Human lymphocytes exposed to low doses of ionizing radiations become refractory to high doses of radiation as well as to chemical mutagens that induce double-strand breaks in DNA

Human lymphocytes exposed to low doses of ionizing radiation from incorporated tritiated thymidine or from X-rays become less susceptible to the induction of chromatid breaks by high doses of X-rays. This response can be induced by 0.01 Gy (1 rad) of X-rays, and has been attributed to the induction of a repair mechanism that causes the restitution of X-ray-induced chromosome breaks. Because the major lesions responsible for the induction of chromosome breakage are double-strand breaks in DNA, attempts have been made to see if the repair mechanism can affect various types of clastogenic lesions induced in DNA by chemical mutagens and carcinogens. When cells exposed to 0.01 Gy of X-rays or to low doses of tritiated thymidine were subsequently challenged with high doses of tritiated thymidine or bleomycin, which can induce double-strand breaks in DNA, or mitomycin C, which can induce cross-links in DNA, approximately half as many chromatid breaks were induced as expected. When, on the other hand, the cells were challenged with the alkylating agent methyl methanesulfonate (MMS), which can produce single-strand breaks in DNA, approximately twice as much damage was found as was induced by MMS alone. The results indicate that prior exposure to 0.01 Gy of X-rays reduces the number of chromosome breaks induced by double-strand breaks, and perhaps even by cross-links, in DNA, but has the opposite effect on breaks induced by the alkylating agent MMS. The results also show that the induced repair mechanism is different from that observed in the adaptive response that follows exposure to low doses of alkylating agents.     

Chromosome and DNA damage and their repair in higher plants irradiated with short-wave ultraviolet light

In this review the authors present only their own results. They include the determination of the duration of the different stages of the cell cylce in UV-irradiated barley cells, the effect of different UV doses on the frequency of chromosome aberrations in barley, the increase in UV-induced chromosome aberration frequency induced in barley by caffeine and the effect of UV doses on the induction of pyrimidine dimers and sites sensitive to UV-endonuclease action (ESS) in barley cells and Nicotina tabacum protoplasts. In addition, the excision of pyrimidine dimers and ESS after irradiation with various doses of UV, unscheduled DNA synthesis in N. tabacum protoplasts and the correlation between the induction of pyrimidine dimers in DNA and the frequency of chromosome aberrations are reported. Data demonstrating that photoreactivation decrease the number of DNA lesions and chromosome aberrations induced by UV are also presented.     

The relationship between DNA and chromosome damage after bleomycin treatment: dose-response measurements

The molecular basis for chromosome aberration formation has been studied using the sensitive techniques of premature chromosome condensation and DNA alkaline elution. The dose response of Chinese hamster ovary cells to bleomycin treatment at the DNA and chromosome levels was compared. Each DNA elution curve showed a 2-component profile, with a more sensitive component apparent at low doses. The chromosome aberration curves also exhibited a 2-component profile when determined in G2-PCC; however, this phenomenon was less apparent when chromosome damage was enumerated in mitotic figures. These results suggest that differential sensitivity to bleomycin exists within the cellular chromatin. The effect of dose rate on aberration formation was examined by administering bleomycin at 2 concentrations that, with different treatment times, yielded equivalent amounts of DNA damage. The chromatid exchange rate was independent of dose rate, suggesting that rapidly repaired DNA lesions are not involved in the formation of exchanges.     

Localization of UvrA and Effect of DNA Damage on the Chromosome of Bacillus subtilis

We found that the nucleotide excision repair protein UvrA, which is involved in DNA damage recognition, localizes to the entire chromosome both before and after damage in living Bacillus subtilis cells. We suggest that the UvrA(2)B damage recognition complex is constantly scanning the genome, searching for lesions in the DNA. We also found that DNA damage induces a dramatic reconfiguration of the chromosome such that it no longer fills the entire cell as it does during normal growth. This reconfiguration is reversible after low doses of damage and is dependent on the damage-induced SOS response. We suggest that this reconfiguration of the chromosome after damage may be either a reflection of ongoing DNA repair or an active mechanism to protect the cell's genome. Similar observations have been made in Escherichia coli, indicating that the alteration of chromosome structure after DNA damage may be a widespread phenomenon.     

