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Experimental studies on multidrug resistance in human leukaemia: role of cellular heterogeneity for daunorubicin kinetics
Linköping University, Department of Medicine and Care, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
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 [en]
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: urn:nbn:se:liu:diva-31913Local ID: 17746ISBN: 91-85299-11-1 (print)OAI: oai:DiVA.org:liu-31913DiVA: diva2:252736
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
List of papers
1. Levels of mdr1 and mrp mRNA in leukaemic cell populations from patients with acute myelocytic leukaemia are heterogenous and inversely correlated to cellular daunorubicin accumulation
Open this publication in new window or tab >>Levels of mdr1 and mrp mRNA in leukaemic cell populations from patients with acute myelocytic leukaemia are heterogenous and inversely correlated to cellular daunorubicin accumulation
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1996 (English)In: British Journal of Haematology, ISSN 0007-1048, E-ISSN 1365-2141, Vol. 92, no 4, 847-854 p.Article in journal (Refereed) Published
Abstract [en]

Multidrug resistance gene (mdr1) expression is associated with a poor prognosis in acute myelocytic leukaemia (AML). Whether expression of the recently described multidrug resistance-associated gene (mrp) has any prognostic importance in AML is still unclear. The aim of the present study was to investigate the functional role of the mdr1 and mrp mRNA levels in peripheral leukaemic cell populations from patients with AML. Peripheral leukaemic cells from 10 patients with AML were incubated with daunorubicin (DNR). Cellular DNR content was analysed with a fluorescence-activated cell sorter (FACS). From each cell population the 20–25% cells with the lowest and highest DNR content were sorted out, and mdr1 and mrp RNA were quantified in these subpopulations with competitive polymerase chain reaction. The ratio between the mean DNR content in the cell populations with high and low DNR content varied between 1.9 and 6.6. The cell fraction with low DNR content had higher (3.8–40 times) mdr1 mRNA levels in 10/10 patients and higher (1.4–26 times) mrp mRNA levels in 8/10, as compared to the cell fraction with high DNR accumulation.

In conclusion, mdr1 and mrp mRNA expressions are heterogenous in leukaemic cell populations from patients with AML. The mdr1 expression, and to some extent mrp expression, is inversely correlated to DNR accumulation in vitro.

Keyword
acute myelocytic leukaemia; multidrug resistance; mRNA; cell sorting; PCR
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84232 (URN)10.1046/j.1365-2141.1996.425963.x (DOI)8616077 (PubMedID)
Available from: 2012-10-02 Created: 2012-10-02 Last updated: 2017-12-07Bibliographically approved
2. Intraclonal heterogeneity in the in vitro daunorubicin-induced apoptosis in acute myeloid leukemia
Open this publication in new window or tab >>Intraclonal heterogeneity in the in vitro daunorubicin-induced apoptosis in acute myeloid leukemia
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1999 (English)In: Leukemia and Lymphoma, ISSN 1042-8194, E-ISSN 1029-2403, Vol. 32, no 3-4, 309-316 p.Article in journal (Refereed) Published
Abstract [en]

Leukemic cells from ten patients with acute myeloid leukemia (AML) were sorted on the basis of in vitro daunorubicin (DNR) uptake. The obtained subpopulations with high and low DNR accumulation were compared with regard to induction of apoptosis, expression of bcl-2 and p53. Heterogeneous induction of apoptosis, confined to subpopulations with high DNR uptake, was observed. The size of the DNR-induced apoptotic fraction (4% to 16%) within a given AML blast population was determined by intracellular drug accumulation and was not related to the level of bcl-2 expression. All tested leukemic samples displayed expression of p53 in a growth promoter orientation, i.e. PAb1620-/PAb240+. In two samples, however, sub-populations expressing a growth suppressor orientation of p53, i.e. PAb1620+/PAb240-, were also present. These subpopulations were confined to high-DNR-uptake fractions and associated with the induction of apoptosis. We conclude that intraclonal heterogeneity in the intracellular drug accumulation and subsequently in DNR-induced apoptosis might allow the selection of inherently drug-resistant AML clones thus contributing to relapse of leukemia.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26976 (URN)10.3109/10428199909167391 (DOI)11610 (Local ID)11610 (Archive number)11610 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
3. Heterogeneity of isolated mononuclear cells from patients with acute myeloid leukemia affects cellular accumulation and efflux of daunorubicin
Open this publication in new window or tab >>Heterogeneity of isolated mononuclear cells from patients with acute myeloid leukemia affects cellular accumulation and efflux of daunorubicin
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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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26978 (URN)11612 (Local ID)11612 (Archive number)11612 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
4. Different effects of metabolic inhibitors and cyclosporin A on daunorubicin transport in leukemia cells from patients with AML
Open this publication in new window or tab >>Different effects of metabolic inhibitors and cyclosporin A on daunorubicin transport in leukemia cells from patients with AML
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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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26979 (URN)10.1016/S0145-2126(02)00093-0 (DOI)11613 (Local ID)11613 (Archive number)11613 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved

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