| 1. |
- 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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| 2. |
- Gregers, Jannie, 1964-
(författare)
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Pharmacogenetic studies in childhood acute lymphoblastic leukaemia with primary focus on methotrexate
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Childhood acute lymphoblastic leukaemia is the most common type of cancer in children. Improvement in treatment has increased survival to approximately 85 per cent. Pharmacogenetics can influence the disposition of anticancer agents and can ideally be used as tool to further improve treatment based on the individual child’s pharmacogenetic profile.The hypothesis in this thesis was that polymorphisms in genes responsible for MTX influx (SLC19A1), efflux (ABCB1, studies with MTX monotherapy have demonstrated effect of variations in this gene) or other MTX pathways (ATIC, MTHFR and SHMT) could have impact on efficacy in childhood acute lymphoblastic leukaemia.The uptake of MTX and impact of SLC19A1 80G>A was investigated in vitro and showed that SLC19A1 80GG had decreased uptake in CD+ T cells and B cells caused by reduced capacity on receptor-to-receptor basis.In more than 500 patients the clinical effect of SLC19A1 80G>A genotype was evaluated and showed that patients with the SLC19A1 80AA had better survival, more bone marrow toxicity, but less liver toxicity than patients with SLC19A1 80GG or 80GA variants. Furthermore, it was demonstrated that SLC19A1 80G>A interacts with chromosome 21 copy number in the leukemic clone.The clinical impact of ABCB1 1199G>A, 1236C>T, 2677GT on the treatment was evaluated. Patients with either the 1199GA or the 3435CC variant had increased risk of relapse compared to patients with the 1199GG or 3435CT/TT variants, respectively. Toxicity was also affected by the ABCB1 polymorphisms.No association between polymorphisms in the ATIC, MTHFR and SHMT genes and outcome was seen. However the 677C>T and 1298 C>A in the MTHFR gene were associated with toxicity.The genotype frequencies between healthy donors and patients were compared, but no association to risk of developing cancer was seen in the investigated polymorphisms.The results in this thesis emphasise the importance of including pharmacogenetic markers in attempts to improve outcome and reduced side effects in childhood ALL.
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| 3. |
- Haglund, Sofie
(författare)
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Interindividual differences in thiopurine metabolism : studies with focus on inflammatory bowel disease
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The thiopurines, 6-mercaptopurine and its prodrug azathioprine, are used in the treatment of inflammatory bowel disease, ulcerative colitis and Crohn´s disease. The main active metabolites are the phosphorylated thioguanine nucleotides (6-TGNs) and methylated thioinosine monophosphate (meTIMP). Both groups contribute to the immunomodulatory effects. About 30-40% of patients fail to benefit from thiopurine treatment. A well-known cause of adverse reactions is decreased or absent thiopurine S-methyltransferase (TPMT) activity. Low TPMT activity is inherited as an autosomal codominant recessive trait and is present in approximately 10% of the population. Although several clinical issues can be solved from determination of TPMT activity, there are cases where it is not possible. In Sweden approximately 25% of IBD-patients display suboptimal 6-TGN concentrations and unexpectedly high concentrations of meTIMP despite a normal TPMT activity. A high meTIMP/6-TGN concentration ratio has been associated with both unresponsiveness to therapy and emergence of adverse reactions. Inosine 5’-monophosphate dehydrogenase (IMPDH) may constitute a candidate gene to explain this metabolite profile, as it is strategically positioned in the metabolic pathway of thiopurines where it competes with TPMT for their common substrate 6-TIMP.In paper I a pyrosequencing method was developed for genotyping of at that time all known genetic variants of TPMT. The concordance between genotype and phenotype in 30 individuals was 93%. The allele frequencies of TPMT*3A, *3B, *3C and *2 in a Swedish background population (n=800) were in agreement with those in other Caucasian or European populations. In Paper II-IV we explored the molecular basis of different metabolite profiles, i.e. low, normal and high meTIMP/6-TGN concentration ratios. The activity of IMPDH was measured in mononuclear cells (MNC). Patients with high metabolite ratios had lower IMPDH activity than patients with normal or low ratios, explained by an inverse correlation to red blood cells concentration of meTIMP. No correlation to 6-TGN was observed. Downregulation of IMPDH activity in HEK293 cells with genetically engineered TPMT activity was associated with an increase in meTIMP, but unexpectedly also of 6-TGN, irrespective of the TPMT status. These results suggest effects of pharmacogenes other than TPMT and IMPDH. A whole genome expression analysis was performed, (1) to identify new candidate genes that could explain differences in metabolite profiles, and (2) to study genes with known associations to the metabolic pathway of (thio)purines. The whole genome expression analysis did not identify any significant group differences. In analysis of the thiopurine related genes, three clusters of co-regulated genes were defined. A co-operation between expression levels of SLC29A1 and NT5E in explaining the meTIMP/6-TGN concentration ratio was observed, and individually SLC29A1 and NT5E correlated to 6-TGN and meTIMP, respectively.Pysosequencing is a convenient and flexible method which is now run in parallel to phenotyping in our laboratory. Our results also illustrate the complexity of the thiopurine metabolism and suggest that differences between metabolite profiles are explained either by interactions between several genes, each with a small contribution, or at the post-transcriptional level. Search for more precise tools to explain differences in metabolite profiles is needed. Furthermore, in order to investigate small effects it is necessary to analyse metabolite concentrations and gene expression levels, as well as enzyme activities in the target cells of therapy (MNC).
