Clinical application of a rapid, functional assay for multidrug resistance based on accumulation of the fluorescent dye, fluo-3

Wall, Dominic; Hu, Xiangdong; Nadalin, Gabriella; Zalcberg, John; Parkin, John; Weyden, Martin Van Der; Marschner, Ian C.

doi:10.1016/s0959-8049(05)80216-3

Cited by 17 publications

(9 citation statements)

References 18 publications

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“…These results are in accordance with studies of other authors who had used either the extrusion of dyes [10][11][12] or monoclonal antibodies as markers of Pgp [3]. Our findings, like these studies, are consistent with the supposition that Pgp can be expressed intrinsically in B-CLL cells.…”

Section: Discussionsupporting

confidence: 94%

“…These results were not seen when we compared this last parameter to anthracycline accumulation. Wall et al [12], in experiments using fluo-3 dye, also found that this test had a significantly greater sensitivity than the anthracycline doxorubicin when this assay was compared to monoclonal antibody labeling.…”

Section: Discussionmentioning

confidence: 96%

“…In B-CLL, some studies [10][11][12], using various methods of detection, demonstrated different levels of Pgp or mdr-1 expression. The correlation between these markers and clinical data is not clear.…”

Section: Introductionmentioning

confidence: 99%

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Comparison between Anthracyclines and Rhodamine-123 Accumulation in Chronic Lymphoid Leukemia: Effect of Cyclosporin A and Verapamil

Maia

Vasconcelos²,

Harab³

et al. 1998

Tumor Biol

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Multidrug resistance in leukemic cells is associated with decreased drug accumulation. A resistant cell line and cells from 11 patients with chronic lymphoid leukemia B were used for the evaluation of intracellular accumulation of daunorubicin (DNR), idarubicin (IDA), epirubicin (EPI) and rhodamine-123 (Rh-123). Cyclosporin A (CSA) and verapamil were used to test their modulatory effects on anthracyclines and the fluorescent dye. In leukemic samples there was a tendency for a lower accumulation index in samples tested with Rh-123 as compared to anthracyclines. IDA was a poorer substrate to P-glycoprotein (Pgp) than two of its analogues, e.g. DNR and EPI. A good correlation (80%) was found between Rh-123 accumulation and Pgp expression by phosphatase-anti-alkaline phosphatase. A strict correlation (100%) was found between modulation by CSA of Rh-123 accumulation and immunoreactivity to Pgp. Two discordant results were seen suggesting that other mechanisms of resistance could be present. The Rh-123 accumulation test seems to give a better indication than anthracyclines, however, it is not selective and may allow the detection of other drug-transport pumps.

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Section: Discussionsupporting

confidence: 94%

Section: Discussionmentioning

confidence: 96%

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Comparison between Anthracyclines and Rhodamine-123 Accumulation in Chronic Lymphoid Leukemia: Effect of Cyclosporin A and Verapamil

Maia

Vasconcelos²,

Harab³

et al. 1998

Tumor Biol

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“…A variety of studies have confirmed that malignant lymphocytes from patients with CLL commonly express both Pgp and mrp and are functionally multidrug resistant (Wall et al, 1993). Pgp has been detected by both immunoassay and functional assays and is accompanied by an increase in mdr1 mRNA.…”

Section: Discussionmentioning

confidence: 98%

“…In contrast, CLL cells reproducibly display the multidrug resistance phenotype. They frequently express Pgp at diagnosis (Herweijer et al, 1990;Cumber et al, 1990;Shustik et al, 1991;Michieli et al, 1991) and display functional drug efflux in vitro (Wall et al, 1993). In addition, based upon in-vitro drug sensitivity testing, CLL cells are drug resistant to anthracyclines Perri et al, 1990;Sparrow et al, 1993).…”

mentioning

confidence: 99%

Expression of mdr1 and mrp in the normal B‐cell homologue of B‐cell chronic lymphocytic leukaemia

et al. 1997

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Summary. B-cell chronic lymphocytic leukaemia (CLL) cells commonly express the multidrug resistance phenotype. The aim of this study was to establish whether the normal homologue in B-cell ontogeny of B-CLL also expressed the multidrug resistance (mdr) phenotype. Human tonsillar lymphocytes were sorted to yield two B-cell subsets based on the expression of CD19, CD5 and CD10. The normal homologue was represented by a population of B cells that was CD19 positive, CD10 negative and weakly expressed CD5. Based upon functional analysis and the detection of mdr1 mRNA by semi-quantitative PCR, these cells expressed the mdr phenotype. In contrast, functional multidrug resistance could not be demonstrated in CD19-positive CD10-positive cells with strong expression of CD5, nor could mdr1 mRNA be found in these cells. MRP was variably expressed in both B-cell subsets with no discernable differences in the pattern of expression. We conclude that normal B cells with a phenotype resembling that of B-CLL cells express the multidrug resistance phenotype.

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

Frey

Yue²,

Haugland³

1995

Cytometry

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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...

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Clinical application of a rapid, functional assay for multidrug resistance based on accumulation of the fluorescent dye, fluo-3

Cited by 17 publications

References 18 publications

Comparison between Anthracyclines and Rhodamine-123 Accumulation in Chronic Lymphoid Leukemia: Effect of Cyclosporin A and Verapamil

Comparison between Anthracyclines and Rhodamine-123 Accumulation in Chronic Lymphoid Leukemia: Effect of Cyclosporin A and Verapamil

Expression of mdr1 and mrp in the normal B‐cell homologue of B‐cell chronic lymphocytic leukaemia

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

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