Gene mutations, chromosome aberrations and survival after the x-ray irradiation of a Chinese hamster cell culture protected by cysteamine

The protective effect of cysteamine against 6-thioguanine-resistance (TCr) mutations, chromosome aberrations and inactivation caused by X-ray in cultured cells of Chinese hamster (clone 431) has been studied. The dose-effect curves have been obtained under irradiation condition without protector and with it. Dose-modifying factor of 2 was calculated for chromosome aberrations and cells inactivation and 2,8 for TCr mutations. It is supposed that the cysteamine acts on the general mechanisms involved in damages realization which results in gene mutations, chromosome aberrations and cell inactivation.     

Mutagenic activity of anticancer agent cis-dichlorodiammine platinum-II.

cis-Dichlorodiamminoplatinum-II (cis-DDP) has been widely used as an anticancer chemotherapeutic agent. The mutagenicity of cis-DDP was investigated in vitro and in vivo using sister-chromatid exchange analysis and the analysis of chromosomal aberrations. Parallel human lymphocyte cultures were incubated with and without the addition of BrdU at 4 concentrations of cis-DDP. Significant increases in SCE rate were observed at 0.25 micrograms/ml and higher, showing a clear dose-response relation between SCE rate and cis-DDP concentration. A significant increase in chromosome breakage and tetraradial figures was observed in BrdU free cultures treated with cis-DDP again showing a dose dependency. Analysis of the distribution of cells in the first, second and third division in cis-DDP treated cultures demonstrated the depressing effect of the drug on mitotic activity. In vivo analysis of SCE and chromosome aberrations in mouse showed that 13.85 mg/kg i.p. of cis-DDP produces significant increases in the rate of SCE and chromosome aberrations in bone-marrow cells.     

Possible mechanisms of the occurrence of chromosome aberrations. II. The formation of aberrations induced by UV irradiation

The report is concerned with one of possible mechanisms of emergence of chromosome aberrations after UV-irradiation of mammalian cells. The process is initiated by DNA cross-links following thymine dimerization, and is completed during mitosis. A model to account for formation of chromosome aberrations has been offered. It is compatible with the modern concept of a scaffolding model for metaphase chromosome structure in which the scaffold organizes DNA into loops along its length. The model predicts the importance of a process of mitotic chromosome isolation during aberration formation (in addition to the processes of DNA replication, reparation and chromosome association). Another feature of the model is an attempt to describe formation of aberrations under conditions of true DNA reparation.     

The action of N-methyl-N-nitrosourea on non-established human cell lines in vitro. II. Non-random distribution of chromatid aberrations in diploid and Down's cells.

Chromatid gaps, breaks and aberrations involved in interchanges induced by N-methyl-N-nitrosourea (MNU) were found non-randomly distributed on individual chromosomes and chromosome segments (G bands) both in human diploid fibroblasts with trisomy 21 cultured in vitro. Aberration events were located exclusively in pale G bands. Considering cells in the first post-treatment mitosis, the pattern of aberration distribution, as revealed by the position of hot spots, varied with recovery time and was different in diploid and Down's cells. In comparison with diploid cells, the X chromosomes of Down's cells were not involved in aberrations. Despite the higher aberration frequencies of Down's cells, the number of hot spots and the proportion of aberrations located in hot spots were not increased in this cell type. Therefore, the increased chromosomal sensitivity to MNU of Down's cells does not reflect an increased sensitivity of special chromosomes or chromosome sites.     

The number of aberrations in aberrant cells as a parameter of chromosomal instability. 1. Characterization of dose dependency

Analysis of chromosome instability (CI) is of great importance in view of pollution of the environment by genotoxic factors. Frequency of aberrant cells, spectrum of chromosome aberrations, damages of aberrant cell and distribution of aberrations in the cells are the most conventional parameters of CI. We have carried out the comparative analysis of the frequency of aberrant cells and the dynamics of aberrant cell damages induced by different mutagenic factors (alpha-irradiation from 241Am, gamma-irradiation from 60Co and tioTEPA) in Allium-test. This comparative analysis denotes that the studied parameters have different dynamics characterizing different mechanisms of CI in Allium cepa L.     

Protoplasmic Incompatibility and Cell Lysis in PODOSPORA ANSERINA. I. Genetic Investigations on Mutations of a Novel Modifier Gene That Suppresses Cell Destruction

The distribution of chromosomal aberrations between and within chromosomes of male D, melanogaster somatic cells after treatment with UV has been analyzed. -- Distribution of the breaks between chromosomes was largely nonrandom since we found a higher aberration frequency than that expected on the Y chromosome. Moreover, within the chromosomes the aberrations are clustered in the pericentromeric heterochromatic regions. The above distribution is compared with that of the breaks induced by X rays and methyl-methane-sulphonate (MMS) which were distributed in a different pattern.     

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.     

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.     

Chromosome rearrangements associated with CAD gene amplification. Experiments with cell hybrids.

