Clonally expanded T-cell populations in atomic bomb survivors do not show excess levels of chromosome instability

Radiation-induced genomic instability has been studied primarily in cultured cells, while in vivo studies have been limited. One major obstacle for in vivo studies is the lack of reliable biomarkers that are capable of distinguishing genetic alterations induced by delayed radiation effects from those that are induced immediately after a radiation exposure. Here we describe a method to estimate cytogenetic instability in vivo using chromosomally marked clonal T-cell populations in atomic bomb survivors. The basic idea is that clonal translocations are derived from single progenitor cells that acquired an aberration, most likely after a radiation exposure, and then multiplied extensively in vivo, resulting in a large number of progeny cells that eventually comprise several percent of the total lymphocyte population. Therefore, if chromosome instability began to operate soon after a radiation exposure, an elevated frequency of additional but solitary chromosome aberrations in clonal cell populations would be expected. In the present study, six additional translocations were found among 936 clonal cells examined with the G-band method (0.6%); the corresponding value with multicolor FISH analysis was 1.2% (4/333). Since these frequencies were no higher than 1.2% (219/17,878 cells), the mean translocation frequency observed in control subjects using the G-band method, it is concluded that chromosome instabilities that could give rise to an increased frequency of persisting, exchange-type aberrations were not commonly generated by radiation exposure.     

Chromosome aberrations of clonal origin in irradiated and unexposed individuals: assessment and implications.

Chromosome painting has proven useful for the detection of chromosomal rearrangements, although the presence of cells containing clonal aberrations can have an effect on the outcome of cytogenetic analyses (e.g. aberration frequency and chromosomal distribution studies). Cells with clonal chromosomal changes have been found in studies of both radiation-exposed Chernobyl cleanup workers ("liquidators") and healthy unexposed human subjects. We have used a simple statistical method to aid in the identification of individuals from distinct Chernobyl radiation-exposed and unexposed control populations who may possess cells containing clonal rearrangements. A chi2 value determined from the observed number of aberrations and the expected number based on chromosome length that corresponds to a probability less than 0.005 appears to be an indicator of clonality. These selected individuals can be analyzed further for clonality, thereby sparing detailed examination of the entire population. Here we present an analysis of individuals possessing clonal aberrations to assess the influence of clonality on the results of cytogenetic studies. Our results show that the subtraction of clonal events from the chi2 calculation for the "outliers" results in nearly all of these values losing their statistically significant deviation from proportionality. These adjustments can also be made to prevent the overestimation of frequencies of chromosome aberrations for biodosimetry. The frequency of clonal aberrations appears to increase as a function of age in control subjects, whereas an age effect was not evident in Chernobyl liquidators. This suggests that spontaneous and radiation-induced clonal expansion are occurring in control subjects and liquidators, respectively.     

Chromosome analysis in 31 cases of benign and malignant breast tumors: a study in Brazil

Cultures of 31 breast tumors, being 20 carcinomas and 11 benign lesions, were cytogenetically analysed. Clonal chromosome aberrations were detected in 16 carcinomas and in 4 benign lesions. Nine carcinomas and 2 benign lesions had multiple cytogenetically unrelated and related clones, whereas a single abnormal clone was observed in 7 carcinomas and in 2 benign lesions. Polyploid clones were found in 7 carcinomas and in 2 benign lesions. The presence of clonal chromosome aberrations and polyploid cells was not associated with the clinicopathologic parameters tested. Carcinomas had more clonal changes than benign lesions (p = 0.031), showing that cytogenetic features are of diagnostic value and that different chromosome anomalies might have different pathogenetic and prognostic significance.     

