Induction of chromosome aberrations in unirradiated chromatin after partial irradiation of a cell nucleus.

PURPOSE: It is generally accepted that chromosome exchanges in irradiated cells are formed through interactions between separate DNA double-strand breaks (DSB). Here we tested whether non-irradiated DNA participates in the formation of chromosome aberrations when complex DNA DSB are induced elsewhere in the nucleus. MATERIALS AND METHODS: Synchronized Chinese hamster cells containing an X chromosome with a late replicating q arm (X(q) domain) were labelled with 125I-iododeoxyuridine (125IdUrd) in a period of S-phase when the vast majority of the X(q) domain was not replicating. DNA damage from 125I decay was accumulated at the G1/S border while the cells were stored in liquid nitrogen. Decay of 125I induced DSB in the immediate vicinity of the 125I atom. Chromosome aberrations involving what is essentially the 125I-free X domain were scored at the first mitosis after cell thawing. As a positive control, cells were treated with 125IdUrd at a later period in S-phase when the X(q) domain replicates, yielding a labelled X(q) domain. RESULTS: The 125I-free X(q) domain exhibited chromosome aberrations (exchanges and fragments). The frequency of these aberrations was linearly dependent on the number of 125I decays elsewhere in the cell nucleus. The efficiency of formation of chromosome aberrations by the 125I-free X(q) domain was approximately half of that observed in the 125I-labelled X(q) domain. CONCLUSIONS: The involvement of the 125I-free X(q) domain in chromosome aberrations suggests that DNA not damaged by the decay of incorporated 125I can interact with damaged DNA, indicating the existence of an alternative pathway for the formation of chromosome aberrations.

A method for the selective irradiation of part of a genome.

We developed a method for partial irradiation of cell nuclei and for highlighting the irradiated chromatin domain(s) in both interphase nuclei and metaphase chromosomes. The method involves the use of the replication program of chromosomes and consists of three major steps: I) selection of a suitable chromatin domain, II) damage induction by 125I, and III) visualization of the domain. Here, the first step of the method, applied to Chinese hamster HA-1 cells, is described. Using pulse labelling with the replication marker IUdR, it was shown that Xq does not replicate at early S-phase and that the replication timing of Xq can be highly effectively synchronized with hydroxyurea in a whole cell population. Thus, the replication timing of Xq may be used to exclude or to incorporate 125I into the Xq. Other chromatin can be selected and targeted with 125I in a similar way. Examples of possible applications of the method are given.

Correlation between cell reproductive death and chromosome aberrations assessed by FISH for low and high doses of radiation and sensitization by iodo-deoxyuridine in human SW-1573 cells.

PURPOSE: To study the relationship between cell reproductive death and exchange frequency in SW-1573 human lung tumour cells with and without incorporated iodo-deoxyuridine (IdUrd) following irradiation of plateau-phase cultures with y-rays. METHOD: Linear-quadratic (LQ) analysis was performed for the data on clonogenic survival and on the frequency of chromosomal exchanges studied with fluorescence in situ hybridization in chromosomes X and 2. RESULTS: Differences in the LQ parameters alpha and beta of both non-sensitized and sensitized chromosomes were found. In both chromosomes an increase in the number of chromosomal exchanges in IdUrd-radiosensitized cells compared with non-sensitized cells was observed. The alpha-enhancement factors of 1.7 and 1.9 for the X-chromosome and for chromosome 2, respectively, are similar. For the X-chromosome, the beta coefficient increased by a factor of 3.9 and for chromosome 2 by a factor of 1.4. After correction to a full genome equivalence, no significant difference in alpha was found between chromosomes X and 2 for both control and sensitized cells. In contrast, an almost 2.8 times higher beta was found for the sensitized X-chromosome compared to this value for chromosome 2. CONCLUSIONS: It can be concluded that the linear-quadratic analysis of dose-response relationships offers insights into the correlation between cell survival and induction of exchanges in non-sensitized and radiosensitized cells.

Increased chromosome exchange frequencies in iodo-deoxyuridine-sensitized human SW-1573 cells after gamma-irradiation.

