Cancer Chemotherapy and Pharmacology

, Volume 27, Issue 6, pp 445–450

Detection of multidrug resistance and quantification of responses of human tumour cells to cytotoxic agents using flow cytometric spectral shift analysis of Hoechst 33342-DNA fluorescence

  • Paul J. Smith
  • Sally A. Morgan
  • James V. Watson
Original Articles Multidrug Resistance, Human Tumour Cells, Hoechst 33342

Summary

We describe the application of a flow cytometric technique for assessing the radiation or drug sensitivity characteristics of human tumour cells. The technique makes use of the phenomenon that a red shift occurs in the fluorescence emission spectrum of a DNA-specific dye (Hoechst 33342) as an increasing number of dye molecules bind to nuclear DNA. Intact, viable cells undergo a time-dependent spectral shift that can be distinguished from the rapid shift observed in cells with damaged membranes by the use of multiparametric flow cytometry. The responses of various human cell lines were compared, namely, those of normal and ataxia-telangiectasia (A-T) lymphoblastoid lines, a small-cell lung carcinoma line and its (in vitro) derived multidrug-resistant variants. A close correlation was found between dye toxicity and the degree of DNA binding of Hoechst 33342 independent of cellular DNA content, with lymphoblastoid and multidrug-resistant small-cell lung cancer cells showing enhanced and restricted dye-binding rates, respectively. VP16-and radiation-induced cell kill was found to result in a quantifiable increase in the fraction of cells undergoing a rapid spectral shift and was capable of detecting the increased radiation sensitivity of A-T-derived cells. Spectral shift analysis provides a rapid method for assessing the responses of tumour cells to cytotoxic agents and for determining the general ability of cells to protect cellular DNA from a model DNA-binding agent (Hoechst 33342) that participates in the multidrug resistance phenotype.

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References

  1. 1.
    Bhuyan BK, Luoghman BE, Fraser TJ, Day KJ (1976) Comparison of different methods of determining cell viability after exposure to cytotoxic compounds. Exp Cell Res 97:275Google Scholar
  2. 2.
    Bontemps J, Houssier C, Fredericq E (1975) Physico-chemical study of the complexes of “33 258 Hoechst” with DNA and nucleohistone. Nucleic Acids Res 2:971Google Scholar
  3. 3.
    Dive C, Watson JV, Workman P (1990) Multiparametric analysis of cell membrane permeability by two colour flow cytometry with complementary fluorescent probes. Cytometry 11:244Google Scholar
  4. 4.
    Durand RE, Olive PL (1982) Cytotoxicity mutagenicity and DNA damage by Hoechst 33 342. J Histochem Cytochem 30:111Google Scholar
  5. 5.
    Ellwart JW, Dormer P (1990) Vitality measurement using spectrum shift in Hoechst 33 342 stained cells. Cytometry 11:239Google Scholar
  6. 6.
    Endicott JA, Ling V (1989) The biochemistry of P-glycoprotein-mediated multidrug resistance. Annu Rev Biochem 58:137Google Scholar
  7. 7.
    Krishan A (1987) Effect of drug efflux blockers on vital staining of cellular DNA with Hoechst 33 342. Cytometry 8:642Google Scholar
  8. 8.
    Lalande ME, Ling V, Miller RG (1981) Hoechst 33 342 dye uptake as a probe of membrane permeability changes in mammalian cells. Proc Natl Acad Sci USA 78:363Google Scholar
  9. 9.
    Latt SA (1979) Fluorescent probes of DNA microstructure and synthesis. In: Melamed MR, Mullaney PF, Mendelsohn ML (eds) Flow cytometry and sorting. Wiley, New York, p 263Google Scholar
  10. 10.
    Latt SA, Stetten G (1976) Spectral studies on 33 258 Hoechst and related bis-benzimidazole dyes useful for fluorescent detection of DNA synthesis. J Histochem Cytochem 24:24Google Scholar
  11. 11.
    Morgan SA, Watson JV, Twentyman PR, Smith PJ (1989) Flow cytometric analysis of Hoechst 33 342 uptake as an indicator of multi-drug resistance in human lung cancer. Br J Cancer 60:282Google Scholar
  12. 12.
    Reeve JG, Rabbitts PH, Twentyman PR (1989) Amplification and expression of mdr 1 gene in a multidrug resistant variant of small cell lung cancer cell line NCI-H69. Br J Cancer 60:339Google Scholar
  13. 13.
    Smith PJ (1984) Relationship between a chromatin anomaly in ataxia-telangiectasia cells and enhanced sensitivity to DNA damage. Carcinogenesis 5:1345Google Scholar
  14. 14.
    Smith PJ, Nakeff A, Watson JV (1985) Flow cytometric detection of changes in the fluorescent emission spectrum of a vital DNA-specific dye in human tumour cells. Exp Cell Res 159:37Google Scholar
  15. 15.
    Smith PJ, Lacy M, Debenham PG, Watson JV (1988) A mammalian cell mutant with enhanced capacity to dissociate a bis-benzimidazole dye-DNA complex. Carcinogenesis 9:485Google Scholar
  16. 16.
    Stokke T, Steen H (1986) Fluorescence spectra of Hoechst 33 258 bound to chromatin. Biochim Biophys Acta 868:17Google Scholar
  17. 17.
    Teng M, Usman N, Frederick CA, Wang AH-J (1988) The molecular structure of the complex of Hoechst 33 258 and the DNA dodecamer d(CGCGAATTCGCG). Nucleic Acids Res 16:2671Google Scholar
  18. 18.
    Twentyman PR, Fox NE, Wright KA, Bleehen NM (1986) Derivation and preliminary characterisation of doxorubicin resistant lines of human lung cancer cells. Br J Cancer 53:529Google Scholar
  19. 19.
    Watson JV, Nakeff A, Chambers SH, Smith PJ (1985) Flow cytometric fluorescence emission spectrum analysis of Hoechst 33 342-stained DNA in chicken thymocytes. Cytometry 6:310Google Scholar
  20. 20.
    Watson JV, Horsnell TS, Smith PJ (1988) Data compression: 8-dimensional flow cytometric data processing with 28K addressable computer memory. J Immunol Methods 113:205Google Scholar
  21. 21.
    Zimmer C, Wahnert U (1986) Nonintercalating DNA-binding ligands: specificity of the interaction and their use as tools in biophysical, biochemical and biological investigations of the genetic material. Prog Biophys Mol Biol 47:31Google Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • Paul J. Smith
    • 1
  • Sally A. Morgan
    • 1
  • James V. Watson
    • 1
  1. 1.Medical Research Council, Clinical Oncology and Radiotherapeutics UnitMRC CentreCambridgeUK

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