Characteristics of genomic instability in clones of TK6 human lymphoblasts surviving exposure to 56Fe ions

Genomic instability in the human lymphoblast cell line TK6 was studied in clones surviving 36 generations after exposure to accelerated 56Fe ions. Clones were assayed for 20 characteristics, including chromosome aberrations, plating efficiency, apoptosis, cell cycle distribution, response to a second irradiation, and mutant frequency at two loci. The primary effect of the 56Fe-ion exposure on the surviving clones was a significant increase in the frequency of unstable chromosome aberrations compared to the very low spontaneous frequency, along with an increase in the phenotypic complexity of the unstable clones. The radiation-induced increase in the frequency of unstable chromosome aberrations was much greater than that observed previously in clones of the related cell line, WTK1, which in comparison to the TK6 cell line expresses an increased radiation resistance, a mutant TP53 protein, and an increased frequency of spontaneous unstable chromosome aberrations. The characteristics of the unstable clones of the two cell lines also differed. Most of the TK6 clones surviving exposure to 56Fe ions showed unstable cytogenetic abnormalities, while the phenotype of the WTK1 clones was more diverse. The results underscore the importance of genotype in the characteristics of instability after radiation exposure.     

Chromosome aberrations of clonal origin are present in astronauts' blood lymphocytes

Radiation-induced chromosome translocations remain in peripheral blood cells over many years, and can potentially be used to measure retrospective doses or prolonged low-dose rate exposures. However, several recent studies have indicated that some individuals possess clones of cells with balanced chromosome abnormalities, which can result in an overestimation of damage and, therefore, influence the accuracy of dose calculations. We carefully examined the patterns of chromosome damage found in the blood lymphocytes of twelve astronauts, and also applied statistical methods to screen for the presence of potential clones. Cells with clonal aberrations were identified in three of the twelve individuals. These clonal cells were present in samples collected both before and after space flight, and yields are higher than previously reported for healthy individuals in this age range (40-52 years of age). The frequency of clonal damage appears to be even greater in chromosomes prematurely condensed in interphase, when compared with equivalent analysis in metaphase cells. The individuals with clonal aberrations were followed-up over several months and the yields of all clones decreased during this period. Since clonal aberrations may be associated with increased risk of tumorigenesis, it is important to accurately identify cells containing clonal rearrangements for risk assessment as well as biodosimetry.     

Does disturbance enhance genotypic diversity in clonal organisms? A field test in the marine angiosperm Zostera marina

Physical disturbance has often been invoked to control genotypic diversity in sessile clonal organisms, yet experimental evidence is lacking. I studied the effects of physical disturbance on genet dynamics and genotypic diversity in a clonal marine angiosperm, Zostera marina (eelgrass). In replicated plots of 1 m2, the vegetation canopy was removed in gaps of zero (control), 25%, 50% and 75% of the area (n = 6 replicates). Before removal and during two consecutive years, the genotypic composition was determined using genetic markers (DNA microsatellites) in a 5 x 5 pixel grid per plot. An aggregate index of genet dynamics summarizing recruitment, increase, loss and decrease of clones was maximal at intermediate disturbance levels (quadratic polynomial P = 0.02). Physical disturbance also increased the occurrence of new genotypes, possibly reflecting recruitment (linear model, P < 0.05). Contrary to expectations, there was no competitive advantage of more heterozygous genotypes over less heterozygous ones. In the absence of disturbance, in particular, clones with lower individual heterozygosity were more likely to increase in area over a 1-year time period than more heterozygous ones, while there was no such correlation in plots with disturbance (logistic model, P(disturbance x heterozygosity) = 0.036). Undisturbed plots revealed background recruitment independent of canopy gaps, suggesting that Z. marina exhibits a strategy of continual recruitment. Effects of experimental disturbance (linear or quadratic) on clonal diversity were not detectable. Instead, initial (pretreatment) clonal diversity accounted for between 68% and 91% of the variance in diversity, indicating remarkable resilience of genotypic diversity in the face of physical disturbance.     