Shedding Light on the DNA Damage Checkpoint

DNA damage checkpoint genes are required to restrain cell cycle progression during DNA repair and to maintain chromosome stability. Checkpoint mutants are highly sensitive to killing by UV light, so the responses mediated by these genes are likely to be essential for survival during exposure to solar radiation. Yet it is still unclear exactly how checkpoint responses coordinate the cell cycle with DNA repair in the presence of UV lesions. At high doses, the UV response shares features with the ionizing radiation response, such as G1/S and G2/M checkpoints. At lower doses, only a postreplication checkpoint is evident. In this perspective we attempt to reconcile these observations and address their physiological meaning, with an emphasis on insights gained from direct cell-cycle measurements and recent studies in yeast.     

The repair of bleomycin-induced DNA damage and its relationship to chromosome aberration repair.

Previous studies using the technique of premature chromosome condensation indicated that nearly one-half of the bleomycin-induced chromatid breaks and gaps in CHO cells could be repaired within 1 h (repair starting at 30 min) after treatment. Cycloheximide and streptovitacin A (but not hydroxyurea or hycanthone) inhibited chromosome repair. The purpose of this study was to measure the kinetics of DNA repair after bleomycin treatment using the alkaline elution technique and to determine whether various inhibitors could block this repair. After bleomycin treatment, the major proportion of the repair of DNA damage occurred within 15 min, with significant repair evident by 2 min. This fast repair component was inhibited by 0.2% EDTA. A slower repair component was observed to occur up to 60 min after bleomycin treatment. None of the inhibitors tested were found to have a significant effect on the repair of bleomycin damage at the DNA level. Since chromosome breaks were observed not to begin repair until after 30 min while over 50% of the DNA was repaired by 15 min, these results suggest that the DNA lesions that are repaired quickly are not important in the formation of chromosome aberrations. Further, since cycloheximide and streptovitacin A blocked chromosome repair but had little measurable effect on DNA repair, these results suggest that the DNA lesions responsible for chromosome damage represent only a small proportion of the total DNA lesions produced by bleomycin.     

Tolerating DNA damage during eukaryotic chromosome replication

In eukaryotes, the evolutionarily conserved RAD6/RAD18 pathway of DNA damage tolerance overcomes unrepaired DNA lesions that interfere with the progression of replication forks, helping to ensure the completion of chromosome replication and the maintenance of genome stability in every cell cycle. This pathway uses two different strategies for damage bypass: translesion DNA synthesis, which is carried out by specialized polymerases that can replicate across the lesions, and DNA damage avoidance, a process that relies on a switch to an undamaged-DNA template for synthesis past the lesion. In this review, we summarise the current knowledge on DNA damage tolerance mechanisms mediated by RAD6/RAD18 that are used by eukaryotic cells to cope with DNA lesions during chromosome replication.     

A model of chromosome aberration induction and chronic myeloid leukaemia incidence at low doses

Some chromosome aberration types, generally translocations, are correlated with specific cancers. An example is provided by chronic myeloid leukemia (CML) cells, most of which carry a translocation involving the ABL gene on chromosome 9 and the BCR gene on chromosome 22. The hypothesis of a causal relationship between CML and the chimeric protein product of the BCR-ABL translocation has recently received strong support. In this framework, a mechanistic model and Monte-Carlo code simulating radiation-induced chromosome aberrations in human lymphocytes will be presented. The current version of the model can predict dose-response curves for the main aberration types following acute irradiation with gamma rays and light ions of different energies. The model is based on the assumption that only clustered DNA lesions can lead to aberrations and that only lesion free ends in neighbouring chromosome territories can join and form exchanges. Such lesions are distributed within the cell nucleus according to the radiation track structure, i.e. randomly for low-LET radiation and along straight lines for high-LET light ions. Interphase chromosome territories are explicitly simulated and background aberrations are taken into account. Very good agreement was found with experimental data taken from the literature that provided a further validation of the model. As an application, yields of BCR-ABL translocations were calculated. Preliminary results led to a CML induction dose-response that is approximately quadratic below 0.1 Gy and essentially linear at higher doses up to 1 Gy. The numerical values obtained for the probability of CML induction are consistent with values obtained by other groups with different approaches.Dedicated to Herwig Paretzke on the occasion of his 60th birthday.     

Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on repair of DNA breaks, interphase chromatin breaks, and potentially lethal damage in plateau-phase CHO cells.

There is evidence suggesting that radiosensitization induced in mammalian cells by substitution in the DNA of thymidine with BrdU has a component that relies on inhibition of repair and/or fixation of radiation damage. Here, experiments designed to study the mechanism of this phenomenon are described. The effect of BrdU incorporation into DNA was studied on cellular repair capability, rejoining of interphase chromosome breaks, as well as induction and rejoining of DNA double- and single-stranded breaks (DSBs and SSBs) in plateau-phase CHO cells exposed to X rays. Repair of potentially lethal damage (PLD), as measured by delayed plating of plateau-phase cells, was used to assay cellular repair capacity. Rejoining of interphase chromosome breaks was assayed by means of premature chromosome condensation (PCC); induction and rejoining of DNA DSBs were assayed by pulsed-field gel electrophoresis and induction and rejoining of DNA SSBs by DNA unwinding. A decrease was observed in the rate of repair of PLD in cells grown in the presence of BrdU, the magnitude of which depended upon the degree of thymidine replacement. The relative increase in survival caused by PLD repair was larger in cells substituted with BrdU and led to a partial loss of the radiosensitizing effect compared to cells tested immediately after irradiation. A decrease was also observed in the rate of rejoining of interphase chromosome breaks as well as in the rate of rejoining of the slow component of DNA DSBs in cells substituted with BrdU. The time constants measured for the rejoining of the slow component of DNA DSBs and of interphase chromosome breaks were similar both in the presence and in the absence of BrdU, suggesting a correlation between this subset of DNA lesions and interphase chromosome breaks. It is proposed that a larger proportion of radiation-induced potentially lethal lesions becomes lethal in cells grown in the presence of BrdU. Potentially lethal lesions are fixed via interaction with processes associated with cell cycle progression in cells plated immediately after irradiation, but can be partly repaired in cells kept in the plateau-phase. It is hypothesized that fixation of PLD is caused by alterations in chromatin conformation that occur during normal progression of cells throughout the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Radiation-induced mitotic and meiotic aneuploidy in the yeast Saccharomyces cerevisiae.

A number of genetic systems are described which in yeast may be used to monitor the induction of chromosome aneuploidy during both mitotic and meiotic cell division. Using these systems we have been able to demonstrate the induction of both monosomic and trisomic cells in mitotically dividing cells and disomic spores in meiotically dividing cells after both UV light and X-ray exposure. The frequency of UV-light-induced monosomic colonies were reduced by post-treatment with photoreactivity light and both UV-light- and X-ray-induced monosomic colonies were reduced by liquid holding post-treatment under non-nutrient conditions. Both responses indicate an involvement of DNA-repair mechanisms in the removal of lesions which may lead to monosomy in yeast. This was further confirmed by the response of an excision-defective yeast strain which showed considerably increased sensitivity to the induction of monosomic colonies by UV-light treatment at low doses. Yeast cultures irradiated at different stages of growth showed variation in their responses to both UV-light and X-rays, cells at the exponential phase of growth show maximum sensitivity to the induction of monosomic colonies at low doses whereas stationary phase cultures showed maximum induction of monosomic colonies at high does. The frequencies of X-ray-induced chromosome aneuploidy during meiosis leading to the production of disomic spores was shown to be dependent upon the stage of meiosis at which the yeast cells were exposed to radiation. Cells which had proceeded beyond the DNA synthetic stage of meiosis were shown to produce disomic spores at considerably lower radiation doses than those cells which had only recently been inoculated into sporulation medium. The results obtained suggest that the yeast sustem may be suitable for the study of sensitivities of the various stages of meiotic cell division to the induction of chromosome aneuploidy after radiation exposure.     

