, prolonged incubation of the mutant cells at 37°C led to abrogation of G₂ arrest, and simultaneously the cells started to lose viability. A sharp increase in chromosome instability followed the abrogation of G₂ arrest. In orc1-4/orc1-4 rad9Δ/rad9Δ diploid cells grown at 37°C, G₂ arrest and induction of cell death were suppressed, while chromosome instability was synergistically augmented. These findings indicated that DNA lesions caused by a defect in Orc1p function trigger the RAD9-dependent checkpoint control, which ensures genomic integrity either by stopping the cell cycle progress until lesion repair, or by inducing cell death when the lesion is not properly repaired. At semirestrictive temperatures, orc2-1/orc2-1 diploid cells demonstrated G₂ arrest and loss of cell viability, both of which require RAD9-dependent checkpoint control. However, chromosome instability was not induced in orc2-1/orc2-1 cells, even in the absence of the checkpoint control. These data suggest that once cells lose the damage checkpoint control, perturbation of origin firing can be tolerated by the cells. Furthermore, although a reduction in origin-firing capacity does not necessarily initiate chromosome instability, the Orc1p possesses a unique function, the loss of which induces instability in the chromosome.     

Irradiation of synchronized cell cultures

Summary When HeLa cells are synchronized by the double thymidine block method, it is shown that the mitotic delay produced by 450 R X-rays is longer when cells are irradiated in G₂ than in G₁ but longer still when they are irradiated in the early part of S. DNA synthesis, measured by³H-Thymidine incorporation, is not reduced considerably (± 25%) and only for a few hours when 600 R are given at this time. A more pronounced inhibition occurs when irradiation is given later in S. There is also no decrease in incorporation of³H-orotic acid in RNA when the cells are irradiated in S or in G₂. These results are discussed and it is concluded that the long mitotic delays cannot he due to a modification of nucleic acids metabolism.Paper read at the 6th Annual Meeting of the European Society for Radiobiology, Interlaken, 5.–8. June, 1968. Round Table: Radiation Effectsin vitro andin vivo. Correlations and Discrepancies.     

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     

Ultraviolet Light-Induced Division Delay in Synchronized Chinese Hamster Cells

The age-dependent, ultraviolet light (UVL) (254 nm)-induced division delay of surviving and nonsurviving Chinese hamster cells was studied. The response was examined after UVL exposures adjusted to yield approximately the same survival levels at different stages of the cell cycle, 60% or 30% survival. Cells irradiated in the middle of S suffered the longest division delay, and cells exposed in mitosis or in G₁ had about the same smaller delay in division. Cells irradiated in G₂, however, were not delayed at either survival level. It was further established, after exposures that yielded about 30% survivors at various stages of the cycle, that surviving cells had shorter delays than nonsurvivors. This difference was not observed for cells in G₂ at the time of exposure; i.e., neither surviving nor nonsurviving G₂ cells were delayed in division. The examination of mitotic index vs. time revealed that most cells reach mitosis, but all of the increase in the number of cells in the population can be accounted for by the increase of the viable cell fraction. These observations suggest strongly that nonsurviving cells, although present during most of the experiment, are stopped at mitosis and do not divide. Cells in mitosis at the time of irradiation complete their division, and in the same length of time as unirradiated controls. Division and mitotic delays after UVL are relatively much larger than after X-ray doses that reduce survival to about the same level.     

Analysis of radiation‐induced changes to human melanoma cultures using a mathematical model

