Evidence that unrejoined DNA double-strand breaks are not predominantly responsible for chromosomal radiosensitivity of AT fibroblasts

To examine more fully the nature of chromosomal radiosensitivity in ataxia telangiectasia (AT) cells, we employed 24-color combinatorial painting to visualize 137Cs gamma-ray-induced chromosome-type aberrations in cells of two AT and one normal primary human fibroblast strains irradiated in log-phase growth. As a measure of misrejoined radiation-induced DSBs, we quantified exchange breakpoints associated with both simple and complex exchanges. As a measure of unrejoined DSBs, we quantified breakpoints from terminal deletions as well as deletions associated with incomplete exchange. For each of these end points, the frequency of damage per unit dose was markedly higher in AT cells compared to normal cells, although the proportion of total breaks that remained unrejoined was rather similar. The majority of breakpoints in both cell types were involved in exchanges. AT cells had a much higher frequency of complex exchanges compared to normal cells given the same dose, but for doses that resulted in approximately the same level of total breakpoints, the relative contribution from complex damage was also similar. We conclude that although terminal deletions and incomplete exchanges contribute to AT cell radiosensitivity, their relative abundance does not-in apparent contrast to the situation in lymphoblastoid cells-overwhelmingly account for the increased damage we observed in cycling AT fibroblasts. Thus, from a cytogenetic perspective, a higher level of unrepaired DSBs does not provide a universal explanation for the radiation-sensitive AT phenotype.     

Hypotonic treatment leads to chromosomal aberrations but not to sister-chromatid exchanges in human lymphocytes

Human peripheral lymphocytes were isolated from whole blood and exposed to culture medium of reduced osmolality. This hypotonic treatment led to a significant increase in the frequencies of chromosomal aberrations when the osmolality was reduced to 60 mOsm/kg H2O and below. Maximum damage occurred when the hypotonic treatment was done 27 or 30 h after starting the cultures. We also looked for the induction of sister-chromatid exchanges (SCE) by hypotonic culture conditions, but the SCE frequencies were not influenced.     

Securin is not required for chromosomal stability in human cells

Abnormalities of chromosome number are frequently observed in cancers. The mechanisms regulating chromosome segregation in human cells are therefore of great interest. Recently it has been reported that human cells without an hSecurin gene lose chromosomes at a high frequency. Here we show that, after hSecurin knockout through homologous recombination, chromosome losses are only a short, transient effect. After a few passages hSecurin^−/− cells became chromosomally stable and executed mitoses normally. This was unexpected, as the securin loss resulted in a persisting reduction of the sister-separating protease separase and inefficient cleavage of the cohesin subunit Scc1. Our data demonstrate that securin is dispensable for chromosomal stability in human cells. We propose that human cells possess efficient mechanisms to compensate for the loss of genes involved in chromosome segregation.     

Non-linear chromosomal inversion response in prostate after low dose X-radiation exposure

Somatic intrachromosomal recombination can result in inversions and deletions in DNA, which are important mutations in cancer. The pKZ1 chromosomal inversion assay is a sensitive assay for studying the effects of DNA damaging agents using chromosomal inversion as a mutation end-point. We have previously demonstrated that the chromosomal inversion response in pKZ1 spleen after single low doses of X-radiation exposure does not follow the linear no-threshold dose–response model. Here, we optimised a chromosomal inversion screening method to study the effect of low dose X-radiation exposure in pKZ1 prostatic tissue. In the present study, a significant induction in inversions was observed after ultra-low doses of 0.005–0.01 mGy or after a high dose of 1000 mGy, whereas a reduction in inversions to below the sham-treated frequency was observed between 1 and 10 mGy exposure. This is the first report of a reduction to below endogenous frequency for any mutation end-point in prostate. In addition, the doses of radiation studied were at least three orders of magnitude lower than have been reported in other mutation assays in prostate in vivo or in vitro. In sham-treated pKZ1 controls and in pKZ1 mice treated with low doses of 1–10 mGy the number of inversions/gland cross-section rarely exceeded three. Up to 4 and 7 inversions were observed in individual prostatic gland cross-sections after doses ≤0.02 mGy and after 1000 mGy, respectively. The number of inversions identified in individual cross-sections of prostatic glands of untreated mice and all treated mice other than the 1000 mGy treatment group followed a Poisson distribution. The dose–response curves and fold changes observed after all radiation doses studied were similar in spleen and prostate. These results suggest that the pKZ1 assay is measuring a fundamental response to DNA damage after low dose X-radiation exposure which is independent of tissue type.     

