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Both Pgp and MRP1 activities using calcein-AM contribute to drug resistance in AML
O Legrand1, G Simonin, J Y Perrot
1EA1529, Université Paris VI, Formation de Recherche Claude Bernard, France.
Abstract:
Thirteen cell lines with different levels of Pgp and MRP1 expression were used to assess the ability of calcein-AM uptake and calcein efflux to measure Pgp and MRP1 functions, respectively. There was a good correlation between MRP1 expression and the modulatory effect of probenecid (a specific modulator of MRP1) on the calcein efflux (r = 0.91, p = 0.0003) and between Pgp expression and the modulatory effect of CsA on calcein-AM uptake (r = 0.96, p < 0.0001). On light of the high correlations for both proteins, we tested calcein-AM uptake and efflux in fresh myeloid leukemic cells. In 53 AML patients, there was also a good correlation between MRP1 expression (measured by RT/PCR and by MRPm6 expression by flow cytometry) and the modulatory effect of probenecid on the calcein fluorescence (r = 0.92, p < 0.0001) and between Pgp expression as measured by UIC2 antibody binding on flow cytometry and the modulatory effect of CsA on calcein-AM uptake (r = 0.83, p < 0.0001). Pgp activity was higher in CD34+ leukemia than in CD34- leukemia (2.26 +/- 1.50 vs 1.46 +/- 1.21 respectively, p = 0.003) and MRP1 activity was higher in CD34- leukemia than in CD34+ leukemia (1.77 +/- 0.40 vs 1.4 +/- 0.29 respectively, p = 0.004). Pgp expression and activity (p = 0.004 and p = 0.01, respectively), MRP1 activity (p = 0.03) but not MRP1 expression were prognostic factors for achievement of CR. The effect of probenecid and CsA together were higher than the effect of either probenecid or CsA alone on calcein-AM uptake. These results suggest that functional testing (with calcein-AM +/- modulators) for the presence of both MRP1 and Pgp activities is of prognostic value and that MRP1 contributes to drug resistance in AML.
Insights
Functional testing using calcein-AM uptake and efflux accurately measures P-glycoprotein (Pgp) and multidrug resistance-associated protein 1 (MRP1) functions in acute myeloid leukemia (AML). These functional assays, along with modulators like probenecid and CsA, show prognostic value for predicting complete remission (CR) in AML patients.
Area of Science:
- Pharmacology and Toxicology
- Cancer Biology
- Hematology
Background:
- P-glycoprotein (Pgp) and multidrug resistance-associated protein 1 (MRP1) are key efflux transporters implicated in multidrug resistance (MDR) in cancer.
- Accurate functional assessment of Pgp and MRP1 is crucial for understanding drug resistance mechanisms and developing effective therapeutic strategies in acute myeloid leukemia (AML).
- Existing methods for assessing transporter function may not always correlate directly with their clinical impact.
Purpose of the Study:
- To evaluate the utility of calcein-AM uptake and calcein efflux assays for measuring Pgp and MRP1 functions, respectively.
- To correlate these functional assessments with Pgp and MRP1 expression levels in cell lines and primary AML samples.
- To determine the prognostic value of Pgp and MRP1 activity in predicting treatment outcomes, specifically complete remission (CR), in AML patients.
Main Methods:
- Utilized thirteen cell lines with varying Pgp and MRP1 expression levels to validate calcein-AM uptake (for Pgp) and calcein efflux (for MRP1) assays.
- Assessed the modulatory effects of cyclosporine A (CsA) on calcein-AM uptake and probenecid on calcein efflux to confirm transporter function.
- Analyzed fresh leukemia cells from 53 AML patients, measuring Pgp and MRP1 expression (RT/PCR, flow cytometry) and function (calcein assays with modulators).
Main Results:
- High correlations were observed between Pgp expression and CsA-modulated calcein-AM uptake (r = 0.96, p < 0.0001) and between MRP1 expression and probenecid-modulated calcein efflux (r = 0.91, p = 0.0003) in cell lines.
- Similar strong correlations were found in AML patients: Pgp expression correlated with CsA effect on calcein-AM uptake (r = 0.83, p < 0.0001), and MRP1 expression correlated with probenecid effect on calcein fluorescence (r = 0.92, p < 0.0001).
- Pgp activity was significantly higher in CD34+ AML cells, while MRP1 activity was higher in CD34- AML cells. Pgp expression/activity and MRP1 activity were identified as significant prognostic factors for achieving CR.
Conclusions:
- Calcein-AM uptake and efflux assays, in conjunction with modulators like CsA and probenecid, provide reliable functional measurements of Pgp and MRP1 activity.
- Functional assessment of Pgp and MRP1 exhibits significant prognostic value for predicting complete remission in acute myeloid leukemia patients.
- MRP1 plays a contributing role in mediating drug resistance within the AML context, highlighting its importance as a therapeutic target.