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The role of deoxyguanosine kinase for nucleoside analog activation in leukemic and solid tumor cell lines
Linköpings universitet, Institutionen för medicin och hälsa, Klinisk farmakologi. Linköpings universitet, Hälsouniversitetet.
Cancer Center Karolinska Department of Oncology and Pathology, Karolinska University Hospital, SE-171 76 Stockholm.
Linköpings universitet, Institutionen för medicin och hälsa, Klinisk farmakologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk farmakologi.
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
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.

Nyckelord [en]
Deoxyguanosine kinase, melanoma, leukemia, nucleoside analog, RNAi
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
URN: urn:nbn:se:liu:diva-64066OAI: oai:DiVA.org:liu-64066DiVA, id: diva2:385949
Tillgänglig från: 2011-01-12 Skapad: 2011-01-12 Senast uppdaterad: 2011-01-12Bibliografiskt granskad
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: 2018-01-12Bibliografiskt granskad

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Fyrberg, AnnaLotfi, Kourosh

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