Chromosomal abnormalities in lymphocytes from a patient with Werner's syndrome in intact culture and after treatment with halogenated analogs of thymidine

Four balanced chromosomal translocation, deletion of chromosome 15, and a break in chromosome 11 were detected in 100 G-banded metaphases of cultured lymphocytes of a patient with Werner's syndrome. We observed aneuploidy that included both trisomies and monosomies for various chromosomes. Halogenated analogs of thymidine in low doses increased significantly the incidence of chromosome aberrations accompanied by fragments. 5-Iododeoxyuridine induced lesions in centromeric regions of B-group chromosomes in 44.4% of all the cases of breaks. A hypothesis is proposed about the existence of a special mechanism for genetic control in changes in the cell nucleus and mitotic chromosome transformation. This mechanism can be manifested after the application of halogenated analogs of thymidine. The mutation involved in Werner's syndrome is presumably related to this mode of genetic control.     

Cytogenetic abnormalities in a patient with hypercalcemia and papillary thyroid carcinoma

Summary Cytogenetic examinations on multiple peripheral blood cultures of a patient with papillary thyroid carcinoma and hypercalcemia revealed the following features: (1) The average frequency of cells with aberrations was 11.6%, considerably higher than in controls. Among metaphases with chromosomal abnormalities, 4.5% had chromosome-type aberrations. (2) One homolog of chromosome 11 showed a fragile site in the proximal end of the long arm, and in three metaphases the segment distal to the fragile site showed branched morphology. (3) The rate of sister chromatid exchanges was within normal limits (8.78/metaphase). (4) The patient's two sons showed 7.0% and 5.0% abnormal metaphases, in the high normal range.     

Chromosome aberrations in peripheral lymphocytes and radiation dose to active bone marrow in patients treated for cancer of the cervix

An international study of cervical cancer patients reported a doubling of the risk for leukemia following radiotherapy. To evaluate the extent of residual chromosome damage in circulating T-cell lymphocytes in this population, approximately 200 metaphases were examined from each of 96 irradiated and 26 nonirradiated cervical cancer patients treated more than 17 years ago (average 23 years). Radiation dose averaged over the total red bone marrow was estimated to be 8.1 Gy. The type and frequency of stable and unstable chromosome aberrations were quantified in 24,117 metaphases. Unstable aberrations did not differ significantly between irradiated and nonirradiated patients (P greater than 0.5). Stable aberrations (i.e., translocations, inversions, or chromosomes with deleted segments), however, were significantly higher among irradiated (2.8 per 100 cells) compared to nonirradiated (0.7 per 100 cells) women (P less than 10(4). The frequency of these stable aberrations was found to increase significantly with increasing dose to the bone marrow. These data indicate that a direct relationship between radiation dose and extent of damage to somatic cells persists in populations and can be detected many years after partial-body radiation exposure. The stable aberration rate in irradiated cervical cancer patients was 50 to 75% lower than those observed 25 years or more after radiation exposure in atomic bomb survivors and in ankylosing spondylitis patients treated with radiotherapy. The average marrow dose was only 1 Gy in the examined atomic bomb survivors and 3.5 Gy in the ankylosing spondylitis patients. It appears, then, that a very high dose delivered to the pelvic cavity in fractionated doses resulted in far fewer persistent stable aberrations than lower doses delivered either in acute whole-body exposure or in fractionated doses to the spinal column and sacroiliac joints. The higher radiation dose and the concentration of that dose in a smaller area of the body appear to be responsible for the lower rate of persistent aberrations observed in cervical cancer patients.     

Cytogenetic findings in pernicious anaemia. Comparison between results obtained with chromosome studies and the micronucleus test.

To elucidate whether the micronucleus test may be a sensitive test for the demonstration of the occurrence of spontaneous structural chromosomal aberrations in human disease, bone-marrow smears and chromosome preparations were studied from ten patients with pernicious anaemia. An increased incidence of metaphases with structural chromosomal aberrations was seen in three of the patients, whereas an increased number of bone-marrow cells containing micro-nuclei was present in eight of the ten patients. The micronucleus test may thus be a rapid and sensitive test to demonstrate whether spontaneous structural aberrations of the chromosomes are present in a group of patients suffering from various diseases.     

Clonal structural chromosomal rearrangements in lymphocytes of four patients with Werner's syndrome

Multiple numerical and structural chromosome abnormalities were found in cultured lymphocytes of four patients with Werner's syndrome. The proportion of metaphases with structural and/or numerical aberrations varied from 30 to 44% and several of them were clonal. These results confirm definitively that Werner's syndrome is a chromosome rearrangement syndrome and that these non-constitutional chromosome changes are not exclusive of cultured fibroblasts but present also in lymphocytes.     