The induction of chromosome exchanges was investigated in SW-1573 human lung tumour cells radiosensitized with iododeoxyuridine (IdUrd) and irradiated with gamma-rays. Following treatment chromosome 2 and X were analyzed using fluorescence in situ hybridization (FISH) with chromosome-specific DNA libraries. The yield of chromosome exchanges involving chromosome 2 was higher than those involving chromosome-X. On the basis of the DNA content the relative involvement of the X-chromosome in exchange frequencies after 2 Gy was much higher than of chromosome 2. After 4 Gy the relative involvement of both chromosomes in exchanges is approximately equal. After radiosensitization, increased chromosome exchange frequencies are observed in both studied chromosomes. For the total chromosome exchange frequencies the sensitizer enhancement ratio (SER) at 2 Gy is 1.8 and 1.3 for chromosome 2 and X respectively. The SER at 4 Gy for total exchange frequencies is 1.6 and 1.9 chromosome 2 and X respectively. For reciprocal exchanges at 2 Gy higher SER values and at 4 Gy lower SER values were observed for both chromosomes.

The effect of 125I decay at different stages of S-phase on survival, expression of micronuclei and chromosome aberrations in CHO cells.

Chinese hamster ovary (CHO) cells were synchronized in M phase by mitotic selection, and then re-synchronized with aphidicolin at the G1/S phase border. The cells were labelled in early-S phase by 10 min exposure to 125I-iododeoxyuridine and then cultured (chased) in non-radioactive medium for 0.5, 3 or 5h, followed by harvesting and freezing to accumulate the desired number of 125I decays. Cell damage was assessed by evaluating colony formation, micronucleus formation and chromosome aberrations. These biological estimators of damage showed that the cytocidal effect of 125I decay increased with the duration of the post-labelling chase period: the highest level of damage was found in cells from the 5 h chase period and the lowest in the cells from the 0.5 h chase period. Survival curves for the three chase periods displayed low-dose hyper-radiosensitivity for 0 to 20 125I decays cell-1. The results indicate that the repair of DNA double-strand breaks (DSBs) may depend on the maturation stage of chromatin and an explanation of this finding is proposed which invokes the homologous recombination model for DSB repair.

Kinetics of micronuclei induced by 125IdU in cells of two lines.

The kinetics of the formation of cells carrying micronuclei (MN) after one doubling time (td) incorporation of 125I-iododeoxyuridine (125IdU) to Chinese hamster ovary (CHO) and rat anterior pituitary tumor (GC) cells was studied. Uptake of 125IdU by cells of both cell lines was linearly dependent on the concentration of extracellular 125I activity. The postlabeling time-dependent decrease in cellular activity of 125IdU was exponential in CHO cells and approximately linear in GC cells. The maximum yield of MN was seen during the second and third td after 125IdU incorporation. The frequency of cells with micronuclei increased monotonically with dose in the interval (1, 40) 125I decays cell-1td-1. The dose-response relationship could be fitted by straight lines with slopes of 1.0 (CHO) and 1.2 (GC) on the subinterval (1, 10) and of 0.6 or 0.5, respectively, for the subinterval (10, 40). Below one 125I decay cell-1td-1, the mean frequency of micronucleated binuclear cells was significantly lower than (CHO) or equal to (GC) the control. On average, one 125I decay/cell induced 0.95 +/- 0.5% (CHO) or 1.0 +/- 0.5% (GC) of micronucleated binuclear cells.

A two-compartment model of micronucleus formation in erythrocytes and its application to mouse bone marrow. I. Rectangularly transient action of clastogen in stationary conditions.