Multiple pathways cooperate to facilitate DNA replication fork progression through alkylated DNA

Eukaryotic genomes are especially vulnerable to DNA damage during the S phase of the cell cycle, when chromosomes must be duplicated. The stability of DNA replication forks is critical to achieve faithful chromosome replication and is severely compromised when forks encounter DNA lesions. To maintain genome integrity, replication forks need to be protected by the S-phase checkpoint and DNA insults must be repaired. Different pathways help to repair or tolerate the lesions in the DNA, but their contribution to the progression of replication forks through damaged DNA is not well known. Here we show in budding yeast that, when the DNA template is damaged with the alkylating agent methyl methanesulfonate (MMS), base excision repair, homologous recombination and DNA damage tolerance pathways, together with a functional S-phase checkpoint, are essential for the efficient progression of DNA replication forks and the maintenance of cell survival. In the absence of base excision repair, replication forks stall reversibly in cells exposed to MMS. This repair reaction is necessary to eliminate the lesions that impede fork progression and has to be coordinated with recombination and damage tolerance activities to avoid fork collapse and allow forks to resume and complete chromosome replication.     

Facilitated detection of chromosome break and repair at low levels of ionizing radiation by addition of wortmannin to G1-type PCC fusion incubation

We have measured rejoining kinetics of chromosome breaks using a modified cell fusion-based premature chromosome condensation (PCC) technique in confluent cultures of normal human fibroblasts irradiated at low doses of X-rays. In order to enhance the sensitivity of the fusion-based PCC assay, we added a DNA double strand break (DSB) repair inhibitor wortmannin during the incubation period for PCC/fusion process resulting in a significantly higher yield of G1-type chromosome breaks. The initial number of chromosome breaks (without repair) gave a linear dose response with about 10 breaks per cell per Gy which is about two times higher than the value with the conventional G1-type PCC method. The chromosome rejoining kinetics at 0.5 and 2.0 Gy X-rays reveal a bi-phasic curve with both a fast and a slow component. The fast component (0-30 min) is nearly identical for both doses, but the slow component for 2 Gy kinetics is much slower than that for 0.5 Gy, indicating that the process occurring during this period may be crucial for the ultimate fate of irradiated cells. The chromosome rejoining kinetics obtained here is similar to that of other methods of detecting DNA DSB repair such as the gammaH2AX assay. Our chromosome repair assay is useful for evaluating the accuracy of other assays measuring DNA DSB repair at doses equal or less than 0.5 Gy of ionizing radiation.     

Chromosome rearrangements and survival of androgenetic rainbow trout (Oncorhynchus mykiss)

The purpose of this work was to quantify the impact of spontaneous and X-radiation-induced chromosome rearrangements on survival rate of androgenetic rainbow trout (Oncorhynchus mykiss). Various doses of X irradiation (50, 150, 250, 350 Gy) were used for inactivation of nuclear DNA in oocytes. After the irradiation, eggs were inseminated with normal sperm from 4 males derived from a strain characterized by Robertsonian rearrangements and length polymorphism of the Y chromosome. The haploid zygotes were exposed to a high hydrostatic pressure (7000 psi) to duplicate the paternal DNA. Neither Robertsonian chromosome polymorphism nor the Y chromosome morphology impaired the viability of the androgenetic embryos and alevins. Moreover, survival of eyed embryos of the androgenetic rainbow trout increased significantly with increasing doses of oocyte X irradiation. After 6 months of rearing, only specimens from the 250 and 350 Gy variants survived. The number of fingerlings with remnants of the maternal genome in the forms of chromosome fragments was higher in the 250 Gy group. Intraindividual variation of chromosome fragment number was observed, and some individuals exhibited haploid/diploid mosaicism and body malformations. Individuals irradiated with less than 250 Gy died, presumably because of the conflict between intact paternally derived chromosomes and the residues of maternal genome in the form of chromosome fragments.     

