Chromosome aberrations induced by high-LET carbon ions in radiosensitive and radioresistant tumour cells

Chromosome aberration formation was analysed in two human tumour cell lines displaying different radiosensitivity. Aberrations involving chromosomes 2, 4, and 5 were studied in one radioresistant cell line (WiDr) and in one radiosensitive cell line (MCF-7). Chromosome aberrations were studied by application of single-colour FISH. We studied the effects of monoenergetic 100 MeV/u carbon ions and carbon ions from extended Bragg peak. Chromosome aberrations induced by carbon ions were compared with aberrations induced by standard 200 kV X-rays. In both tumour cell lines, carbon ions induced aberrations more effectively than X-rays. The radioresistance and radiosensitivity of the corresponding cell lines, as observed for X-rays, were also found after carbon ion irradiation. In both cell lines, the typical effects of ion irradiation were an increased proportion of cells containing complex aberrations, and an increased complexity of these complex exchanges. However, comparable effects were induced in MCF-7 cells by a much lower dose than in WiDr cells. Insertions were also induced more efficiently in MCF-7 cells than in WiDr cells.     

Kinetics of induction of sister-chromatid exchanges by X-rays through two cell cycles

V79 Chinese hamster cells were exposed to X-rays at various times through the two cell cycles required to obtain harlequin-stained chromosomes. A two-fold SCE enhancement was found between the first and the second G1 phase when BrdUrd was incorporated during the first S phase only. This BrdUrd effect was not found when MNNG was used. Furthermore, the kinetics of SCE and aberrations were different, suggesting two separate mechanisms for their formation: SCE activity takes place when DNA damage occurs before the DNA replication, and aberration activity when the DNA damage occurs chiefly after the DNA replication.     

An explanation of interspecific differences in sensitivity to X-ray-induced chromosome aberrations and a consideration of dose-response curves

Using 1-β- -arabinofuranosylcytosine (AraC) which is an inhibitor of DNA-repair resynthesis, previous studies have shown that the frequency of chromosome-type aberrations is influenced by the rate of repair of araC-inhibitable DNA damage. The experiments described here are a further test of this hypothesis and also an attempt to determine if the different sensitivities of lymphocytes of different species to X-ray-induced aberrations are related to the rate of endonucleolytic incision during repair of DNA damage. Unstimulated lymphocytes from 4 species were exposed to an X-ray dose of 200 rad, and then incubated with araC for 0, 1, 2, 3 or 4 h. The aberration frequencies increased in all species up to 3–4 h. It was also clear that the rate of increase was different between species and was approximately proportional to the ratios of X-ray-induced aberrations observed in the absence of araC. For example, human lymphocytes are approximately twice as sensitive as rabbit lymphocytes to the induction of aberrations by X-rays and the rate of increase of aberrations in the presence of araC was about twice as great in human as rabbit lymphocytes. In addition, using 50, 100, 200 or 300 rad of X-rays and treating human lymphocytes for 0, 1, 2 or 3 h in araC post-irradiation, we have shown that the rate of increase in aberrations is proportional to the amount of araC-inhibitable DNA damage; with a limiting dose at about 50 rad. These results appear to provide a basis for interpreting differences in sensitivities to aberration induction among mammalian species.     

Heterogeneous chromosomal radiosensitivity of phytohaemagglutinin-stimulated human blood lymphocytes in culture

Human blood lymphocytes were irradiated with hard X-rays, stimulated with phytohaemagglutinin (PHA), and grown in presence of amethopterin to accumulate the responding cells at the GI/S boundary of the first cell cycle in vitro. After reversal of the GI/S block with exogenous thymidine, the frequencies of asymmetric chromosome exchanges in relation to the position of metaphases within the first generation mitotic wave were compared. Significant differences of aberration yields within replicate culture series were found in several experiments. A gradual increase of aberration frequencies with increasing duration of S + G2 phases was the most constant feature encountered. In addition, in two parallel series of cultures from one donor, the highest frequency of aberrations was found in samples corresponding to the shortest S+G2 phase duration. A significant contribution of selective mitotic delay of aberration-carrying cells to the distribution of aberration frequencies was excluded. Therefore, it was inferred that the results reflect a true variability of radiosensitivity among the PHA-responsive cells, probably of a discontinuos character, ranging over the ratio of two.     