Evidence for chromosomal replicons as units of sister chromatid exchanges

Chromosomal replicons have been described as the cytological counterpart of DNA replicon clusters and have previously been studied in vitro using premature chromosome condensation-sister chromatid differentiation (PCC-SCD) techniques. Chromosomal replicons are visualized as small SCD segments in S-phase cells, and measurement of these segments can provide estimates of relative chromosomal replicon size corresponding to DNA replicon clusters functioning coordinately in S-phase. Current hypotheses of sister chromatid exchange (SCE) formation postulate that sites of SCE induction are associated with active replicons or replicon clusters. We have applied the PCC-SCD technique to in vivo studies of mouse bone marrow cells that have been treated with cyclophosphamide (CP) for two cell cycles. We have been able to visualize chromosomal replicons, as well as SCEs which have been induced in vivo by CP treatment, simultaneously in the same cells. Chromosomal replicons visualized as small SCD segments were measured in PCC cells classified at early or late S-phase based on SCD segment size prevalence. Early S-phase (E/S) PCC cells contained 90% of the SCD segments measured clustered in a segment size range of 0.1 to 0.8 m with a peak value around 0.3 to 0.6 m regardless of CP treatment. As the cells progressed through S-phase, late S-phase (L/S) PCC cells were characterized by the appearance of larger SCD segments and even whole SCD chromosomes in addition to small SCD segments. A concentration of units around 0.4 to 1.0 m was found for L/S SCD segment size distributions regardless of CP treatment with an apparent bimodal profile. Our in vivo data support the existence of a subunit organization of chromosomal replication with a basic functional unit being 0.3 to 0.6 m in size. In addition, we have found that this chromosomal unit of replication or chromosomal replicon does not seem to be functionally perturbed by the mutagen CP. We also found that small SCD segments of 0.4 to 0.7 m in length were involved in the formation of an SCE, suggesting that both spontaneous and CP-induced SCEs occur between chromosomal replicons. These findings provide direct cytogenetic evidence to support a replicon cluster/chromosomal replicon model for SCE formation.     

Microsatellite length differences between humans and chimpanzees at autosomal Loci are not found at equivalent haploid Y chromosomal Loci

When homologous microsatellites are compared between species, significant differences in mean length are often noted. A dominant cause of these length differences is ascertainment bias due to selection for maximum repeat number and repeat purity when the markers are being developed. However, even after ascertainment bias has been allowed for through reciprocal comparisons, significant length differences remain, suggesting that the average microsatellite mutation rate differs between species. Two classes of mechanism have been proposed: rapid evolution of enzymes involved in the generation and repair of slippage products (enzyme evolution model) and heterozygote instability, whereby interchromosomal events at heterozygous sites offer extra opportunities for mutations to occur (heterozygote instability model). To examine which of these hypotheses is most likely, we compared ascertainment bias and species length differences between humans and chimpanzees in autosomal and Y chromosomal microsatellites. We find that levels of ascertainment bias are indistinguishable, but that interspecies length differences are significantly greater for autosomal loci compared with haploid Y chromosomal loci. Such a pattern is consistent with predictions from the heterozygote instability model and is not expected under models of microsatellite evolution that do not include interchromosomal events such as the enzyme evolution model.     

Influence of dose rate on the induction of simple and complex chromosome exchanges by gamma rays

