Gene-Locus Mutation Assays in Diploid Human Lymphoblast Lines

  • W. G. Thilly
  • J. G. DeLuca
  • E. E. Furth
  • H. HoppeIV
  • D. A. Kaden
  • J. J. Krolewski
  • H. L. Liber
  • T. R. Skopek
  • S. A. Slapikoff
  • R. J. Tizard
  • B. W. Penman


Our primary reasons for using diploid human lymphoblast lines in studies of mutagenesis are that they can be grown in free suspension; they appear to be genetically stable for chromosome number, as well as immortal; and they are readily derived from humans of different genetic backgrounds and possibly different sensitivities to mutagenic agents. Of these properties, the most important in assessing the mutagenicity of a large number of suspect chemicals is the lymphoblasts' ability to grow in suspension culture. This characteristic offers the potential for preprogrammed, automatic handling, which probably could not be achieved with anchorage-dependent cells. In this chapter, we will introduce the basics of lymphoblast husbandry and some protocols (tricks) that we have used to facilitate their use in studying genetic change.


Phenotypic Expression Feeder Layer Selective Condition Chinese Hamster Cell Mutation Assay 
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  1. 1.
    C. F. Arlett and S. A. Harcourt, The induction of 8 azaguanine-resistant mutants in cultured Chinese hamster cells by ultraviolet light. The effect of changes in post-irradiation conditions, Mutat. Res. 14, 431 (1972).CrossRefGoogle Scholar
  2. 2.
    C. F. Arlett and S. A. Harcourt, Expression time and spontaneous mutability in the estimation of induced mutation frequency following treatment of Chinese hamster cells by ultraviolet light, Mutat. Res. 16, 301 (1972).CrossRefGoogle Scholar
  3. 3.
    C. F. Arlett, D. Turnbull, S. A. Harcourt, A. R. Lehman, and C. M. Colella, A comparison of the 8-azaguanine and ouabain-resistance systems for the selection of induced mutant Chinese hamster cells, Mutat. Res. 33, 261 (1975).CrossRefGoogle Scholar
  4. 4.
    J. C. Asquith, The effect of dose fractionation on 7-radiation induced mutations in mammalian cells, Mutat. Res. 43, 91 (1977).CrossRefGoogle Scholar
  5. 5.
    R. M. Baker, D. M. Brunette, R. Mankovitz, L. H. Thompson, G. F. Whitmore, L. Siminovitch, and J. E. Till, Oubain-resistant mutants of mouse and hamster cells in culture, Cell 1, 9 (1974).CrossRefGoogle Scholar
  6. 6.
    B. A. Bridges and J. Huckle, Mutagenesis of cultured mammalian cells by X-irradiation and ultraviolet light, Mutat. Res. 10, 141 (1970).CrossRefGoogle Scholar
  7. 7.
    L. Chasin, The effect of ploidy on chemical mutagenesis in cultured Chinese hamster cells, J. Cell. Physiol. 82, 299 (1973).CrossRefGoogle Scholar
  8. 8.
    E. H. Y. Chu and H. V. Mailing, Mammalian cell genetics. II. Chemical induction of specific locus mutations in Chinese hamster cells in vitro, Proc. Natl. Acad. Sci. (U.S.A.) 61, 1306 (1968).CrossRefGoogle Scholar
  9. 9.
    D. Clive, W. G. Flamm, M. R. Machesko, and N. J. Bernheim, A mutational assay system using the thymidine kinase locus in mouse lymphoma cells, Mutat. Res. 16, 77 (1972).CrossRefGoogle Scholar
  10. 10.
    D. Clive, W. G. Flamm, and J. B. Patterson, Specific-locus mutational assay systems for mouse lymphoma cells, in: Chemical Mutagens: Principles and Methods for Their Detection, Vol. 3 (A. E. Hollaender, ed), p. 79, Plenum Press, New York (1973).Google Scholar
  11. 11.
    P. Coffino, R. Baumal, R. Laskov, and M. D. Scharff, Cloning of mouse myeloma cells and defection of rare variants,/. Cell. Physiol. 79, 429 (1972).CrossRefGoogle Scholar
  12. 12.
    J. Cole and C. F. Arlett, Ethyl methanesulphonate mutagenesis with L5178Y mouse lymphoma cells: A comparison of ouabain, thioguanine and excess thymidine resistance, Mutat. Res. 34, 507 (1976).CrossRefGoogle Scholar
  13. 13.
    J. Cole, C. F. Arlett, and M. H. L. Green, The fluctuation test as a more sensitive system for determining induced mutation in L5178Y mouse lymphoma cells, Mutat. Res. 41, 377.Google Scholar
  14. 14.
