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The pattern of deoxycytidine- and deoxyguanosine kinase activity in relation to messenger RNA expression in blood cells from untreated patients with B-cell chronic lymphocytic leukemia
Linköpings universitet, Institutionen för medicin och hälsa, Klinisk farmakologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk farmakologi.
Östergötlands Läns Landsting, Kirurgi- och onkologicentrum, Hematologiska kliniken US.
Linköpings universitet, Institutionen för medicin och hälsa, Klinisk farmakologi. Linköpings universitet, Hälsouniversitetet.
Lund University Hospital.
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2006 (Engelska)Ingår i: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 71, nr 6, s. 882-890Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) catalyze the first step in the intracellular cascade of fludarabine (2-fluoroadenine-β- d-arabinofuranoside) and cladribine (2-chlorodeoxyadenosine) phosphorylation, which leads to activation of these prodrugs, commonly used for treatment of chronic lymphocytic leukemia (CLL). Thus, resistance to nucleoside analogues may primarily be due to low levels of deoxynucleoside kinase activity. The purpose of this study was to investigate the activity profiles of dCK and dGK and characterize the possible relationship between the levels of dCK enzymatic activities and mRNA levels in B-CLL cells from untreated patient samples in an attempt to determine the best approach for predicting sensitivity to nucleoside analogues and thereby optimizing treatment of CLL. For this purpose, dCK and dGK analyses were done in blood cells from 59 untreated symptomatic patients with CLL. The dGK activity towards 2-chlorodeoxyadenosine was significantly lower than of dCK (median 73 pmol/mg protein/min (85-121, 95% CI) versus 353 pmol/mg protein/min (331-421)). The median dCK mRNA level was 0.107 (0.096-0.120, 95% CI). There was a lack of correlation between the activities of dCK and dGK, which indicates that these proteins are regulated independently. We also found that the dCK and dGK activity measurement towards their endogenous substrates were comparable to the nucleoside analogues tested. Such variations in enzyme activities and mRNA levels may well explain differences in clinical responses to treatment. There was no correlation between the levels of dCK mRNAs and enzymatic activities using a quantitative real-time PCR procedure. Sequencing of dCK mRNA did not reveal alternate splicing or mutations in the coding region. The relation between activity and mRNA levels was studied by short interfering RNA (siRNA) method, which showed that in the siRNA treated cells the down-regulation of dCK expression, and activity followed each other. However, in control cells the mRNA levels remained stable but the protein activity markedly decreased. These data demonstrate that the dCK activity is not reflected by dCK mRNA expression that indicates a post-translational mechanism(s). © 2005 Elsevier Inc. All rights reserved.

Ort, förlag, år, upplaga, sidor
2006. Vol. 71, nr 6, s. 882-890
Nyckelord [en]
Purine analogues; Deoxycytidine kinase; Deoxyguanosine kinase; Chronic lymphocytic leukemia
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
URN: urn:nbn:se:liu:diva-36125DOI: 10.1016/j.bcp.2005.12.007Lokalt ID: 30004OAI: oai:DiVA.org:liu-36125DiVA, id: diva2:256973
Tillgänglig från: 2009-10-10 Skapad: 2009-10-10 Senast uppdaterad: 2018-03-21
Ingår i avhandling
1. Nucleoside analoge cytotoxicity-focus on enzyme regulation, metabolism, and mechanisms of resistance
Öppna denna publikation i ny flik eller fönster >>Nucleoside analoge cytotoxicity-focus on enzyme regulation, metabolism, and mechanisms of resistance
2010 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

The aim of this thesis was to determine the role of nucleoside analog activating and deactivating enzymes in nucleoside analog metabolism and resistance development. Nucleoside analogs are anti-cancer drogs and are often used to treat different leukemias, attributably to presence of high levels of nucleoside analog activating enzymes in hematopoietic cells. More recently some of the newer analogs have been used  successfully to treat solid tumors as well.

We have used human leukemic cell lines, and isolated cells from patients with leukemia, to investigate the nucleoside analog activating enzymes deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) and some of the deactivating enzymes called 5'nucleotidases (5'-NTs). We have measured mRNA expressions and enzymatic activities and correlated them with the cytotoxic response to nuc1eoside analogs and changes in cell cycle progression. We optimized and evaluated a siRNA-transfection method and decreased the activities of dCK and dGK in two different cell lines in order to find out more about their respective contribution to activation of these drogs. An expression microarray analysis of a nucleoside analog resistant cell line was also performed in order to clarify which genes are involved in development of resistance.

We found that expressions and activities of dCK and dGK were not correlated. The enzyme activities of activating and deactivating enzymes changed during cell cycle progression, giving actively proliferating cells a more favorable enzymatic profile with regard to nucleoside analog cytotoxicity.

The activities of dCK and dGK could be reduced transiently in leukemic and solid tumor cell lines, thereby confer either resistance or increased sensitivity to nucleoside analogs to variable degrees. Expression microarray analysis was used to evaluate the effect of the transfection method and the specificity of siRNA. We concluded that cells tolerated the transfection weIl without major effects on gene expression, and considered the siRNA used to be specific to its target.

An expression microarray experiment on a nucleoside analog-induced resistant cell line revealed a hypomethylating capacity of the drog and induction of fetal hemoglobin and a multidrog resistance efflux pump as a result of the hypomethylation. This pump should not be affected by nucleoside analoges since they are not a substrate of it, and upregulation of the pump unfortunately renders the cells highly cross-resistant to different types of drogs. Our preliminary data supports our theory that it may be upregulated in order to help excrete hemoglobin that otherwise would be toxic to the cells.

Ort, förlag, år, upplaga, sidor
Linköping: Linköping University Electronic Press, 2010. s. 72
Serie
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1209
Nationell ämneskategori
Farmakologi och toxikologi
Identifikatorer
urn:nbn:se:liu:diva-63247 (URN)978-91-7393-310-0 (ISBN)
Disputation
2010-12-10, Eken, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2011-01-12 Skapad: 2010-12-13 Senast uppdaterad: 2020-02-26Bibliografiskt granskad

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Lotfi, KouroshKarlsson, KarinFyrberg, AnnaJuliusson, GunnarPeterson, CurtAlbertioni, Freidoun

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Klinisk farmakologiHälsouniversitetetKlinisk farmakologiHematologiska kliniken USKlinisk farmakologi
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