The micronucleation process of mouse erythrocytes caused by a clastogen has been described by means of a two-compartment model in which the bone marrow pool of polychromatic erythrocytes (PCE) and of normochromatic erythrocytes (NCE) are considered to be the compartments. The kinetic processes involved in this system and its rates (in parentheses) are: the generation of PCE (kg), the micronucleation of erythrocytes (kMN), the maturation of PCE to NCE (km) and the removal of NCE (k(out)) and of micronucleated NCE (k(out)MN). It is assumed that kMN = k(inj) kg, where k(inj) is an injury factor which may depend on the clastogen concentration. The model has been applied to the data published by Hayashi et al. (1984) for the frequency of micronucleated PCE under the assumptions that the clastogen "dose" was constant during a period T and the PCE generation was steady and unperturbed by the clastogen. For the three chemicals used--mitomycin C (MMC), 6-mercaptopurine (6-MP) and 1-beta-D-arabinofuranosylcytosine (Ara-C)--the following parameters were obtained: k(inj) = 5.4%, km = 0.069 hr-1, T = 24 hr (MMC); k(inj) = 7.0%, km = 0.075 hr-1, T = 24 hr (6-MP) and k(inj) = 4.3%, km = 0.083 hr-1, T = 22 hr (Ara-C). The Ara-C data set best fitted the two-compartment model under the assumptions stated above. It has been suggested that the deviations from theoretical predictions may be due to varying with time clastogen concentration and non-steady PCE generation.

Kinetics of micronucleus induction by 125I-labelled thyroid hormone in hormone-responsive cells.

Two cell lines, CHO and GC, different in their tissue origin, were investigated with the aim of discovering the correlation between the level of 125I-T3 binding and chromosomal damage induced by 125I decay. Incubation of cells with 125I-T3 has been performed in two exposure schedules: continuous incubation for one to six cell cycles and a pulse-chase schedule involving exposure for one cell cycle. The cellular uptake of 125I-T3, its compartmentization and kinetics were different in the two cell lines. GC cells contained about 7 times more 125I-T3 than CHO cells when incubated with the same external 125I activity concentration (74 kBq of 125I-T3 ml-1 medium). Approximately 70% of the cellular 125I-T3 was found in nuclei of GC cells and only 5% in the nuclei of CHO cells. During the long-term incubation of GC cells with 74 kBq of 125I-T3 ml-1 medium, the 125I activity concentration in cells and their nuclei initially decreased by a half, and thereafter reached a plateau after the third doubling time. In CHO cells and nuclei a very slow linear increase of 125I activity was observed. In GC cells, micronucleus frequency was found to be correlated with nuclear 125I activity. One cell cycle pulse labelling with 74 kBq of 125I-T3 ml-1 medium caused a significant enhancement of micronucleus frequency above the control level during six doubling times, with a maximum at the first post-labelling doubling time. In GC cells continuously incubated with 74 kBq of 125I-T3 ml-1 medium, the micronucleus frequency increased with the incubation time. A model of T3 receptor-dependent dose delivery to nuclei of GC cells continuously incubated with 125I-T3 is proposed. The frequency of micronuclei in the CHO cell line continuously incubated with 125I-T3 did not differ significantly from the control, whereas in the pulse-chase schedule the mean frequency of micronucleated binuclear cells was lower during 4 post-labelling doubling times (significantly at the first and second post-labelling doubling time and insignificantly at the later doubling times) than in the control. Incubation of GC cells with various activity concentrations in medium for four cell cycles resulted in a linear increase of 125I activity in cells and nuclei; however, with a saturation in the region of highest 125I-T3 concentrations used. The frequency of binuclear cells bearing micronuclei was linearly dependent on the nuclear 125I-T3 concentration.

Microscopic and flow cytometric study of micronuclei in iododeoxyuridine labelled cells irradiated with soft x-rays.

Iododeoxyuridine labelled (IUdR(+)) and unlabelled (IUdR(-)) CHO cells irradiated with 2 Gy of soft x-rays showed only minor differences in the kinetics of micronuclei formation during the first 20 hours postirradiation period. Between 20 to 40 hours, the IUdR(-) cells showed approximately a constant number # of micronuclei while the number of micronuclei in IUdR(+) cells was still increasing. The frequency of micronuclei was higher in IUdR(+) cells compared to IUdR(-) cells at 24 hours after irradiation with various doses up to 4.0 Gy. Dose modifying factors were found to be 1.3 (microscopic evaluation) and 1.8 (flow cytometric evaluation). Flow cytometry with use of two parameters, fluorescence from propidium iodide and light scattering, seems to be a good tool to estimate the frequency of micronuclei in CHO cells in the dose range up to about 4 Gy. At higher doses perturbation of the cell cycle and the appearance of dying cells will influence the results.