Ethyl methanesulphonate mutagenesis with L5178Y mouse lymphoma cells: A comparison of ouabain, thioguanine and excess thymidine resistance

Cole, J.; Arlett, C.F.

doi:10.1016/0027-5107(76)90226-8

Cited by 71 publications

(12 citation statements)

References 18 publications

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“…As additional in vitro mammalian test systems were developed and investigated, it became apparent that the time allowed for expression was most critical in determining the actual observed mutant frequencies. Several factors, including the specific mutagen [Myhr and DiPaolo, 19751, dose of mutagen [Carver et al, 1976;Cole and Arlett, 1976;Arlett and Harcourt, 1972;Huang, 1977;van Zeeland and Simons, 1976;Capizzi et al, 19741, and locus evaluated (see below) can profoundly affect the time required for full expression. For the asparagine marker, both L5 178Y mouse lymphoma cells [Summers, 19731 and Jenson sarcoma cells [Morrow et al, 19761 require expression times of 1 or 2 days.…”

Section: Introductionmentioning

confidence: 99%

The quantitation of TK^−/− and HGPRT⁻ mutants of L5178Y/TK^+/− mouse lymphoma cells at varying times post‐treatment

Moore

Clive

1982

Environ. mutagen

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The quantitation of newly induced TFT-resistant and TG-resistant mutants in TK"--3.7.2C mouse lymphoma cells was analyzed using conventional soft agar cloning and a newly developed technique that allowed for the sequestering, expression, and selection (SES) of mutants in Linbro wells. The conventional method of cloning in semi-soft-agar-supplemented nutrient medium was found to provide maximum mutant frequencies at 1-3 days post-treatment for TFT resistance and at 5-8 days for TG resistance. The length of time required to reach maximum expression was both mutagen-and dose-dependent. Following complete expression, TKdeficient mutants induced by some mutagens (eg, CH,I and 2-AAF) declined in frequency.The SES procedure, which avoids the potential changes in mutant frequency due to differential growth between mutants and the total population, was applied to assess the ability of conventional cloning methods to completely detect induced mutant frequencies. We determined that at the TK locus, and perhaps also at the HGPRT locus, EMS induced several times more mutants than are detected by conventional soft agar cloning. At least part of this difference in detected frequency appeared to be due to a significant population of slowly growing mutants which declined in frequency (because of their slow growth) during the time allowed for expression in the conventional assay procedure. An analysis of cells from mutant colonies isolated from both conventional cloning and the SES procedure revealed that TK-deficient mutants could be roughly divided into three categories: (1) tiny colony ( 7 ) mutants which were exceedingly slow-growing and were quantitated only in the SES analysis; (2) small colony (0) mutants which were slow growing; and Abbreviations: 2-AAF, 2-acetylaminofluorene; BUdR, bromodeoxyuridine; CH,l, methyl iodide; DMN, dimethylnitrosamine; EMS, ethyl methanesulfonate; G,, the post-DNA synthesis phase of interphase; HGPRT, hypoxanthineguanine phosphoribosyltransferase; IUdR, iododeoxyuridine; X, large colony (TK+ or TIT?; u, small colony (TK+ or TFT?; SES, sequester, express, and select; T, tiny colony (TK-Ior TFT'); TIT, trifluorothymidine; THMG, thymidine + hypoxanthine + methotrexate + glycine; TG, 6-thioguanine; TK, thymidine kinase.

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Section: Introductionmentioning

confidence: 99%

The quantitation of TK^−/− and HGPRT⁻ mutants of L5178Y/TK^+/− mouse lymphoma cells at varying times post‐treatment

Moore

Clive

1982

Environ. mutagen

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“…Sobrero & Bertino (1986) been extablished. However similar problems have been reported with the measurement of methotrexate resistance using soft agar cloning techniques (Cole & Arlett, 1976 complicating factor in the in vitro chemosensitivity measurements in human tumour stem cell assays. Metabolites from dying cells may also modulate the cytotoxicity of other antimetabolites, such as 5-fluorouracil and cytosine arabinoside when similar culture conditions are utilised.…”

Section: Discussionmentioning

confidence: 68%

Modulation of antifolate cytotoxicity by metabolites from dying cells in a lymphocyte clonal assay

Hughes

deFazio²,

Tattersall³

1988

Br J Cancer

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“…In contrast, ouabainresistant colonies obtained by Cole and Arlett [1976] in a different L5178Y cell line, one presumably more like the 3.7.2C subclone's progenitor, were all large. No chromosomal analyses were made on the ouares clones in this study, but a specific chromosomal mechanism for the induction of ouabain resistance is highly unlikely although chromosome 11 anomalies conceivably could be present.…”