Frequency and distribution of aneuploidy in human female gametes

Summary During the past 6 years, 14 cytogenetic studies on human oocytes recovered during in vitro fertilization procedures have been published; they report contradictory results. The present survey has pooled the more than 1500 oocyte chromosome complements examined to date, in order to determine generalized trends in chromosomal abnormalities of female gametes. The overall frequency of abnormalities in mature oocytes is 24.0% with a large majority of aneuploidies (22.8%) over structural aberrations (1.2%), which could be explained by the difficulty in the detection of structural abnormalities in oocyte chromosome sets. An analysis of the distribution of non-disjunction among all chromosomes was also performed. In the A, C, D, and especially in the G groups, there is a significant difference between the observed non-disjunction and the frequencies expected from an equal partitioning of non-disjunction among all chromosomes. These data are discussed with reference to the differences obtained from cytogenetic studies on human sperm and from investigations on spontaneous abortion.     

Cytogenetic analyses of a murine carcinoma cell line and six metastatic derivatives with different degrees of radioresistability

Summary We reported that a murine carcinoma (DEN3) and its six pulmonary metastases (M2, M4C, M4D, M4E, M4F, and M6) exhibited different degrees of radioresistability (In Vitro Cell. Dev. Biol. 26:222–228; 1990). While the M2, M4C, M4E, and M4F cultured cells survived up to 2.5 Gy, the cells of DEN3 and M6 tolerated up to 5.0 Gy, and the M4D cells could withstand up to 10.0 Gy of X-irradiation. In the present investigation, the cytogenetic features of these cell lines were examined: (a) to determine the degree of cytogenetic heterogeneity among these cell lines, and (b) to investigate whether any association between the cytogenetic anomaly and the degree of radioresistability could be established. Heterogeneous cytogenetic aberrations were detected in all of the above lines. Karyotype analysis of the M4D and M6 cell lines displayed both numerical and structural abnormalities. The gain and loss of chromosomal copies were observed. Structural aberrations, such as translocation and deletion appeared in both cell lines. However, correlation between the cytogenetic abnormality and the degree of radioresistability was not demonstrated except for a dramatic reduction in one or more copies of the X-chromosome that occurred in 86% and 93% of the M6 and M4D cells, respectively. The results suggest heterogeneous cytogenetic aberrations among these cell lines and a possible association between the loss of X-chromosome and radioresistability of these tumor cells.     

Results of dynamic cytogenetic study of children and teenagers living in areas, radioactive by contaminated after the Chernobyl accident

Results of multiyear cytogenetic study of children and teenagers living in areas, radioactive by contaminated after Chernobyl accident, were adduced. Mean density of 137Cs contamination in two compared living areas were 111 and 200 kBq/m2 and mean external doses accumulated for 1986-2001 were 6.7 and 11.4 mGy correspondingly. Averaged thyroid doses receives by subjects of all age groups in the second area were approximately 1.5 times higher than in the first area; in the youngest group (0-1 year) the doses were 114.3 and 174.3 mGy. During 17 years cytogenetic investigation approximately from 30% to 60% of examined persons were observed the increased level of chromosome aberrations in lymphocytes of peripheral blood. Average frequency of unstable aberrations (acentrics, dicentrics and centric rings) constituted about 0.4 per 100 cells (0.22 per 100 cells in controls) during all period of observation. Level of marker aberrations (dicentrics and centric rings) was increased almost all times of study and varied within 0.04-0.19 per 100 cell (0.03 in control group). The parallel investigation of frequency of stable aberrations by FISH method showed up their level about 3 times exceeding observed dicentrics level. Comparably higher indexes of cytogenetic disturbances were revealed in group exposed in utero during period of accident.     

Apparent “in situ” clone of cytogenetically marked ataxia-telangiectasia lymphocytes

Summary Six adjacent metaphases, each with the same cytogenetic aberration of a group D chromosome, most probably a No. 14, were observed in a field of a slide from a 96-hour culture of lymphocytes from an individual with ataxia-telangiectasia (AT). None of the 304 orther metaphases examined from this or other simultaneous cultures of this individual showed such an aberration. It seems most likely that an in situ marked clone has been observed and this supports interpretation of consistent cytogenetic abnormalities in those with AT as having clonal origin. The method of slide preparation employed which involves placing, rather than dropping, the cell suspension on the slide may facilitate detection of in situ clones.     

