Jelena Klawitter

Abnormalities in glucose uptake and metabolism in imatinib-resistant human BCR-ABL-positive cells.

The development of imatinib resistance has become a significant therapeutic problem in which the etiology seems to be multifactorial and poorly understood. As of today, clinical criteria to predict the development of imatinib resistance in chronic myelogenous leukemia (CML), other than rebound of the myeloproliferation, are under development. However, there is evidence that the control of glucose-substrate flux is an important mechanism of the antiproliferative action of imatinib because imatinib-resistant gastrointestinal stromal KIT-positive tumors reveal highly elevated glucose uptake in radiologic images. We used nuclear magnetic resonance spectroscopy and gas chromatography mass spectrometry to assess 13C glucose uptake and metabolism (glycolysis, TCA cycle, and nucleic acid ribose synthesis) during imatinib treatment in CML cell lines with different sensitivities to imatinib. Our results show that sensitive K562-s and LAMA84-s BCR-ABL–positive cells have decreased glucose uptake, decreased lactate production, and an improved oxidative TCA cycle following imatinib treatment. The resistant K562-r and LAMA84-r cells maintained a highly glycolytic metabolic phenotype with elevated glucose uptake and lactate production. In addition, oxidative synthesis of RNA ribose from 13C-glucose via glucose-6-phosphate dehydrogenase was decreased, and RNA synthesis via the nonoxidative transketolase pathway was increased in imatinib-resistant cells. CML cells which exhibited a (oxidative/nonoxidative) flux ratio for nucleic acid ribose synthesis of >1 were sensitive to imatinib. The resistant K562-r and LAMA84-r exhibited a (oxidative/nonoxidative) flux ratio of <0.7. The changes in glucose uptake and metabolism were accompanied by intracellular translocation of GLUT-1 from the plasma membrane into the intracellular fraction in sensitive cells treated with imatinib, whereas GLUT-1 remained located at the plasma membrane in LAMA84-r and K562-r cells. The total protein load of GLUT-1 was unchanged among treated sensitive and resistant cell lines. In summary, elevated glucose uptake and nonoxidative glycolytic metabolic phenotype can be used as sensitive markers for early detection of imatinib resistance in BCR-ABL–positive cells.

Quantification of 15-F2t-isoprostane in human plasma and urine: results from enzyme-linked immunoassay and liquid chromatography/tandem mass spectrometry cannot be compared

Quantification of F(2)-isoprostanes is considered a reliable index of the oxidative stress status in vivo. Several immunoassays and chromatography/mass spectrometry-based assays are available for 15-F(2t)-isoprostane quantification. However, it remains unclear if results of immunoassays using different assays can be compared with those of liquid chromatography/mass spectrometry (LC/MS) assays. Previous studies comparing enzyme-linked immunosorbent assay (ELISA) and more specific gas chromatography/mass spectrometry assays have already indicated that ELISAs may overestimate 15-F(2t)-isoprostane concentrations in human plasma. Concentrations of 15-F(2t)-isoprostane in 25 human plasma and urine samples were measured by three commercially available ELISA assays (Assay Designs, Cayman Chemical and Oxford Biomedical Research) and compared with the concentrations measured with a validated, semi-automated high-throughput HPLC tandem mass spectrometry assay (LC/LC-MS/MS). All three ELISAs measured substantially higher 15-F(2t)-isoprostane concentrations (2.1-182.2-fold higher in plasma; 0.4-61.9-fold higher in urine) than LC/LC-MS/MS. Utilization of solid-phase extraction (SPE) columns, especially isoprostane affinity purification columns, brought ELISA isoprostane urine concentrations closer to the LC/LC-MS/MS results. However, SPE did not have much of an effect on ELISA plasma concentrations which remained significantly higher than corresponding LC/LC-MS/MS results. A poor correlation not only between LC/LC-MS/MS and immunoassay results, but also among the immunoassays was found. Especially in plasma, ELISAs grossly overestimate 15-F(2t)-isoprostane concentrations and are not comparable with each other or with LC/LC-MS/MS. It is most disturbing that a sample with relatively high concentrations measured with one ELISA may show low concentrations with another ELISA, and vice versa, potentially affecting the conclusions drawn from such data. The use of specific mass spectrometry-based assays seems advisable.

Urine metabolites reflect time-dependent effects of cyclosporine and sirolimus on rat kidney function.

