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Heterogeneity of isolated mononuclear cells from patients with acute myeloid leukemia affects cellular accumulation and efflux of daunorubicin
Linköping University, Department of Medicine and Care, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
Departments of Pathology, Karolinska Hospital, Stockholm, Sweden.
Division of Hematology, Karolinska Hospital, Stockholm, Sweden.
Division of Hematology, Karolinska Hospital, Stockholm, Sweden.
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2000 (English)In: Haematologica, ISSN 0390-6078, E-ISSN 1592-8721, Vol. 85, no 2, 124-132 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND AND OBJECTIVE: Pharmacologic studies on blasts from patients with leukemia are generally performed on density gradient isolated blood or bone marrow cells. Thereby, cellular drug accumulation and efflux are determined as mean values of the entire cell population. The objective of the present study was to characterize the heterogeneity in the accumulation and efflux of daunorubicin in various subpopulations of mononuclear cells isolated from patients with acute myeloid leukemia (AML).

DESIGN AND METHODS: Mononuclear cells from 33 patients with AML were isolated from peripheral blood by density gradient centrifugation on Lymphoprep (1. 077 g/mL). Cellular accumulation of fluorescent daunorubicin was determined by flow cytometry after incubation of the cells at +37C for 1 hour. Thereafter, the cells were washed and reincubated in drug-free medium. Kinetics of drug efflux were determined by frequent determination of cellular fluorescence during 30 min. Daunorubicin accumulation and efflux were compared in the total isolated mononuclear cell population and in the various blast cell populations gated on FSC/SSC according to the results of immunophenotyping.

RESULTS: In 8 of these 33 (24%) patient samples, two distinct blast cell populations could be identified. In 7 out of 8 these cases the more immature blasts had a lower drug accumulation and in 6 out of the 8 cases also a higher efflux rate than the differentiating cell population. Cyclosporin A increased daunorubicin accumulation and reduced efflux in the immature blast population. In the differentiating cell population cyclosporin A increased both the accumulation and the efflux. In patients with a single blast cell population, the gated blast cells had a significantly lower drug accumulation but also a lower drug efflux rate than the total cell population.

INTERPRETATION AND CONCLUSIONS: The results imply that drug transport studies on cells isolated from patients with AML give somewhat different results depending on the cell population studied. Some, but not all, of these differences in daunorubicin accumulation and efflux as well as in the effect of cyclo-sporin A can be explained by a heterogenous expression of the mdr1-gene. The observed heterogeneity may be of special relevance with regard to drug resistance. The presence of even a small resistant cell clone may jeopardize the effect of the chemotherapy due to expansion resulting in relapse of disease.

Place, publisher, year, edition, pages
2000. Vol. 85, no 2, 124-132 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-26978Local ID: 11612OAI: oai:DiVA.org:liu-26978DiVA: diva2:247529
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)
Opponent
Supervisors
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-10-02Bibliographically approved
2. Heterogeneity of leukemic cells with regard to daunorubicin accumulation and efflux: potential role in drug resistance
Open this publication in new window or tab >>Heterogeneity of leukemic cells with regard to daunorubicin accumulation and efflux: potential role in drug resistance
2000 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Cellular resistance to chemotherapy is a major cause of treatment failure in acute myelocytic leukemia (AML) and other malignancies. Drug resistance is multifactorial; the most studied mechanism being multidrug resistance (MDR), mediated by the P-glycoprotein (PPG), a transmembrane transport protein with a pump function, encoded by the mdr 1 gene. MDR is characterised by cross resistance to a wide range of chemotherapeutics of natural origin e.g. anthracyclines, vinca alkaloids, epipodophyllotoxins and taxanes. The aim of the present study was to elucidate transport kinetics of the anthracycline daunorubicin (Dnr) and to study various forms of heterogeneity 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 on the basis of accumulation of the autofluorescent Dnr with flow cytometry. Gene expression of the Pgp and the multidrug resistance-associated protein (MRP) in sorted subpopulations were analysed with polymerase chain reaction (PCR). Drug accumulation and efflux in leukemic cell populations, apoptosis, expression of p53 and bcl-2 in the sorted subpopulations were studied with flow cytometry.

Gene expressions of mdrl and mrp were shown to be heterogeneous in the leukemic samples and drug accumulation correlated inversely to the gene expression. The blast cell population differed in drug accumulation and efflux compared to the total mononuclear cell population that contains also normal lymphocytes and monocytes. In leukemic samples with two blast cell populations the more immature blast cells accumulated less drug and had a higher efflux rate than the differentiating blast cells. Cell populations with higher drug accumulation entailed more apoptosis.

The results suggest that, since several resistance factors can occur in the same patient heterogeneity in the leukemic cell population ought to be taken into account whenever resistance patterns are studied.

Place, publisher, year, edition, pages
Linköping: Linköping University, 2000. 41 p.
Series
Linköping Studies in Health Sciences. Thesis, ISSN 1100-6013 ; 46
Keyword
Drug resi stance. acute myeloid leukemia, flow cytometry, gene expression, transport kinetics, apoptosis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-28317 (URN)13449 (Local ID)91-7219-580-0 (ISBN)13449 (Archive number)13449 (OAI)
Presentation
2000-04-14, Lab ettans Sal1, Hälsouniversitetet, Linköping, 13:00 (Swedish)
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2013-07-10Bibliographically approved

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