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On mechanisms of drug resistance in acute myeloid leukemia
Örebro University, School of Health and Medical Sciences.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis focus has been to increase the knowledge and understanding of some of the mechanisms responsible for drug resistance in acute myeloid leukemia, as well as identify possibilities to predict drug resistance at diagnosis.

We have studied the intracellular behavior of cytostatic drugs and their main metabolites (paper I) and the cellular response to cytostatic drugs (paper III). A new flow cytometry in vitro chemosensitivity assay was developed, to enable identification of viable myeloid cells and determination of drug sensitivity (paper II). Finally, possible new markers involved in drug resistance were investigated (paper IV).

In conclusion we found that idarubicin and daunorubicin are equally toxic at the same intracellular concentrations. The contribution of the main metabolites to the cytotoxic effects of idarubicin and daunorubicin, in both drug sensitive and drug resistant human myeloid leukemia cells, is low. It is most likely the pharmacokinetic properties of idarubicin and daunorubicin that confer their main cytotoxic effect. With the new flow cytometry chemosensitivity assay we selectively identified viable CD13/CD33 expressing myeloid cells and found that the cytotoxicity results correlated to clinical parameters, such as secondary AML and resistant disease. Short-term exposure of leukemia cell lines with different levels of drug resistance to ara-C revealed that Pgp mRNA and protein ex-pression levels, as well as GSTπ mRNA levels, were rapidly up-regulated. Clinically, this up-regulation may be of importance for the sequential scheduling of daunorubicin and ara-C during the induction treatment of AML. CRIM1 has

never been studied in the context of drug resistance before. We show for the first time that baseline expression of CRIM1 mRNA is much higher in drug resistant leukemia cells compared to drug sensitive cells. We also found a co-variance between CRIM1 and Pgp mRNA expression levels in leukemia cell lines with different levels of drug resistance, suggesting that CRIM1 may be useful as a marker of drug resistance.

Place, publisher, year, edition, pages
Örebro: Örebro universitet , 2010. , 87 p.
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 45
National Category
Medical and Health Sciences
Research subject
Biomedicine
Identifiers
URN: urn:nbn:se:oru:diva-10603ISBN: 978-01-7668-729-1 OAI: oai:DiVA.org:oru-10603DiVA: diva2:317650
Public defence
2010-06-04, Wilandersalen, Universitetssjukhuset, Örebro, 15:54 (Swedish)
Opponent
Supervisors
Available from: 2010-05-05 Created: 2010-05-04 Last updated: 2011-04-26Bibliographically approved
List of papers
1. Comparison of idarubicin and daunorubicin and their main metabolites regarding intracellular uptake and effect on sensitive and multidrug-resistant HL60 cells
Open this publication in new window or tab >>Comparison of idarubicin and daunorubicin and their main metabolites regarding intracellular uptake and effect on sensitive and multidrug-resistant HL60 cells
1996 (English)In: Cancer Chemotherapy and Pharmacology, ISSN 0344-5704, E-ISSN 1432-0843, Vol. 38, no 5, 87 p.476-480 p.Article in journal (Refereed) Published
Abstract [en]

To study the effect of the main metabolites on the cytotoxic effect of daunorubicin and idarubicin in human HL-60 cells, drug-sensitive and multidrug-resistant HL60 cells were incubated with idarubicin and daunorubicin and their metabolites idarubicinol and daunorubicinol over a wide range of concentrations. The intracellular uptake of the drugs was determined by photofluorometry, and the cytotoxic effect in vitro was determined by a bioluminescence assay of intracellular adenosine triphosphate (ATP) after 4 days of culture in liquid medium. The effect of intracellular drugs was calculated from the incubation-concentration versus intracellular-uptake and cytotoxic-effect curves. The intracellular uptake of idarubicin was 6 times that of daunorubicin in drug-sensitive cells and 25 times higher in resistant cells. For idarubicinol as compared with daunorubicinol the corresponding factors were 25 and 7, respectively. As compared with the parent substances, the uptake of idarubicinol and daunorubicinol was 16% and 4%, respectively, in sensitive cells and 40% and >100%, respectively, in resistant cells. An intracellular concentration of 0.5 nmol/mg protein of both parent substances caused a 50% growth inhibition in drug-sensitive cells as compared with 10 nmol/mg protein for drug-resistant cells. For the metabolites an intracellular concentration of 0.4 nmol/mg protein of idarubicinol and 2.0 nmol/mg protein of daunorubicinol was required to inhibit cells’ growth by 50% in drug-sensitive HL60 cells. In the resistant HL60 cells the corresponding values were 30 nmol/mg protein for idarubicinol and 40 nmol/mg protein for daunorubicinol. These results confirm that idarubicinol may significantly contribute to the clinical effect of idarubicin. However, in combination with previous results that have shown low intracellular concentrations of the metabolites in vivo, it appears that the pharmacokinetic properties of the mother substances provide the major explanation for the clinical effect of idarubicin.