G0 chromosomal radiosensitivity in ataxia telangiectasia lymphocytes

Contrary to an earlier report, peripheral lymphocytes from 4 AT patients were not found to exhibit higher yields of unequivocal chromosome type aberrations following irradiation in the G₀ phase of the cell cycle, providing that only first post-irradiation metaphases were included in the samples (ensured by 5-bromodeoxyuridine (BrdU) incorporation and differential fluorescence or Giemsa staining). We were able, however, to confirm the earlier-reported increase in chromatid-type aberrations in the G₀-irradiated cells. AT lymphocytes were found to experience more cell-cycle delay following G₀ irradiation than normal cells. These observations appear consistent with the damaged base excision DNA-repair defect reported for AT cells.     

Analysis of chromosome aberrations in lymphocytes of long-term heavy smokers

We assessed the incidence of structural chromosome aberrations in 500 diploid first-division metaphases from 48-h lymphocyte cultures from each of 6 non-smokers and from 6 persons who had smoked a minimum of 1 pack of cigarettes per day for at least 20 years. Cytogenetic analyses of coded slides revealed a single dicentric chromosome with its accompanying fragment and two symmetrical chromatid exchanges in 3000 metaphases from the non-smokers. In contrast, 9 dicentric chromosomes, 8 translocations or inversions, and 7 chromatid exchanges were observed in 3000 metaphases from lymphocyte cultures from the 6 heavy smokers. A total of 13 metaphases having chromosome-type inter- or intra-changes was noted including 9 with a single aberration, and 4 with 2 or more. Our findings provide additional evidence of the in vivo clastogenicity of cigarette smoke in long-term heavy smokers, and further demonstrate that the distribution of chromosome-type exchange aberrations is overdispersed relative to that expected based on Poisson assumptions.     

The analysis of chromosome lesions in lymphocytes of Hodgkin's lymphoma patients after chemotherapy and radiation therapy

The analysis of chromosome lesions in peripheral blood lymphocytes of Hodgkin's lymphoma (HL) patients after chemotherapy and chemotherapy with the subsequent course of radiation therapy is carried out. Is shown, that the mean aberration frequency was significantly higher in HL patients after chemotherapy (7.20 +/- 0.58 per 100 metaphases) than in non-treated HL patients (4.80 +/- 0.54, p < 0.01). The subsequent carrying out of radiation therapy enlarges number of chromosome aberrations on 100 metaphases up to 46.7 +/- 10.7 (p < 0.05), of which chromosome-type aberrations (43.2 +/- 10.3 on 100 metaphases) averaged 92.5%. In lymphocytes of 37 out of 43 HL antitumoral treatment patients, we found, in addition to ordinary aberrant cells, a large number of multiaberrant (MA-cells) cells, i.e. metaphases carrying multiple (at least four) chromosome-type exchange aberrations. In 30 non-treated HL patients only one MA-cell was found. From 171 MA-cells which were in 43 HL patients after antitumoral treatment, 114 MA-cells were found at inspection of 9766 diploid metaphases, and the remaining 57 MA-cells were found at inspection of 196 polyploid metaphases. The carrying out after chemotherapy of radiation therapy enlarges in lymphocytes frequency of appearance of MA-cells. The analysis of MA-cells in diploid and polyploid metaphases shown, that the MA-cells could be formed both in vivo, and in vitro in absence of influence of clastogenic factors, and could survive at least two rounds of in vitro replication.     

The pattern of radiation-induced transmissible aberrations in a human cell culture

Summary The G-band pattern in 445 metaphases obtained seven weeks after irradiation (600 rad gamma-ray) was analysed. Approximately 37% of these cells had one or more structural aberrations. The majority of the aberrant events was reciprocal translocation followed by inversion and deletion in the proportion of 9:1:1 respectively. Statistical analyses (Chi-square tests) on the distribution of breakpoints among chromosomes showed an excess number of breaks in chromosomes 1,7,and 12. Chromosomes 1 and 12 were particularly involved in cells carrying multiple aberrations while chromosome 7 was preferentially involved in deletion. Within chromosomes a significantly large number of breaks were located in(a) the light bands and (b) the terminal segments. The significance of these findings is discussed.     