The effect of X-irradiation on cell cycle progression and chromatid aberrations in stimulated human lymphocytes using cohort analysis studies.

Stage sensitivity for the production of chromatid-type aberrations and mitotic delay has been investigated in a stimulated human lymphocyte population, following an absorbed dose of 1.5 Gy 250 kVp X-rays. BrdU replication banding was used to obtain a fine analysis of the cell cycle and to permit cohort analysis. Fluctuations in yield with sample time were found for all aberration categories, but these could not be related simply to either the developmental stage of the cells at time of exposure, or to the time-to-run to metaphase. In general G2 and late S cells had higher aberration yields than early S and pre S cell populations. Mitotic delay and perturbation at this dose extends to all sub-phases of S and is as great, if not greater, in the earliest S cells as it is in G2.     

Relationships between chromosome damage, cell cycle delay and cell killing induced by bleomycin or X-rays

The extent of mitotic delay and chromosome aberration induction by X-rays and bleomycin has been compared in normal human foetal fibroblasts at doses giving approximately equal levels of cell killing, assayed as colony-forming ability. Bleomycin induced much less G2 delay and chromosome damage than X-rays. We conclude that the major mechanism of cell killing by bleomycin does not involve chromosome damage but the cells pass through a number of division cycles before dying and a common DNA lesion is involved in G2 delay and chromosome damage.     

Effect of linear energy transfer (LET) on the complexity of alpha-particle-induced chromosome aberrations in human CD34+ cells

The aim of this study was to assess the relative influence of the linear energy transfer (LET) of alpha particles on the complexity of chromosome aberrations in the absence of significant other differences in track structure. To do this, we irradiated human hemopoietic stem cells (CD34+) with alpha particles of various incident LETs (110-152 keV/microm, with mean LETs through the cell of 119-182 keV/microm) at an equi-fluence of approximately one particle/cell and assayed for chromosome aberrations by mFISH. Based on a single harvest time to collect early-division mitotic cells, complex aberrations were observed at comparable frequencies irrespective of incident LET; however, when expressed as a proportion of the total exchanges detected, their occurrence was seen to increase with increasing LET. Cycle analysis to predict theoretical DNA double-strand break rejoining cycles was also carried out on all complex chromosome aberrations detected. By doing this we found that the majority of complex aberrations are formed in single non-reducible cycles that involve just two or three different chromosomes and three or four different breaks. Each non-reducible cycle is suggested to represent "an area" of finite size within the nucleus where double-strand break repair occurs. We suggest that the local density of damage induced and the proximity of independent repair areas within the interphase nucleus determine the complexity of aberrations resolved in metaphase. Overall, the most likely outcome of a single nuclear traversal of a single alpha particle in CD34+ cells is a single chromosome aberration per damaged cell. As the incident LET of the alpha particle increases, the likelihood of this aberration being classed as complex is greater.     

Cytogenetic effects of low-dose radiation with different LET in human peripheral blood lymphocytes

Chromosome damage and the spectrum of aberrations induced by low doses of γ-irradiation, X-rays and accelerated carbon ions (195 MeV/u, LET 16.6 keV/μm) in peripheral blood lymphocytes of four donors were studied. G₀-lymphocytes were exposed to 1–100 cGy, stimulated by PHA, and analyzed for chromosome aberrations at 48 h post-irradiation by the metaphase method. A complex nonlinear dose–effect dependence was observed over the range of 1 to 50 cGy. At 1–7 cGy, the cells showed the highest radiosensitivity per unit dose (hypersensitivity, HRS), which was mainly due to chromatid-type aberration. According to the classical theory of aberration formation, chromatid-type aberrations should not be induced by irradiation of unstimulated lymphocytes. With increasing dose, the frequency of aberrations decreased significantly, and in some cases it even reached the control level. At above 50 cGy the dose–effect curves became linear. In this dose range, the frequency of chromatid aberrations remained at a low constant level, while the chromosome-type aberrations increased linearly with dose. The high yield of chromatid-type aberrations observed in our experiments at low doses confirms the idea that the molecular mechanisms which underlie the HRS phenotype may differ from the classical mechanisms of radiation-induced aberration formation. The data presented, as well as recent literature data on bystander effects and genetic instability expressed as chromatid-type aberrations on a chromosomal level, are discussed with respect to possible common mechanisms underlying all low-dose phenomena.     