Resistance to phosphonacetyl-L-aspartate (PALA) is caused by CAD gene amplification. The marker chromosome of a PALA-resistant cell line containing a homogeneously staining region with amplified CAD gene was introduced into PALA-sensitive Chinese hamster cells by microcell-mediated chromosome transfer. Two monochromosomal hybrids containing the marker chromosome in addition to the normal chromosome complement of sensitive cells and 1 tetraploid hybrid containing the complete genomes of donor (resistant) and recipient (sensitive) cells were studied in detail. It was shown that (i) the presence of the marker chromosome was both a necessary and a sufficient condition for the expression of the PALA-resistant phenotype; (ii) the marker chromosome underwent rearrangements in the monochromosomal hybrids, with preferential loss of non-amplified chromosomal regions, while it was not rearranged in the tetraploid hybrid; (iii) unlike the original PALA-resistant cells obtained after long-term selection in the presence of PALA, the PALA-resistant hybrids did not show chromosomal aberrations of other than the marker chromosome. This result indicates that chromosomal aberrations may be due to the selective procedure and is not an inherent property of cells containing amplified genes.     

Congenital chromosome breakage clusters within Giemsa-light bands and identifies sites of chromatin instability

Analysis of the distribution of published chromosome breaks in cells with constitutional chromosome aberrations showed a nonrandom distribution of breaks among chromosomes and chromosome regions. A significant amount of breakage occurred at Giemsa-negative bands. In addition, chromosome sites associated with a number of fragile sites and cellular oncogene sites were affected nonrandomly. The data are consistent with the hypothesis that chromosome breakage occurs in somatic or germ cells as a result of recombinational errors involving actively transcribing chromatin regions or regions of unstable DNA sequence structure placed in proximity during interphase.     

A medico-genetic study of isolates in Uzbekistan. III. Results of a cytogenetic study]

The cytogenetic examination of the population of the villages of Karakent and Ishan consisted of two directions: the study on the frequency of chromosome aberrations in the culture of lymphocytes and the diagnostics of anomalies and variants of karyotype. The frequency of chromosome aberrations of 40 individuals was studied. It is shown that the frequency of cells with chromosome aberrations of the individuals in different villages does not differ and on the average is 1,4%. The distribution of lymphocyte culture according to the number of aberrations corresponds to that of theoretical Puassonian. Chromatide breaks are the main type of the aberrations. Among 250 karyotypically examined individuals no evident structure reformations were found. In the caryotype of a 7-year old girl trisomia of the 21st autosome was detected. On the basis of differential staining of chromosomes by Hiemse's dye 18 individuals (7,2%) were diagnosed to have insignificant changes in morphology of some chromosomes, considered as variants of caryotype. In the majority of cases these changes were of a family nature. On the medical examination mnay individuals were diagnosed to have different pathological features, though we did not succeed in revealing any correlation between a certain microanomaly of chromosomes and a pathological feature.     

Chromosome aberrations and reproductive death of mammalian cells. Quantitative relations between these effects

The data reported in the literature concerning the relationship between the yield of chromosome aberrations and the number of cells without aberrations, on the one hand, and the survival rate of mammalian cells, on the other, with a reference to different types of radiation are reviewed in this article. It is shown that the number of chromosome aberrations per one lethal damage, as to the results obtained by different authors, ranges from 0.5 to 1.5, this discrepancy is mainly due to different methods applied by different authors, and at least one chromosome aberration corresponds to a lethal damage caused by irradiation in the G1 phase.     

Enhancing effects of heterocyclic amines and beta-carbolines on the induction of chromosome aberrations in cultured mammalian cells.

The effects of post-treatment with heterocyclic amines and beta-carbolines on the induction of chromosome aberrations were studied in Chinese hamster CHO K-1 cells and SV40-transformed excision repair-deficient human XP2OSSV cells. The number of chromosome aberrations induced by UV and MMC were increased by post-treatment with Trp-P-1 and Trp-P-2, in both the presence and the absence of S9 mix. A alpha C, MeA alpha C, Glu-P-1, Glu-P-2, IQ, MeIQ, harman and harmine increased chromosome aberrations only in the presence of S9 mix. Glu-P-2, IQ, MeIQ, harman, and harmine did not induce chromosome aberrations by themselves at the concentrations used in this study. Trp-P-1, Trp-P-2, A alpha C, MeA alpha C and Glu-P-1 were weak clastogens by themselves, but at much higher concentrations than those at which they increased the induction of chromosome aberrations in cells pretreated with UV or MMC. Therefore, the increases in chromosome aberrations were not considered to be additive.     

Structure of the interphase nucleus and formation of chromosome aberrations in Crepis capillaris]

A distribution of chromosome X-rays induced aberrations was studied in 2n and 4n root cells of Crepis capillaris at different stages of the mitotic cycle. Participation of various chromosomes in aberrations was found to be different from theoretically expected one found from the chromosome length. The chromosome A was characterized by excession of intrachromosome aberrations (terminal deletions, isolocuses, duplications and rings). The chromosome D very often took part in the formation of rings. The chromosome C was characterized by an excession of interchromosome aberrations and the lack of intrachromosome ones. The data obtained support the model of interphase nucleus proposed by Wagenaar. The frequency of exchanges of homologues in 2n and 4n cells well correlated with theoretically expected. The inhibition of mitosis increased the exchanges of homologues. In 2n and 4n cells asymmetrical translocations predominated over symmetrical ones.     