Spontaneous chromosomal aberrations in Fanconi anaemia,ataxia telangiectasia fibroblast and Bloom's syndrome lymphoblastoid cell lines as detected by conventional cytogenetic analysis and fluorescence in situ hybridisation (FISH) technique

Several primary and transformed human cell lines derived from cancer prone patients are employed routinely for biochemical and DNA repair studies. Since transformation leads to some chromosomal instability a cytogenetic analysis of spontaneous chromosome aberrations in fibroblast cell lines derived from patients with Fanconi anaemia (FA), ataxia telangiectasia (AT), and in lymphoblastoid cell lines derived from patients with Bloom's syndrome (BS), was undertaken. Unstable aberrations were analysed in Giemsa stained preparations and the chromosome painting technique was used for evaluating the frequencies of stable aberrations (translocations). In addition, the frequency of sister-chromatid exchanges (SCEs) was determined in differentially stained metaphases. The SV40-transformed fibroblasts from these cell lines have higher frequencies of unstable aberrations than the primary fibroblasts. In the four lymphoblastoid cell lines derived from BS patients higher frequencies of spontaneously occurring chromosomal aberrations in comparison to normal TK6wt cells were also evident. The frequency of spontaneously occurring chromosome translocations was determined with fluorescence in situ hybridisation (FISH) and using DNA libraries specific for chromosomes 1, 2, 3, 4, 7, 8, 11, 14, 19, 20 and X. The translocation levels were found to be elevated for primary FA fibroblasts and lymphoblastoid cells derived from BS patients in comparison with control cell lines, hetero- and homozygote BS cell lines not differing in this respect. The SV40-transformed cell lines showed very high frequencies of translocations independent of their origin and almost every cell contained at least one translocation. In addition, clonal translocations were found in transformed control TK6wt and AT cell lines for chromosomes 20 and 14, respectively. The spontaneous frequencies of SCEs were similar in transformed fibroblasts derived from normal individuals and AT patients, whereas in SV40-transformed FA cells these were higher (4-fold). Among cell lines derived from BS patients, heterozygote lines behaved like control, whereas in homozygote cell lines very high frequencies of SCEs (about 12-fold) were evident.     

The cytogenetics of acute leukemia

Methodological prerequisites for the culture of leukaemic blasts and the fluorescence banding of spread metaphases are represented in detail. By using an improved method the percentage of aneuploid karyotype findings increased in AML from 49% to 68% and ALL from 65% to 81% of all evaluable patients. Specific aberrations known up to the present day are discussed. They could be diagnosed in 46% of aneuploid AML-clones and in 59% of ALL-clones. The fluorescence banding according to the three-colour technique is demonstrated by three examples of "non-specific" aberrations: a) Tandem translocation of 1q and 7 q (64a, male, FAB:M5B), b) Karyotype instability with variability of chromosome numbers arising from trisomy, tetrasomy and polyploidisation of single normal as well as marker chromosomes (57a, female, FAB:M6), c) Identification of a marker chromosome as a derivative chromosome 16 (33a, female, FAB:L2, c-ALL).     

The mechanisms of adaptive response. Estimation of capacity of human blood lymphocytes to radiation adaptive response using different criteria

Blood lymphocytes of 15 healthy donors have been investigated for the ability to decrease their radiosensitivity after treatment with low dose irradiation named radioinduced adaptive response (AR). The unstable chromosome aberrations were used to evaluate the radiosensitivity change after irradiation of cells with low adaptive dose (5 cGy) and subsequent high challenge dose (1.0 Gy) in comparison with the effect of challenge irradiation only. Three indexes were used: the frequency of cells with aberrations in all analyzed cells (A), the number of chromosome aberrations per cell (B) and the number of chromosome aberrations per one aberrant cell (C). It has been discovered that all donors examined can be divided into four groups: 1--individuals which cells did not show AR by all indexes used; 2--individuals which cells showed AR by indexes A and B, but not C; 3--AR was demonstrated by indexes B and C; 4--AR was confirmed by all three indexes. Generally accepted repair model for AR formation explains only the case of donor groups 3 and 4, but can not explain the mechanism leading to the case of group 2. For understanding this mechanism, the distribution of metaphases by the number of chromosome aberrations per cell was analyzes for each donor. It was shown that the part of cells without aberrations in group 2 donors increased significantly after treatment with the adaptive and challenge irradiation in comparison with that after irradiation with challenge dose only. The conclusion is that in this case AR is formed as a result of change in the frequency 0 cell class--population shift. The analogous shift was observed in the distributions of metaphases for all donors of the group 4, but was absent in the group 3 donors. The data obtained suggest that AR of blood lymphocytes might be a result of several processes: activation of submutational genome damage repair; population shifts manifested by the change in the part of undamaged cells; and, possibly, activation of apoptotic cell death. The complex nature of AR affects each of radiosensitivity evaluation criteria to a different extent.     

