Chromosome aberrations induced by aphidicolin

The expression of aphidicolin (apc)-produced common fragile sites and chromosome aberrations observed 24 h after apc treatment was studied in a normal individual. The chromosome lesions (gaps and breaks) induced by apc are expressed as full chromosomal aberrations in later cell divisions. We compared chromosome rearrangements or anomalies induced by apc (detected in 45.4% of metaphases analyzed) with those present in human neoplasia or involved in primate evolution. We found that 55.7% of deletions observed in our study coincided with deletions implicated in several types of neoplasia. However, none of 49 translocations observed in our study coincided with those described as recurrently associated with human neoplasia, probably due to their unbalanced nature. When chromosome aberrations detected in our study (only deletions and inversions were taken into account) were compared to those involved in primate evolution, we found a low rate of coincidence. The low coincidence between chromosome alterations in neoplasia and evolution and those observed in our study could be explained because we analyzed chromosome alterations that had not been selected, whereas those present in chromosome evolution and in neoplasia had been subjected to a selection process.     

Spontaneous and induced chromosome aberrations in the bone marrow cells of different strains of mice during aging

Sensitivity of bone marrow cell chromosomes to alkylating agent thiophosphamide and to gamma-irradiation has been studied in the course of ageing in 101/H, A/He, CBA, BALB/c and C57BL/6 mice. The effects of both the kinds of mutagenic treatment and of the genotype of the animals on the age-dependent changes in sensitivity of bone marrow cell chromosomes were found. Following gamma-irradiation under our experimental conditions, no variation in the output of chromosomal aberrations was observed between the strains studied. Following thiophosphamide treatment, aged mice of strains 101/H, A/He and CBA showed an increased chromosome instability as compared to young ones. In C57BL/6 mice the level of induced chromosome aberrations was found to be age-independent. Following thiophosphamide treatment, cells with multiple chromosome lesions were found in the bone marrow. The higher instability of aged animals in some strains was mainly due to a sharp increase in the number of such cells. In the intact mice of all the strains studied no age-dependent increase in the number of cells showing structural chromosome aberrations was observed, while accumulation of aneuploid cells varied with genotype.     

The repair of gamma-ray-induced chromosomal damage in human lymphocytes after exposure to extremely low frequency electromagnetic fields

G(0) human blood lymphocytes were irradiated with 2.0 Gy gamma-rays and cultured to metaphase whilst held in a 50-Hz power frequency magnetic field of 0.23, 0.47 or 0.7 mT. No differences were found in the frequencies of gamma-induced chromosome aberrations observed in cells held in the EM fields compared with replicates held in a sham coil. Similar field conditions have been reported to increase the frequency of gamma-induced HPRT mutations, leading to a suggestion that the EM fields alter the fidelity of repair of genomic lesions. This was not confirmed by the chromosome aberration assay described here.     

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.     

Increased bleomycin-induced chromosome damage in lymphocytes of patients with common variable immunodeficiency indicates an involvement of chromosomal instability in their cancer predisposition

Summary To study mutagen-induced chromosome instability in cancer disposition, late S and G₂ lymphocytes of 15 patients with common variable immunodeficiency and 14 healthy controls were exposed to bleomycin in vitro. The groups did not differ in the frequency of spontaneous chromosome aberrations. In bleomycin-treated samples we found higher numbers of break events per cell and increased frequency of cells with aberrations compared to the control group. A slightly reduced breakage of chromosome group D was noted in patients. These results support the hypothesis that a higher incidence of cancer in patients with genetically determined immunodeficiencies may be explained by an increased mutagen-induced chromosome instability in at least some of them.     

Screening human populations for chromosome aberrations

In order to determine the usefulness of micronuclear counts (MNC) for identifying people with relatively high frequencies of chromosome aberrations we have examined factors that influence the MNC in a learning set of blood samples obtained from 28 adults. The presence of cells with chromosome aberrations among approximately 170 metaphase cells per sample was the most important factor. Controlling for the effect of chromosome aberrations we found that age had a significant effect on MNC, but that donor sex, the mitotic index, the per cent of metaphase cells in the second or third division or the frequency of abnormal anaphase cells did not. Using logistic regression analysis we found that MNC was an excellent predictor of the presence of cells with chromosome aberrations among both the learning set and a test set of 17 additional blood samples.     

