Flow cytometric functional analysis of multidrug resistance by Fluo-3: a comparison with rhodamine-123

Koizumi, Satoshi; Konishi, Masayoshi; Ichihara, Tomofumi; Wada, Hideo; Matsukawa, Harumi; Goi, Kumiko; Mizutani, Shigehiko

doi:10.1016/0959-8049(95)00288-t

Cited by 23 publications

(14 citation statements)

References 16 publications

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“…Previously, fluorescent indicator such as fura-2 and fluo-3 have been used as model anions for identifying and studying the mechanisms of organic anion transport activity in different cell types [3,10,17,21,38]. However, the use of fluo-3 alone poses problems, since fluorescence intensity increases more than 40-fold upon calcium binding (K D =0.4 µM) [42].…”

Section: Discussionmentioning

confidence: 99%

Identification of an organic anion transport system in the human colon carcinoma cell line HT29 clone 19A

Abrahamse

Rechkemmer²

2001

Pfl�gers Archiv European Journal of Physiology

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The human colon carcinoma cell line HT29 c1.19A was studied for organic anion transporter activity by determining intracellular fluo-3 and fura-red accumulation and by measuring fluo-3 efflux. Modulators of organic anion transport systems were used to identify the transporters that are involved in dye extrusion. Addition of probenecid to the dye-loading medium, containing 10 microM fluo-3/AM and fura-red/AM, resulted in a dose-dependent increase in fluo-3 and fura-red accumulation in the cells. The increase in fluo-3 accumulation in the cells in the presence of probenecid was explained by the inhibitory effect of this compound on fluo-3 efflux. Fluo-3 efflux from the cells was also inhibited by sulfinpyrazone, another inhibitor of organic anion transport. Substrates of renal probenecid-sensitive organic anion exchange mechanisms as well as modulators of multidrug resistance associated protein (MRP) activity did not influence fluo-3 extrusion rates. However, reducing intracellular ATP contents completely blocked fluo-3 extrusion. Moreover, MK571, an inhibitor of MRP, significantly stimulated dye accumulation, whereas inhibitors of the multidrug resistance gene (MDR1) product Pglycoprotein, cyclosporin A and verapamil, did not. As probenecid inhibits fluo-3 efflux across the apical membrane of cells grown on permeable supports, we conclude that a probenecid-sensitive organic anion transporter is present in the apical membrane of HT29 c1.19A cells. This organic anion transport system differs from MDRI and MRP2.

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Section: Discussionmentioning

confidence: 99%

Identification of an organic anion transport system in the human colon carcinoma cell line HT29 clone 19A

Abrahamse

Rechkemmer²

2001

Pfl�gers Archiv European Journal of Physiology

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“…Although they share numerous common substrates (22), MRP1 and MDR1 can be distinguished by either specific substrates or specific inhibitors. MDR1, unlike MRP1, can mediate the transport of rhodamine 123 (23,24), a dye widely used to detect MDR1 expression (25)(26)(27) even if expressed at a low level. GF120918, a potent inhibitor of MDR1, was selected by our laboratory from a chemical optimization program of acridone derivatives (28).…”

mentioning

confidence: 99%

Multidrug Resistance P-Glycoprotein Is Not Involved in Cholesterol Esterification

Issandou¹,

Grand-Perret²

2000

Biochemical and Biophysical Research Communications

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“…P‐glycoprotein is located at the plasma membrane, and is expressed in almost every cell type, including human kidney and neuroblastoma cells [28–30]. This pump is able to excrete many different fluorescent probes [22,31]. Our data imply that the fluorescent probe JC‐1 accumulates in cells as a consequence of P‐glycoprotein inhibition, resulting in an apparent increase in Δ Ψ m .…”

Section: Discussionmentioning

confidence: 82%

Cyclosporin A‐induced oxidative stress is not the consequence of an increase in mitochondrial membrane potential

et al. 2007

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Cyclosporin A induces closure of the mitochondrial permeability transition pore. We aimed to investigate whether this closure results in concomitant increases in mitochondrial membrane potential (DW m ) and the production of reactive oxygen species. Fluorescent probes were used to assess DW m (JC-1, 5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethyl-benzimidazolyl-carbocyanine iodide), reactive oxygen species [DCF,[5][6]in human kidney cells (HK-2 cells) and in a line of human small cell carcinoma cells (GLC4 cells), because these do not express cyclosporin A-sensitive P-glycoprotein. We used transfected GLC4 cells expressing P-glycoprotein as control for GLC4 cells. NIM811 (N-methyl-4-isoleucinecyclosporin) and PSC833 (SDZ-PSC833) were applied as selective mitochondrial permeability transition pore and P-glycoprotein blockers, respectively. To study the effect of cyclosporin A on mitochondrial function, we isolated mitochondria from fresh pig livers. Cyclosporin A and PSC833 induced a more than two-fold increase in JC-1 fluorescence in HK-2 cells, whereas NIM811 had no effect. None of the three substances induced a significant increase in JC-1 fluorescence in GLC4 cells. Despite this, cyclosporin A, NIM811 and PSC833 induced a 1.5-fold increase in DCF fluorescence (P < 0.05) and a two-fold increase in Fluo-3 fluorescence (P < 0.05). Studies in isolated mitochondria showed that blockage of mitochondrial permeability transition pores by cyclosporin A affected neither DW m , ATP synthesis, nor respiration rate. The mitochondrial permeability transition pore blockers cyclosporin A and NIM811, but also the non-mitochondrial permeability transition pore blocker PSC833, induced comparable degrees of reactive oxygen species production and cytosolic [Ca 2+ ]. Neither mitochondria, effects on P-glycoprotein nor inhibition of

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Flow cytometric functional analysis of multidrug resistance by Fluo-3: a comparison with rhodamine-123

Cited by 23 publications

References 16 publications

Identification of an organic anion transport system in the human colon carcinoma cell line HT29 clone 19A

Identification of an organic anion transport system in the human colon carcinoma cell line HT29 clone 19A

Multidrug Resistance P-Glycoprotein Is Not Involved in Cholesterol Esterification

Cyclosporin A‐induced oxidative stress is not the consequence of an increase in mitochondrial membrane potential

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