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| 4. |
- Knaust, Eva, 1944-
(författare)
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Experimental studies on multidrug resistance in human leukaemia : role of cellular heterogeneity for daunorubicin kinetics
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cellular resistance to chemotherapy is a major cause of treatment failure in acute myeloid leukaemia (AML) and still the majority of the patients die from their disease. Drug resistance 1s multifactorial, the most studied mechanism being multidrug resistance (MDR), mediated by the P-glycoprotein (Pgp). Pgp is an energy-dependent transport protein, encoded by the mdr1 gene, with the power to extrude the cytotoxic drugs out of the cells; thus causing reduced effect of the drug on the leukaemic cells. MDR is characterised by cross-resistance to a wide range of chemotherapeutics of natural origin. Other transport proteins, involved in drug resistance, are the multidrug resistance associated protein (MRP) and the lung resistance protein (Lrp).The aims of this thesis were to elucidate transport kinetics of the anthracycline, daunorubicin, (Dnr) and to investigate the effects of reversing agents on heterogeneity of drug accumulation in cells from patients with AML. The ultimate goal is to improve treatment based on each patient's individual resistance patterns.Density gradient isolated mononuclear cells from patients with AML were incubated with Dnr. Incubated cells were sorted with flow cytometry (FC) on the basis of accumulation levels of the autofluorescent Dnr. Gene expression of the Pgp and the MRP in sorted subpopulations were analysed with polymerase chain reaction (PCR). Apoptosis, expression of p53 and bcl-2 in the sorted subpopulations were determined with monoclonal antibodies and FC. Drug accumulation and efflux, with/without the resistance modifier Cyclosporin A (CyA) and energy-depleting metabolic inhibitors (MJ), were also determined in the leukaemic cell populations with FC.Gene expressions of mdr1 and mrp1 were shown to be heterogeneous in the leukaemic samples and drug accumulation correlated inversely to the gene expression. Cell populations with the higher drug accumulation entailed more apoptosis. The leukaemic cell lopulation, defined by immunopenotyping, differed in drug accumulation an efflux compared to the total mononuclear cell population that also contains normal lymphocytes and monocytes. In leukaemic samples with two blast cell populations, the more immature blast ceUs accumulated drug to a lesser extent and bad a higher efflux rate than the differentiating blast cells. CyA reduced Dnr efflux more efficiently than MI, but MJ increased drug accumulation in leukaemic cells more than CyA.In conclusion: analysis of the total mononuclear population does not give an accurate picture of the leukaemic cell population as concerns resistance mechanisms. Heterogeneity in the leukaemic cell population ought to be taken into account since two or more leukaemic cell populations often exist. The most immature blast cell population should be analysed as relapse usually derives from this population. Furthermore the role of Pgp in MDR is not conclusive as results with reversing agents differed from what was expected.
