Irena Bubko

A study of molecular changes relating to energy metabolism and cellular stress in people with Huntington’s disease: looking for biomarkers

Huntington’s disease (HD) is a neurodegenerative disorder characterized by a progressive motor and cognitive decline and the development of psychiatric symptoms. The origin of molecular and biochemical disturbances in HD is a mutation in the HTT gene, which is autosomally dominantly inherited. The altered huntingtin protein is ubiquitously expressed in the CNS, as well as in peripheral tissues. In this study we measured the metabolism changes in gene transcription in blood of HD gene carriers (premanifest and manifest combined) versus 28 healthy controls. The comparison revealed statistically significant Global Pattern Recognition Fold Change (FC) for 6 mRNA transcripts, reflecting an increase of: MAOB (FC = 3.07; p = 0.0005) which encodes an outer mitochondrial membrane-bound enzyme called monoamine oxidase type B; TGM2 (FC = 1.8; p = 0.02) encoding a transglutaminase 2 that mediates cellular stress; SLC2A4 (FC = 1.64; p = 0.02) solute carrier family 2 (facilitated glucose transporter) member 4; branched chain ketoacid dehydrogenase kinase (BCKDK) (FC = 1.34; p = 0.02); decrease of LDHA (FC = −1.16; p = 0.03) lactate dehydrogenase A; and brain-derived neurotrophic factor (BDNF) (FC = −2,11; p = 0.03). These distinguished changes coincided with HD progress. The analyses of gene transcription levels in sub-cohorts confirmed these changes and also revealed 28 statistically significant FCs of gene transcripts involved in ATP production and BCAA metabolism.

Effect of structural modification at the 4, 3', and 2' positions of doxorubicin on topoisomerase II poisoning, apoptosis, and cytotoxicity in human melanoma cells.

Abstract.Introduction:The mechanism of the cytotoxicity of anthracyclines is pleiotropic and its significance in cell growth inhibition seems to be highly specific and dependent on cell type and anthracycline drug. Resistance and the high cardiotoxicity of anthracyclines have stimulated many studies aimed at identifying critical substituents required for optimal activity. Many authors point to the fact that the double-strand breaks, the consequence of the activity of topoisomerase II poisons, and the inability of cells to repair the DNA lesions are the signal for apoptosis. The aim of this study was to define the influence of 4-demetoxy 2′-halogenated analogs with altered basicity at the 3′-position on topoisomerase II and the relationship of that interaction with apoptosis and the cytotoxicity of these novel anthracyclines. Parental human ME18 melanoma cells and the ME18/R subline, obtained experimentally, resistant to doxorubicin (DOX), exposed to 1.7 and 8.6 µM DOX or its analogs, annamycin and WP903 (both 0.3 and 3.0 µM) were studied.Materials and Methods:The MTT test was used to assay cytotoxicity. Interaction of the drugs with topoisomerase II and apoptosis were done by Western blot and fluorescence microscopy using Hoechst 33342.Results:The structural changes at positions 4, 2′, and 3′ can influence topoisomerase II interaction and apoptotic activity, although correlation between these events and cytotoxic consequences has not been proved.Conclusions:The biological response of the cells to the structurally similar anthracyclines may be variable and probably depends on the cell type which seems to be an additional problem in the multifactorial resistance of tumor cells to anthracyclines.

Up-regulation of glutathione-related genes, enzyme activities and transport proteins in human cervical cancer cells treated with doxorubicin.

Doxorubicin (DOX), one of the most effective anticancer drugs, acts in a variety of ways including DNA damage, enzyme inhibition and generation of reactive oxygen species. Glutathione (GSH) and glutathione-related enzymes including: glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferases (GST) may play a role in adaptive detoxification processes in response to the oxidative stress, thus contributing to drug resistance phenotype. In this study, we investigated effects of DOX treatment on expression and activity of GSH-related enzymes and multidrug resistance-associated proteins in cultured human cervical cancer cells displaying different resistance against this drug (HeLa and KB-V1). Determination of expression level of genes encoding GST isoforms and MRP proteins (GCS, GPX, GSR, GSTA1-3, GSTM1, GSTP1, ABCC1-3, MGST1-3) was performed using StellARray™ Technology. Enzymatic activities of GPX and GSR were measured using biochemical methods. Expression of MRP1 was examined by immunofluorescence microscopy. This study showed that native expression levels of GSTM1 and GSTA3 were markedly higher in KB-V1 cells (2000-fold and 200-fold) compared to HeLa cells. Resistant cells have also shown significantly elevated expression of GSTA1 and GSTA2 genes (200-fold and 50-fold) as a result of DOX treatment. In HeLa cells, exposure to DOX increased expression of all genes: GSTM1 (7-fold) and GSTA1-3 (550-fold, 150-fold and 300-fold). Exposure to DOX led to the slight increase of GCS expression as well as GPX activity in KB-V1 cells, while in HeLa cells it did not. Expression of ABCC1 (MRP1) was not increased in any of the tested cell lines. Our results indicate that expression of GSTM1 and GSTA1-3 genes is up-regulated by DOX treatment and suggest that activity of these genes may be associated with drug resistance of the tested cells. At the same time, involvement of MRP1 in DOX resistance in the given experimental conditions is unlikely.