Identification of radiation,chemical and combined effects on natural populations of aquatic organisms

Approaches to the identification of radiation, chemical and combined effects on natural populations of aquatic organisms are proposed. These approaches are based on generalization of our previous experimental data concerning separate and combined effects of ionizing radiation and chemical mutagens (heavy metals and chlororganic compounds) on crustaceans and fishes embryos, as well as on results of field studies carried out before and after the Chernobyl NPP accident. For the identification of damaged factors it is proposed to use the following criteria: a comparison of cytogenetic effects in mutagen equivalent doses, a distribution of chromosome aberrations in cells and a number of aberrations per an aberrant cell.     

Number of chromosome aberrations induced by chemical carcinogens in the cells of patients with 2 forms of xeroderma pigmentosum

A study was made of the effects of a chemical mutagen of the "gamma-type"--methylmethansulfonate (MMS) and of mutagen of the "UV-type"--4-nitroquinolin-1-oxide (NQO) and 7-brommethylbenz(alpha)antracen (BMBA) exerted on chromosome aberration frequency in lymphocytes of patients with classical Xeroderma pigmentosum and with a so-called form II of the disease on different stages of the cell cycle. Mutagens were added to PHA stimulated lymphocyte cultures every 3 hours, simultaneously with pulse 3H-thymidine labelling, to fix the stage of the cell cycle at the moment of treatment. NQO and BMBA treatments were found to increase the frequency of chromosome aberrations in classical XP cells, whereas MMS was not found to. In the XP II cells, defective in repair of both UV and gamma damaged DNA, chromosome aberrations yield is higher than in normal cells after all the three mutagens treatment. The data obtained show the correlation between DNA repair and chromosome aberrations yield.     

Cell survival and radiation induced chromosome aberrations

Human peripheral lymphocytes were irradiated in whole blood with 0.5-4.0 Gy of 220 kVp X-rays and the frequency of chromosome aberrations was determined in 1st or 2nd division metaphases discriminated by fluorescence plus giemsa staining. Using the empirical distributions of aberrations among cells, cell survival and transmission of aberrations were investigated. Considering both daughter cells, we found that 20% of fragments and 55% of dicentrics or ring chromosomes are lost during the 1st cell division; i.e. cell survival rate from 1st to 2nd generation is mainly influenced by anaphase bridging of these two-hit aberrations. Cell survival to 2nd mitosis was calculated considering this situation and compared with the survival derived from the fraction of M 1 cells without unstable aberrations. The resulting shouldered survival curves showed significantly different slopes, indicating that cell reproductive death is overestimated in the latter approach.     

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.     

Aberrant and multiaberrant (rogue) cells in peripheral lymphocytes of Hodgkin's lymphoma patients after chemotherapy

We analyzed spontaneous chromosome lesions in peripheral lymphocytes cultured from Hodgkin's lymphoma (HL) patients before and after cytostatic chemotherapy. 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), and in non-treated patients than in healthy subjects (2.12+/-0.13). In lymphocytes of HL patients, who received chemotherapy, we found, in addition to ordinary aberrant cells, a large number of multiaberrant (or rogue) cells, i.e. metaphases carrying multiple (at least four) chromosome-type exchange aberrations. Rogue cells were found in 15 out of 18 chemotherapeutically treated HL patients (in total, 60 rogue cells per 5,568 scored cells), whereas in 30 non-treated patients only 1 rogue cell was found (per 4,988 scored cells). No correlation was found between the yield of rogue cells and the aberration frequency in ordinary aberrant cells. Aberration spectra (ratios of chromatid- to chromosome-type aberrations and of breaks to exchanges) were essentially different in ordinary aberrant and multiaberrant cells. These data, as well as analysis of cellular distributions of aberrations, implied independent induction of chromosome damage in ordinary aberrant and rogue cells. Analysis of aberration patterns in diploid and polyploid rogue metaphases belonging to the first, second, and third in vitro division indicated that rogue cells could be formed both in vivo and in vitro, and could survive at least two rounds of in vitro replication, given blocked chromosome segregation. These results suggested that formation of rogue cells, unlike ordinary aberrant cells, was triggered by events other than direct DNA and/or chromosome lesions. A hypothesis regarding disrupted apoptosis as a candidate mechanism for rogue cell formation seems to be most suitable for interpretation of our data. Cultured lymphocytes of chemotherapeutically treated HL patients may represent a model system for further examination of the multiaberrancy phenomenon.     