Place, publisher, year, edition, pages
Berlin: Springer, 1996. 87 p.
Keyword
Idarubicin, idarubicinol, daunorubicin, daunorubicinol, intracellular uptake, in vitro effect.
National Category
Medical and Health Sciences
Research subject
biomedicin
Identifiers
urn:nbn:se:oru:diva-10451 (URN)10.1007/s002800050514 (DOI)978-91-7668-729-1 (ISBN)
Available from: 2010-05-03 Created: 2010-04-22 Last updated: 2013-06-12Bibliographically approved
2. In vitro chemosensitivity testing of selected myeloid cells in acute myeloid leukemia 
Open this publication in new window or tab >>In vitro chemosensitivity testing of selected myeloid cells in acute myeloid leukemia 
Show others...
2003 (English)In: Leukemia and Lymphoma, ISSN 1042-8194, Vol. 44, no 5, 783-789 p.Article in journal (Refereed) Published
Abstract [en]

In several studies different chemosensitivity assays have been examined in acute myeloid leukemia (AML). Some have shown that in vitro chemosensitivity testing is an independent prognostic factor but so far no one has been able to show that the use of these methods can improve treatment outcome. In an attempt to improve in vitro chemosensitivity testing in AML we wanted to establish and evaluate a new flow cytometry chemosensitivity assay. After 4 days of incubation viable mononuclear myeloid cells were identified by the exclusion of propidium iodide in CD13 or CD33 positive cells. Sixty-eight samples from 64 AML patients were included. In this study, we showed that the flow cytometry method is feasible in AML and we also found some correlations to clinical data. The secondary AML at diagnosis showed an in vitro resistance to etoposide and amsacrine that was significantly higher compared to de novo AML at diagnosis (p = 0.04 and p = 0.02). When AML patients at diagnosis were compared to resistant disease/relapse patients there was a significantly higher effect of ara-C in the diagnosis group (p = 0.03). Responders and non-responders were compared in vitro but we found no significant differences. In vitro mitoxantrone was more effective in multidrug resistance (MDR) negative cells compared to MDR positive cells (p < 0.01). This new method is feasible and makes it possible to selectively evaluate the effect of cytotoxic drugs in myeloid cells. Further studies with a larger group of patients are needed to evaluate the predictive value of the assay.

Place, publisher, year, edition, pages
Taylor & Frances healthsciences, 2003
Keyword
Chemosensitivity, cytotoxicity, drug resistance, flow cytometry, acute myeloid leukemia
National Category
Medical and Health Sciences
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-10578 (URN)10.1080/1042819031000067594 (DOI)12802914 (PubMedID)
Available from: 2010-05-03 Created: 2010-05-03 Last updated: 2011-05-06Bibliographically approved
3. Rapid Induction of P-Glycoprotein mRNA and Protein Expression by Cytarabine in HL-60 Cells
Open this publication in new window or tab >>Rapid Induction of P-Glycoprotein mRNA and Protein Expression by Cytarabine in HL-60 Cells
Show others...
2009 (English)In: Anticancer Research, ISSN 0250-7005, E-ISSN 1791-7530, Vol. 29, no 10, 4071-4076 p.Article in journal (Refereed) Published
Abstract [en]

Background: Overexpression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and glutathione-S-transferase π (GSTπ) is associated with drug resistance in acute myeloid leukemia (AML). The short-term effects of drug exposure on their expression levels were investigated.

Materials and Methods: HL-60 cells and drug-resistant sublines were cultured with or without daunorubicin (DNR) and cytarabine (Ara-C). At several time-points the expression levels of P-gp, BCRP and GSTπ were determined.

Results: After exposure to Ara-C, P-gp mRNA rapidly increased in all the cell lines. P-gp protein was detected in the sensitive cells after 8 h exposure to Ara-C. GSTπ mRNA increased in the resistant cells, but no change in BCRP mRNA was observed. Exposure to DNR revealed rapidly increased P-gp and GSTπ mRNA in the resistant cells.

Conclusion: Ara-C rapidly increases P-gp mRNA and protein expression in sensitive and resistant cells, and GSTπ mRNA in resistant cells, in vitro. This may be of clinical importance during AML induction chemotherapy.

Place, publisher, year, edition, pages
Athens: International Institute of Anticancer Research, 2009
Keyword
Drug resistance, P-glycoprotein, daunorubicin, cytarabine, HL-60 cells, glutathione-S-transferase pi
National Category
Medical and Health Sciences
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-10580 (URN)000271487400051 ()19846953 (PubMedID)2-s2.0-71949122848 (Scopus ID)
Available from: 2010-05-03 Created: 2010-05-03 Last updated: 2017-02-20Bibliographically approved
4. CRIM1 is expressed at higher levels in drug resistant than in drug sensitive myeloid leukemia HL60 cells
Open this publication in new window or tab >>CRIM1 is expressed at higher levels in drug resistant than in drug sensitive myeloid leukemia HL60 cells
(English)Manuscript (preprint) (Other academic)
Keyword
CRIM1, cytarabine, daunorubicin, drug resistance, HL60 cells
National Category
Medical and Health Sciences
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-10582 (URN)
Available from: 2010-05-03 Created: 2010-05-03 Last updated: 2016-12-12Bibliographically approved

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