Chromosomal damage in sperm of patients surviving Hodgkin's disease following MOPP (nitrogen mustard,vincristine, procarbazine,and prednisone) therapy with and without radiotherapy

Following fusion with hamster eggs, human sperm chromosomes from six Hodgkin's disease patients were analyzed to determine the genotoxic effects of therapy. Each patient had received two to six cycles of MOPP (nitrogen mustard, vincristine, procarbazine, and prednisone), with or without radiotherapy, from 3 to 20 years before the study. A total of 571 cells from the six patients were analyzed; 9.8% of the cells had structural aberrations, and 1.6% were hyperhaploid. Analysis of 5998 metaphases from a control group of 24 male donors revealed only 6.9% of cells with structural aberrations and 0.8% aneuploidy. The increase in hyperhaploidy in the patients was statistically significant. Thus, results of this study suggest that the MOPP regimen, with or without radiotherapy, is capable of causing chromosome abnormalities in the sperm of Hodgkin's disease patients.     

Chromosome aberrations in relation to radiation dose following partial-body exposures in three populations

Structural chromosome aberrations were evaluated in peripheral blood samples obtained from three populations exposed to partial-body irradiation. These included 143 persons who received radiotherapy for enlarged thymus glands during infancy and 50 sibling controls; 79 persons irradiated for enlarged tonsils and 81 persons surgically treated for the same condition during childhood; and 77 women frequently exposed as young adults to fluoroscopic chest X rays during lung collapse treatment for tuberculosis (TB) and 66 women of similar ages treated for TB with other therapies. Radiation exposures occurred 30 and more years before blood was drawn. Doses to active bone marrow averaged over the entire body were 21, 6, and 14 cGy for the exposed thymic, tonsil, and TB subjects, respectively. Two hundred metaphases were scored for each subject, and the frequencies of symmetrical (stable) and asymmetrical (unstable) chromosome aberrations were quantified in 97,200 metaphases. Cells with stable aberrations were detected with greater frequency in the irradiated subjects compared with nonirradiated subjects in all three populations, and an overall test for an association between stable aberrations and partial-body ionizing radiation was highly significant (P less than 0.001). We found no evidence that radiation-induced aberrations varied by age at exposure. These data show that exposure of children or young adults to partial-body fractionated radiation can result in detectable increased frequencies of stable chromosome aberrations in circulating lymphocytes 30 years later, and that these aberrations appear to be informative as biological markers of population exposure.     

Karyologic research on murine B-cell hybridomas

Karyotypes of 10 murine hybridomas producing monoclonal antibodies to microbal antigenes were examined using chromosome slides stained with Azur-eosine. Hybrid origin of all the cell clones was confirmed. The cultures differed from each other in modal chromosome numbers, in novel markers that were absent from cells of the parental myeloma X63.Ag8.653, in the frequency of metaphases with double minute chromosomes and in the level of cells with chromosome aberrations. The results obtained enable us to recommend a cytogenetic analysis for the identification of hybridomas. The following observations point out to a relative instability of the chromosomal apparatus of hybridomas: chromosome numbers varied significantly from cell to cell within one and the same clone; modal chromosome counts decreased in 3 of 5 hybridomas that were studied repeatedly within 1-2 months; in some hybridomas unstable chromosome aberrations were found in 18-38% of cells.     

Fragility and spiralization anomalies of the chromosomes in three cases, including fraternal twins, with Fanconi's anemia, type Estren-Dameshek

Fraternal twins, offspring of consanguineous parents, developed pancytopenia, the boy at 7, the girl at 12 years of age. A third patient became anemic at 3 years. All three are free of associated malformations. In blood cultures the incidence of chromatid breaks, exchanges, and chromosome-type aberrations was elevated to 24%, 18%, and 28%, respectively. In addition, in a low number of mitotic cells unusual observations, pointing to profound disturbances of chromosome structure, were made. It is suggested that these patients have a genetic defect impairing the normal process of mitotic chromosome condensation and decondensation.     