The effects of radon daughter alpha-particle irradiation in K1 and xrs-5 CHO cell lines

We investigated the radiobiological effects of the radon daughter bismuth-212 (212Bi) in Chinese hamster ovary (CHO) K1 cells and in xrs-5 cells, which are X-ray sensitive and deficient in the ability to rejoin DNA double-strand breaks. The cells were exposed to 250 kVp X-rays or to 212Bi chelated to diethylene triamine pentaacetic acid (DTPA); chelation of 212Bi to DTPA prevented its attachment to or entry into the cells. Cytotoxic, clastogenic, and mutagenic responses of the cells were measured and RBEs (D10, 2 chromatid aberrations/cell and 10 induced 6-thioguanine-resistant mutants) were calculated to be 3.8, 3.5, and 3.9, respectively for K1, and 1.4, 0.8, and 5.1, respectively, for xrs-5. With the exception of the RBE of less than 1 for alpha-induced aberrations in xrs-5, the results are consistent with the following conclusions: (1) alpha-particles are in general more effective cytotoxic, clastogenic and mutagenic agents than X-rays; (2) the primary lethal and clastogenic lesion induced by both X-rays and alpha-particles is probably a DNA double-strand break; (3) DNA double-strand breaks induced by alpha-radiation are less well repaired than those induced by X-rays, although a portion of alpha-induced damage is repairable; and (4) deficiencies in rejoining DNA double-strand breaks affect the clastogenic and cytotoxic effects of X-rays and alpha-radiation, not their mutagenic effects. The RBE of 0.8 for aberration induction in xrs-5 cells could reflect a deficiency in the ability of these cells to convert alpha-induced damage to chromosome aberrations. Alternatively, the RBE of less than 1 might reflect an unusual sensitivity of xrs-5 cells to alpha-induced G2 delays.     

The effect of functional inactivation of TP53 by HPV-E6 transformation on the induction of chromosome aberrations by gamma rays in human tumor cells

The influence of expression of TP53 (formerly known as p53) on the induction of chromosome aberrations by gamma rays was examined in an isogenic pair of human tumor cell lines where TP53 expression was normal or inactivated by human papillomavirus (HPV) type 16 E6 expression. Plateau-phase cultures were exposed to 0-8 Gy gamma rays and then either immediately released by subculture or held for 24 h prior to subculture and subsequent cytogenetic analysis. Aberration frequency was determined only in cells entering their first mitosis after irradiation, and cells were sampled over a 48-h period to include cells whose progression into mitosis was delayed. While aberration frequencies were similar at early harvest times, there was evidence for a subpopulation of more heavily damaged cells in the E6-transformed cells that cycled into late mitosis. Holding cells noncycling for 24 h to allow repair of potentially lethal damage eliminated this subpopulation of more heavily damaged cells. The E6-transformed cells also had higher levels of chromatid-type aberrations and sister chromatid exchanges, consistent with an additional defect in kinetics of repair of base damage that is associated with the E6 transformation. Holding cells noncycling for 24 h eliminated the elevated levels of chromatid-type aberrations and sister chromatid exchanges. These studies demonstrate that E6 transformation of human tumor cells will influence both the frequency and types of chromosome aberrations observed after radiation exposure, and that these effects are related to the expression of potentially lethal damage.     

Frequencies of chromosomal aberrations induced in human blood lymphocytes by low doses of X-rays

The dose-response for radiation-induced chromosome aberrations in human lymphocytes is usually fitted to the quadratic model. This assumes that the slope is essentially linear at low doses. Empirical observations of linearity at less than 200 mGy are, however, sparse. Some data have been published indicating a non-linear (threshold) response and these are reviewed. In particular one study with X-rays showed a plateau in response up to 50 mGy and with a significant dip below the control level at 4 mGy. The mechanism proposed to explain non-linearity is that low doses stimulate the enzymic repair capability of lymphocytes. Preliminary data are presented from a large experiment by six laboratories in which the low dose-response for X-rays has been re-examined. The plateau in the dose-response relationship, if it exists, does not extend to doses above approximately 10 mGy. No irradiated cells yielded aberration levels significantly below the control. Over the range 0-300 mGy the response can be fitted to a linear regression. There are, however, variations in sensitivity between cells from different donors. An unexpected finding was that some lymphocytes contained greater than 1 exchange aberrations. This may indicate a small subset of cells that are especially susceptible to the induction of aberrations by low doses.     

