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Altered expression of multidrug resistance (mDR1) gene in cells selected for resistance to 9-ß-D-Arabinofuranosylguanine
Linköping University, Department of Medicine and Care, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
Department of Medicine, Division of Clinical Pharmacology, Karolinska Hospital, Stockholm, Sweden.
Linköping University, Department of Medicine and Care, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
Department of Medicine, Division of Clinical Pharmacology, Karolinska Hospital, Stockholm, Sweden.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

9-ß-D-Arabinofuranosylguanine (AraG) is an important and relatively new guanine nucleoside analogue that is highly toxic to T-cell maliguancies. Two resistant sublines with low activity of nucleoside phosphorylating enzymes, deoxycytidine kinase (dCK) (50-70% reduction) and deoxyguanosine kinase (dGK, 70-80% reduction), were generated from a MOLT-4 cell line with stepwise-increasing concentrations of AraG. As expected, the resistant sublines were highly cross-resistance to analogues that are activated by dCK and dGK such as cytosine arabinoside, cladribine, and fludarabine. Surprisingly, the resistant cells were siguificantly less sensitive to anthracyclines and podophyllotoxin derivatives resulting from decreased cellular drug accumulation restored by cyclosporin A, as determined by calcein uptake and flow cytometry studies. To clarify the mechanisms of this resistance, the gene expression of various multidrug resistance proteins, e.g. the multidrug resistance gene (mdr1), multidrng resistance-associated protein, lung resistance-associated protein and topoisomerase IIα or IIß were investigated. We found that the resistant cells overexpressed the mdr1 gene, as assessed by a real-time PCR, and contained higher levels of P-glycoprotein, as assessed by western blotting. The expression of other multidrug resistance proteins was not affected in the AraG-resistant sublines. These fmdings may be useful in the clinical trials of AraG singly and in combination with anthracyclines and related agents.

Keyword [en]
leukaemia, 9-ß-D-arabinofuranosylguanine, multidrug resistance, cytotoxicity
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-80287OAI: oai:DiVA.org:liu-80287DiVA: diva2:546409
Available from: 2012-08-23 Created: 2012-08-23 Last updated: 2012-08-23Bibliographically approved
In thesis
1. Pharmacology and resistance mechanisms of nucleoside analogues and topoisomerase II interactive agents: studies on human leukemia cells with a focus on cross-resistance
Open this publication in new window or tab >>Pharmacology and resistance mechanisms of nucleoside analogues and topoisomerase II interactive agents: studies on human leukemia cells with a focus on cross-resistance
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The purpose of this thesis was to elucidate mechanisms of action and resistance of clinically relevant nucleoside analogues and topoisomerase interactive agents in human leukemia cell lines and leukernia cells isolated from peripheral blood of leukemia patients. Interactions and cross-resistance patterns of these different cytotoxic drug families were also studied since these drugs are usually administrated in combination in the clinic.

Two novel nucleoside analogues, clofarabine (2-chloro-2'-arabino-fluoro 2'-deoxyadenosine, CAFdA) and nelarabine (9-ß-D-arabinofuranosylguanine, AraG) were studied regarding cellular activation and mechanisms of resistance. Compared to cladribine (2-chloro- 2'-deoxyadenosine, CdA), CAFdA was more effective due to better stability and more efficient phosphorylation by deoxycytidine kinase (dCK). The mechanism of resistance to CAFdA was decreased activity of dCK. The most important mechanism contributing to resistance to AraG seems to be the deficiency of the activating enzymes dCK and deoxyguanosine kinase (dGK), as measured by enzyme activity assays, Western blotting, and real-time polymerase chain reaction. Unexpected cross-resistance between topoisomerase interactive agents and nucleoside analogues was identified in CEM and MOLT-4 cell lines developed for resistance to etoposide (VP) and AraG, respectively, by means of a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide viability assay. Resistance to VP was due to a decrease in the activity and amount of topoisomerase TI. Major cause of resistance to the nucleoside analogues CdA and AraC was metabolic alterations producing increased activity of 5'-nucleotidase and higher level of endogenous deoxycytidine triphosphate. The AraG-resistant cells showed also classical multidrug resistance (MDR) phenomena. The accumulation and cytotoxicity of daunorubicin (Dnr) were studied in AraG-resistant cells and in response to the resistance modifiers, such as cyclosporin A. The level of mdr1 mRNA and its product, P-glycoprotein, was increased. The topoisomerase interactive agent, idarubicin (Ida), a semisynthetic derivative of Dnr, was more effective in inducing apoptosis as determined by the Annexin V -FITC method, and Ida-resistant cells did not show any classical MDR phenomena.

Thus, these studies suggest that anticancer agents from the same class of cytostatics could have important differences in effectivity and mechanisms of resistance. These results confirm the possibility of coexpression of multiple mechanisms of resistance in human leukemic cells, which have been selected by exposure to a single-dmg. The generally assumed lack of crossresistance between nucleoside analogues and topoisomerase interactive agents is questionable.

The rationale for combination therapy should be based on biological properties and cross-resistance analyses of the included drugs.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2001. 75 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 698
Keyword
Nucleoside analogue, cross-resistance, multidrug resistance, deoxycytidine kinase, leukemia, anthracyclines, P-glycoprotein
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27518 (URN)12174 (Local ID)91-7373-140-4 (ISBN)12174 (Archive number)12174 (OAI)
Public defence
2001-11-16, Berzeliussalen, Universitetssjukhuset, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-11-02Bibliographically approved

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