Cytogenetic alterations in field workers routinely exposed to pesticides in Bogota flowers farms

Frequency of cytogenetic alterations (micronuclei and chromosome aberrations), DNA repair deficiencies and acetylcholinesterase activity was determined for field workers in Bogotá, Colombia. These workers were regularly exposed to organophosphate and carbamate insecticides while employed on farms for flower growing. Interviews were conducted with 31 workers associated with occupational risk of pesticides exposure and 30 without exposure. A standard cytogenetic assay was used to determine chromosome aberrations and micronuclei frequencies. In addition, a challenge assay assessed response to gamma-rays as an indication of DNA repair deficiencies--cells were exposed to gamma-rays in vitro and the frequencies of chromosome aberrations in post-irradiation metaphase cells were quantified. The data were evaluated for percentage of aberrant cells, cells with chromosome aberrations and frequencies of chromatid breaks per 100 metaphase cells in each worker. The exposed group had a significantly higher frequency of cells with chromosome aberrations and micronuclei as compared with the non-exposed group (p = 0.02). However, the challenge assay did not indicate a significant difference (p > 0.1). These findings require confirmation by further analytical studies involving larger sample. Cytogenetic and toxicological studies, in conjunction with thorough clinical examination are recommended.     

Cellular DNA region involved in induction of thymic lymphomas (Mlvi-2) maps to mouse chromosome 15.

Two cellular DNA regions representing common domains for proviral DNA integration ( Mlvi -1 and Mlvi -2) have been identified in Moloney murine leukemia virus-induced rat thymic lymphomas. Cellular sequences which were free of repeated DNA derived from a clone that defines the Mlvi -2 integration domain (lambda Cl228 ) were found to be highly conserved in a variety of vertebrate species that we examined, including mice, hamsters, cats, and humans. In this study, we identified the chromosomal map location of the Mlvi -2 homologous sequences in mice by using hamster-mouse somatic cell hybrids. The results show that Mlvi -2 is present on mouse chromosome 15 but is unrelated to the c-myc and c-sis proto-oncogenes, which map on the same chromosome. Since aberrations on chromosome 15 have been observed reproducibly in mouse thymomas, our data suggest that Mlvi -2 may define a novel sequence involved in the induction or progression of murine thymic lymphomas.     

Spontaneous chromosome aberrations in human somatic cells

Conclusion In conclusion it is necessary to say that at present, we cannot consider whether there may be a geographical difference in frequency of spontaneous chromosome aberrations in somatic cells. For that purpose, a very abundant experimental material is required, as well as an improvement in methodology: what causes the difference in the results of various investigators; what methodical principles should be used for collection of data.Important factors in the differences of frequencies of spontaneous chromosome aberrations are likely to be the conditions of cultivation and making the preparations, as well as the methods of scoring the chromosome aberrations. The international standardisation of the cultivation conditions and of the estimates of chromosome aberrations is needed for the further study of the rate and reasons of the spontaneous mutation process in somatic cells.     

3-Aminobenzamide does not affect X-ray-induced cytogenetic damage in G0 human lymphocytes

The inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide (3AB) has been reported to have very different effects on X-ray-induced chromosome aberrations in G0 human lymphocytes. One group of investigators observed a 2-3-fold increase in the yield of rings, dicentrics and chromosome breaks after X-irradiation and 3AB treatment, whereas another group found that 3AB had no effect on X-ray-induced chromosome aberrations. To resolve this discrepancy, we repeated the experiments as described by both groups and found no effect of 3 mM or 5 mM 3AB on the frequency of chromosome aberrations induced by either 1 Gy or 2 Gy of X-rays. Furthermore, we found no effect of 3AB on X-ray-induced aberration yields in C-banded prematurely condensed chromosome preparations from unstimulated human lymphocytes. These results indicate that poly(ADP-ribose) polymerase is not involved in the repair of cytogenetic damage in G0 human lymphocytes.     

Revision of th distribution of chromosome aberrations induced by chemical mutagens using the BUDR label

Cell distribution was analysed with the help of the BrDU label for the number of chromosome aberrations and breaks induced by one-center (thiophosphamide and phosphamide) and two-center (dipine and fotrine) mutagens at the stage G0 in the Ist mitosis of human lymphocytes harvested at different times of culturing (from 56 to 96 h). The comparison was made between the type of aberration distribution in cells and the dependence of their frequency on the harvesting point for various mutagens. Poisson aberration distribution in cells for two-center mutagens was found to correspond to their constant frequency observed at different times of harvesting. On the other hand, for one-center mutagens, a geometrical distribution of chromosome breaks corresponded to an exponential decrease in their frequency in time. It is suggested that two-center chemical mutagens and ionizing radiation cause largely short-live damages which are realized into chromosome aberrations rather quickly (during one cell cycle). One-center mutagens, however, cause such damages that the probability of their transformation into chromosome aberrations is decreasing rather slowly in time, under the exponential law, and their realization into chromosome aberrations can occur in subsequent cell cycle.     