An autoradiographic study of unscheduled DNA synthesis in the germ cells of male mice treated with X-rays and methyl methanesulfonate

Unscheduled DNA synthesis (UDS) in the germ cells of male mice after in vivo treatment with X-rays or methyl methanesulfonate (MMS) was assayed by use of a quantitative autoradiographic procedure. MMS induced UDS in meiotic through type III elongating spermatid stages, whereas X-rays induced UDS in meiotic through round spermatid stages. No UDS was detected in the most mature spermatid stages present in the testis with either MMS or X-rays. Taking into account differences in DNA content of the various germ-cell stages studied, we concluded that X-rays induced a maximum UDS response in spermatocytes at diakinesis--metaphase I. The level of UDS induced by MMS was about the same in all the stages capable of repair. Chromosome damage and UDS were measured simultaneously in the same spermatocytes at diakinesis 90 min after X-irradiation or MMS treatment. The level of UDS in most of the X-irradiated cells paralleled the extent of chromosome damage induced. A statistical analysis of these results revealed a positive correlation. As expected, MMS induced no chromosome aberrations above control levels. Therefore no correlation was determined between UDS and chromosome damage in this case. The distribution of UDS over the chromosomes treated at diakinesis with MMS or X-rays was studied. It was found that UDS occurred in clusters in the irradiated cells, whereas it was uniformly distributed in the MMS-treated cells.     

Genetic Damage at Specific Gene Loci in Drosophila with Betatron X-Rays

The mutation rate was determined for mature sperm at eight specific gene loci on the third chromosome of Drosophila melanogaster using the low ion density radiations of 22 Mev betatron X-rays. A dose of 3000 rads of betatron X-rays produced a mutation rate of 4.36 x 10^-8 per rad/locus. Among the mutations observed, 66% were recessive lethals and 34% viable when homozygous. Only one of the 24 viable mutations was associated with a chromosome aberration. Among the 47 recessive lethals, no two-break aberrations were detected in 48.9% of the lethals, deletions were associated with 42.2%, inversions with 6.7% and translocations with 2.2%.—When these genetic results are compared to those for 250 KV X-rays, the mutation rate for betatron treatments was slightly lower (.76), the recessive lethal rate among induced mutations was higher, and the chromosome aberrations among lethal mutations were slightly lower than with 250 KV X-rays. Although the two types of irradiations differ by an ion density of approximately ten, the amount and types of inheritable genetic damage induced by the two radiations in mature sperm were not significantly different.     

新制癌菌素和平阳霉素诱发蚕豆染色体畸变的比较研究

本文报道了新制癌菌素(NCS)能诱发植物染色体畸变,同时观察了利用咖啡因后处理对NCS、PYM诱发染色体畸变的影响,研究了PYM切断DNA断头的性质。结果表明,NCS切割DNA产生3'-羟基末端和3'-磷酸末端;咖啡因能封闭3'-羟基末端抑制DNA的修复,从而提高诱变频率。PYM加咖啡因后处理,其染色体畸变频率与PYM单独处理无明显差异。说明PYM切断DNA所得到的产物,不是3'-羟基末端,而是3'-磷酸末端。     

Chromosomal instability in an oxygen-tolerant variant of Chinese hamster ovary cells

