title: Highly sensitive and specific detection of P-glycoprotein function for haematological and solid tumour cells using a novel nucleic acid stain.
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.
authors.affiliation: Department of Medical Oncology, Academisch Ziekenhuis Vrije Universiteit, Amsterdam, The Netherlands.
authors.forename: H J
authors.initials: HJ
authors.lastname: Broxterman
authors.forename: G J
authors.initials: GJ
authors.lastname: Schuurhuis
authors.forename: J
authors.initials: J
authors.lastname: Lankelma
authors.forename: J W
authors.initials: JW
authors.lastname: Oberink
authors.forename: C A
authors.initials: CA
authors.lastname: Eekman
authors.forename: A M
authors.initials: AM
authors.lastname: Claessen
authors.forename: K
authors.initials: K
authors.lastname: Hoekman
authors.forename: M
authors.initials: M
authors.lastname: Poot
authors.forename: H M
authors.initials: HM
authors.lastname: Pinedo
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis*; D000208:Acute Disease; D015703:Antigens, CD; D004273:DNA, Neoplasm / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D007624:KB Cells; D007951:Leukemia, Myeloid / Q000378:metabolism*; D051917:Leukosialin; D018613:Microscopy, Confocal; D012334:RNA, Neoplasm / Q000378:metabolism; D012509:Sarcoma / Q000737:chemistry*; D012680:Sensitivity and Specificity; D012795:Sialoglycoproteins / Q000032:analysis*; D013194:Staining and Labeling / Q000379:methods
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D015703:Antigens, CD; D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D051917:Leukosialin; D012334:RNA, Neoplasm; C465014:SPN protein, human; D012795:Sialoglycoproteins