Cytogenetic investigations on children with acute non-lymphocytic leukemia

Cytogenetic data from 30 children with acute non-lymphocytic leukemia (ANLL) are evaluated in connection with patient's age, morphological type of leukemia and prognosis. In 20 out of 30 patients clonal chromosome aberrations were found. The frequency of chromosome aberrations and the prognostic parameters in the various morphological and age groups proved to be different and no direct relationship could be found in a given group between the frequency of aberrations and the prognosis. A more detailed analysis of data, however, provided some evidence that chromosome aberrations observed at diagnosis had a prognostic value independent of age and the morphological properties of blast cells: the normal karyotype and the pseudodiploidy proved to be of a favorable value but the hyperdiploidy and polyploidy an unfavorable prognostic parameter. Besides the known cytogenetic differences between childhood and adult ANLL, some similarities are also emphasized.     

Cytogenetic studies in Dupuytren contracture.

Dupuytren contracture is a connective tissue disease mainly confined to Caucasians. It is characterized by nodular growth and proliferation of collagen in the palmar and plantar fascias. Autosomal dominance with variable penetrance is considered the most likely mode of inheritance. The goal of the present study was to examine the cytogenetics of this common benign neoplasia. Chromosome studies were performed on the nodular growth of eight patients with Dupuytren contracture, all of whom showed chromosome abnormalities that included numerical and structural clones, random numerical and structural aberrations, prophasing, and premature centromere separation. Numerical clones of trisomies 7 and/or 8, as well as some random structural aberrations, were considered to represent in vivo abnormalities, whereas most structural clones appeared likely to be the results of rapid and selective in vitro growth of particular cells. The disease process occurring in Dupuytren contracture was found to involve marked chromosome instability, as well as some in vivo clonal formation. Transverse fascial tissue, usually considered to be uninvolved in the disease process, unexpectedly showed all the same types of abnormalities as the nodular tissue. This indicates a more widespread distribution of disease in the tissues than previously suspected. The findings in the present study are similar to those in various malignant and benign types of tumorous growth and suggest the importance of further cytogenetic investigation into other conditions of benign growth.     

Chromosome 11 aberrations in small colony L5178Y TK-/- mutants early in their clonal history

We have developed a cytogenetic technique that allows observation of chromosome rearrangements associated with TK-/- mutagenesis of the L5178Y/TK+/-3.7.2C cell line early in mutant clonal history. For a series of mutagenic treatments we show that the major proportion (93%) of small-colony (sigma) mutants studied have chromosome 11 rearrangements (the chromosome containing the thymidine kinase gene) while large-colony (lambda) mutants do not have detectable chromosome rearrangements. In addition, we find among the chromosome abnormalities in sigma mutants a significant proportion (34%) with dicentric chromosomes involving chromosome 11. These potentially unstable chromosome rearrangements may help to explain the karyotypic instability and heterogeneity among chromosome 11 aberrations previously noted in sigma mutants when they are analyzed later in their clonal history.     

FISH chromosome analysis of plutonium workers from the Sellafield nuclear facility

Chromosome analysis using a single-color FISH technique to paint three pairs of chromosomes was undertaken on a group of 46 retired plutonium workers with assessed bone marrow doses >60 mSv, 34 of whom were categorized as having robust dosimetry and 12 for whom internal doses were considered less reliable. Comparisons were made with a group of 34 workers with negligible radiation exposure and a group of 34 workers with similar recorded external gamma-ray doses but negligible internal dose. The simple translocation frequency of 17.65 +/- 1.96 x 10(-3) per genome equivalent for the 34 plutonium workers with robust dosimetry was significantly increased in comparison with that of 10.06 +/- 1.16 x 10(-3) per genome equivalent for the unirradiated control group (P = <0.001) and that of 13.55 +/- 1.43 x 10(-3) per genome equivalent for the group with similar external gamma-ray exposure (P = 0.012). Thus, although in vitro studies have indicated that the majority of alpha-particle-irradiated cells suffer complex non-transmissible chromosome damage, in vivo a significant proportion survive with simple exchanges that can be passed on to descendant cells. In contrast, the three groups demonstrated no significant differences in stable complex aberrations. No evidence of an increase in dicentrics or unstable complex aberrations associated with plutonium exposure was observed, and it can therefore be assumed that there is little, if any, ongoing irradiation of mature lymphocytes. The translocation frequency of 12.08 +/- 1.92 x 10(-3) per genome equivalent for the group of 12 plutonium workers with less reliable internal dosimetry could adequately be accounted for by age and external dose and indicates that the internal bone marrow doses are likely to have been overestimated. Cytogenetic analysis can therefore make a valuable contribution to the validation of internal doses from plutonium deposition.     