Background levels of chromosomal aberrations and sister-chromatid exchanges (SCEs) were determined in CHO-99 cells, an oxygen-tolerant variant substrain of Chinese hamster ovary (CHO-20) cells capable of stable proliferation under an atmosphere of 99% O2/1% CO2, a level of hyperoxia at which cultured mammalian cells normally cannot survive. The mean chromosomal aberration frequency in CHO-99 cells was as high as 1 aberration per cell (mainly chromatid and chromosome gaps and breaks) versus 0.05 aberration/cell in CHO-20 cells, while the SCE frequency was 1.7- to 2.1-fold increased. While most aberrations were apparently distributed at random over the chromosomes, up to 31% of the aberrations appeared to be involved in site-specific fragility at a homologous site in chromosomes Z3 and Z4. Immediately upon shifting CHO-99 cells to air-equilibrated conditions their SCE frequency decreased to the control level, whereas the aberration rate persisted at a still elevated level of 0.16-0.31 aberration per cell, even after a culture period of 14 weeks under normoxia. This indicates that at least part of the chromosomal instability is a constitutional property of the variant cells, i.e., not directly dependent upon hyperoxic stress. In CHO-99 X CHO-20 hybrids the occurrence of chromatid-type aberrations and fragile site but not that of chromosome-type aberrations was suppressed under normoxic conditions, suggesting that chromatid-type aberrations and fragile site expression on the one hand and chromosome-type aberrations on the other hand are mediated by different constitutional defects in CHO-99 cells. No gross alterations in (deoxy)ribonucleoside triphosphate pools were detected in CHO-99 cells that could be held responsible for their chromosomal instability. In addition, no increased level of DNA damage was detected by the technique of alkaline elution. The excessive chromosomal instability in CHO-99 cells, as observed under hyperoxic conditions, may originate from reactive intermediates giving rise to DNA double-strand breaks and/or a type of DNA lesion that is resistant to the conditions of the alkaline elution technique. However, alternative mechanisms based upon reactive species interfering with DNA replication/repair processes cannot be excluded.     

Modulation of restriction enzyme-induced damage by chemicals that interfere with cellular responses to DNA damage: a cytogenetic and pulsed-field gel analysis

The electroporation of restriction enzymes into mammalian cells results in DNA double-strand breaks that can lead to chromosome aberrations. Four chemicals known to interfere with cellular responses to DNA damage were investigated for their effects on chromosome aberrations induced by AluI and Sau3AI; in addition, the number of DNA double-strand breaks at various times after enzyme treatment was determined by pulsed-field gel electrophoresis (PFGE). The poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide (3AB) dramatically increased the yield of exchanges and deletions and caused a small but transitory increase in the yield of double-strand breaks induced by the enzymes. 1-beta-D-Arabinofuranosylcytosine, which can inhibit DNA repair either by direct action on DNA polymerases alpha and delta or by incorporation into DNA, potentiated aberration induction but to a lesser extent than 3AB and did not affect the amount of DNA double-strand breakage. Aphidicolin, which inhibits polymerases alpha and delta, had no effect on AluI-induced aberrations but did increase the aberration yield induced by Sau3AI. The postreplication repair inhibitor caffeine had no effect on aberration yields induced by either enzyme. Neither aphidicolin nor caffeine modulated the amount of DNA double-strand breakage as measured by PFGE. These data implicate poly(ADP-ribosyl)ation and polymerases alpha and delta as important components of the cellular processes required for the normal repair of DNA double-strand breaks with blunt or cohesive ends. Comparison of these data with the effect of inhibitors on the frequency of X-ray-induced aberrations leads us to the conclusion that X-ray-induced aberrations can result from the misjoining or nonrejoining of double-strand breaks, particularly breaks with cohesive ends, but that this process accounts for only a portion of the induced aberrations.     

Chromosomal radiosensitivity of human leucocytes in relation to sampling time

Frequencies of chromosomal aberrations after irradiation with X-rays of peripheral blood lymphocytes in vitro were determined at different times after initiation of cultures. In each culture, the kinetics of cell multiplication was followed by using BrdU labelling and differential staining of chromosomes.The results indicate that the mixing up of first and second cell cycle cells at later sampling times cannot explain the observed variation in the frequencies of chromosomal aberrations but that donor-to-donor variation is a predominant factor influencing yields of aberrations. The condition of a donor seems to be most important because repeats on the same donor also showed marked variability.     