The clinical use of the immunosuppressant calcineurin inhibitor cyclosporine is limited by its nephrotoxicity. This is enhanced when combined with the immunosuppressive mTOR inhibitor sirolimus. Nephrotoxicity of both drugs is not yet fully understood. The goal was to gain more detailed mechanistic insights into the time-dependent effects of cyclosporine and sirolimus on the rat kidney by using a comprehensive approach including metabolic profiling in urine ((1)H NMR spectroscopy), kidney histology, kidney function parameters in plasma, measurement of glomerular filtration rates, the oxidative stress marker 15-F(2t)-isoprostane in urine, and immunosuppressant concentrations in blood and kidney. Male Wistar rats were treated with vehicle (controls), cyclosporine (10/25 mg/kg/day), and/or sirolimus (1 mg/kg/day) by oral gavage once daily for 6 and 28 days. Twenty-eight day treatment led to a decrease of glomerular filtration rates (cyclosporine, -59%; sirolimus, -25%). These were further decreased when both drugs were combined (-86%). Histology revealed tubular damage after treatment with cyclosporine, which was enhanced when sirolimus was added. No other part of the kidney was affected. (1)H NMR spectroscopy analysis of urine (day 6) revealed time-dependent changes of 2-oxoglutarate, citrate, and succinate concentrations. In combination with increased urine isoprostane concentrations, these changes indicated oxidative stress. After 28 days of cyclosporine treatment, urine metabonomics shifted to patterns typical for proximal tubular damage with reduction of Krebs cycle intermediates and trimethylamine-N-oxide concentrations, whereas acetate, lactate, trimethylamine, and glucose concentrations increased. Again, sirolimus enhanced these negative effects. Our results indicate that cyclosporine and/or sirolimus induce damage of the renal tubular system. This is reflected by urine metabolite patterns, which seem to be more sensitive than currently used clinical kidney function markers such as creatinine concentrations in serum. Metabolic profiling in urine may provide the basis for the development of toxicodynamic monitoring strategies for immunosuppressant nephrotoxicity.

Effects of lovastatin on breast cancer cells: a proteo-metabonomic study

IntroductionStatins are cholesterol-lowering drugs with pleiotropic activities including inhibition of isoprenylation and reduction of signals driving cell proliferation and survival responses.MethodsIn this study we evaluated the effects of lovastatin acid and lactone on breast cancer MDAMB231 and MDAMB468 cells using a combination of proteomic and metabonomic profiling techniques.ResultsLovastatin inhibited proliferation of breast cancer cell lines. MDAMB231 cells were more sensitive to its effects, and in most cases lovastatin acid showed more potency towards the manipulation of protein expression than lovastatin lactone. Increased expression of Rho inhibitor GDI-2 stabilized the non-active Ras homolog gene family member A (RhoA) leading to a decreased expression of its active, membrane-bound form. Its downstream targets cofilin, CDC42 and G3BP1 are members of the GTPase family affected by lovastatin. Our data indicated that lovastatin modulated the E2F1-pathway through the regulation of expression of prohibitin and retinoblastoma (Rb). This subsequently leads to changes of E2F-downstream targets minichromosome maintenance protein 7 (MCM7) and MutS homolog 2 (MSH2). Lovastatin also regulated the AKT-signaling pathway. Increased phosphatase and tensin homolog (PTEN) and decreased DJ-1 expression lead to a down-regulation of the active pAkt. Lovastatins involvement in the AKT-signaling pathway was confirmed by an upregulation of its downstream target, tumor progressor NDRG1. Metabolic consequences to lovastatin exposure included suppression of glycolytic and Krebs cycle activity, and lipid biosynthesis.ConclusionsThe combination of proteomics and metabonomics enabled us to identify several key targets essential to the antitumor activity of lovastatin. Our results imply that lovastatin has the potential to reduce the growth of breast cancer cells.

Toxicodynamic therapeutic drug monitoring of immunosuppressants: promises, reality, and challenges.

