Advertisement

The Human T-Cell Cloning Assay: Identifying Genotypes Susceptible to Drug Toxicity and Somatic Mutation

  • Sai-Mei Hou
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1105)

Abstract

Humans exhibit marked genetic polymorphisms in drug metabolism that contribute to high incidence of adverse effects in susceptible individuals due to altered balance between metabolic activation and detoxification. The T-cell cloning assay, which detects mutations in the gene for hypoxanthine-guanine phosphoribosyl transferase (HPRT), is the most well-developed reporter system for studying specific locus mutation in human somatic cells. The assay is based on a mitogen- and growth factor-dependent clonal expansion of peripheral T-lymphocytes in which the 6-thioguanine-resistant HPRT mutants can be selected, enumerated, and collected for molecular analysis of the mutational nature. The assay provides a unique tool for studying in vivo and in vitro mutagenesis, for investigating the functional impact of common polymorphism in metabolism and repair genes, and for identifying risk genotypes for drug-induced toxicity and mutagenicity. This chapter presents a simple and reliable method for the enumeration of HPRT mutant frequency induced in vitro without using any source of recombinant interleukin-2. The other main feature is that only truly induced and unique mutants are collected for further analysis.

Key words

Genetic polymorphism Human lymphocytes HPRT Gene mutation T-cell cloning assay 

References

  1. 1.
    Morley AA, Trainor KJ, Seshadri R, Ryall RG (1983) Measurement of in vivo mutations in human lymphocytes. Nature 302:155–156PubMedCrossRefGoogle Scholar
  2. 2.
    Cariello NF, Douglas GR, Dycaico MJ, Gorelick NJ, Provost GS, Soussi T (1997) Databases and software for the analysis of mutations in the human p53 gene, the human hprt gene and both the lacI and lacZ gene in transgenic rodents. Nucleic Acids Res 25:136–137PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Hou S-M (2000) Somatic mutations and aging, methods for molecular analysis of HPRT mutations. In: Barnett YA, Barnett CR (eds) Aging, methods and protocols. Humana, Totowa, NJ, pp 189–197CrossRefGoogle Scholar
  4. 4.
    Cole J, Skopek TR (1994) Somatic mutant frequency, mutation rates and mutational spectra in the human population in vivo. Mutat Res 304:33–105PubMedCrossRefGoogle Scholar
  5. 5.
    Hou SM, Falt S, Steen AM (1995) Hprt mutant frequency and GSTM1 genotype in non-smoking healthy individuals. Environ Mol Mutagen 25:97–105PubMedCrossRefGoogle Scholar
  6. 6.
    Hou SM, Falt S, Yang K, Nyberg F, Pershagen G, Hemminki K, Lambert B (2001) Differential interactions between GSTM1 and NAT2 genotypes on aromatic DNA adduct level and HPRT mutant frequency in lung cancer patients and population controls. Cancer Epidemiol Biomarkers Prev 10:133–140PubMedGoogle Scholar
  7. 7.
    Andersson B, Falt S, Lambert B (1992) Strand specificity for mutations induced by (+)-anti BPDE in the hprt gene in human T-lymphocytes. Mutat Res 269:129–140PubMedCrossRefGoogle Scholar
  8. 8.
    Bastlova T, Podlutsky A (1996) Molecular analysis of styrene oxide-induced hprt mutation in human T-lymphocytes. Mutagenesis 11: 581–591PubMedCrossRefGoogle Scholar
  9. 9.
    McGregor WG, Maher VM, McCormick JJ (1994) Kinds and locations of mutations induced in the hypoxanthine-guanine phosphoribosyltransferase gene of human T-lymphocytes by 1-nitrosopyrene, including those caused by V(D)J recombinase. Cancer Res 54: 4207–4213PubMedGoogle Scholar
  10. 10.
    Noori P, Hou SM (2001) Mutational spectrum induced by acetaldehyde in the HPRT gene of human T lymphocytes resembles that in the p53 gene of esophageal cancers. Carcinogenesis 22:1825–1830PubMedCrossRefGoogle Scholar
  11. 11.
    Hou SM, Van Dam FJ, de Zwart F, Warnock C, Mognato M, Turner J, Podlutskaja N, Podlutsky A, Becker R, Barnett Y, Barnett CR, Celotti L, Davies M, Huttner E, Lambert B, Tates AD (1999) Validation of the human T-lymphocyte cloning assay–ring test report from the EU concerted action on HPRT mutation (EUCAHM). Mutat Res 431:211–221PubMedCrossRefGoogle Scholar
  12. 12.
    Norimura T, Maher VM, McCormick JJ (1990) A quantitative assay for measuring the induction of mutations in human peripheral blood T-lymphocytes. Mutat Res 230: 101–109PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press 2014

Authors and Affiliations

  1. 1.Department of BiosciencesKarolinska InstituteHuddingeSweden

Personalised recommendations