Possible mechanisms of the occurrence of chromosome restructurings. IV. Chromosome restructurings in spontaneous mutagenesis

An attempt was undertaken to modify the spontaneous mutation process by varying its conditions in somatic cells of different species and tissues. The rate of chromosome aberrations and their types were studied in anaphase and metaphase. Under normal conditions, chromosome breaks were only found to occur. Breakage of chromosomes occurs during interphase, and as a result, acentric fragments are located outside the equatorial plate during metaphase. This process of chromosome breakage leads to elimination of some genetic material, without concomitant exchanges, and therefore, it has been named "elimination" process. Spontaneous chromosome mutagenesis manifesting itself at cytogenetic level was concluded to be an elimination process directed to elimination of a portion of chromatin from chromosomes. When the conditions of spontaneous mutagenesis are altered, in particular, by cardiovascular diseases in man, by partial inhibition of DNA repair in mice and pea cells, by transformation of Chinese hamster cells, upon ageing of pea seeds-qualitative changes in the chromosomal aberrations are registered, connected with the appearance of chromosome exchanges and acentric fragments situated within the equatorial plate during metaphase. These two types of chromosome aberrations are proposed to be considered as new criteria of pathology. A system of processes was suggested to exist, preventing the appearance of aberrations during mitosis, and it is supposed to be one of the most significant homeostatic systems.     

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.     

Number of aberrations per cell as a parameter of chromosome instability. 2. Comparative analysis of the factors of different nature

The average number of aberrations per aberrant cell was concluded to carry out information on chromosome instability peculiarities induced by different mutagens as it was shown in our previous work. The purpose of the current study was to present comparative analysis of intercellular distribution of number of aberrations and their theoretical approximations. Distribution of numbers of aberrations per cell in Allium cepa L. and Allium fistulosum L. root tip cells induced by different mutagenic factors (gamma-irradiation, thiotepa, formaldehyde and seed aging) have been studied. The results were approximated to theoretical Poisson, geometric and negative binomial distributions. The intercellular distribution of aberrations did not correspond to any of the used theoretical distributions when A. cepa seeds were gamma-irradiated. There was some, but not regular, accordance with theoretical distributions when chemical mutagens thiotepa in A. cepa and formaldehyde in A. fistulosum and seed aging in both species were evaluated. During seed aging frequency of aberrant cells increased more quickly in A. fistulosum in comparison with A. cepa.     

Possible mechanisms of the occurrence of chromosome aberrations. I. Patterns in the occurrence of aberrations induced by ultraviolet irradiation

The frequency of chromosome aberrations induced by UV light at wavelengths 254, 265, 280 and 302 using doses 2-10 J/m2 in the primary culture of mouse embryonic fibroblasts during the G1, S and G2 phases was studied at metaphase of the first mitosis. Two classes of chromosome aberrations were distinguished. These classes differ in the time intervals of the final establishment of the cell cycle. The aberrations of the class 1 emerge before the beginning of prometaphase (possibly, at interphase). Formation of the second class aberrations is completed during the metaphase. It is shown that the class 1 aberrations occur with almost the same rate in approx. 7% of cells, irrespective of the cell cycle, irradiation dose and wavelength. It is suggested that these aberrations arise as a result of indirect UV action on the chromosome structures; the mechanism of their emergence does not depend on DNA replication. The class 2 aberrations do not appear after UV irradiation during the post-DNA-synthetic G2 phase of the cell cycle. However, after UV treatment at the G1 or S periods, they represent the majority of aberrations and their rate increases almost monotonously with the radiation dose. The UV action spectrum for these aberrations coincides with the adsorption spectrum of thymidine and the action spectrum for DNA cross-links. Thus, it may be inferred that formation of DNA cross-links following thymine dimerization is the first step in formation of UV-induced aberrations of the class 2. The passage of cells through DNA replication is a very important step in the process of their emergence.     

Mechanism of formation of chromatid exchanges

Chinese hamster cells with different patterns of distribution of 5-bromodeoxyuridine (BrdUrd) between chromosome subunits were subjected, during the G2 stage, to UV irradiation, which only produced breaks in BrdUrd substituted DNA. The frequency of chromatid and subchromatid interchanges as well as isochromatid aberrations was estimated. It was found that only BrdUrd containing chromatids were involved into aberrations; this result challenges the so called "molecular theory" for aberration production proposed by Leenhouts and Chadwick. A very small increase of the aberration yield in chromosomes without BrdUrd may be connected with the action of UV on the frequency of recombination. The observed frequency of interchanges was not proportional to the BrdUrd content in chromosomes and depended on the time of its incorporation: more exchanges were induced in the chromatids incorporating BrdUrd during the last round of replication. These regularities may be connected with some molecular peculiarities of chromosome structure and function.