High chromosomal sensitivity of Chinese hamster xrs 5 cells to restriction endonuclease induced DNA double-strand breaks

The cytogenetic effects of restriction endonucleases (RE) and X-rays were examined in the radiosensitive mutant Chinese hamster cell line xrs 5 and its normal parental line CHO K1. Cells were permeabilized with Sendai virus and exposed to Pvu II and Eco RV which induce blunt-ended double-strand breaks (dsb) in the DNA of cells, or Bam H1 and Eco R1 which induce cohesive-ended dsb with a four-base overlap. Treated cells were then assayed for the presence of metaphase chromosomal aberrations by sampling at multiple fixation times and in experiments where cells were exposed to graded series of RE concentrations. Exposure to X-rays or RE causing blunt-ended dsb was found to be between two and three times more effective in xrs 5 than in CHO K1 cells. We interpret this higher chromosomal sensitivity of xrs 5 cells as reflecting the reported defect in dsb repair in xrs 5. Both xrs 5 and CHO K1 cells yielded less aberrations after exposure to Bam H1 or Eco R1 than after exposure to Pvu II or Eco RV, confirming our previous results and demonstrating that cohesive-ended dsb are less damaging than blunt-ended dsb. Multiple fixation time experiments showed that the higher sensitivity of xrs 5 was evident at several different sampling times after treatment. Similarly the low yield of aberrations after exposure of cells to Bam H1 was evident at all sampling times. Overdispersion of chromosomal aberrations was observed in samples exposed to RE. This is thought to be due to a non-uniform permeabilization of the cell population to RE. Our results indicate that RE-induced dsb are handled by cells in a similar way to those arising during X-ray exposure.     

FISH-based analysis of 10- and 25-kV soft X-ray–induced DNA damage in 184A1 human mammary epithelial cells

Over the past years, several in vitro studies have been performed on DNA damage induced by soft X-rays, especially in the energy range below 50 keV. Radiation effects originating from such low-energy photons are relevant in the context of medical diagnostics, for example, mammography, or of accidental exposure to scattered radiation. The present study was initiated to investigate the X-ray energy-dependent induction of stable and unstable chromosomal aberrations in the human mammary epithelial cell line 184A1. Three colour fluorescence in situ hybridisation was applied to identify chromosomal damage in chromosomes 1, 8 and 17, induced by 10-kV or 25-kV soft X-rays as well as by 200-kV X-rays as a reference quality. The overall results confirm the X-ray energy dependencies published for human lymphocytes showing increasing chromosomal aberration frequencies and higher aberration complexity with decreasing X-ray energy and increasing dose. Comparing the obtained dose dependencies, ratios of 0.84 ± 0.09 and 1.22 ± 0.18 were revealed for stable translocations induced by 25- and 10-kV X-rays, respectively, using 200-kV X-rays as reference. Moreover, the analysis of the minimum number of breaks required to form the visible chromosomal damage resulted in similar ratios of 0.93 ± 0.07 for 25-kV X-rays and 1.25 ± 0.10 for 10-kV X-rays relative to 200-kV X-rays. In addition, non-DNA-proportional contributions of chromosomes 8 and 17 to the whole DNA damage and deviations from the expected 1:1 ratio of translocations and dicentrics were observed for cell line 184A1.     

Induction of chromosome damage by restriction enzymes during mitosis.

Once electroporated into the nucleus of eukaryotic cells, restriction enzymes will bind at specific DNA sequences and cleave DNA to make double-strand breaks. These induced breaks can lead to chromosome aberrations and consequently offer one approach to determining the mechanism(s) of aberration formation. Because the higher-order structure of DNA in eukaryotic cells might influence the ability of restriction enzymes to locate their recognition sequence, bind, and cleave DNA, we have investigated whether enzymes will cut DNA during metaphase when the chromosomes are most condensed. Chinese hamster ovary cells synchronized in mitosis and treated with either AluI or Sau3AI showed few chromosome aberrations when held in mitosis for 1, 2, or 3 h after enzyme treatment. However, some disruption of chromosome morphology was seen, especially after exposure to Sau3AI. When cells were allowed to complete one cell cycle after enzyme treatment in the preceding mitosis, there was extensive chromosome damage, with the most abundant type of lesion being the interstitial deletion. It appears that restriction enzymes will cleave the highly condensed DNA in mitotic cells but that decondensation, DNA replication, and recondensation are required before the aberrations are manifested.