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On the mechanism of 9-ß-D-arabinofuroanosylguanine resistance in human leukemic cells
Division of Clinical Pharmacology, Department of Medicine, Karolinska Hospital, Stockholm.
Linköping University, Department of Medicine and Care, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
Department of Clinical Microbiology, Karolinska Hospital, Stockholm.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The present study was undertaken to elucidate the mechanism of resistance to a relatively new nucleoside, 9-ß-D-arabinofuranosylguanine (Ara-G), which is highly toxic to T-cell malignancies. In order to do this we generated two stable resistant cell lines, MOLT-4/AraG500 (100-fold resistant) and MOLT-4/Ara-G900 (180-fold resistant). The presumed limiting step in the activation of Ara-G is catalyzed by both deoxycytidine kinase (dCK) and the mitochondrial deoxyguanosine kinase (dGK). Cross-resistance was noted to analogues such as cytosine arabinoside (Ara-C), cladribine, and fludarabine, but not to difluorodeoxycytidine. HPLC measurements of intracellular triphosphates of Ara-C and Ara-G showed that resistant cells generated significantly lower levels of metabolites. The activity of dGK (MOLT-4/AraG500, 79%, and MOLT-4/Ara-G900, 83%) and dCK (MOLT-4/Ara-G500, 54%, and MOLT- 4/Ara-G900, 73%), as well as protein and mRNA levels were significantly reduced in the resistant cell lines compared to the wild type. The resistant cells also showed decreased activity as well as decreased mRNA expression of the cytosolic 5'-nucleotidase (5'-NT), compared to the wild type. The reduced levels of enzyme activity in the resistant cells may be a consequence of reduced numbers of chromosomes carrying the genes for dGK, dCK and 5 'NT. Unexpectedly, the resistant cells showed higher activity of the mitochondrial enzyme thymidine kinase 2, probably resulting from down-regulation of dGK. No mutations were found in the dCK gene ofMOLT-4/Ara-G500 but in 2 of 13 clones ofMOLT-4/Ara-G900, we found two point mutations, one T to C and other A to G, which gave rise to changes in the amino acid sequence and affected the catalytic activity. The intracellular dNTP levels were determined and a nearly unchanged dCTP pool was observed in the resistant cells, while the other dNTP pools were significantly reduced compared to those in the wild type. These results show that altered activity of the deoxyribonucleoside kinases confer resistance to Ara-G in these cell lines.

Keyword [en]
Leukemia, 9-ß-D-arabinofuroanosylguanine, deoxycytidine kinase, deoxyguanosine kinase, resistance and cytotoxicity
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-80283OAI: diva2:546403
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.
Linköping University Medical Dissertations, ISSN 0345-0082 ; 698
Nucleoside analogue, cross-resistance, multidrug resistance, deoxycytidine kinase, leukemia, anthracyclines, P-glycoprotein
National Category
Medical and Health Sciences
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)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-11-02Bibliographically approved

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