Highly sensitive and specific detection of P-glycoprotein function for haematological and solid tumour cells using a novel nucleic acid stain

H. J. Broxterman, G. J. Schuurhuis, J. Lankelma, J. W. Oberink, C. A. Eekman, Ame Claessen, K. Hoekman, M. Poot, H. M. Pinedo

Research output: Contribution to journalArticle

Abstract

Progress in our understanding of the contribution of P-glycoprotein (P-gp)-mediated resistance to chemotherapy failure in haematological as well as solid tumours has been hampered by the lack of highly sensitive, reliable methods for the detection of P-gp function in fresh human tumour cells. The present study identifies the novel nucleic acid stain SYTO16 as a highly sensitive and specific dye to assess P-gp function. The effect of P-gp is expressed here as the ratio of dye fluorescence (RF) from cells incubated with dye with or without 2 μM of the P-gp inhibitor PSC 833. Using flow cytometric analysis, an RF of 0.9 was found for SYTO16 in the KB3-1 (P-gp-) and 1.6 in KB8 (P-gp+) cells. Three types of patients' cells were studied: (1) in haematopoietic CD34+ cells, which are known to express P-gp, the RF was 6.0 for SYTO16 compared with 2.5 for rhodamine 123 and 1.3 for daunorubicin (mean of five individuals); (2) in acute myeloid leukaemia cells, the RF for SYTO16 was 1.0 in P-gp- and 4.5 in P-gp+ samples; (3) for the first time, we have quantitated P-gp function in fresh human solid tumour (sarcoma) cells. We found, in a P-gp+ leiomyosarcoma, an RF of 16 for SYTO16 and 2.7 for daunorubicin. This means that complete inhibition of P-gp function in these sarcoma cells would lead to an increase of daunorubicin accumulation with 170% compared with 30% in the CD34+ cells. Next, we showed that SYTO16 could be fixed in nuclei by 3.6% formaldehyde treatment, allowing quantification of the nuclear fluorescence on cytospins by laser scanning microscopy. In conclusion, SYTO16 proved to have a combination of favourable properties: it can be excited at 488 nm and has large fluorescence enhancement upon binding to nucleic acids, allowing the use of low, nontoxic (<10 nM) concentrations. Because the RF for SYTO16 is much higher than for daunorubicin, it can be applied for the determination of P-gp function in relatively small numbers of low-P-gp-expressing tumour cells by laser scanning microscopy. Individual sarcomas were found to have high P-gp function compared with CD34+ cells. This assay may be used to select patients for P-gp modulation protocols.

Original languageEnglish (US)
Pages (from-to)1029-1034
Number of pages6
JournalBritish Journal of Cancer
Volume76
Issue number8
StatePublished - 1997
Externally publishedYes

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Keywords

  • CD34
  • DNA binding
  • Fluorescence detection
  • P-glycoprotein
  • PSC 833
  • Sarcoma
  • SYTO16

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Broxterman, H. J., Schuurhuis, G. J., Lankelma, J., Oberink, J. W., Eekman, C. A., Claessen, A., Hoekman, K., Poot, M., & Pinedo, H. M. (1997). Highly sensitive and specific detection of P-glycoprotein function for haematological and solid tumour cells using a novel nucleic acid stain. British Journal of Cancer, 76(8), 1029-1034.