Analysis of streptozotocin-induced incomplete chromosome elements and excess acentric fragments in Chinese hamster cells using a telomeric PNA probe

Fluorescence in situ hybridization (FISH) with a telomeric peptide nucleic acid (PNA) probe was employed to analyze the induction of incomplete chromosome elements (ICE, i.e., unjoined or “open” chromosome elements with telomeric signal at only one end) and excess acentric fragments (i.e., in excess of fragments resulting from the formation of dicentric and ring chromosomes) by the methylating agent streptozotocin (STZ) in a Chinese hamster embryo (CHE) cell line. CHE cells were treated with 0–4 mM STZ and chromosomal aberrations were analyzed in the first mitosis after treatment using the telomeric probe. Centric (incomplete chromosomes) and acentric (terminal fragments) ICE were the only unstable chromosome-type aberrations induced by STZ in CHE cells. The induction of these aberrations exhibited a curvilinear concentration–response relationship. About 40% of the metaphases present in cell cultures treated with STZ contained one or more pairs of ICE. In STZ-treated cells, ICE were always observed as pairs consisting of an incomplete chromosome and a terminal fragment. Moreover, all of the excess acentric fragments induced by STZ were of terminal type. These results indicate that chromosomal incompleteness is a very common event following exposure to STZ and suggest that all of the excess acentric fragments induced by STZ originate from terminal deletions.     

Cytogenetics and acute non lymphocytic leukemia

The authors report haematologic and cytogenetic data from 47 patients with ANLL, demonstrating the usefulness of cytogenetic studies for the classification as well as for the prognosis of this disorder. Chromosome studies also permitted the classification of marrow cellularity in: all diploid metaphases (NN), diploid and aneuploid metaphases (AN), and all aneuploid metaphases (AA). The remission rate for patients in whom only normal metaphases were detected (NN patients) was 83% while the remission rates were 67% and 33% respectively for patients in whom both normal and abnormal metaphases were seen (AN patients) and for those in whom only abnormal metaphases were noted (AA patients). In all FAB subgroups, complete remission was related to chromosomal abnormalities, except for M4 patients who evidenced a large number of complete remissions, although presenting more chromosomal abnormalities. The longer survival in this subgroup may be related to rearrangements of chromosome 16, which is associated with a better prognosis.     

Persistence of chromosome rearrangements in peripheral lymphocytes from patients treated with melphalan for ovarian carcinoma

Summary Chromosome aberrations were studied in peripheral lymphocytes from 50 patients treated with melphalan against ovarian carcinoma. The chromosome analyses were carried out 4–132 months (mean 57 months) after the end of melphalan therapy. Most of the patients were studied several times during four years. The mean frequency of cells with chromosome and chromatid aberrations was 5.4% in the patients and 2.3% in an untreated control group. The highest aberration frequency (average 18%) was found in a patient who later developed gastric carcinoma. The dominating types of berrations in the patients were chromosome exchanges occurring as single marker chromosomes or as multiple chromosome rearrangements. These types of aberrations were found in only 0.3% of the control cells as compared to 3.8% of the patient cells. Patients with a high total dose of melphalan (above 420 mg) and a long duration of the therapy (average 22.5 months) had a higher frequency of cells with aberrations (6.3%) than patients with a lower total dose (below 420 mg) and a shorter therapy (12 months) (4.2%). No additive effect of radiation therapy was observed on the aberration frequency.This work was supported by grants from the Swedish Cancer Society (1179), and the Swedish Medical Research Council (3681)     

Chromosome analysis of human sperm

Summary A modified technique has been developed for the visualization of the chromosomes in human sperm. The cytogenetic analysis of 129 G-banded human sperm metaphases of 6 normal donors showed an incidence of structural and numerical chromosome abnormalities of 7.8%. Two out of 129 spermatozoa were aneuploid (1.6%). The frequency of sperms with chromatid-type aberrations was 2.3% (3/129). Chromosome-type aberrations were found in 5 out of 129 (3.9%) spermatozoa. X to Y ratio did not differ significantly from the expected one-to-one ratio. Twenty-six sperm complements from a patient 18–20 months after testes exposure to 30 Gy were examined. A significant increase of numerical and structural chromosome abnormalities was not observed. Chromatidtype aberrations were found in two sperm complements (7.7%) and chromosome-type aberrations in one sperm complement (3.9%). The cytogenetic analysis of 15 human sperms from a cancer patient 26 months after chemotherapy showed an increased frequency of aberrant sperm complements (33.4%). One chromatid-type (6.7%), three chromosometype aberrations (20.0%) and one (6.7%) hyperploid sperm complement could be observed. The sample size is still too small to answer the question whether chemical mutagens may increase the frequency of chromosomal abnormalities in human sperm.