    R. P. Cox and W. K. Mason, X-Ray dose response for mutation to fructose utilization in cultured diploid human fibroblasts, Nature 252, 308 (1976).CrossRefGoogle Scholar
  15. 15.
    R. P. Cox, M. R. Krauss, M. E. Balis, and J. Dancis, Communication between normal and enzyme deficient cells in tissue culture, Exp. Cell. Res. 74, 251 (1972).CrossRefGoogle Scholar
  16. 16.
    R. P. Cox, M. R. Krauss, M. E. Balis, and J. Dancis, Metabolic cooperation in cell culture and studies of the mechanisms of cell interaction, J. Cell Physiol. 84, 237 (1974).CrossRefGoogle Scholar
  17. 17.
    J. G. DeLuca, D. A. Kaden, J. Krolewski, T. R. Skopek, and W. G. Thilly, Comparative mutagenicity of ICR-191 to S. typhimurium and diploid human lymphoblasts, Mutat. Res. 46, 11 (1977).Google Scholar
  18. 18.
    J. G. DeLuca, J. Krolewski, T. R. Skopek, D. A. Kaden, and W. G. Thilly, 9-Amino- acridine—A frameshift mutagen for Salmonella typhimurium TA 1537 inactive at the hgprt locus in human lymphoblasts, Mutat. Res. 42, 327 (1977).CrossRefGoogle Scholar
  19. 19.
    E. Eisenstadt and A. Gold, Cyclopenta[c,£/]pyrene: A highly mutagenic polycyclic aromatic hydrocarbon, Proc. Natl. Acad. Sci. (U.S.A.) 75, 1667 (1978).CrossRefGoogle Scholar
  20. 20.
    S. M. Elsevier, R. S. Kucherlapati, E. A. Nichols, R. P. Creagan, R. E. Giles, F. H. Ruddle, K. Willecke, and J. K. McDougall, Assignment of the gene for galactokinase to human chromosome 17 and its regional localization to band q21 –22, Nature 251, 633 (1974).CrossRefGoogle Scholar
  21. 21.
    W. Y. Fujimoto, J. H. Subak-Sharpe, and J. W. Seegmiller, Hypoxanthine-guanine phos- phoribosyltransferase deficiency: Chemical agents selective for mutant or normal fibroblasts in mixed and heterozygote cultures, Proc. Natl. Acad. Sci. (U.S.A.) 68, 1516 (1971).CrossRefGoogle Scholar
  22. 22.
    Y. Fujiwara, T Oki, and C. Heidelberger, Flourinated pyrimidines. XXXVII. Effects of 5- triflourourethyl-2′ -deoxyuridine on the synthesis of deoxyribonucleic acid of mammalian cells in culture, Mol. Pharmacol. 6, 273 (1970).Google Scholar
  23. 23.
    F. D. Gillin, D. J. Roufa, A. L. Beaudet, and C. T. Caskey, 8-Azaguanine resistance in mammalian cells. I. Hypoxanthine-guanine phosphoribosyltransferase, Genetics 72, 239 (1972).Google Scholar
  24. 24.
    M. Z. Gilman and W. G. Thilly, Cytotoxicity and mutagenicity of hyperthermia for diploid human hymphoblasts, J. Thermal Biol. 2, 95 (1977).CrossRefGoogle Scholar
  25. 25.
    B. Goz and W. H. Prusoff, Pharmacology of viruses, Ann. Rev. Pharmacol. 10, 143 (1970).CrossRefGoogle Scholar
  26. 26.
    H. Hoppe IV, T. R. Skopek, H. L. Liber, and W. G. Thilly, Alkyl methane sulfonate mutation of diploid human lymphoblasts and Salmonella typhimurium, Cancer Res. 38, 1595 (1978).Google Scholar
  27. 27.
    A. W. Hsie, P. A. Brimer, T. J. Mitchell, and D. G. Gosslee, The dose-response relationship for ultraviolet-light-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells, Somat. Cell Genet. 1, 383 (1975).CrossRefGoogle Scholar
  28. 28.
    E. M. Jensen, R. J. LaPolla, P. E. Kirby, and S. R. Hayworth, In vitro studies of chemical mutagens and carcinogens. I. Stability studies in cell culture medium, J. Natl. Cancer Inst. 59, 941 (1977).Google Scholar
  29. 29.
    D. A. Kaden, R A. Hites, and W. G. Thilly, Mutagenicity of soot and associated polycyclic aromatic hydrocarbons, Cancer Res. 39, 4152–4159 (1979).Google Scholar
  30. 29a.
