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Nucleoside analoge cytotoxicity-focus on enzyme regulation, metabolism, and mechanisms of resistance
Linköping University, Department of Medicine and Health Sciences, Clinical Pharmacology . Linköping University, Faculty of Health Sciences.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2010. , 72 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1209
National Category
Pharmacology and Toxicology
Identifiers
URN: urn:nbn:se:liu:diva-63247ISBN: 978-91-7393-310-0 (print)OAI: oai:DiVA.org:liu-63247DiVA: diva2:377210
Public defence
2010-12-10, Eken, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2011-01-12 Created: 2010-12-13 Last updated: 2011-01-12Bibliographically approved
List of papers
1. 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
Open this publication in new window or tab >>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
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2006 (English)In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 71, no 6, 882-890 p.Article in journal (Refereed) 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.

Keyword
Purine analogues; Deoxycytidine kinase; Deoxyguanosine kinase; Chronic lymphocytic leukemia
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-36125 (URN)10.1016/j.bcp.2005.12.007 (DOI)30004 (Local ID)30004 (Archive number)30004 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13
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4. The role of deoxyguanosine kinase for nucleoside analog activation in leukemic and solid tumor cell lines
Open this publication in new window or tab >>The role of deoxyguanosine kinase for nucleoside analog activation in leukemic and solid tumor cell lines
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Screening malignant melanoma cell lines against nucleoside analogs revealed as high sensitivity to fludarabine, clofarabine, and gemcitabine as to leukemic cells, and especially in those cells expressing high levels of the mitochondrial enzyme deoxuguanosine kinase (dGK). This enzyme, together with the cytosolic deoxycytidine kinase (dCK), and the mitochondrial thymidine kinase 2 (TK2) contributes to the activation of natural deoxyribonucleosides and nucleoside analogs to phosphorylated compounds. dCK is the most prominent enzyme in hematopoietic cells, while dGK may be high in cells harbouring many mitochondria, such as neurons and melanocytes. We found that dGK mRNA and protein expression was considerably higher in melanoma cells than in a leukemic cell line, while the difference at the activity level was less profound.

Downregulation of dGK in the melanoma cell line RaH5 using siRNA led to a compensatory increase in TK2 activity, which led to significantly increased sensitivity of the cells to gemcitabine. In contrast, downregulation of dGK in the human leukemic CEM cell line decreased TK2 activity, and rendered the cells more resistant to the drugs. The compensatory regulation of deoxynucleoside kinases with over-lapping substrate specificity differed in leukemic and melanoma cell lines probably because they preferably rely on different deoxynucleoside kinases for nucleoside and nucleoside analog activation. dGK and TK2 that are both located in the mitochondria, seems to be able to compensate for each other to a higher extent in the dGK-dependent melanoma cells compared to CEM cells that possess high dCK activity. Solid tumors, such as melanoma, expressing high levels of dGK should be considered for nucleoside analog therapy preferably in combination with their standard treatment.

Keyword
Deoxyguanosine kinase, melanoma, leukemia, nucleoside analog, RNAi
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-64066 (URN)
Available from: 2011-01-12 Created: 2011-01-12 Last updated: 2011-01-12Bibliographically approved
5. Induction of fetal hemoglobin and ABCB1 gene expression in 9-β-D-arabinofuranosylguanine-resistant MOLT-4 cells
Open this publication in new window or tab >>Induction of fetal hemoglobin and ABCB1 gene expression in 9-β-D-arabinofuranosylguanine-resistant MOLT-4 cells
2011 (English)In: Cancer Chemotherapy and Pharmacology, ISSN 0344-5704, E-ISSN 1432-0843, Vol. 68, no 3, 583-591 p.Article in journal (Refereed) Published
Abstract [en]

PURPOSE: To characterize resistance mechanisms to the nucleoside analog 9-β-D-arabinofuranosylguanine (AraG) in the T-cell acute lymphoblastic leukemia cell line MOLT-4 and its AraG-resistant variant.

METHODS: A gene expression microarray analysis was performed, as well as gene expression and enzyme activity measurements of key enzymes in the activation of AraG. Cytotoxicity of AraG and cross-resistance to other compounds were evaluated using a standard cytotoxicity assay.

RESULTS: Gene expression microarray analysis revealed that fetal hemoglobin genes and the multidrug resistance ABCB1 gene, encoding the drug efflux pump P-gp, were the most highly upregulated genes in the resistant cells, while genes traditionally associated with nucleoside analog resistance were not. Fetal hemoglobin and ABCB1 induction can be due to global DNA hypomethylation. This phenomenon was studied using AraG during a period of 4 weeks in MOLT-4 cells and the lung adenocarcinoma cell line A549, leading to up-regulation of hemoglobin gamma and ABCB1 as well as DNA hypomethylation. Inhibiting P-gp in the AraG-resistant MOLT-4 cells led to decreased proliferation, reduced hemoglobin expression, and highly induced ABCB1 expression.

CONCLUSIONS: We show that AraG can cause hypomethylation of DNA and induce the expression of the fetal hemoglobin gamma gene and the ABCB1 gene. We speculate that the induction of ABCB1/P-gp may occur in order to help with excretion of hemoglobin degradation products that would otherwise be toxic to the cells, and we present data supporting our theory that P-gp may be linked to the induction of hemoglobin.

Place, publisher, year, edition, pages
Springer, 2011
Keyword
9-β-D-arabinofuranosylguanine - Fetal hemoglobin - P-glycoprotein - Microarray - Hypomethylation
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-63246 (URN)10.1007/s00280-010-1524-5 (DOI)000294345400004 ()21110023 (PubMedID)
Note
Funding Agencies|Swedish Cancer Foundation||Swedish Childhood Cancer Foundation||Signe and Olof Wallentin Foundation||Capios Research Foundation||County Council of Ostergotland||Swedish Fund for Research without Animal Experiments||Available from: 2010-12-13 Created: 2010-12-13 Last updated: 2017-12-11

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