Single-color painting of whole chromosomes, or protocols in which only a few chromosomes are distinctively painted, will always fail to detect a proportion of complex exchanges because they frequently produce pseudosimple painting patterns that are indistinguishable from those produced by bona fide simple exchanges. When 24-color multi-fluor FISH (mFISH) was employed for the purpose of distinguishing (truly) simple from pseudosimple exchanges, it was confirmed that the acute low-LET radiation dose-response relationship for simple exchanges lacked significant upward curvature. This result has been interpreted to indicate that the formation of simple exchanges requires only one chromosome locus be damaged (e.g. broken) by radiation to initiate an exchange-not two, as classical cytogenetic theory maintains. Because a one-lesion mechanism implies single-track action, it follows that the production of simple exchanges should not be influenced by changes in dose rate. To examine this prediction, we irradiated noncycling primary human fibroblasts with graded doses of (137)Cs gamma rays at an acute dose rate of 1.10 Gy/min and compared, using mFISH, the yield of simple exchanges to that observed after exposure to the same radiation delivered at a chronic dose rate of 0.08 cGy/min. The shape of the dose response was found to be quasi-linear for both dose rates, but, counter to providing support for a one-lesion mechanism, the yield of simple aberrations was greatly reduced by protracted exposure. Although chronic doses were delivered at rates low enough to produce damage exclusively by single-track action, this did not altogether eliminate the formation of complex aberrations, an analysis of which leads to the conclusion that a single track of low-LET radiation is capable of inducing complex exchanges requiring up to four proximate breaks for their formation. For acute exposures, the ratio of simple reciprocal translocations to simple dicentrics was near unity.     

Sister-chromatid exchanges are increased in epileptics, but not by sodium valproate.

Sister-chromatid exchange (SCE) frequency has been studied from peripheral blood lymphocyte cultures of 21 patients with epilepsy on sodium valproate, 20 patients who had not started therapy (untreated) and 20 normal healthy controls. Treated and untreated patients with epilepsy were observed to have higher SCE frequencies (mean 9.05 and 9.82 respectively) than healthy controls (mean 4.8; P < 0.001). There was no significant difference in SCE frequency between treated and untreated patients. This suggests that the disease itself may be associated with an increased frequency of SCEs.     

Constraints in chromosomal inversions in Escherichia coli are not explained by replication pausing at inverted terminator-like sequences

Regions close to the replication terminus of the Escherichia coli chromosome are strongly refractory to genomic inversions. Since these regions also harbour polar replication terminator-like sequences or pause sites, we have investigated the possibility that slowing of replication as a result of pausing at inverted pause sites is responsible for inability to isolate stable inversions affecting these regions. A mutation in the tus gene is known to abolish replication pausing at terminators. We show here that the distribution of invertible and noninvertible segments along the chromosome is not affected by tus mutations. This observation eliminates replication pausing as a cause for the reduced fitness of bacteria harbouring certain chromosomal inversions.     

The radiation response of asynchronous cells at low dose: evidence of substructure

Flow cytometry and cell sorting techniques have been used together with repeated measurement in an attempt to define better the radiation survival response of asynchronously dividing Chinese hamster V79-171 cells under aerobic and hypoxic conditions. Although the first two decades of cell inactivation have been examined, particular attention has been given to the low-dose range of a few grays, as used in individual radiation therapy treatments. A single linear-quadratic dose-response function was consistently unable to fit both the low-dose and high-dose data satisfactorily, suggesting a two-component response. Separate fitting of the low-dose and high-dose portions of the response yielded alpha and beta values which differed significantly (P = 0.001 to 0.002). The data are consistent with the hypothesis that the observed substructure simply reflects the presence of subpopulations of sensitive (G1-, G2-phase) and resistant (late S-phase) cells, which are resolved in these measurements. These results may have significance for certain situations in radiation therapy and in biophysical modeling of the radiation response.     

Two strands of DNA are not equivalent as probes in hybridizations at low stringency

Only one of the two complementary strands of a restriction fragment hybridizes under low stringency conditions to a cloned Arabidopsis thaliana genomic DNA fragment. We propose that this effect is caused by the energetic nonequivalence of the two possible mismatched duplexes, resulting from the accumulation of mismatches and extrahelical bases. These mismatches will differ between the two duplexes. The choice of probe strand may therefore be important for the success of heterologous hybridizations utilizing single-stranded probes.     

Neutron dose effect relationships at low doses

Summary Stimulated by recent observations of non-linearity in the dose effect relationship for the transformation of mammalian cells in vitro by fission neutron irradiation and the reverse dose rate effect in this system, the data for mutation induction in the stamen hairs ofTradescantia occidentalis has been re-examined. The non-linear dose effect relationships suggested in the original reports are confirmed both by the dose effect relationships themselves and by an examination of the statistics of the results. This non-linearity also appears to be present in the more recent observations of other workers. It is suggested that the non-linearity in the system may be due to a sub-population of cells in a particularly sensitive phase of the cell cycle at the time of irradiation. There is a possibility that the data also indicate a qualitative difference in the underlying biophysical actions of neutron and photon radiations.Dedicated to Prof. W. Jacobi on the occasion of his 60th birthday     