Quantitative description of the process of radiation inactivation of cells. VII. Nature of primary radiation lesions leading to reproductive cell death]

The entity of radiation damage of viruses, bacteria and cells is defined by the organization of genetic structures. Asimmetrical chromosome exchanges have been proposed as the main reason of inactivation of di- and polyploid eukaryotic cells. If a single molecule of DNA is taken for the core of chromosome, the exchange is believed to be a consequence of cross-polymerization of two polypeptid strands of the single DNA molecule. Thus, the double strand break of DNA is necessary to produce aberration. A hypothesis is put forward on the identity of primary lesion of chromosome with the double strand break. The experimental survival curve is approximated according to the formula derived from the model. The yield of primary lesions of chromosomes is proposed to be equal to that of double strand breaks of chromosomes in order to examine the validity of the hypothesis. The optimal interaction distance of primary lesions in correspondence with parameters of the survival curve is equal to 0.8 mkm. This estimation is in good agreement with the microdosimetrical data, and the proposed hypothesis is not contradicted.     

Evidence that the adaptive response of human lymphocytes to ionizing radiation acts on lethal damage in nonaberrant cells

In a previous study we found that a cytogenetic adaptive response could lead to increases in survival if there was a sufficient increase in nonaberrant cells (Shadley and Dai, 1992). Since the high challenge doses used produced mainly multiply aberrant cells, we suggested using challenge doses that gave mainly singly aberrant cells in order to improve detection of a survival adaptive response. To test this, human lymphocytes from 6 donors were exposed in the first G₁ phase to 5 cGy of X-rays, followed by 100 cGy 6 h later. Nearly all of the aberrant cells bore only one chromosome aberration with this challenge dose, and in agreement with our proposal, survival adaptive responses were seen in 4 of 6 donors. A near 1:1 relationship between the % nonaberrant cells and % survival was found with 100 cGy, suggesting that the lymphocyte populations scored in the survival and aberration assays were representative of each other. However, the increase in nonaberrant cells was not sufficient to account for the increase in survival. Thus, a large fraction of the increase in survival was due to a decrease in lethal damage in cytologically nonaberrant cells. Such damage could range sub-microscopic lesions, to larger alterations not visible in Giemsa-stained cells. In conjunction with adaptive response studies of others, these results intimate that the adaptive response affects damage at different levels of chromosomal hierarchy (i.e. from the chromosome to DNA). The process(es) responsible for the effects observed in this study may act on lethal, rather than mutagenic lesions.     

Cytogenetic insights into DNA damage and repair of lesions induced by a monomethylated trivalent arsenical

Arsenic is a human carcinogen, and only recently animal models have been developed that are useful in investigating its carcinogenic mode of action (MOA). However, how arsenic induces cancer is still an open question. In a previous paper, we proposed a model detailing how arsenic might induce DNA lesions leading to cytogenetic damage [A.D. Kligerman, A.H. Tennant, Toxicol. Appl. Pharmacol. 222 (2007) 281–288]. In this model we hypothesized that arsenic does not induce chromosome damage via DNA adduction but induces short-lasting lesions from the action of reactive oxygen species (ROS). These lesions cause single-strand breaks (SSB) that induce chromosome breakage when treatment is in late G₁- or S-phase. However, if treatment is confined to the G₀- or early G₁-phase of the cell cycle, it is predicted that little or no cytogenetic damage will result at the subsequent metaphase. Here, we describe the results from testing this model using monomethylarsonous acid (MMA^III) and cytosine arabinoside (araC), a DNA chain terminator, to extend the time that DNA lesions remain open during repair to allow the lesions to reach S-phase or interact to form DNA exchanges that would lead to exchange aberrations at metaphase. The results of our study only partially confirmed our hypothesis. Instead, the results indicated that the lesions induced by MMA^III are quickly repaired through base excision repair, that there is little chance for araC to extend the life of the lesions, and thus the DNA damage induced by arsenicals that leads to chromosome aberrations is very short lived.     