Equal induction and persistence of chromosome aberrations involving chromosomes with heterogeneous lengths and gene densities

Little is known about the factors modulating the initial induction and persistence of chromosome aberrations. Chromosome length and gene density have been proposed to play a significant role. We have therefore analyzed the induction and persistence of gamma-ray-induced aberrations involving four human chromosomes (1, 4, 18, and 19) with highly heterogeneous lengths and gene densities. Multicolor FISH was performed on a wild-type lymphoblastoid cell line 1, 3, 7, 14, 28, 42, and 56 d after gamma-irradiation. The frequency of induced chromosomal aberrations was proportional to the length of the chromosomes. Complex aberrations, dicentrics, and fragments were highly unstable and disappeared during the first week after treatment and with similar kinetics for all four chromosomes. The frequency of translocations decreased with time and followed an exponential decline. Thirty percent of the gamma-ray-induced translocations were stable over the entire study period, irrespective of the length and the gene density of the chromosome involved. Accordingly, we concluded that the induction of chromosome aberrations is proportional to the length of the chromosome, that gene density makes no measurable contribution to induction, and that neither length nor gene density influences the persistence of chromosome aberrations.     

Report on the Eleventh International Workshop on the Identification of Transcribed Sequences 2001. November 9-11, 2001. Washington, DC, USA

Glioblastoma multiforme (GBM) is characterized by intratumoral heterogeneity as to both histomorphology and genetic changes, displaying a wide variety of numerical chromosome aberrations the most common of which are monosomy 10 and trisomy 7. Moreover, GBM in vitro are known to have variable karyotypes within a given tumor cell culture leading to rapid karyotype evolution through a high incidence of secondary numerical chromosome aberrations. The aim of our study was to investigate to what extent this mitotic instability of glioblastoma cells is also present in vivo. We assessed the spatial distribution patterns of numerical chromosome aberrations in vivo in a series of 24 GBM using two-color in situ hybridization for chromosomes 7/10, 8/17, and 12/18 on consecutive 6-microm paraffin-embedded tissue slides. The chromosome aberration patterns were compared with the histomorphology of the investigated tumor assessed from a consecutive HE-stained section, and with the in vitro karyotype of cell cultures established from the tumors. All investigated chromosomes showed mitotic instability, i.e., numerical aberrations within significant amounts of tumor cells in a scattered distribution through the tumor tissue. As to chromosomes 10 and 17, only monosomy occurred, as to chromosome 7 only trisomy/polysomy, apparently as a result of selection in favor of the respective aberration. Conversely, chromosomes 8, 12, and 18 displayed scattered patterns of monosomy as well as trisomy within a given tumor reflecting a high mitotic error rate without selective effects. The karyotypes of the tumor cell cultures showed less variability of numerical aberrations apparently due to clonal adaptation to in vitro conditions. We conclude that glioblastoma cells in vivo are characterized by an extensive tendency to mitotic errors. The resulting clonal diversity of chromosomally aberrant cells may be an important biological constituent of the well-known ability of glioblastomas to preserve viable tumor cell clones under adaptive stress in vivo, in clinical terms to rapidly recur after antitumoral therapy including radio- or chemotherapy.     

Recurrent genomic rearrangements are not at the fragile sites on chromosomes 3 and 5 in human renal cell carcinomas

Summary It has been suggested that fragile sites on human chromosomes predispose to specific rearrangements seen in cancer. Renal cell carcinoma is characterised by recurrent aberrations of chromosome 3p and frequent rearrangements of chromosome 5q. To investigate whether there might be an association between fragile sites and recurrent breakpoints in renal cell carcinoma, we have determined the breakpoints observed in 50 tumours and compared them to the known fragile sites on chromosomes 3 and 5. No correlation between fragile sites and cancer-related breakpoints in renal cell carcinomas was found.     

No evidence for constitutional chromosome instability in testicular cancer

Summary Chromosome aberrations in 20 lymphocytes of 20 patients with testicular germ cell tumors (TGCT) treated with surgery alone were compared with those of 20 cells from 20 healthy controls using standard G-banding technique. No increase in structural aberrations was found in the cancer group. An unexpected finding was that of more cells with losses of chromosomes being present in the control group. These losses predominantly affected small chromosomes in the control group, whereas the pattern of chromosome loss was different in the cancer group. The literature claiming increased chromosome instability in TGCT patients is reviewed. Point estimates and 95% confidence intervals to exclude such a hypothesis based on our results were calculated.