Although current immunosuppressive protocols have dramatically decreased acute rejection episodes, there has been less progress in terms of long-term graft survival after kidney transplantation over the last 2 decades. The key to reducing the damage to a transplanted organ as caused by chronic processes is early detection. Modern screening technologies in the fields of genetics, genomics, protein profiling (proteomics), and biochemical profiling (metabolomics) have opened new opportunities for the development of sensitive and specific diagnostic tools. Metabolic profiling appears to be a promising strategy because changes in the cell biochemistry are ultimately responsible for the histologic and pathophysiologic changes of the transplanted kidney and are most likely already detectable before histologic and pathophysiologic changes occur. Using truly no-targeted screening technologies as clinical diagnostic tools is not yet feasible, mostly because of the complexity of the data generated and the lack of algorithms to convert this information into clinically applicable information. A realistic and powerful targeted approach is the development of combinatorial biomarkers. These are biomarker patterns that typically consist of five or more individual parameters. Combined biomarker patterns confer significantly more information than a single measurement and, thus, can be expected to have better specificity and sensitivity. A series of studies in rats and healthy individuals evaluating the effects of immunosuppressants on urine metabolite patterns showed that immunosuppressant-induced changes of metabolite patterns in urine were associated with a combination of changes in glomerular filtration, changes in secretion/absorption by tubulus cells, and changes in kidney cell metabolism. These studies suggested that a combination of biomarkers that can be used for toxicodynamic therapeutic drug monitoring of immunosuppressants should include urine metabolites that constitute valid surrogate markers of these kidney functions.

Metabolic characteristics of imatinib resistance in chronic myeloid leukaemia cells

Background and purpose:  Early detection of resistance development is crucial for imatinib‐based treatment in chronic myeloid leukaemia (CML) patients. We aimed to distinguish metabolic markers of cell resistance to imatinib.

Toxicodynamic effects of ciclosporin are reflected by metabolite profiles in the urine of healthy individuals after a single dose

WHAT IS ALREADY KNOWN ABOUT THE SUBJECT * Ciclosporins nephrotoxicity initially targets the proximal tubule and is, at least in part, driven by increased formation of oxygen radicals. * (1)H-nuclear magnetic resonance spectroscopy (NMR)- and mass spectrometry (MS)-based biochemical profiling (metabolomics) allows for the sensitive detection of metabolite pattern changes in urine. * In systematic studies in rats we showed that ciclosporin caused urine metabolite pattern changes typical for proximal tubule damage and that these pattern changes seemed to be more sensitive than established clinical kidney function markers such as serum creatinine concentrations. WHAT THIS PAPER ADDS * This study showed that urine metabolite pattern changes as assessed by (1)H-NMR and HPLC-MS are sensitive enough to detect the effect of ciclosporin as early as 4 h after a single oral dose. * In our previous rat studies, changes in urine metabolite pattern in response to ciclosporin translated into healthy humans, indicating the involvement of the same toxicodynamic mechanisms. * The results provide proof of concept for further development of this combination molecular marker strategy into diagnostic tools for the detection and monitoring of drug nephrotoxicity. AIMS The immunosuppressant ciclosporin is an efficient prophylaxis against transplant organ rejection but its clinical use is limited by its nephrotoxicity. Our previous systematic studies in the rat indicated urine metabolite pattern changes to be sensitive indicators of the negative effects of ciclosporin on the kidney. To translate these results, we conducted an open label, placebo-controlled, crossover study assessing the time-dependent toxicodynamic effects of a single oral ciclosporin dose (5 mg kg(-1)) on the kidney in 13 healthy individuals. METHODS In plasma and urine samples, ciclosporin and 15-F(2t)-isoprostane concentrations were assessed using HPLC-MS and metabolite profiles using (1)H-NMR spectroscopy. RESULTS The maximum ciclosporin concentrations were 1489 +/- 425 ng ml(-1) (blood) and 2629 +/- 1308 ng ml(-1) (urine). The increase in urinary 15-F(2t)-isoprostane observed 4 h after administration of ciclosporin indicated an increase in oxidative stress. 15-F(2t)-isoprostane concentrations were on average 2.9-fold higher after ciclosporin than after placebo (59.8 +/- 31.2 vs. 20.9 +/- 19.9 pg mg(-1) creatinine, P < 0.02). While there were no conclusive changes in plasma 15-F(2t)-isoprostane concentrations or metabolite patterns, non-targeted metabolome analysis using principal components analysis and partial least square fit analysis revealed significant changes in urine metabolites typically associated with negative effects on proximal tubule cells. The major metabolites that differed between the 4 h urine samples after ciclosporin and placebo were citrate, hippurate, lactate, TMAO, creatinine and phenylalanine. CONCLUSION Changes in urine metabolite patterns as a molecular marker are sufficiently sensitive for the detection of the negative effects of ciclosporin on the kidney after a single oral dose.

Association of immunosuppressant-induced protein changes in the rat kidney with changes in urine metabolite patterns: a proteo-metabonomic study.