Influence of light and nitrate assimilation on the growth strategy in clonal weed <Emphasis Type="Italic">Eichhornia crassipes</Emphasis>

We studied as to how the inter-connected modular architecture of clonal Eichhornia crassipes allows nutrient to transfer from established ramets to developing ramets, and nitrate translocation within clonal system and how such a strategy may play an important role in successful establishment and expansion of this clonal plant. Using this stoloniferous E. crassipes as a model, we studied the effects of light and nitrate availability on growth and nitrate assimilation in inter-connected parent and offspring ramets. Our results showed that increase in light and nitrate availability significantly increased growth rate of the whole clonal fragments and reproduction of offspring ramets in E. crassipes. In addition, increases in nitrate reductase (NR) activity and glutamine synthetase (GS) activity were observed in both parent and offspring ramets with increase in light density and nitrate supply. We also found that nitrate translocation is greater in offspring ramets than in parent ramets under abundant light and nitrate environment in this fast-growing clonal plant. Consequently, majority of nitrate assimilation in offspring ramets is beneficial to the growth of whole clonal system, as indicated by a close correlation between nitrate assimilation in offspring ramets and RGR of whole clonal fragments. We strongly contend that nitrate translocation and assimilation within clone system is important for efficient utilization of nitrogen in alien clonal plant E. crassipes during establishment and expansion, and thus for increase in its invasiveness in natural water columns.     

利用杀配子染色体2C诱导中国春-黑麦二体附加系染色体畸变的研究

将中国春-黑麦(1R-7R)二体附加系与中国春-2C(Aegilops cylindrica)二体附加系杂交,获得F1,对F1体细胞染色体进行C分带鉴定和花粉母细胞减数分裂行为的观察与分析,发现减数分裂行为异常。对自交获得的430株F2进行单株染色体C分带和荧光原位分子杂交鉴定,检测到易位、缺失、等臂染色体、双着丝点染色体等染色体畸变类型。此外还检测到2C与小麦2A、2B、2D染色体的二体或单体自发代换系。杂交F。染色体畸变的规律与频率如下：研究共得到含黑麦染色体的变异22株,变异频率为5,1％。其中含黑麦染色体的易位系为10株,占2,3％;缺失12株,占2．79％;黑麦的等臂染色体3株,占O．7％。易位染色体既有含小麦着丝点的(大部分),也含有黑麦着丝点的(仅1例)。黑麦的染色体畸变中,发生于不同同祖群的频率不同,1R为5个,2R为3个;3R为1个;4R为3个;5R为6个;6R为4个。易位多为端部易位。共鉴定出小麦的缺失系54株,其中A基因组有27个,占6.27％;B基因组有20个,占4,65％;D基因组有7个,占1.66％。对杀配子染色体对小麦及黑麦不同同祖群染色体作用的差异性及作用特点进行了探讨。     

Increased risk of cancer in radon-exposed miners with elevated frequency of chromosomal aberrations