Effects of low dose rate (0.003-0.025 Gy/h) chronic X-irradiation on radioresistant and radiosensitive L5178Y mouse lymphoma cells

Cultures of radioresistant (LY-R) and radiosensitive (LY-S) strains of L5178Y mouse lymphoma cells were exposed continuously to X-rays delivered at dose rates ranging from 0.003 to 0.025 Gy/h for up to 35 days. Populations of both strains proliferated actively during the exposure, but the growth rates were reduced in a dose rate-dependent manner. The reduction of growth rate occurred for strain LY-S earlier during the exposure and at lower dose rates than for strain LY-R. The survival (as measured by colony forming ability) of strain LY-R was affected only slightly at all dose rates applied. For strain LY-S, a decrease in the surviving fraction was observed in the initial part of the exposure. This decrease was followed by a plateau and eventually by an increase, in some cases to values close to the control level. The increase in the surviving fraction indicated that the radioresistance of the exposed LY-S cells had increased. This pattern was particularly clear for dose rates greater than 0.014 Gy/h. The pre-irradiated cells exhibited radioresistance when exposed to acute X-radiation after termination of the chronic exposure. The increase in radiation resistance was stable for at least 70 days after termination of the protracted exposure. These results show that mutagenic and/or selective phenomena leading to an increase in radiation resistance of mammalian cells can be caused by protracted exposures to X-rays at dose rates permitting active proliferation.     

Determination of the chromosomal site for the human radiosensitive ataxia telangiectasia gene by chromosome transfer

The chromosomal localization of the gene which complements radiation hypersensitivity of AT cells was studied by microcell-mediated chromosome transfer. A 6-thioguanine-resistant derivative of an immortalized AT cell line, AT2KYSVTG, was used as a recipient for microcell-mediated chromosome transfer from 4 strains of mouse A9 cells, 3 of which carried a human X/11 recombinant chromosome containing various regions of chromosome 11, while the other carried an intact X chromosome. HAT-resistant microcell hybrids were isolated and examined for their radiosensitivity and chromosome constitution. The microcell hybrid clones obtained from the transfer of an intact X chromosome or an X/11 chromosome bearing the pter → q13 region of chromosome 11 did not show a difference in radiosensitivity from parental AT cells, while those obtained from the transfer of X/11 chromosomes bearing either the p11 → qter or the pter → q23 region of chromosome 11 exhibited a marked radioresistance which was comparable to normal human fibroblasts. A HAT-resistant but radiosensitive variant was further obtained from the microcell fusion with an A9 cell strain carrying an X/11 chromosome bearing the 11p11 → qter region, in which a deletion at the 11q23 region was found. The results indicate that the gene which complements a radiosensitive phenotype of AT is located at the q23 region of chromosome 11.     

Ectopically hTERT expressing adult human mesenchymal stem cells are less radiosensitive than their telomerase negative counterpart

During the past several years increasing evidence indicating that the proliferation capacity of mammalian cells is highly radiosensitive, regardless of the species and the tissue of origin of the cells, has accumulated. It has also been shown that normal bone marrow cells of mice have a similar radiosensitivity to other mammalian cells so far tested. In this study, we investigated the genetic effects of ionizing radiation (2.5-15 Gy) on normal human mesenchymal stem cells and their telomerised counterpart hMSC-telo1. We evaluated overall genomic integrity, DNA damage/repair by applying a fluorescence-detected alkaline DNA unwinding assay together with Western blot analyses for phosphorylated H2AX and Q-FISH was applied for investigation of telomeric damage. Our results indicate that hMSC and TERT-immortalized hMSCs can cope with relatively high doses of gamma-rays and that overall DNA repair is similar in the two cell lines. The telomeres were extensively destroyed after irradiation in both cell types suggesting that telomere caps are especially sensitive to radiation. The TERT-immortalized hMSCs showed higher stability at telomeric regions than primary hMSCs indicating that cells with long telomeres and high telomerase activity have the advantage of re-establishing the telomeric caps.     