Section: Discussionmentioning

confidence: 73%

“…With the discovery of small ouares colonies in this study and the previously reported recovery of a large proportion of small colonies in nonselective medium (ie, not TK-/-) soon after treatment with a high concentration of MMS in a study of TFTreS and colony size [Amacher and Paillet, 19811, it is clear that small colony formation in soft-agar medium is not restricted to TFT or bromodeoxyuridine (BUdR) resistance, but may be a general characteristic of 3.7.2C cells when manipulated or stressed and then cloned 0-2 days later in soft-agar medium. In contrast, ouabainresistant colonies obtained by Cole and Arlett [1976] in a different L5178Y cell line, one presumably more like the 3.7.2C subclone's progenitor, were all large. No chromosomal analyses were made on the ouares clones in this study, but a specific chromosomal mechanism for the induction of ouabain resistance is highly unlikely although chromosome 11 anomalies conceivably could be present.…”

Section: Discussionmentioning

confidence: 82%

Mutagenesis at the ouabain‐resistance locus of 3.7.2c l5178y cells by chromosomal mutagens

Amacher

Dunn

1985

Environ. mutagen

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Chemical mutagens including methyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, iodomethane, and epichlorohydrin have been classified as "chromosomal mutagens" in the L5178Y/thymidine kinase (TK) gene mutation assay by Clive and coworkers [Mutat Res 59:61-108, 1979; and "The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation." Amsterdam: Elsevier/North Holland, pp 103-123, 1980] who observed mutagen-dependent increases in small TK-deficient mutant colonies with detectable damage to the chromosome (11) that carries the TK locus. In this study, we tested these four chemicals for the induction of gene mutations at the ouabain-resistance (ouares) locus of 3.7.2C L5178Y cells to determine if presumptive chromosomal mutagens would go undetected at a gene locus that is unresponsive to chromosomal damage. A final concentration of 375 micrograms/ml ouabain in soft-agar medium selected against the ouabain-sensitive phenotype without loss of the mutagen-induced ouabain-resistant phenotype. Verification of the mutant phenotype was completed for six individual soft-agar ouares colonies derived from mutagen-treated cultures via growth for 10-11 days in nonselective medium followed by retesting for colony formation in selective soft-agar medium. Dose-related reproducible increases in the frequency of ouabain-resistant mutants were observed for 3.7.2C L5178Y cells that had been exposed for 3 hr to 24-46 micrograms/ml epichlorohydrin, 1.9-3.6 micrograms/ml iodomethane, 0.006-0.011 micrograms/ml N-methyl-N'-nitro-N-nitrosoguanidine and and 2.0-5.4 micrograms/ml methyl methanesulfonate. Also, treatments with EMS, which induced sufficient numbers of ouares colonies to permit analysis of colony size distribution, showed the existence of a bimodal size distribution similar to those reported for TK-deficient mutants. This discovery suggests that mutant colony size in this cell line may be independent of specific gene locus effects. We conclude that (1) chemicals that induce a high proportion of chromosomal mutants, as detected at the TK locus in earlier studies, also induce single gene mutations at the ouabain-resistance locus and (2) a bimodal distribution of mutant colony sizes in soft-agar medium after short expression periods may be a distinctive characteristic of the 3.7.2C L5178Y cell line and is not confined to the TK locus.

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Ethyl methanesulphonate mutagenesis with L5178Y mouse lymphoma cells: A comparison of ouabain, thioguanine and excess thymidine resistance

Cited by 71 publications

References 18 publications

The quantitation of TK^−/− and HGPRT⁻ mutants of L5178Y/TK^+/− mouse lymphoma cells at varying times post‐treatment

The quantitation of TK^−/− and HGPRT⁻ mutants of L5178Y/TK^+/− mouse lymphoma cells at varying times post‐treatment

Modulation of antifolate cytotoxicity by metabolites from dying cells in a lymphocyte clonal assay

Mutagenesis at the ouabain‐resistance locus of 3.7.2c l5178y cells by chromosomal mutagens

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Ethyl methanesulphonate mutagenesis with L5178Y mouse lymphoma cells: A comparison of ouabain, thioguanine and excess thymidine resistance

Cited by 71 publications

References 18 publications

The quantitation of TK−/− and HGPRT− mutants of L5178Y/TK+/− mouse lymphoma cells at varying times post‐treatment

The quantitation of TK−/− and HGPRT− mutants of L5178Y/TK+/− mouse lymphoma cells at varying times post‐treatment

Modulation of antifolate cytotoxicity by metabolites from dying cells in a lymphocyte clonal assay

Mutagenesis at the ouabain‐resistance locus of 3.7.2c l5178y cells by chromosomal mutagens

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The quantitation of TK^−/− and HGPRT⁻ mutants of L5178Y/TK^+/− mouse lymphoma cells at varying times post‐treatment

The quantitation of TK^−/− and HGPRT⁻ mutants of L5178Y/TK^+/− mouse lymphoma cells at varying times post‐treatment