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| 5. |
- Skoglund, Karin, 1982-
(författare)
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Influence of CYP3A enzymes and ABC transporters on the activity of tyrosine kinase inhibitors in chronic myeloid leukemia
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The introduction of imatinib, a tyrosine kinase inhibitor (TKI), in the treatment of chronic myeloid leukemia (CML) was a major break-through and the first drug that was successfully designed to target the specific mechanism of a malignant disease. Imatinib still remains as the standard treatment of newly diagnosed CML patients although a second generation of TKIs has also been approved for first-line CML treatment.Most patients achieve a good therapeutic effect with imatinib, but some patients are resistant to the drug and are at greater risk of disease progression. In order to further improve CML treatment, a better understanding of the underlying reasons for variable responses to imatinib and the second generation TKIs is important.A number of potential determinants of imatinib response have been suggested, including interindividual variability in pharmacokinetics. Variations in drug metabolism and cellular transport might contribute to the large variations observed in imatinib plasma concentrations and might, therefore, affect the amount of drug that reaches target CML cells. Imatinib is primarily metabolized by the CYP3A hepatic enzymes that are known to be highly variable in activity between different individuals. Imatinib is also a substrate of the ABCB1 and ABCG2 efflux pumps that potentially regulate the elimination of imatinib from the plasma. The ABCB1 and ABCG2 genes are polymorphic and contain single nucleotide polymorphisms (SNPs) that might influence the transport capacity of these proteins. The primary aim of the present thesis was to investigate the influence of CYP3A metabolic activity and cellular transport mediated by genetic variants of ABCB1 and ABCG2 on the response to imatinib and the second generation TKIs used for CML therapy.In vivo CYP3A activity and plasma concentrations of imatinib and its pharmacologically active metabolite CGP74588 were analyzed in CML patients treated with imatinib. CYP3A phenotypes were correlated to plasma concentrations and imatinib outcome 12 months after initiation of treatment. The influence of ABC transport on TKI efficacy was evaluated in vitro by the transduction of genetic variants of ABCB1 and ABCG2 into the CML cell line K562. Functionality of the transport proteins was evaluated by measuring protein expression levels on the cell surface, the intracellular accumulation of TKIs, and the ability of ABCB1 and ABCG2 variants to protect cells from TKI cytotoxicity.We found that CYP3A metabolic activity does not influence the drug plasma concentrations or the therapeutic outcome of imatinib in CML patients. These findings indicate that even though imatinib is primarily metabolized by CYP3A this metabolic activity is not the rate-limiting step in imatinib elimination. CYP3A activity, therefore, is not a suitable predictive marker of imatinib outcome. The in vitro studies revealed that the ABCB1 variants investigated here do not alter the transport of imatinib, CGP74588, dasatinib, or nilotinib. In contrast, the ABCG2 SNPs 421C>A, 623T>C, 886G>C, and 1574T>G significantly impaired the cellular efflux of imatinib, CGP74588, dasatinib, and nilotinib and could possibly influence transport of these TKIs in vivo. It was also found that CGP74588 is by far a better substrate than imatinib for both ABCB1 and ABCG2, and this might have implications in patients with high levels of CYP3A activity. In conclusion, our studies show that ABCG2 SNPs might be important for prediction of imatinib outcome in vivo. On the other hand, CYP3A activity and the ABCB1 SNPs investigated in this study are not likely to be useful as predictors of imatinib outcome.
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| 6. |
- Vikingsson, Svante, 1983-
(författare)
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Development of new methodology for therapeutic drug monitoring of thiopurine treatment
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The three thiopurine drugs azathioprine (AZA), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are used to treat several diseases, including inflammatory bowel disease (IBD). They are pro-drugs and are believed to act through the formation of thioguanine nucleotides (TGNs). Other important metabolites are the methylthioinosine nucleotides (meTINs). These metabolites are active in the white blood cells (WBCs).Most patients respond well to the thiopurine drugs but up to a third have to modify or discontinue their treatment due to adverse events or a lack of therapeutic effects. This could be caused by inter-patient variability in the metabolism of the drugs. Therapeutic drug monitoring (TDM) of thiopurine nucleotides in red blood cells (RBCs) is used to guide treatment. Current routine assays measure the nucleotides after hydrolysation to nucleic bases and are therefore unable to distinguish between mono-, di-, and triphosphates. Recently it was shown that these assays failed to predict the clinical outcome in about 40% of the patients. It has been suggested that measuring thioguanosine triphosphate (TGTP) (believed to be the most active of the TGNs) separately might increase the clinical value.An assay suitable for measuring thioguanosine mono- (TGMP) and diphosphate (TGDP) and TGTP, as well as methylthioinosine mono- (meTIMP), di- (meTIDP) and triphosphate (meTITP) separately in RBCs in clinical samples has been developed. In clinical studies of 82 IBD patients, we found no correlation between the thiopurine dose and metabolite levels in RBCs, thus illustrating the importance of metabolite measurements in the TDM of thiopurines.The TGN peak measured by the routine assay during TDM of patients treated with thiopurines consisted of TGTP and TGDP with a small contribution from TGMP. The meTIN also consisted of mono-, di- and triphosphates, but in different proportions, indicating differences in the formation. The inter-individual differences in nucleotide distribution were very small and a strong correlation between the different nucleotides and their respective sums was observed. As a consequence, measuring the mono-, di- and triphosphates separately was not beneficial in predicting remission, which was confirmed by the results from the clinical study.Further research into the metabolism and mode of action of thiopurine drugs is needed to understand the inter-patient variability in response and metabolite formation. An assay suitable for such studies, measuring TGNs and meTINs in cultured cells, has also been developed.
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