In spite of the extensive use of cytogenetic analysis of human peripheral blood lymphocytes in the biomonitoring of exposure to various mutagens and carcinogens, the long-term effects of an increased frequency of chromosomal aberrations in individuals are still uncertain. Few epidemiologic studies have addressed this issue, and a moderate risk of cancer in individuals with an elevated frequency of chromosomal aberrations has been observed.In the present study, we analyzed data on 1323 cytogenetic assays and 225 subjects examined because of occupational exposures to radon (range of exposure from 1.7 to 662.3 working level month (WLM)). Seventy-five subjects were non-smokers. We found 36 cases of cancer in this cohort.Chromatid breaks were the most frequently observed type of aberrations (mean frequency 1.2 per 100 cells), which statistically significantly correlated with radon exposure (Spearman's correlation coefficient R=0.22, P<0.001). Also, the frequency of aberrant cells (median of 2.5%) correlated with radon exposure (Spearman's correlation coefficient R=0.16, P<0.02). Smoking and silicosis were not associated with results of cytogenetic analyses.The Cox regression models, which accounted for the age at time of first cytogenetic assay, radon exposure, and smoking showed strong and statistically significant associations between cancer incidence and frequency of chromatid breaks and frequency of aberrant cells, respectively. A 1% increase in the frequency of aberrant cells was paralleled by a 62% increase in risk of cancer (P<0.000). An increase in frequency of chromatid breaks by 1 per 100 cells was followed by a 99% increase in risk of cancer (P<0.000). We obtained similar results when we analyzed the incidence of lung cancer and the incidence other than lung cancer separately.Contrary to frequency of chromatid breaks and frequency of aberrant cells, the frequency of chromatid exchanges, and chromosome-type aberrations were not predictive of cancer.     

EVOLUTIONARY INTERACTIONS BETWEEN SEXUAL AND ALL-FEMALE TAXA OF CYPRINOTUS (OSTRACODA: CYPRIDIDAE)

Freshwater ostracodes show both an exceptionally high incidence of transitions to unisexuality and, in some cases, an extraordinary level of clonal diversity. There is no understanding of the agents promoting these transitions to thelytoky, although it has been suggested that their frequency may set the stage for sexual taxa to infuse clonal diversity into unisexuals. This study examines the nature and origins of clonal diversity in the unisexual ostracode Cyprinotus incongruens. A combination of allozyme and cytogenetic studies revealed the presence of two diploid clones of this species at three temperate sites and ten clones at one arctic site including three diploids, five triploids, and two tetraploids. The low heterozygosity (0%–20%) of its diploid clones suggests that parthenogenesis has arisen spontaneously in C. incongruens rather than through hybridization, as in vertebrate asexuals. Polyploid clones appear to owe their origin to genome additions from sexual taxa, although subsequent mutational divergence has played a role in further enhancing diversity. Two triploid clones have apparently originated from the incorporation of a haploid genome from the sexually reproducing C. glaucus, as evidenced by their high heterozygosity and possession of alleles otherwise found only in that species. Other polyploid clones have likely arisen as a result of interbreeding between bisexual and unisexual C. incongruens. These results suggest that both the incidence of spontaneous transitions to clonality and the frequency of interbreeding with relatives may be the key processes that govern clonal diversity in unisexual ostracodes.     

To the importance of genetic diversity of Phragmites australisin the development of reed stands

Reed stands in Germany and Europe were investigatedwith respect to their genetic diversity by means ofRandom Amplified Polymorphic DNA (RAPD) via thePolymerase Chain Reaction (PCR-fingerprinting).Different types of clonal distribution could bedistinguished: monoclonal reed stands and polyclonalreed stands, and among the latter, stands with low orhigh numbers of different clones as well as standswith mosaic-like coexisting or intermingling clones.Furthermore, clonal distribution and the expansion ofdifferent clones was observed within a newlydeveloping reed population. The findings appear tosupport a model of colonization postulating thatpopulations initiated by seeds are initiallygenetically diverse, but over time become dominated byone or a few clones adapted to the prevailing siteconditions. Thus, low genetic diversity seems to bethe result of a natural selection process. Clonaldiversity within a reed population and the type ofclonal distribution are discussed with regard to theirimportance for the reed stand's ability to adapt tochanging site conditions and eventually for itssurvival or die-back.