Dyes providing increased sensitivity in flow‐cytometric dye‐efflux assays for multidrug resistance

Abstract

In an effort to improve detection of P-glycoprotein-mediated multidrug resistance (mdr), several dyes were compared to rhodamine 123 (R123) in efflux assays. Two dyes (Sy-38 and SY-3150) were found that provided better sensitivity. These dyes were effluxed by a cell line known to be mdr-positive (P388ht84) but not by an mdr-negative cell line (P388). Efflux was blocked by both verapamil and cyclosporine A. Efflux from KGla cells was less than from P388ht84, just as has been seen with R123 and daunomycin. In further experiments, a model sysMany tumors do not respond to a wide variety of chemotherapeutic agents, a phenomenon known as multidrug resistance (mdr). One cause of mdr is the activity of a pump that removes chemotherapeutic drugs from the cell. This pump is a protein known as P-glycoprotein, Pgp, or gp170 (7). Fluorescent drugs (and dyes) fail to accumulate in cells with mdr activity, because they are pumped out (effluxed) nearly as rapidly as they enter.There is currently considerable interest in detecting mdr in tumor samples. Information about mdr levels might be used to identify tumors with high mdr activity at presentation. It might also be used to monitor the course of chemotherapy in order to detect mdr activity arising during treatment ( 1,5,15,19,20). Flow cytometry is particularly useful in obtaining this information, because it measures the property of interest on single cells. Performing cell-by-cell measurement allows the detection of a small population of abnormal cells that does not create large changes in the bulk sample average. Flow cytometry also allows surface immunophenotype to be correlated with mdr activity (2,4).Several different classes of fluorescent substrates have been used to measure the efflux activity of P-glycoprotein. One class of useful substrates is the naturally fluorescent chemotherapeutic drugs, including daunomycin and doxorubicin (12). A second class of substrates includes dyes such as rhodamine 6G, rhodamine 123, and other assorted dyes (18). The acetoxymethyl esters of various indicator dyes have also been shown to be useful in measuring rndr efflux activity (6,21).tem was used to demonstrate two-color immunofluorescence plus efflux measurements. This was done using a sequential staining method. A procedure was devised that, at least for this model system, allowed single-step staining with both dye and antibody. The sensitivity of the efflux measurement was slightly compromised by using this one-step staining method. o 1995 Wiley-Liss, Inc. Key terms: Drug resistance, immunofluorescence, dye efflux, nucleic acid dyesAlthough Hoechst 33342 has been clearly shown to be a substrate for mdr-mediated efflux ( l l ) , nucleic acidbinding dyes have not been extensively studied for their suitability for dye efflux measurement. Nucleic acidbinding dyes might be expected to be useful, because they have many binding sites per cell, reasonable quantum yields, and very large changes in fluorescence between bound and free dye. Hoechst 33342 has not been widely used for mdr meas...

Stemness and chemoresistance are imparted to the OC cells through TGFβ1 driven EMT

“…Rh123 is a known substrate of the ABC transporter family, especially ABCG2, which has an efflux property in breast, and OC tissues. 33,39,40 Verapamil, an inhibitor of these transport pumps, binds to them and prevents their activity, 41 thus helping in drug retention in these chemoresistant cells and sensitizing them towards cytotoxicity in response to platinum drugs like cisplatin (Figure 5). Among the different EMT promoting transcription factors, we found ZEB1 to be of prime importance in the gain of stemness in the OC cells.…”

DRAQ5 Labeling of Nuclear DNA in Live and Fixed Cells

“…The introduction of the cell-permeant cyanine SYTO nucleic-acid stains (e.g., SYTO 17; Frey, 1995;Frey et al, 1995) has provided reagents with convenient red-shifted spectral characteristics. These agents passively diffuse through the membrane of most cells and can be excited by UV or visible light, but stain RNA and DNA in both live and dead eukaryotic cells.…”

1H and 13C NMR Assignments for the Cyanine Dyes SYBR Safe and Thiazole Orange

“…A compound with structure 1 (CAS 167504-51-0) has been referenced twice in Chemical Abstracts. The first is a 1995 article from a Becton Dickinson/Molecular Probes group that examined its use in flow cytometric studies of multi-drug resistance in various cell lines . The other is the 2005 Molecular Probes patent describing a number of tests indicative of the safety of 1 as well as it applicability in nucleic acid detection .…”