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

Br J Cancer. 1997;76(8):1029-34. doi: 10.1038/bjc.1997.503.

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 microM 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / analysis*
  • Acute Disease
  • Antigens, CD*
  • DNA, Neoplasm / metabolism
  • Flow Cytometry
  • Fluorescent Dyes*
  • Humans
  • KB Cells
  • Leukemia, Myeloid / metabolism*
  • Leukosialin
  • Microscopy, Confocal
  • RNA, Neoplasm / metabolism
  • Sarcoma / chemistry*
  • Sensitivity and Specificity
  • Sialoglycoproteins / analysis*
  • Staining and Labeling / methods

Substances

  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Antigens, CD
  • DNA, Neoplasm
  • Fluorescent Dyes
  • Leukosialin
  • RNA, Neoplasm
  • Sialoglycoproteins
  • UN1 sialoglycoprotein, human