Cytogenetic changes in primary, immortalized and malignant mammalian cells

Kirchner, Stephan; Stopper, Helga; Papp, Thilo; Eckert, Inge; Yoo, Hee J.; Vig, Baldev K.; Schiffmann, Dietmar

doi:10.1016/0378-4274(93)90062-3

Cited by 12 publications

(6 citation statements)

References 8 publications

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“…One could expect that this continuous cell line would give limited intra-and inter-laboratory variation in the number of spontaneous MN. However, there is a good correlation between the mean 406 spontaneous MN frequency in our studies and those obtained by Kirchner et al (1993); 4.5 Ϯ 2.4/2000 mononucleate cells (range 0-10 MN/2000 mononucleate cells) and 3.7 Ϯ 0.6/2000 mononucleate cells, respectively. This rate is lower than the baseline level observed in cultured human T lymphocytes.…”

Section: Discussionsupporting

confidence: 80%

“…As a matter of fact, a recent evaluation of the MLA database by the Environmental Protection Agency's Gene-Tox Mouse Lymphoma Committee gave no support to Storer's contention that the assay gives positive results for a large number of non-carcinogens (Clark et al, 1998). These mouse lymphoma cells show several other advantages, such as: growth in suspension (no need for trypsinization), rapid growth (doubling time of~10-12 h), unsynchronized division, stable karyotype and low and reproducible incidence of spontaneous MN (Kirchner et al, 1993). One could expect that this continuous cell line would give limited intra-and inter-laboratory variation in the number of spontaneous MN.…”

Section: Discussionmentioning

confidence: 99%

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A micromethod for the in vitro micronucleus assay

Nesslany

Marzin

1999

Mutagenesis

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A micromethod for the in vitro micronucleus assay was developed using L5178Y cells to enable the rapid screening of a large number of molecules. The method is quick, simple to perform and needs very small amounts of compound, i.e. <10 mg. In this methodology, three types of treatment were carried out in parallel, enabling an optimal detection of both aneugenic and clastogenic compounds: two treatments without metabolic activation with or without a recovery period after a 24 h continuous treatment and one treatment with metabolic activation by Aroclor 1254-induced rat or hamster liver S9 mix. Seventeen known genotoxins (12 clastogens and five aneugens) and seven known non-genotoxins were tested. The in vitro micronucleus micromethod using L5178Y cells exhibited good sensitivity (16 positive/ 17 known genotoxins tested) and specificity (7 negative/7 known non-genotoxins tested) for the 24 test compounds studied with or without metabolic activation. Furthermore, this test showed a good correlation with other in vitro micronucleus tests performed using macromethods with various mammalian cell cultures. We conclude that the in vitro micronucleus micromethod with L5178Y cells could be used in the earliest stages of development of new molecules as a preliminary short-term screening assay before starting regulatory tests. IntroductionThe increasing numbers of new molecules synthesized in the pharmaceutical, chemical and cosmetic industries requires the use of assays providing rapid results and requiring small amounts of test material. As regulatory tests cannot reasonably be performed at an early stage of chemical development due to their complex protocols (duration, cost and need for large amounts of compound), we tried to miniaturize a chromosomal mutation assay, the in vitro micronucleus (MN) test. In fact, the in vitro MN test is known to be well adapted to the prescreening stage as it requires lower quantities of compound (500 mg are generally sufficient for the evaluation) and slide reading is easily and quickly performed. Moreover, it is able to demonstrate structural and numerical chromosomal aberrations in the same assay (Marzin, 1997). In fact, this methodology can simultaneously detect mitotic delay, apoptosis, chromosome breakage, chromosome loss and nondisjunction .The first requirement was to use microplates in order to decrease the amount of test compound. Thus, cells growing in suspension, which do not need trypsinization, were chosen. It

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Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

A micromethod for the in vitro micronucleus assay

Nesslany

Marzin

1999

Mutagenesis

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“…If primary cultures of lymphocytes and fibroblasts are irradiated, mitotic nondisjunction becames the basic mechanism of aneuploidy [67]. In nondisjunction an extra chromosome remains in one of the daughter cells.…”

Section: Aneuploidymentioning

confidence: 99%

“…It has been discovered that accelerated decondensation of the chromatin of individual chromosomes in connection with a disturbance in the kinetochore organization is apparently also accompanied by aneuploidy [76]. Aneuploidy may by induced by changes in the length of the cell cycle, a disturbance in the synchronic-ity of cleavage of the centromeric regions of daughter chromosomes [67], and by nondisjunction of chromosomes [78]. (6) A change in the interaction between the centromeric regions of daughter chromatids in the process of mitosis.…”

Section: Aneuploidymentioning

confidence: 99%

Cytogenetic anomalies and causes for their occurrence in somatic cells

Kovaleva

2008

Cytol. Genet.

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Different types of cytogenetic anomalies used in classical cytogenetics for estimating the level of damage to the chromosome apparatus are considered. Possible causes for the occurrence of different types of cytogenetic anomalies as well as the range of methods of micronucleus testing are discussed. It is shown that different levels of organization of genetic material (nucleotide, chromosome, or suprachromosome material) have an effect on processes involved in realization of a defect in the nucleotide sequence into a cytogenetic anomaly.

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“…In addition to SHE cells which we have previously used , we chose L5178Y mause cells because of their low incidence of spontaneaus micronuclei (Kirchner et al, 1992) and because we have previously shown that 5-azacytidine induces TFf resistance in these cells (McGregor et al, 1989). In this paper we report that approximately 80% of the 5-azacytidine induced micronuclei did not stain for kinetochores and for centromeric heterochromatic DNA suggesting that 5-azacytidine is primarily a clastogen.…”

mentioning

confidence: 99%

Cell-cycle dependent micronucleus formation and mitotic disturbances induced by 5-azacytidine in mammalian cells

Stopper¹,

Körber²,

Schiffmann³

et al. 1993

Mutation Research/Genetic Toxicology

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Summary 5-Azacytidine was originally developed to treat human myelogenous leukemia. However, interest in this compound has expanded because of reports of its ability to affect cell differentiation and to alter eukaryotic gene expression. In an ongoing attempt to understand the biochemical effects of this compound, we examined the effects of 5-azacytidine on mitosis and on micronucleus formation in mammalian cells. In L5178Y mouse cells, 5-azacytidine induced micronuclei at concentrations at which we and others have already reported its mutagenicity at the tk locus. Using CREST staining and C-banding studies, we showed that the induced micronuclei contained mostly chromosomal fragments although some may have contained whole chromosomes. By incorporating BrdU into the DNA of SHE cells, we determined that micronuclei were induced only when the compound was added while the cells were in S phase. Microscopically visible effects due to 5-azacytidine treatment were not observed until anaphase of the mitosis following treatment or thereafter. 5-Azacytidine did not induce micronuclei via interference with formation of the metaphase chromosome arrangement in mitosis, a common mechanism leading to aneuploidy. SupravitalUV microscopy revealed that chromatid bridges were observed in anaphase and, in some cases, were sustained into interphase. In the first mitosis after 5-azacytidine treatment we observed that many cells were unable to perform anaphase separation. All of these observations indicate that 5-azacytidine is predominantly a clastogen through its incorporation into DNA.

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Cytogenetic changes in primary, immortalized and malignant mammalian cells

Cited by 12 publications

References 8 publications

A micromethod for the in vitro micronucleus assay

A micromethod for the in vitro micronucleus assay

Cytogenetic anomalies and causes for their occurrence in somatic cells

Cell-cycle dependent micronucleus formation and mitotic disturbances induced by 5-azacytidine in mammalian cells

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