    D. F. Krahn and C. Heidelberger, Liver homogenate-mediated mutagenesis in Chinese hamster V79 cells by polycyclic aromatic hydrocarbons and aflatoxins, Mutat. Res. 46, 27–44 (1977).Google Scholar
  31. 30.
    J. E. Lever, G. Nuki, and J. E. Seegmiller, Expression of purine overproduction in a series of 8-azaguanine-resistant diploid human lymphoblast lines, Proc. Natl. Acad. Sci. (U.S.A.) 71, 2679 (1974).CrossRefGoogle Scholar
  32. 31.
    J. Miller, P. W. Allderdice, D. A. Miller, W. R. Breg, and B. R. Migeon, Human thymidine kinase gene locus: Assignment to chromosome 17 in a hybrid of man and mouse cells, Science 173, 244 (1971).CrossRefGoogle Scholar
  33. 32.
    N. R. Morris and G. A. Fischer, Studies concerning the inhibition of the synthesis of deoxycytidine by phosphorylated derivatives of thymidine, Biochim. Biophys. Acta 42, 183 (1960).CrossRefGoogle Scholar
  34. 33.
    J. Morrow, Population dynamics of purine and pyrimidine analogue sensitive and resistant mammalian cells grown in culture, Genetics 71, 429 (1972).Google Scholar
  35. 34.
    J. A. Nelson, J. W. Carpenter, L. M. Rose, and D. J. Adamson, Mechanisms of action of 6- thioguanine, 6-mercaptopurine, and 8-azaguanine, Cancer Res. 35, 2872 (1975).Google Scholar
  36. 35.
    B. W. Penman and W. G. Thilly, Concentration-dependent mutation of diploid human lymphoblasts by methylnitronitrosoguanidine: The importance of phenotypic lag, Somat. Cell Genet. 2, 235 (1976).CrossRefGoogle Scholar
  37. 36.
    B. W. Penman, M. V. Wong, and W. G. Thilly, Mutagenicity of 5-halodeoxyuridines to diploid human lymphoblasts, Life Sci. 19, 563 (1976).CrossRefGoogle Scholar
  38. 37.
    B. W. Penman, H. Hoppe IV, and W. G. Thilly, Concentration-dependent mutation by alkylating agents in human lymphoblasts and Salmonella typhimurium: Methylni- trosourethane and beta-propiolactone,/. Natl. Cancer Inst. 63, 903–907 (1979).Google Scholar
  39. 37.
    B. W. Penman, H. Hoppe IV, and W. G. Thilly, Concentration-dependent mutation by alkylating agents in human lymphoblasts and Salmonella typhimurium: Methylni- trosourethane and beta-propiolactone,/. Natl. Cancer Inst. 63, 903–907 (1979).Google Scholar
  40. 39.
    G. P. Prado, M. L. Lee, and R. A. Hites, in: Sixteenth International Symposium on Combustion, The Combustion Institute, Cambridge, Massachusetts (1973).Google Scholar
  41. 40.
    Proceedings of Symposium, Airlie, Virginia, Birth Defects: Ong. Artie. Ser. (D. Bergsma, ed.), 9, 1, The National Foundation, New York (1972).Google Scholar
  42. 41.
    P. Reichard, Z. N. Canellakis, and E. S. Canellakis, Regulatory mechanisms in the synthesis of deoxyribonucleic acid in vitro, Biochim. Biophys. Acta 41, 558 (1960).CrossRefGoogle Scholar
  43. 42.
    P. Reichard, Z. N. Canellakis, and E. S. Canellakis, Studies on a possible regulatory mechanism for the biosynthesis of deoxyribonucleic acid,/. Biol. Chem. 236, 2514 (1961).Google Scholar
  44. 43.
    P. Reyes and C. Heidelberger, Fluorinated pyrimidines. XXVI. Mammalian thymidylate synthetase: Its mechanism of action and inhibition by fluorinated nucleotides, Mol. Pharmacol. /, 14 (1965).Google Scholar
  45. 44.
    H. Sato, R. S. Slesinski, and J. W. Littlefield, Chemical mutagenesis at the phosphoribosyl- transferase locus in cultured human lymphoblasts, Proc. Natl. Acad. Sci. (U.S.A.) 69, 1244 (1972).CrossRefGoogle Scholar
  46. 45.
    T. R. Skopek, H. L. Liber, B. W. Penman, and W. G. Thilly, Isolation of a human lympho- blastoid line heterozygous at the thymidine kinase locus: Possibility for a rapid human cell mutation assay, Biochem. Biophys. Res. Commun. 84, 411 (1978).CrossRefGoogle Scholar
  47. 46.
