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Pharmacological basis for cladribine resistance in a human acute T lymphoblastic leukaemia cell line selected for resistance to etoposide
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.
Oncology Department, Mayo Clinic, Rochester, Minnesota, USA.
Department of Oncology-Pathology, Karolinska Hospital Stockholm, Sweden.
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2001 (English)In: British Journal of Haematology, ISSN 0007-1048, E-ISSN 1365-2141, Vol. 113, no 2, 339-346 p.Article in journal (Refereed) Published
Abstract [en]

Cross-resistance between different classes of anti-neoplastic agents can jeopardize successful combination cancer chemotherapy. In this study, we observed an unexpected cross-resistance between the podophyllotoxine derivative etoposide (VP) and the nucleoside analogue cladribine (CdA) in CCRF-CEM cells developed for resistance to VP. The resistant cells also displayed 14- and twofold resistance to cytarabine (ara-C) and gemcitabine respectively. Closer analysis of these cells showed that they contained lower amounts of topoisomerase (topo) IIα (P < 0·001) and β protein (P < 0·026), formed substantially lower amounts of the topo II–DNA complex, and had a markedly decreased level of Fas (CD95/APO-1)-ligand mRNA expression. Interestingly, Fas expression in the resistant cells did not differ from that in the parental cell line. No differences were observed in the accumulation/efflux of daunorubicin or in the gene expressions of P-glycoprotein, multidrug resistance-associated protein and the lung resistance-related protein. The activity of deoxycytidine kinase (dCK), responsible for activation of CdA and ara-C, was the same for resistant and wild-type cells. However, there was an increase in the activity of the cytosolic 5′-nucleotidases (5′-NT), responsible for deactivation of nucleotides, amounting to 206% (P < 0·001) for the high Km and 134% (P < 0·331) for the low Km 5′-NT in resistant cells. The high Km 5′-NT is probably responsible for the decreased amount of the active metabolite CdA 5′-triphosphate [40% decreased (P < 0·045)], as well as for other purine ribonucleosides and deoxyribonucleosides triphosphates in the resistant cells. In contrast, a significantly higher deoxycytidine triphosphate (dCTP) level (167%, P < 0·001) was observed in the resistant cells. Thus, this study suggests that the major cause of resistance to the nucleoside analogues CdA and ara-C in cells selected for resistance to VP is a result of metabolic alterations producing increased activity of 5′-NT and higher dCTP levels. Furthermore, these results indicate that there is a common factor in the regulation of nucleotide-degrading enzymes and DNA topoisomerases, which may be altered in cross-resistant cells.

Place, publisher, year, edition, pages
2001. Vol. 113, no 2, 339-346 p.
Keyword [en]
etoposide, cladribine, CD95, deoxycytidine kinase, 5′-nucleotidase
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-26977DOI: 10.1046/j.1365-2141.2001.02751.xLocal ID: 11611OAI: diva2:247528
Available from: 2009-10-08 Created: 2009-10-08 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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