Objectives: Tumour cells respond to ionizing radiation by cycle arrest, cell death or repair and possible regrowth. We have developed a dynamic mathematical model of the cell cycle to incorporate transition probabilities for entry into DNA replication and mitosis. In this study, we used the model to analyse effects of radiation on cultures of five human melanoma cell lines. Materials and methods: Cell lines were irradiated (9 Gy) prior to further culture and harvesting at multiple points up to 96 h later. Cells were fixed, stained with propidium iodide and analysed for G₁‐, S‐ and G₂/M‐phase cells by flow cytometry. Data for all time points were fitted to a mathematical model. To provide unique solutions, cultures were grown in the presence and absence of the mitotic poison paclitaxel, added to prevent cell division. Results: The model demonstrated that irradiation at 9 Gy induced G₂‐phase arrest in all lines for at least 96 h. Two cell lines with wild‐type p53 status additionally exhibited G₁‐phase arrest with recovery over 15 h, as well as evidence of cell loss. Resumption of cycling of surviving cells, as indicated by increases in G₁/S and G₂/M‐phase transitions, was broadly comparable with results of clonogenic assays. Conclusions: The results, combined with existing data from clonogenic survival assays, support the hypothesis that a dominant effect of radiation in these melanoma lines is the induction of long‐term cell cycle arrest.     

Cellular responses to etoposide: cell death despite cell cycle arrest and repair of DNA damage

The topoisomerase IIα inhibitor etoposide is a ‘broad spectrum’ anticancer agent and a potent inducer of DNA double strand breaks. DNA damage response of mammalian cells usually involves cell cycle arrest and DNA repair or, if unsuccessful, cell death. We investigated these processes in the human colon cancer cell line HT-29 treated with three different etoposide regimens mimicking clinically relevant plasma concentrations of cancer patients. Each involved a period of drug-free incubation following etoposide exposure to imitate the decline of plasma levels between the cycles of chemotherapy. We found a massive induction of double strand breaks that were rapidly and nearly completely fixed long before the majority of cells underwent apoptosis or necrosis. An even greater percentage of cells lost clonogenicity. The occurrence of double strand breaks was accompanied by a decrease in the levels of Ku70, Ku86 and DNA-PK_cs as well as an increase in the level of Rad51 protein. Twenty-four hours after the first contact with etoposide we found a pronounced G₂/M arrest, regardless of the duration of drug exposure, the level of double strand breaks and the extent of their repair. During the subsequent drug-free incubation period, the loss of clonogenicity correlated well with the preceding G₂/M arrest as well as with the amount of cell death found several days after exposure. However, it correlated neither with early apoptosis or necrosis nor with any of the other investigated parameters. These results suggest that the G₂/M arrest is an important determinant in the cytostatic action of etoposide and that the removal of DNA double strand breaks is not sufficient to ensure cell survival.     

Functional and molecular consequences of ionizing irradiation on large conductance Ca2+-activated K+ channels in rat aortic smooth muscle cells

AimsThe goal of this study was to evaluate the influence of γ-irradiation on Ca²⁺-activated K⁺ channel (BK_Ca) function and expression in rat thoracic aorta.Main methodsAortic cells or tissues were studied by the measurement of force versus [Ca²⁺]_i, patch-clamp technique, and RT-PCR.Key findingsStimulation of smooth muscle cells with depolarizing voltage steps showed expression of outward K⁺ currents. Paxilline, an inhibitor of BK_Ca channels, decreased outward K⁺ current density. Outward currents in smooth muscle cells obtained from irradiated animals 9 and 30 days following radiation exposure demonstrated a significant decrease in K⁺ current density. Paxilline decreased K⁺ current in cells obtained 9 days, but was without effect 30 days after irradiation suggesting the absence of BK_Ca channels. Aortic tissue from irradiated animals showed progressively enhanced contractile responses to phenylephrine in the post-irradiation period of 9 and 30 days. The concomitant Ca²⁺ transients were significantly smaller, as compared to tissues from control animals, 9 days following irradiation but were increased above control levels 30 days following irradiation. Irradiation produced a decrease in BK_Ca α- and β₁-subunit mRNA levels in aortic smooth muscle cells suggesting that the vasorelaxant effect of these channels may be diminished.SignificanceThese results suggest that the enhanced contractility of vascular tissue from animals exposed to radiation may result from an increase in myofilament Ca²⁺ sensitivity in the early post-irradiation period and a decrease in BK_Ca channel expression in the late post-irradiation period.