Reference method for detection of Pgp mediated multidrug resistance in human hematological malignancies: A method validated by the laboratories of the French Drug Resistance Network

Huet, Sylvie; Marie, Jean‐Pierre; Gualde, N.; Robert, Jacques

doi:10.1002/(sici)1097-0320(19981215)34:6<248::aid-cyto2>3.0.co;2-x

Cited by 48 publications

(43 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In this paper, we present data that further characterize the transport of several rhodamine analogues. We took advantage of the intrinsic fluorescence of rhodamines and performed a flow-cytometric analysis of dye accumulation in the wild-type drug sensitive K562 that do not express P-gp and its MDR subline which display high level of MDR (the resistance factor for daunorubicin was equal to 20 [31]). The measurements were made in real time using intact cells.…”

Section: Discussionmentioning

confidence: 99%

New insights into the P‐glycoprotein‐mediated effluxes of rhodamines

Loetchutinat

Saengkhae

Marbeuf-Gueye

et al. 2003

European Journal of Biochemistry

View full text Add to dashboard Cite

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma drug transporter P-glycoprotein (P-gp). This protein is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic peptides. Despite the advances of recent years, we still have an unclear view of the molecular mechanism by which P-gp transports such a wide diversity of compounds across the membrane. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. The aim of the present study was to further characterize the transport of several rhodamine analogues, either positively charged or zwitterionic. We took advantage of the intrinsic fluorescence of rhodamines and performed a flow-cytometric analysis of dye accumulation in the wild-type drug sensitive K562 that do not express P-gp and its MDR subline that display high levels of MDR. The measurements were made in real time using intact cells. The kinetic parameter, k a ¼ V M /k m , which is a measure of the efficiency of the P-gp-mediated efflux of a substrate was similar for almost all the rhodamine analogues tested. In addition these values were compared with those determined previously for the P-gp-mediated efflux of anthracycline. Our conclusion is that the compounds of these two classes of molecules, anthracyclines and rhodamines, are substrates of P-gp and that their pumping rates at limiting low substrate concentration are similar. The findings presented here are the first to show quantitative information about the kinetic parameters for P-gp-mediated efflux of rhodamine analogues in intact cells.Keywords: P-glycoprotein; multidrug resistance; membrane transport; rhodamine; efflux. Since 1940, a broad variety of antibiotics active against many infectious organisms were discovered or developed. The widespread, and sometimes uncontrolled, use of these drugs has led to the emergence of defence mechanisms that, at present, are the major drawback of the drug-based treatment of infectious diseases and cancers. Such resistance is not restricted to the drugs (or analogues) used in the treatment but also involves several structurally and functionally unrelated compounds. This phenomenon, which has been termed multidrug resistance (MDR), can be caused by various mechanisms. However, over expression of the P-glycoprotein multidrug transporter (P-gp) in the plasma membrane is believed to be a major cause of resistance to multiple chemotherapeutic drugs [1][2][3][4]. P-gp is an unusual ABC protein in that it appears to be highly promiscuous: hundreds of compounds have been identified as substrates. The spectrum of MDR compounds includes a large number of anticancer drugs (e.g. anthracyclines, vinca alkaloids, taxanes) as well as steroids, fluorescent dyes, rhodamines, and the c-emitting radio pharmaceutical 99m Tc-MIBI. P-gp can transport neutral and positively charged molecules but not negatively charged ones. Despite the advances of recent years, we still h...

show abstract

Section: Discussionmentioning

confidence: 99%

New insights into the P‐glycoprotein‐mediated effluxes of rhodamines

Loetchutinat

Saengkhae

Marbeuf-Gueye

et al. 2003

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…Fresh viable cells (5 Â 10 5 ) (cell lines and patient cells) were incubated with the monoclonal antibody UIC2 (1 mg/ml; Immunotech, France), and then labeled with a secondary antibody IgG2a conjugated with phycoerythrin, as previously described (14). Fluorescence was analyzed on a flow cytometer [at the beginning of the study on a FACSort flow cytometer (Becton Dickinson, France) and more recently with EPICS Altra flow cytometer (Beckman Coulter, France)].…”

Section: P-gp Expressionmentioning

confidence: 99%

“…The cells were washed twice in PBS at 48C and resuspended in Rh 123-free medium for 1 h at 378C with and without the P-gp inhibitor (Cyclosporine A (CsA), 2 mM) (14). Fluorescence was analyzed on a flow cytometer.…”

Section: P-gp Acitivity Detection With Rh 123mentioning

confidence: 99%

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

Chaoui

Faussat

Majdak

et al. 2006

Cytometry Part B Clinical

Self Cite

View full text Add to dashboard Cite

Background: JC-1 probe has been successfully used for the analysis of either apoptosis or P-glycoprotein (P-gp) activity. Therefore, we wanted to see if JC-1 could also simultaneously assess both, P-gp activity and apoptosis, in acute myeloid leukemia (AML) cells.Methods Conclusions: JC-1 can simultaneously evaluate two important parameters involved in drug resistance in AML cells, P-gp activity and apoptosis. q

show abstract

“…10,11 Cell surface marker expression was evaluated by using a panel of murine monoclonal antibodies including CD11b, CD33, CD34, CD13, CD14 and CD15 (Becton Dickinson, Mountain View, CA, USA). Analysis was performed using a Coulter XL Flow Cytometer (Coulter Corporation, Miami, FL, USA).…”

Section: Flow Cytometrymentioning

confidence: 99%

Further elucidation of mechanism of resistance to vincristine in myeloid cells: role of hypochlorous acid in degradation of vincristine by myeloperoxidase

et al. 2000

View full text Add to dashboard Cite

Inherent resistance of myeloblasts to vincristine (VCR) has been related to the activity of myeloperoxidase (MPO) which can degrade VCR in the presence of hydrogen peroxide (H 2 O 2 ). We investigated the relationship between VCR degradation and hypochlorous acid (HOCl) generation from the reaction of H 2 O 2 with chlorine (Cl) as catalyzed by MPO. A cell-free system, three human leukemia cell lines (CEM/CCRF, HL-60, U937) and 15 bone marrow samples from children with acute myeloid leukemia (AML) were studied. VCR cytotoxicity was evaluated by MTT assay and by quantitative measurement of apoptosis. In vitro levels of VCR in cell-free systems were measured by high performance liquid chromatography (HPLC), and intracellular HOCl levels by oxidation of 5-thio-2-nitrobenzoic acid with the accompanying decrease in the absorbency at 412 nm. VCR was degraded by increasing concentrations of HOCl in cell-free systems and this activity was inhibited by taurine, which is known to block HOCl activity. This finding was confirmed by the VCR cytotoxicity studies on cell lines. The HOCl-producing myeloblasts from patients were resistant to VCR. In five samples out of eight HOCl was also detected extracellularly. These results suggest that oxidation by HOCl may be the final step in VCR degradation catalyzed by MPO through its action on intracellular H 2 O 2 and Cl. Leukemia (2000) 14, 47-51.

show abstract

Reference method for detection of Pgp mediated multidrug resistance in human hematological malignancies: A method validated by the laboratories of the French Drug Resistance Network

Cited by 48 publications

References 11 publications

New insights into the P‐glycoprotein‐mediated effluxes of rhodamines

New insights into the P‐glycoprotein‐mediated effluxes of rhodamines

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

Further elucidation of mechanism of resistance to vincristine in myeloid cells: role of hypochlorous acid in degradation of vincristine by myeloperoxidase

Contact Info

Product

Resources

About