Role of thiamine in Huntington’s disease pathogenesis: In vitro studies

BACKGROUND Oxidative stress accompanies neurodegeneration and also causes abnormalities in thiaminedependent processes. These processes have been reported to be diminished in the brains of patients with several neurodegenerative diseases. OBJECTIVES The aim of this work was to conduct a comparative analysis of the impact of supplemented thiamine on the viability of human B lymphocytes with CAG abnormal expanded huntingtin gene (mHTT) (GM13509) and control, B lymphocytes without mHTT (GM14467) through the following studies: determination of the supplemented thiamine concentrations, which are effective for cell growth stimulation after incubation in thiamine deficit conditions; determination of cell capability to intake the exogenous thiamine; evaluation of exogenous thiamine influence on the profile of the genes related to thiamine and energy metabolism; determination of ATP synthesis and activities of thiamine-dependent enzymes, KGDHC and BCKDHC in the intact cells and upon the exogenous thiamine. MATERIAL AND METHODS The following methods were used: EZ4U test for cell growth analysis; HPLC for determination of thiamine intake and ATP synthesis, qRT-PCR for evaluation of the gene profiles and spectrophotometric method for KGDHC and BCKDHC activities determination. RESULTS Maximal cell growth stimulation was observed at 2.5 mM in GM14467 up to 135% of the control culture and at 5.0 mM in GM13509 cells up to 165% of the control culture. Native levels of total ATP and KGDHC and BCKDHC activities in both cell types were comparable and did not changed upon thiamine deficit or supplementation. GM13509 cells showed more of an increase in growth stimulation upon thiamine supplementation than GM14467 cells and this effect was reflected in the increase of intracellular thiamine concentration. CONCLUSIONS The above results and reported changes in expression of GAPDH, IDH1 and SLC19A3 genes observed upon thiamine deficit conditions suggest that intracellular thiamine status and energy metabolism can have a role in HD pathogenesis.

B23 Influence of mycoplasma arginini on inflammatory response and energy metabolism in human cells with and without HTT gene mutation

Background Mycoplasma sp. include the self-replicating bacteria with the smallest genome and cell wall deficiency which need cholesterol for growth, glucose or different metabolites from the host cells. Mycoplasmas can target leukocytes and reach the central nervous system through the blood- brain barrier. Up to now any influence of Mycoplasma on function of cells with HTT mutation is unknown. The aim of the study was to evaluate influence of M. arginini contamination on transcription level of selected 94 genes related to inflammatory response and energy metabolism in human B-lymphoid cells with and without HTT mutation. Material and methods EBV – immortalised human B-lymphoid cells: GM13509 with 70 CAG repeats in HTT and GM14467 unmutated (control) were co-cultured with bacteria (1:1000 for human cell:bacterial cell number ratio) for 2 weeks. Monitoring of the M. arginini, EBV copies and CD19 antigen were measured by qPCR and flow cytometry, respectively. Results Spectacular transcript changes after M. arginini contamination were noted in GM13509 as compared to control cells after the first 72 h of incubation, ie inflammatory response gene transcripts significant decrease, especially of CD36 (from FC = 7.91 in control to −97.86 in mutated cells) and similarly of IL8 (141.26; −244), CBS (86.55; 21.68) and AQP9 (58.66; 2.59). The most increase of SLC1A2 (122.89; 143.20) transcript related to energy metabolism was discerned. However, Antigen CD19 presentation and the EBV copy number in both contaminated cell lines were reduced more effectively than in uncontaminated cells. More increase of M. arginini amount during 2-week co-culture was observed in cells with HTT mutation. Conclusions M. arginini can modulate inflammatory response and energy metabolism on the transcription level in B-lymphoid cells with HTT mutation.

F09 The study on molecular changes related to energy metabolism in Huntington's disease subjects: looking for biomarkers

Huntingtons disease (HD) is a neurodegenerative disorder characterised by a progressive motor and cognitive decline and psychiatric symptoms. The origin of molecular and biochemical disturbances in HD is the genetic defect in the HTT gene, autosomally dominantly inherited. The altered huntingtin protein is ubiquitously expressed in the Central Nervous System as well as in peripheral tissues. The aim of the study was to detect the metabolism changes on the transcription level in blood cells in HD subjects (n=29) with regards to the control, healthy subjects (n=28). The gene expression analysis was performed using quantitative PCR and StellAarray system. A panel of 94 genes involved in glycolysis, Krebs cycle, electron transport chain, BCAAs metabolism and other processes were selected to study. The results calculations were conducted by Global Pattern Recognition software with ΔΔCt type of analysis application. The comparison between HD and control revealed statistically significant Global Pattern Recognition fold change (FC) values for six transcripts, including a decrease of BDNF (−2.11), LDHA (−1.16) and an increase of BCKDK (1.34), MAOB (3.07), SLC2A4 (1.64) and TGM2 (1.8). Multiple analyses in sub-cohorts distinguished by gender, age, BMI, calf circumference, nutrition status, CAG number repeats, TFC, UHDRS motor and HD time from onset also showed 34 transcript FCs. The widest FC ranges decrease concerned to BDNF (from −1.66 to −3.6), BCAT2 (from 1.85 to −1.42) and increase to MAOB (from 1.87 to 7.89) transcript levels that coincided together with the length of HD duration period and the HD progress.