Restriction endonucleases do not induce sister-chromatid exchanges in Chinese hamster ovary cells

Bacterial restriction enzymes offer the unique opportunity to determine the biological and cytogenetic consequences of DNA double-strand breakage. To examine the role of various types of breaks in sister-chromatid exchange (SCE) formation, we used restriction enzymes with different recognition sequences and different cutting frequencies to generate DNA double-strand breaks in Chinese hamster ovary (CHO) cells. The restriction enzymes were introduced by electroporation into exponentially growing cells during the second replication cycle in bromodeoxyuridine, and SCEs were analyzed at mitosis. Contrary to results reported by others, we found no increase in SCE frequency in cells exposed to restriction enzymes despite the presence of numerous cells with chromatid aberrations. These data suggest that DNA double-strand breaks do not lead to SCE formation.     

Monogenic traits are not simple: lessons from phenylketonuria.

The classification of genetic disease into chromosomal, monogenic and multifactorial categories is an oversimplification. Phenylketonuria (PKU) is a classic 'monogenic' autosomal recessive disease in which mutation at the human PAH locus was deemed sufficient to explain the impaired function of the enzyme phenylalanine hydroxylase (enzymic phenotype), the attendant hyperphenylalaninemia (metabolic phenotype) and the resultant mental retardation (cognitive phenotype). In the era of molecular genetics, expectations for a consistently close correlation between the mutant genotype and variant phenotype have been somewhat disappointed, and PKU is used here to illustrate how and why this might be the case. So-called monogenic traits do, indeed, conform to long-accepted ideas about the expression of 'major' loci and their importance in determining parameters of phenotype, but the associated features are as complex, in their own ways, as those in so-called complex traits.     

Radiation-induced chromosomal aberrations in human lymphocytes. I. Dependence on the dose of gamma-rays and an anomaly at low doses

Cultures of human lymphocytes obtained from blood of healthy adult donors were irradiated with different doses of ⁶⁰Co γ-rays and the irradiated cells were analysed in metaphase 50 h after irradiation. The effect (total yield of abberations of chromosome type, or total yield of exchange type abberations) produced by the lowest dose (5 rad) appears to be statistically significant in a sample of 1500 cells. In the usual dose range (25–400 rad), both parabolic and linear-quadratic equations give a satisfactory fit of experimental data (dicentrics, fragments, or all aberrations of chromosome type). Low doses of γ-rays, however, produced more aberrations than expected, if one extrapolates dose-effect curves from higher doses. Both relations should be considered, therefore, merely as empirical equations. Dicentrics show at low doses (10–30 rad) a plateau which appears to be statistically significant. Some indications are obtained that the total number of chromosome-type aberrations is a more reliable criterion of cytogenetic damage than the usually accepted yeild of dicientrics and rings.     

DNA damage in non-proliferating cells subjected to ionizing irradiation at high or low dose rates

Ionizing radiation damage to the genome of a non-cycling mammalian cell is analyzed using continuous time Markov chains. Immediate damage induced by the radiation is modeled as a batch Poisson arrival process of DNA double strand breaks (DSBs). Different kinds of radiation, for example gamma rays or alpha particles, have different batch probabilities. Enzymatic modulation of the immediate damage is modeled as a Markov process similar to the processes described by the master equation of stochastic chemical kinetics. An illustrative example is the restitution/complete exchange model, which postulates that radiation induced DSBs can subsequently either undergo enzymatically mediated repair (restitution) or can participate pairwise in chromosome exchanges, some of which make irremediable lesions such as dicentric chromosome aberrations. One may have rapid irradiation followed by enzymatic DSB processing or have prolonged irradiation with both DSB arrival and enzymatic DSB processing continuing throughout the irradiation period. A complete solution of the Markov chain is known for the case that the exchange rate constant is negligible so that no irremediable chromosome lesions are produced and DSBs are the only damage to the genome. Using PDEs for generating functions, a perturbation calculation is made assuming the exchange rate constant is small compared to the repair rate constant. Some non-perturbative results applicable to very prolonged irradiation are also obtained using matrix methods: Perron-Frobenius theory, variational methods and numerical approximations of eigenvalues. Applications to experimental results on expected values, variances and statistical distributions of DNA lesions are briefly outlined.Continuous time Markov chain models are the most systematic of those current radiation damage models which treat DSB-DSB interactions within the cell nucleus as homogeneous (e.g. ignore diffusion limitations). They contain most other homogeneous models as special cases, limiting cases or approximations. However, applying the continuous time Markov chain models to studying spatial dependence of DSB interactions, which is generally believed to be very important in some situations, presents difficulties.