    S. A. Slapikoff, B. M. Andon, and W. G. Thilly, Comparison of toxicity and mutagenicity of butyl methanesulfonate among human lymphoblast lines, Mutat. Res. 54, 193 (1978).Google Scholar
  48. 47.
    G. H. Strauss and R. J. Albertini, 6-Thioguanine resistant lymphocytes in human peripheral blood, in: Progress in Genetic Toxicology ( D. Scott, B. A. Bridges, and F. H. Sobels, eds.), p. 327, Elsevier/North-Holland Biomedical Press, Amsterdam (1977).Google Scholar
  49. 48.
    W. Szybalski and E. H. Szvbalski, Drug sensitivity as a genetic marker for human cell lines, Univ. Mich. Med. Bull. 28, 277 (1962).Google Scholar
  50. 49.
    W. Szybalski, N. K. Cohn, and C. Heidelberger, A survey of the biochemistry of 5-trifluo- romethyluracil (FST) and 5-trifluoromethyl-2'-deoxyuridine (F3TDR), Fed. Proc. Fed. Am. Soc. Exp. Biol. 22 (Suppl.), 532 (1963).Google Scholar
  51. 50.
    J. Thacker and R. Cox, Mutation induction and inactivation in mammalian cells exposed to ionizing radiation, Nature 258, 429 (1975).CrossRefGoogle Scholar
  52. 51.
    W. G. Thilly, T. S. Nowak, Jr., and G. N. Wogan, Maintaining perpetral synchrony in HeLa S3 culture, Biotechnol. Bioeng. 16, 149 (1974).CrossRefGoogle Scholar
  53. 52.
    W. G. Thilly, D. I. Arkin, T. S. Nowak, Jr., and G. N. Wogan, Maintenance of perpetual synchrony in HeLa suspension cultures: Question of unbalanced growth, Biotechnol. Bioeng. 17, 703 (1975).CrossRefGoogle Scholar
  54. 53.
    W. G. Thilly, J. G. DeLuca, H. Hoppe IV, and B. W. Penman, Mutation of human lym- phoblasts by methylnitrosourea, Chem.-Biol. Interact. 15, 33 (1976).CrossRefGoogle Scholar
  55. 54.
    W G. Thilly, J. G. DeLuca, H. Hoppe IV, H. L. Liber, and B. W. Penman, Mutation assay in diploid human lymphoblasts: Methodological aspects, J. Environ. Pathol Toxicol. 1, 91 (1977).Google Scholar
  56. 55.
    W. G. Thilly, J. G. DeLuca, H. Hoppe IV, and B. W. Penman, Phenotypic lag and mutation to 6-thioguanine resistance in diplid human lymphoblasts, Mutat. Res. 50, 137 (1978).CrossRefGoogle Scholar
  57. 56.
    A. A. van Zeeland and J. W. I. M. Simons, Linear dose-response relationships after prolonged expression times in V-79 Chinese hamster cells, Mutat. Res. 35, 129 (1976).CrossRefGoogle Scholar
  58. 57.
    A. A. van Zeeland, M. C. E. van Diggelen, and J. W. I. M. Simons, The role of metabolic cooperation in selection of hypoxanthine-guanine-phosphoribosyl-transferase (H6-PRT)-defi- cient mutants from diploid mammalian cell strains, Mutat. Res. 14, 355 (1972).CrossRefGoogle Scholar
  59. 58.
    A. A. van Zeeland, Y. C. E. M. de Ruijfer, and J. W. I. M. Simons, The role of 8-azagua- nine in the selection from human diploid cells of mutants deficient in hypoxanthine-guanine- phosphoribosyl-transferase (HGPRT), Mutat. Res. 23, 55 (1974).Google Scholar
  60. 60.
    N. Xeros, Deoxyriboside control and synchronization of mitosis, Nature 194, 682 (1962).CrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1980

Authors and Affiliations

  • W. G. Thilly
    • 1
  • J. G. DeLuca
    • 1
  • E. E. Furth
    • 1
  • H. HoppeIV
    • 1
  • D. A. Kaden
    • 1
  • J. J. Krolewski
    • 1
  • H. L. Liber
    • 1
  • T. R. Skopek
    • 1
  • S. A. Slapikoff
    • 1
  • R. J. Tizard
    • 1
  • B. W. Penman
    • 1
  1. 1.Genetic Toxicology Laboratory, Department of Nutrition and Food ScienceMassachusetts Institute of TechnologyCambridgeUSA

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