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Different effects of metabolic inhibitors and cyclosporin A on daunorubicin transport in leukemia cells from patients with AML
Linköping University, Department of Medicine and Care, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
Department of Pathology, Division of Hematology, Karolinska Hospital and Institutet, Stockholm, Sweden.
Departments of Medicine, Division of Hematology, Karolinska Hospital and Institutet, Stockholm, Sweden.
Departments of Medicine, Division of Hematology, Karolinska Hospital and Institutet, Stockholm, Sweden.
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2003 (English)In: Leukemia research: a Forum for Studies on Leukemia and Normal Hemopoiesis, ISSN 0145-2126, E-ISSN 1873-5835, Vol. 27, no 2, 183-191 p.Article in journal (Refereed) Published
Abstract [en]

The objective of this study was to determine the role of transport proteins in daunorubicin (Dnr) accumulation and efflux in leukemia cells from 36 patients with acute myeloid leukemia (AML). Mononuclear cells were isolated and incubated with 1 μM Dnr with/without addition of 3 μM cyclosporin A (CyA) or metabolic inhibitors (MI). Cellular Dnr concentration in leukemia blast cells was measured with flow cytometry. After washing and reincubation of the cells in drug-free medium, Dnr efflux was followed with/without addition of CyA or MI. Levels of mRNA expression for mdr1, multidrug resistance associated protein (mrp) and lung resistance protein (lrp) were determined with reverse transcriptase-polymerase chain reaction (RT-PCR). MI enhanced cellular Dnr accumulation to a higher extent than CyA whereas CyA reduced Dnr efflux more efficiently than MI (P<0.001). There was a significant difference in Dnr accumulation between samples with low and high mdr1 mRNA levels but only in the presence of MI or CyA. Our results imply that other factors than P-glycoprotein (Pgp) are of major importance for in vitro Dnr accumulation in AML blasts and that the role of Pgp as a drug efflux pump is not conclusive.

Place, publisher, year, edition, pages
2003. Vol. 27, no 2, 183-191 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-26979DOI: 10.1016/S0145-2126(02)00093-0Local ID: 11613OAI: oai:DiVA.org:liu-26979DiVA: diva2:247530
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Experimental studies on multidrug resistance in human leukaemia: role of cellular heterogeneity for daunorubicin kinetics
Open this publication in new window or tab >>Experimental studies on multidrug resistance in human leukaemia: role of cellular heterogeneity for daunorubicin kinetics
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cellular resistance to chemotherapy is a major cause of treatment failure in acute myeloid leukaemia (AML) and still the majority of the patients die from their disease. Drug resistance 1s multifactorial, the most studied mechanism being multidrug resistance (MDR), mediated by the P-glycoprotein (Pgp). Pgp is an energy-dependent transport protein, encoded by the mdr1 gene, with the power to extrude the cytotoxic drugs out of the cells; thus causing reduced effect of the drug on the leukaemic cells. MDR is characterised by cross-resistance to a wide range of chemotherapeutics of natural origin. Other transport proteins, involved in drug resistance, are the multidrug resistance associated protein (MRP) and the lung resistance protein (Lrp).

The aims of this thesis were to elucidate transport kinetics of the anthracycline, daunorubicin, (Dnr) and to investigate the effects of reversing agents on heterogeneity of drug accumulation in cells from patients with AML. The ultimate goal is to improve treatment based on each patient's individual resistance patterns.

Density gradient isolated mononuclear cells from patients with AML were incubated with Dnr. Incubated cells were sorted with flow cytometry (FC) on the basis of accumulation levels of the autofluorescent Dnr. Gene expression of the Pgp and the MRP in sorted subpopulations were analysed with polymerase chain reaction (PCR). Apoptosis, expression of p53 and bcl-2 in the sorted subpopulations were determined with monoclonal antibodies and FC. Drug accumulation and efflux, with/without the resistance modifier Cyclosporin A (CyA) and energy-depleting metabolic inhibitors (MJ), were also determined in the leukaemic cell populations with FC.

Gene expressions of mdr1 and mrp1 were shown to be heterogeneous in the leukaemic samples and drug accumulation correlated inversely to the gene expression. Cell populations with the higher drug accumulation entailed more apoptosis. The leukaemic cell lopulation, defined by immunopenotyping, differed in drug accumulation an efflux compared to the total mononuclear cell population that also contains normal lymphocytes and monocytes. In leukaemic samples with two blast cell populations, the more immature blast ceUs accumulated drug to a lesser extent and bad a higher efflux rate than the differentiating blast cells. CyA reduced Dnr efflux more efficiently than MI, but MJ increased drug accumulation in leukaemic cells more than CyA.

In conclusion: analysis of the total mononuclear population does not give an accurate picture of the leukaemic cell population as concerns resistance mechanisms. Heterogeneity in the leukaemic cell population ought to be taken into account since two or more leukaemic cell populations often exist. The most immature blast cell population should be analysed as relapse usually derives from this population. Furthermore the role of Pgp in MDR is not conclusive as results with reversing agents differed from what was expected.

Place, publisher, year, edition, pages
Linköping: Linköping Universitet, 2005. 81 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 901
Keyword
Acute myeloid leukaemia, heterogeneity, multidrug resistance. transport kinetics flow cytometry, gene expression, mdr1, mrp, lrp, apoptosis. bcl-2, p53
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-31913 (URN)17746 (Local ID)91-85299-11-1 (ISBN)17746 (Archive number)17746 (OAI)
Public defence
2005-06-03, Föreläsningssal Linden, Hälsouniversitetet, Linköping, 10:00 (Swedish)
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Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-10-02Bibliographically approved

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Knaust, EvaPeterson, Curt

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