The basic mechanisms underlying calcineurin inhibitor (CI) nephrotoxicity and its enhancement by sirolimus are still largely unknown. We investigated the effects of CIs alone and in combination with sirolimus on the renal proteome and correlated these effects with urine metabolite pattern changes. Thirty-six male Wistar rats were assigned to six treatment groups (n = 4/group for proteome analysis and n = 6/group for urine (1)H NMR metabolite pattern analysis): vehicle controls, sirolimus 1 mg/kg/day, cyclosporine 10 mg/kg/day, cyclosporine 10 mg/kg/day + sirolimus 1 mg/kg/day, tacrolimus 1 mg/kg/day, tacrolimus 1 mg/kg/day + sirolimus 1 mg/kg/day. After 28 days, 24 h-urine was collected for (1)H NMR-based metabolic analysis and kidneys were harvested for 2D-gel electrophoresis and histology. Cyclosporine affected the following groups of proteins: calcium homeostasis (regucalcin, calbindin), cytoskeleton (vimentin, caldesmon), response to hypoxia and mitochondrial function (prolyl 4-hydroxylase, proteasome, NADH dehydrogenase), and cell metabolism (kidney aminoacylase, pyruvate dehydrogenase, fructose-1,6-bis phosphate). Several of the changes in protein expression, confirmed by Western blot, were associated with and explained changes in metabolite concentrations in urine. Representative examples are an increase in kidney aminoacylase expression (decrease of hippurate concentrations in urine), up regulation of pyruvate dehydrogenase and fructose-1,6-bisphosphatase, (increased glucose metabolism), and down regulation of arginine/glycine-amidino transferase (most likely due to an increase in creatinine concentrations). Protein changes explained and qualified immunosuppressant-induced metabolite pattern changes in urine.

Irradiation Alters Selection for Oncogenic Mutations in Hematopoietic Progenitors

Exposure to ionizing radiation and other DNA-damaging carcinogens is strongly associated with induction of malignancies. Prevailing paradigms attribute this association to the induction of oncogenic mutations, as the incidence of oncogenic events is thought to limit initiation and progression of cancers. On the other hand, random mutagenic and genotoxic effects of irradiation are likely to alter progenitor cell populations and the microenvironment, thus altering the selective effects of oncogenic mutations. Using competitive bone marrow transplantation experiments in mice, we show that ionizing irradiation leads to a persistent decline in the numbers and fitness of hematopoietic stem cells, in part resulting from persistent induction of reactive oxygen species. Previous irradiation dramatically alters the selective effects of some oncogenic mutations, substantially inhibiting clonal expansion and leukemogenesis driven by Bcr-Abl or activated N-Ras oncogenes but enhancing the selection for and leukemogenesis driven by the activated Notch1 mutant ICN. Irradiation-dependent selection for ICN expression occurs in a hematopoietic stem cell-enriched pool, which should facilitate the accumulation of additional oncogenic events at a committed T-progenitor stage critical for formation of T-lymphocytic leukemia stem cells. Enhancement of ICN-driven selection and leukemogenesis by previous irradiation is in part non-cell autonomous, as partial restoration of normal hematopoiesis can reverse these effects of irradiation. These studies show that irradiation substantially alters the adaptive landscape in hematopoietic progenitors and suggest that the causal link between irradiation and carcinogenesis might involve increased selection for particular oncogenic mutations.

Cyclosporine Induces Endothelial Cell Release of Complement-Activating Microparticles

Defective control of the alternative pathway of complement is an important risk factor for several renal diseases, including atypical hemolytic uremic syndrome. Infections, drugs, pregnancy, and hemodynamic insults can trigger episodes of atypical hemolytic uremic syndrome in susceptible patients. Although the mechanisms linking these clinical events with disease flares are unknown, recent work has revealed that each of these clinical conditions causes cells to release microparticles. We hypothesized that microparticles released from injured endothelial cells promote intrarenal complement activation. Calcineurin inhibitors cause vascular and renal injury and can trigger hemolytic uremic syndrome. Here, we show that endothelial cells exposed to cyclosporine in vitro and in vivo release microparticles that activate the alternative pathway of complement. Cyclosporine-induced microparticles caused injury to bystander endothelial cells and are associated with complement-mediated injury of the kidneys and vasculature in cyclosporine-treated mice. Cyclosporine-induced microparticles did not bind factor H, an alternative pathway regulatory protein present in plasma, explaining their complement-activating phenotype. Finally, we found that in renal transplant patients, the number of endothelial microparticles in plasma increases 2 weeks after starting tacrolimus, and treatment with tacrolimus associated with increased C3 deposition on endothelial microparticles in the plasma of some patients. These results suggest that injury-associated release of endothelial microparticles is an important mechanism by which systemic insults trigger intravascular complement activation and complement-dependent renal diseases.