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

Abstract

Summary 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 SYT016 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 gM of the P-gp inhibitor PSC 833. Using flow cytometric analysis, an RF of 0.9 was found for SYT016 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 SYT016 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 SYT016 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.

JC‐1, a sensitive probe for a simultaneous detection of P‐glycoprotein activity and apoptosis in leukemic cells

“…Kuhnel et al (8) have also found a higher sensitivity of the red fluorescence of JC-1 for the changes in P-gp activity. Several other P-gp substrates are being used for the evaluation of P-gp activity: Rh 123, Calcein-AM (Ca-AM), Syto 16 (9,12,17). So far none of them has been described as a substrate specific for P-gp.…”

Large populations of non-clonogenic early apoptotic CD34-positive cells are present in frozen-thawed peripheral blood stem cell transplants

“…This results in steady state cellular accumulation. 25 Incubation was carried out in the presence or absence of 1 m PSC833 to check the effect of P-glycoprotein activity on Syto R 16 accumulation. 25 After washing in PBS/BSA the fluorescence was recorded using flow cytometry.…”

“…25 In addition, recently Frey 12 has shown that Syto dyes can be used to detect apoptosis probably by detection of changes in the structure of DNA and/or RNA. Since plasma membrane changes are among the earliest changes in the apoptotic process, in the present study the question addressed was whether Syto R 16 might be able to detect an early phase of apoptosis, characterised among others by P-glycoprotein activity changes.…”

Towards an understanding of apoptosis detection by SYTO dyes

“…They exhibit very low inherent fluorescence, with strong enhancement upon binding to DNA/ RNA. SYTO-stained eukaryotic cells display both nuclear and diffuse cytoplasmic staining pattern (17,18), the latter shown to be abolished after formaldehyde fixation (19). DNA binding properties of SYTO dyes have been exploited to visualize characteristic nuclear morphology during apoptosis or even to assess level of DNA synthesis after exposure to endocrine disrupting chemicals (18,20,21).…”

“…It should be noted, however, that SYTO probes are not exclusive DNA stains, and thus several of them have been successfully applied to visualize translocation of RNA granules in neurons and allow differential discrimination of nuclear/mitochondrial DNA from cytoplasmic RNA using two-photon lifetime imaging (17,22). Importantly recent reports suggest the reliability of some SYTO dyes as effective substrates in quantitative P-glycoprotein (P-gp) function assessment (14,19,23).…”