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- Fyrberg, Anna, 1981-, et al.
(författare)
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Cell cycle dependent regulation of deoxycytidine kinase, deoxyguanosine kinase, and cytosolic 5′-nucleotidase I activity in MOLT-4 cells
- 2006
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Ingår i: Nucleosides, Nucleotides & Nucleic Acids. - : Informa UK Limited. - 1525-7770 .- 1532-2335. ; 25:9-11, s. 1201-1204
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Tidskriftsartikel (refereegranskat)abstract
- Activation of nucleoside analogues is dependent on kinases and 5′-nucleotidases and the balance between the activity of these enzymes. The purpose of this study was to analyze deoxycytidine kinase, deoxyguanosine kinase, and 4 different 5′-nucleotidases during cell cycle progression in MOLT-4 cells. The activity of both kinases was cell cycle dependent and increased during proliferation while the activity of cytosolic 5′-nucleotidase I decreased. We could show that the kinase activity was higher than the total nucleotidase activity, which was unchanged or decreased during cell cycle progression. These data may be important in designing modern combination therapy with nucleoside analogues. Copyright © Taylor & Francis Group, LLC.
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| 3. |
- Fyrberg, Anna, 1981-
(författare)
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Nucleoside analoge cytotoxicity-focus on enzyme regulation, metabolism, and mechanisms of resistance
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 4. |
- Jakobsen, Ingrid, 1984-
(författare)
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Prognostic markers in acute myeloid leukemia : A candidate gene approach
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The standard treatment of acute myeloid leukemia (AML) consists of induction chemotherapy, most commonly daunorubicin together with the nucleoside analogue cytarabine (Ara-C), followed by consolidation chemotherapy and in selected cases allogenic stem cell transplantation (allo-SCT). Despite a high initial response rate, a considerable proportion of all AML cases eventually suffer from relapse and the five-year overall survival rate in patients >60 years is only around 15%. Based on cytogenetic analysis, patients are divided into low risk, intermediate risk, and high-risk groups. While low risk patients have a high chance of reaching and remaining in remission after standard induction therapy, high-risk patients are likely to suffer from relapse and should be scheduled for allo-SCT when first complete remission is reached. The intermediate risk group consists of normal karyotype (NK) patients and those with karyotypes of uncertain clinical relevance, but the outcomes are heterogeneous. In NKAML patients, risk classification has improved with the addition of molecular markers including FLT3 internal tandem duplications (ITD) and mutations of NPM1 and CEBPA. Despite this development, there is a group of patients lacking reliable prognostic markers and in some cases the outcomes predicted do not match the outcomes observed, highlighting the need for additional markers. ABCB1 encodes a transporter protein responsible for the extrusion of cytotoxic compounds, including daunorubicin, over the cell membrane, and is a known resistance mechanism. Ara-C is subject to both activating and inactivating metabolic enzymes including DCK (activating), CDA and cN-II (inactivating). ABCB1, DCK, CDA and cN-II are all polymorphic, and SNPs affecting enzyme function and/or activity have potential as prognostic markers. In addition, recurrent IDH1/2 mutations lead to the expression of an enzyme with neomorphic activity associated with epigenetic alterations and disturbed differentiation. Mutations as well as a SNP in codon 105 of IDH1 have prognostic implications in AML, although the effects of different IDH mutations have been unclear. The aim of this thesis was to investigate SNPs in ABCB1 and genes associated with Ara-C metabolism, mutations in IDH1/2 and the IDH1 SNP, and their associations with treatment response and survival in AML. We show that the 1236C>T and 2677G>T SNPs in ABCB1 influence in vitro sensitivity towards AML drugs, with corresponding effects on NK-AML patient survival. These survival differences were seen mainly in patients lacking FLT3-ITD, further adding to the risk stratification. In contrast, the CDA SNPs 79A>C and -451C>T appear to influence survival mainly in FLT3-ITD positive cases. In conclusion, the above-mentioned SNPs have the potential to add important information to risk classifications especially in NK-AML patients with the ambiguous FLT3-ITD-/NPM1- or FLT3-ITD+/NPM1+ genotypes. In addition, we have shown that IDH2 R140 mutation is associated with impaired survival in AML, and that the IDH1 codon 105 SNP appears to confer a worse outcome in a subset of intermediate risk patients without FLT3-ITD. With the introduction of next generation sequencing into clinical diagnostics, IDH mutations may not only provide prognostic information but also guide the selection of patients for new drugs targeting the variant enzyme. Our results indicate that in addition to leukemia-specific mutations, constitutional SNPs may prove useful for further individualizing care-taking and should be considered when implementing these new techniques.
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