Study of numerical aberrations of chromosome 1 by fluorescent in situ hybridization and DNA content by densitometric analysis on (pre)-malignant cervical lesions

Summary In an attempt to determine whether the fluorescent in situ hybridization (FISH) can be used as a rapid approach for the identification of aneuploidy in premalignant cervical smears, a centromeric probe for chromosome 1 was used. The results from the FISH experiments were compared with measurements of the overall DNA content obtained by means of an image analysis system. With progression to neoplasia, a decrease of the frequency of cells with two spots was observed, due to an increasing polysomy of chromosome 1. As far as the DNA content was concerned, an increasing DNA index and 5C-exceeding ratio (fraction of cells with a DNA content higher than 5C) was observed. Classification of the FISH results by a linear discriminant analysis revealed that 67.6% of the cases were classified in agreement with the CIN classification. These data suggest that chromosome 1 may be considered as a marker chromosome for pre-malignant cervical lesions and that the DNA content measurements are complementary to the FISH results.     

The relationship of DNA and chromosome damage to survival of synchronized X-irradiated L5178Y cells. II. Repair

The purpose of this study was to investigate the role of DNA and chromosome repair in determining the difference in radiosensitivity between a radiosensitive murine leukemic lymphoblastoid cell line, L5178Y-S, and its radioresistant counterpart, L5178Y-R. Populations of cells in the G1 or G2 phase of the cell cycle were obtained by centrifugal elutriation and irradiated with X-ray doses up to 10 Gy and allowed to repair at 37 degrees C for various periods. The kinetics of DNA double-strand break repair was estimated using the DNA neutral filter elution method, and the kinetics of chromosome repair was measured by premature chromosome condensation. L5178Y-S cells exhibited decreased repair rates and limited repair capacity at both the DNA and chromosome level in both G1 and G2 phases when compared to L5178Y-R cells. For the repair-competent L5178Y-R cells, the rate of DNA repair was similar in G1 and G2 cells and exhibited both fast and slow components. While the kinetics of chromosome break repair in G1 cells was similar to that of DNA repair, chromosome repair in G2 cells had a diminished fast component and lagged behind DNA repair in terms of fraction of damage repaired. Interestingly, concomitant with a diminished repair capacity in L5178Y-S cells, the number of chromatid exchanges in G2 cells increased with time, whereas it remained constant with repair time in L5178Y-R cells. These results suggest that the basis for the exceptional radiosensitivity of L5178Y-S cells is a defect in the repair of both DNA double-strand breaks and chromosome damage.     

The relationship of DNA and chromosome damage to survival of synchronized X-irradiated L5178Y cells. I. Initial damage

We investigated the role of initial DNA and chromosome damage in determining the radiosensitivity difference between the variant murine leukemic lymphoblast cell lines L5178Y-S (sensitive) and L5178Y-R (resistant) and the difference in cell cycle-dependent variations in radiosensitivity of L5178Y-S cells. We measured initial DNA damage (by the neutral filter elution method) and chromosome damage (by the premature chromosome condensation method) and compared them with survival (measured by cloning) for both cell lines synchronized in G1 or G2 phase of the cell cycle (by centrifugal elutriation) and irradiated with low doses of X rays (up to 10 Gy). The initial yield of DNA and chromosome damage in G2 L5178Y-S cells was almost twice that in G1 L5178Y-S cells and G1 or G2 L5178Y-R cells. In all cases DNA damage expressed as relative elution corresponded with chromosome damage (breaks in G1 chromosomes, breaks and gaps in G2 chromosomes). Also we found that the initial DNA and chromosome damage did not determine cell age-dependent radiosensitivity variations in L5178Y-S cells, as there was less initial damage in the more sensitive G1 phase than in the G2 phase. L5178Y-R cells showed only small changes in survival or initial yield of DNA and chromosome damage throughout the cell cycle. Because survival and initial damage in sensitive and resistant cells irradiated in G2 phase correlated, the difference in radiosensitivity between L5178Y-S and L5178Y-R cells might be determined by initial damage in G2 phase only.