Mutation assay at the thymidine kinase locus in diploid human lymphoblasts

Liber, Howard L.; Thilly, William G.

doi:10.1016/0027-5107(82)90308-6

Cited by 234 publications

(101 citation statements)

References 18 publications

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“…This is about 2-fold lower than our measurements in the TK6 cells (10.5 ± 1.5 per 10 6 nt). However, TK6 cells may possess higher endogenous levels of dU due to the presence of AID activity in this B-cell-derived cell line (36).…”

Section: Nucleobase Deamination In Human Cells Exposed To No Under Bimentioning

confidence: 97%

Relatively Small Increases in the Steady-State Levels of Nucleobase Deamination Products in DNA from Human TK6 Cells Exposed to Toxic Levels of Nitric Oxide

Dong

Dedon

2005

Chem. Res. Toxicol.

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Nitric oxide (NO) is a physiologically important molecule that has been implicated in the pathophysiology of diseases associated with chronic inflammation, such as cancer. While the complicated chemistry of NO-mediated genotoxicity has been extensively study in vitro, neither the spectrum of DNA lesions nor their consequences in vivo have been rigorously defined. We have approached this problem by exposing human TK6 lymphoblastoid cells to controlled steady-state concentrations of 1.75 or 0.65 μM NO along with 186 μM O 2 in a recently developed reactor that avoids the anomalous gas-phase chemistry of NO and approximates the conditions at sites of inflammation in tissues. The resulting spectrum of nucleobase deamination products was defined using a recently developed liquid chromatography/mass spectrometry (LC/MS) method and the results were correlated with cytotoxicity and apoptosis. A series of control experiments revealed the necessity of using dC and dA deaminase inhibitors to avoid adventitious formation of 2′-deoxyuridine (dU) and 2′-deoxyinosine (dI), respectively, during DNA isolation and processing. Exposure of TK6 cells to 1.75 μM NO and 186 μM O 2 for 12 hr (1260 μM•min dose) resulted in 32% loss of cell viability measured immediately after exposure and 87% cytotoxicity after a 24 hr recovery period. The same exposure resulted in 3.5-, 3.8-, and 4.1-fold increases in dX, dI and dU, respectively, to reach the following levels: dX, 7 (±1) per 10 6 nt; dI, 25 (±2.1) per 10 6 nt; and dU, 40 (±3.8) per 10 6 per nt. dO was not detected above the limit of detection of 6 lesions per 10 7 nt in 50 μg of DNA. A 12 hr exposure to 0.65 μM NO and 190 μM O 2 (468 μM•min dose) caused 1.7-, 1.8-, and 2.0-fold increases in dX, dI, and dU, respectively, accompanied by a ∼15 (±3.6) % reduction in cell viability immediately after exposure. Again, dO was not detected. These results reveal modest increases in the steady-state levels of DNA deamination products in cells exposed to relatively cytotoxic levels of NO. This could result from limited nitrosative chemistry in nuclear DNA in cells exposed to NO or high levels of formation balanced by rapid repair of nucleobase deamination lesions in DNA.

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Section: Nucleobase Deamination In Human Cells Exposed To No Under Bimentioning

confidence: 97%

Relatively Small Increases in the Steady-State Levels of Nucleobase Deamination Products in DNA from Human TK6 Cells Exposed to Toxic Levels of Nitric Oxide

Dong

Dedon

2005

Chem. Res. Toxicol.

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“…We first compared NO Y -induced cell lethality determined by trypan blue exclusion with that determined by plating efficiency (25) and found that both methods produced similar survival values 8 and 24 h after treatment. Therefore, in this study survival was determined by staining cells with 0.4% trypan blue solution followed by enumeration of those excluding the stain under a phase-contrast microscope.…”

Section: Analysis Of Cell Survivalmentioning

confidence: 99%

“…For plating efficiency analysis, 120 cells in 12 ml of nonselective medium from each group were plated in duplicate in 96-well plates at a density of one cell per 100 l per well. After 2 weeks of incubation, colonies were counted and MF was calculated according to the described method (25). In parallel experiments, to estimate spontaneous mutation rate and to determine response of the HPRT and TK1 genes to treatment with a well characterized mutagen, similar MF analyses were done on untreated cells and cells treated with 4-nitroquinoline 1-oxide (4-NQO) (140 ng͞ml for 1.5 h) as described above.…”

Section: Analysis Of Cell Survivalmentioning

confidence: 99%

Nitric oxide-induced genotoxicity, mitochondrial damage, and apoptosis in human lymphoblastoid cells expressing wild-type and mutant p53

Trudel

Wogan

2002

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

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“…Our difficulty in obtaining HPRT-mutants of MClA-Cl and the subsequent detection of three copies of the X chromosome suggested a different strategy. Since it appeared that MClA-Cl had more than one active X chromosome and more than one active hprt gene, we chose to create a hprt heterozygote, as has been carried out at the tk and aprt loci (Liber and Thilly, 1982;Sebastio et al, 1985). This was done by screening for revertants to HATR.…”

Section: Disaussionmentioning

confidence: 99%

Hprt mutants in a transplantable murine tumour arise more frequently in vivo than in vitro

Wilkinson

Sandhu²,

Breneman³

et al. 1995

Br J Cancer

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Sumnuary A model system was developed to allow investigation of the frequency at which clastogenic and or mutagenic events occur in situ in a transplantable murine fibrosarcoma tumour (MCIA-Cl) compared with in vitro culture. The marker selected for detecting these events was the X-linked hprt (hypoxanthine-guanine phosphonrbosyltransferase) gene. We found that the hprt gene in MCIA-Cl was not suitable for this purpose, most likely because multiple active copies were present. To circumvent the problem, HPRT -[6-thioguanine (6-TG)-resistant] clones were isolated by inactivating all hprt genes with methylnitrosourea. Spontaneous revertants to hypoxanthine/aminopterin/thymidine resistance (HATR) were isolated and found to be approximately 1000 times more sensitive than the parental tumour to induction of 6-TGR mutants by cobalt-60 y-rays. This sensitivity is expected for a heterozygous marker; these revertants may therefore possess only one functional hprt locus but two or more active X chromosomes. A clone with a stable hprt gene was identified and a neo gene was introduced. The resulting cell line (MN-i 1) could be grown as a subcutaneous tumour in syngeneic C57BL/6 animals. The frequency of mutations arising in vivo in the marker hprt gene could be estimated by cultunrng explanted tumour cells in the presence of 6-TG, using G418 selection to distinguish tumour from host cells. The frequency of mutants in MN-lI cells grown as tumours was found to be 3.4-fold higher than in tissue culture for an equivalent period of time. These data provide the first direct evidence for the existence of mutagenic factors in a tumour environment that might contribute to tumour progression.

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Mutation assay at the thymidine kinase locus in diploid human lymphoblasts

Cited by 234 publications

References 18 publications

Relatively Small Increases in the Steady-State Levels of Nucleobase Deamination Products in DNA from Human TK6 Cells Exposed to Toxic Levels of Nitric Oxide

Relatively Small Increases in the Steady-State Levels of Nucleobase Deamination Products in DNA from Human TK6 Cells Exposed to Toxic Levels of Nitric Oxide

Nitric oxide-induced genotoxicity, mitochondrial damage, and apoptosis in human lymphoblastoid cells expressing wild-type and mutant p53

Hprt mutants in a transplantable murine tumour arise more frequently in vivo than in vitro

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