Theoretical and practical considerations for the measurement of P-glycoprotein function in acute myeloid leukemia

Abstract

This paper summarizes experimental data and theoretical conare the level of sensitivity that is deemed relevant for the study The following discussion of detection methods will be in the Materials and methods cells.Keywords: P-glycoprotein; multidrug resistance; leukemia; PSC833; rhodamine 123All cell lines and treatment of AML samples and other methods have been extensively described in our earlier papers. 1-10 Additional specific details are given when considered Introduction useful, in the Results section.It is the purpose of this paper to describe our experience with assays for the P-glycoprotein (Pgp)-mediated multidrug resistFluorescence laser scan microscopy ance (MDR) phenotype in adult acute myeloid leukemia (AML). The emphasis is on the measurement of Pgp function, Cells were incubated with daunorubicin (concentration range in comparison with measurements of Pgp protein expression.0.05-4 M) for 75 min at 37°C in medium with 10% fetal Measurement of 'Pgp function' or 'activity' implies the assesscalf serum (FCS) and 20 mM Hepes (pH = 7.4) as described ment of a drug (or dye) transport-related parameter, such as previously. 1-3 About 0.3 × 10 6 KB cells or 0.6 × 10 6 leukemic uptake, efflux, steady-state accumulation or intracellular discells per ml were incubated with regular shaking. After incutribution. Our experience is built upon extensive comparison bation the cells were washed twice with ice-cold phosphateof methods to determine Pgp MDR in tumor cell lines, also buffered saline (PBS) and cytocentrifuged for 5 min at 350 with low levels of Pgp/MDR1 expression, 1-6 experiments and r.p.m. on slides precoated with 0.1% bovine serum albumin model calculations intended to understand the kinetics of Pgpsolution. Per cytospin 100 l containing 35 000 KB cells or mediated daunorubicin transport 7,8 and the application of this 80 000 leukemic cells were used. After spinning, the cells knowledge to measure Pgp in human AML. 9-11 We emphasize were immediately fixed for 5 min with 3.6% formaldehyde the reasons for choosing certain methods above others and solution at room temperature and washed with water. Excess when appropriate refer to related methods used by other water was removed with tissue and the spins were dried horilaboratories.zontally in the dark. Criteria that need to be determined before starting a study Recording of the fluorescence was done initially on a Zeiss into the clinical relevance of Pgp for chemotherapy resistance laser scanning microscope 41 (Carl-Zeiss, Oberkochen, Germany) using the Argon laser providing a 488 nm beam. Emission was measured at wavelengths longer than 515 nm. 18 or the use of a biotin-streptavidin step 17 in order the possibility of determining heterogeneity in expression. We to be able to measure Pgp in blood cells. 18 mainly used the monoclonal antibodies JSB-1, C219, MRK16(5) The application of neuraminidase to unmask MRK16 epiand occasionally 4E3 or C494 on acetone-fixed cytospins with topes in AML has not been useful in our hands. 9,10 peroxidase or alkaline...

True

“…14,16 This was confirmed in the present group of untreated AMLs (RHOratio vs DNRratio (n = 140): r s = 0.51, (P Ͻ 0.0001); RHOratio vs PGPMRK16 (n = 134): r s = 0.50, (P Ͻ 0.0001)). For this reason and the reasons discussed extensively before, 14,16 we regard the RHOratio parameter as the 'gold standard' for Pgp activity to which other parameters are compared. Eighty-seven percent of the RHOratio values were below 2.0 and 97% below 3.0.…”

“…14,16 SW-1573 and SW-1573/2R120, GLC 4 and GLC 4 /ADR lung carcinoma cell lines are used as controls for the detection of LRP and MRP1.…”

“…[11][12][13] Still the question whether the Pgp-mediated efflux of DNR or other drugs out of leukemic cells is a major contributor to the in vivo antitumor effect of combination chemotherapy or even to the in vitro cytotoxicity of the drug has not been answered. 14 The answer to these questions might be important to understand the relative contribution of Pgp to clinical treatment failure and for the proper planning of clinical trials with Pgp inhibitors or alternative therapies.…”

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

“…Therefore, we compared a CLSM method for the quantification of nuclear-fixed SYTO16 with flow cytometric data. We have previously shown that the low sensitivity of daunorubicin as a P-gp probe precluded the accurate measurement of the PSC 833 effect in KB8 cells, as well as in most AML samples, by CLSM analysis of about 200 cells (Broxterman et al, 1997). Using SYTO16 in KB8 cells, the development of fluorescence in individual cells was first followed qualitatively in real time before and after the addition of PSC 833 and after fixation.…”

“…It is important to realize that, for any new combination of probe and modulator, one should be aware of putative interactions with the fluorescence of the probe not related to pump activity and appropriate controls have to be performed. We have discussed the theoretical and practical considerations of the different probes in a more comprehensive way in Broxterman et al (1997).…”