Measurement of mutagenesis in mammalian cells.

Waldren, Charles A.; Jones, Carol; Puck, Theodore T.

doi:10.1073/pnas.76.3.1358

Cited by 118 publications

(54 citation statements)

References 33 publications

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“…This gene encodes the CD59 cell surface antigen (also known as the S1 antigen) that, in the presence of rabbit serum complement (HRP, Denver, PA), renders A L cells sensitive to killing by the monoclonal antibody E7.1. Antibody E7.1 was produced by a hybridoma culture as described (28,29). A L cells were maintained in Ham F-12 medium supplemented with 8% heatinactivated FBS, 25 g͞ml gentamycin, and 2ϫ normal glycine (2 ϫ 10 Ϫ4 M) at 37°C in a humidified 5% CO 2 incubator and passaged as described (19,20).…”

Section: Methodsmentioning

confidence: 99%

Induction of oxyradicals by arsenic: Implication for mechanism of genotoxicity

Liu¹

2001

Proceedings of the National Academy of Sciences

233

155

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Although arsenic is a well-established human carcinogen, the mechanisms by which it induces cancer remain poorly understood. We previously showed arsenite to be a potent mutagen in humanhamster hybrid (AL) cells, and that it induces predominantly multilocus deletions. We show here by confocal scanning microscopy with the fluorescent probe 5 ,6 -chloromethyl-2 ,7 -dichlorodihydrofluorescein diacetate that arsenite induces, within 5 min after treatment, a dose-dependent increase of up to 3-fold in intracellular oxyradical production. Concurrent treatment of cells with arsenite and the radical scavenger DMSO reduced the fluorescent intensity to control levels. ESR spectroscopy with 4-hydroxy-2,2,6,6-tetramethyl-1-hydroxypiperidine (TEMPOL-H) as a probe in conjunction with superoxide dismutase and catalase to quench superoxide anions and hydrogen peroxide, respectively, indicates that arsenite increases the levels of superoxide-driven hydroxyl radicals in these cells. Furthermore, reducing the intracellular levels of nonprotein sulfhydryls (mainly glutathione) in A L cells with buthionine S-R-sulfoximine increases the mutagenic potential of arsenite by more than 5-fold. The data are consistent with our previous results with the radical scavenger DMSO, which reduced the mutagenicity of arsenic in these cells, and provide convincing evidence that reactive oxygen species, particularly hydroxyl radicals, play an important causal role in the genotoxicity of arsenical compounds in mammalian cells.

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Section: Methodsmentioning

confidence: 99%

Induction of oxyradicals by arsenic: Implication for mechanism of genotoxicity

Liu¹

2001

Proceedings of the National Academy of Sciences

233

155

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“…In most cases, only one and at times no more than two CD59 Ϫ mutants were isolated from each irradiated population for mutant spectrum analysis. Five DNA marker genes on chromosome 11 (Wilms' tumor, Parathyroid Hormone, Catalase, RAS, and Apolipoprotein A-1) were chosen for multiplex PCR analysis because of their mapping positions relative to the CD59 gene, which encodes the CD59 antigen (16,17,23), and the availability of PCR primers for the coding regions of these genes (24-26). PCR amplifications were performed for 30 cycles by using a DNA thermal cycler model 480 (Perkin-Elmer͞Cetus) in 20-l reaction mixtures containing 0.2 g of the EcoRI-digested DNA sample in 1ϫ Stoffel fragment buffer, all four dNTPs (each at 0.2 mM), 3 mM MgCl 2 , 0.2 mM each primer, and 2 units of Stoffel fragment enzyme (19,21).…”

mentioning

confidence: 99%

Induction of a bystander mutagenic effect of alpha particles in mammalian cells

Zhou

Randers-Pehrson

Waldren

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

432

281

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Ever since the discovery of X-rays was made by Rö ntgen more than a hundred years ago, it has always been accepted that the deleterious effects of ionizing radiation such as mutation and carcinogenesis are attributable mainly to direct damage to DNA. Although evidence based on microdosimetric estimation in support of a bystander effect appears to be consistent, direct proof of such extranuclear͞extracellular effects are limited. Using a precision charged particle microbeam, we show here that irradiation of 20% of randomly selected A L cells with 20 alpha particles each results in a mutant fraction that is 3-fold higher than expected, assuming no bystander modulation effect. Furthermore, analysis by multiplex PCR shows that the types of mutants induced are significantly different from those of spontaneous origin. Pretreatment of cells with the radical scavenger DMSO had no effect on the mutagenic incidence. In contrast, cells pretreated with a 40 M dose of lindane, which inhibits cell-cell communication, significantly decreased the mutant yield. The doses of DMSO and lindane used in these experiments are nontoxic and nonmutagenic. We further examined the mutagenic yield when 5-10% of randomly selected cells were irradiated with 20 alpha particles each. Results showed, likewise, a higher mutant yield than expected assuming no bystander effects. Our studies provide clear evidence that irradiated cells can induce a bystander mutagenic response in neighboring cells not directly traversed by alpha particles and that cell-cell communication process play a critical role in mediating the bystander phenomenon.

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“…We have described a mutation assay (the AL system) which permits the measurement in a single in vitro test of point mutations, small and large chromosome deletions, and chromosome loss (nondisjunction) (23)(24)(25). This methodology employs a permanent line of humanChinese hamster hybrid cells ( A L J~) that contains a standard set of CHO-K1 chromosomes plus a single human chromosome, number ll (15,271.…”

mentioning

confidence: 99%

“…The rate of spontaneous loss of the a l marker has been determined by fluctuation analysis (16) to be 7-15 x generatiodcell (13,24) which results in the presence of 1-10 a l -mutants per lo4 survivors in the expanded populations employed in mutation studies (23). This proportion of pre-existing mutants is high enough to limit the detection of induced mutants to frequencies 2-4 times higher than the spontaneous level.…”

mentioning

confidence: 99%

Use of somatic cell hybrids for quantitation of mutagenesis: Reduction in background mutants by fluorescence‐activated cell sorting (FACS)

et al. 1984

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Environmentally induced mutations, especially those involving large scale genetic damage such as deletions and chromosome loss, are of central importance in the production of human genetic disease and cancer. We have developed a methodology, the AL assay, that permits detection of such extensive genetic changes which often escape detection in other systems in which they are lethal. The AL assay employs a human-Chinese hamster ovary cell hybrid that retains a single human chromosome, number 11. A set of specific cell surface antigens result from genes located on opposite arms of this chromosome. Exposure to mutagens produces mutants which form colonies in the presence of complement and specific antiserum that kill nonmutant cells.The frequency and pattern of marker loss provides a measure of single gene mutation, large and small deletion, and loss of the entire chromosome 11. We have employed the indirect fluorescein conjugated isothiocyanate (FITC) technique and fluorescence-activated cell sorting (FACS) to remove spontaneous mutants from the initial population. The 100-fold reduction in background thus far achieved should allow accurate analysis of mutation by ionizing radiation at doses of less than 10 rad.

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Measurement of mutagenesis in mammalian cells.

Cited by 118 publications

References 33 publications

Induction of oxyradicals by arsenic: Implication for mechanism of genotoxicity

Induction of oxyradicals by arsenic: Implication for mechanism of genotoxicity

Induction of a bystander mutagenic effect of alpha particles in mammalian cells

Use of somatic cell hybrids for quantitation of mutagenesis: Reduction in background mutants by fluorescence‐activated cell sorting (FACS)

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