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Intraclonal heterogeneity in the in vitro daunorubicin-induced apoptosis in acute myeloid leukemia
Department of Hematology and Infection, Karolinska Hospital, Stockholm, Sweden.
Department of Clinical Pharmacology, Karolinska Hospital, Stockholm, Sweden.
Department of Hematology and Infection, Karolinska Hospital, Stockholm, Sweden.
Department of Hematology and Infection, Karolinska Hospital, Stockholm, Sweden.
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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.

Place, publisher, year, edition, pages
1999. Vol. 32, no 3-4, 309-316 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-26976DOI: 10.3109/10428199909167391Local ID: 11610OAI: oai:DiVA.org:liu-26976DiVA: diva2:247527
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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Knaust, EvaPeterson, Curt

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