Joanna Głogowska-Ligus

Cell proliferative activity estimated by histone H2B mRNA level correlates with cytogenetic damage induced by radiation in human glioblastoma cell lines

We studied the relationship between proliferative activity and radiation-induced DNA damage in human malignant gliomas in vitro. Nine human glioblastoma established cell lines were γ-irradiated (60Co) over a dose range of 0–10 Gy. H2B and H4 histone mRNA level was assessed with quantitative RT-PCR technique (TaqMan) and histone labeling index (HLI) with in situ hybridization to define proliferation rate, while cytochalasin-block micronucleus assay was performed to measure cytogenetic damage. Micronucleus frequency correlated with H2B mRNA level (Spearman’s R up to 0.82 at 8 Gy), HLI, nuclear division index (NDI) and percentage of binucleated cells (%BNC). There was a high correlation between H2B mRNA level and NDI (R=0.80) as well as %BNC and HLI (R=0.72). Histone H2B and H4 mRNA level (not significant), HLI, NDI, and %BNC (significant) were higher in cell lines sensitive to DNA damage. Proliferative activity correlates with radiation-induced DNA damage in human glioma cell lines. Histone H2B mRNA level and HLI may be a useful molecular predictor of the tumour response to radiation treatment in gliomas of the same histological grade, however the risk of potentially more rapid tumour-cell repopulation must be considered. Presumed protective activity of histones against radiation-induced DNA damage was not confirmed at the transcript level.

Expression analysis of intercellular adhesion molecule-2 (ICAM-2) in the context of classical cardiovascular risk factors in acute coronary syndrome patients

Introduction Cardiopulmonary diseases are the most common cause of hospitalization and death. Often the basic problem is endothelial dysfunction leading to elevated expression of adhesion proteins as well as increased adhesion and aggregation of blood cells. The goal of the study was to assess expression level of intercellular adhesive molecule-2 (ICAM-2) in patients with acute coronary syndrome (ACS). Material and methods The obtained data were analysed in the context of the occurrence of classical cardiovascular risk factors. The two studied groups consisted of 60 ACS patients and 20 healthy individuals who both were qualified based on electrocardiography (ECG), transthoracic echocardiography and biochemical tests. The ACS patients additionally had coronary angiography performed. The number of ICAM-2 gene mRNA molecules was evaluated on the basis of QRT-PCR reaction kinetics. To compare the results the Mann-Whitney U test was used. Results were judged statistically significant if p < 0.05. Results Analysis of the results showed a significantly higher number of ICAM-2 gene mRNA copies in ACS patients compared to healthy subjects (140920 ±105207 and 15023 ±14325, respectively). Furthermore, our results indicate a correlation between obesity (p = 0.012) and positive burdening family history (p = 0.041) and increased ICAM-2 levels in patients with ACS. Conclusions Increased ICAM-2 gene expression in ACS patients is probably symptomatic of endothelium dysfunction and may be responsible for intensified adhesion and aggregation processes as well as for appearance of acute coronary syndrome. These results indicate a correlation between obesity and burdening family history on the one hand, and increased ICAM-2 levels in patients with ACS, on the other.

DNA microarray study of genes differentiating acute myocardial infarction patients from healthy persons

Objective: Using oligonucleotide microarrays HG-U133A, we here studied the expression levels of genes that could differentiate between patients with myocardial infarction (MI) from healthy subjects, as well as to select among such genes those that seem crucial for manifestation of cardiovascular diseases. Materials and methods: The microarray study was conducted using material derived from blood samples collected in 17 individuals. Results: Analysis of gene expression data from 17 microarrays allowed identification of 28 genes strongly differentiating the examined groups. Conclusion: The differentiating genes that we tracked down indicate possible linkage with atherosclerotis and could be a prognostic marker for development of cardiovascular diseases.

Effects of standard treatment on the dynamics of matrix metalloproteinases gene expression in patients with acute coronary syndromes

Inflammation plays a critical role in the pathology of acute coronary syndrome (ACS). Matrix metalloproteinases (MMP)--proteolytic enzymes participating in plaque destabilization--are the crucial effectors of proinflammatory mechanisms leading to plaque rupture. Numerous reports have confirmed the significance of these factors both in circulating blood and locally in the plaque. There is, however, a lack of information on the molecular mechanisms leading to these disturbances, and the effect of standard treatment for ACS on these processes. The aim of the study was to assess the gene expression of MMP-2, -9 and TIMP-2, and the effect of standard treatment on the expression of the studied genes. The study was conducted in 32 patients with ACS and 15 healthy subjects (control group). Monocytes were isolated using Rosette-Sep kits. Gene expression of MMP-2, MMP-9 and TIMP-2 was evaluated on days 1 and 5 in the studied group and once in controls. Total mRNA was extracted from monocytes and the number of mRNA copies was assessed by QRT-PCR. Monocytes of ACS patients present with significantly higher gene expression of MMP-2, -9 and TIMP-2 compared to healthy controls (0.0915 ± 0.037 vs. 0.001 ± 0.0002, p < 0.01; 0.81 ± 0.279 vs. 0.10 ± 0.057, p < 0.05; 0.84 ± 0.140 vs. 0.42 ± 0.126, p < 0.05, respectively). After the 5-day standard treatment, a significant decrease in MMP-2 gene expression was observed. Other studied genes did not show relevant changes during the observation period. No significant correlation was found between classical atherosclerosis risk factors and the expression of the studied genes. Monocytes of ACS patients significantly overexpressed MMP-2, MMP-9 and TIMP-2. Five days of standard treatment resulted in downregulation of the MMP-2 gene. MMP gene overexpression appears to be an independent factor concerning the pathogenesis of ACS.

The transcript expression profile of the leptin receptor-coding gene assayed with the oligonucleotide microarray technique — Could this be an Anorexia nervosa marker?

Anorexia nervosa is a serious eating disorder with the highest mortality rate of any psychiatric disorder. The DSM-IV classification differentiates two AN types: the restricting type (AN-R) and the binge-eating/purging type (AN-BP). Leptin (LEP) levels can be thought of as a signal to the body of its energy reserves. The leptin receptor (including all its mRNA isoforms) is expressed in many tissues. Our aim was to discover the transcript expression profile of the LEP receptor-coding gene in the peripheral blood mononuclears in AN-R and AN-BP patients. Three young women suffering from Anorexia nervosa (one with AN-BP and two with AN-R) took part in the study, along with three non-anorexic subjects as our reference group. LEP receptor gene expression was examined using the oligonucleotide microarray method (HG-U133A, Affymetrix). The results were normalized using RMAExpress. Next, the accumulation analysis method was used (clustering). Hierarchical clustering resulted in three groups of separate clusters. The first group (cluster I) consisted of AN-R patients. The next group (cluster II) consisted of reference group patients suffering from different psychic disorders not related to eating disorders. Cluster III consisted of two patients — the first with AN-BP and the second with an adaptive disorder.

Expression of angiogenesis-related genes in right atrial appendages from patients with advanced myocardial ischemia – preliminary results

Introduction The most frequent cause of myocardial ischemia is atherosclerotic lesions which narrow coronary vessels leading to impaired blood flow or their total occlusion. Material and methods Using HG-U133A oligonucleotide microarrays (Affymetrix) we studied the expression levels of angiogenesis-related genes in patients with different types of heart failure. We analyzed the RNA from right atrial appendages from patients: 1) with advanced coronary disease and myocardial infarction history, 2) with advanced stable coronary disease and no infarction history, and 3) after surgery due to mitral stenosis, but with no coronary vessel lesions. Results Analysis of the data from oligonucleotide microarrays allowed identification of 2 genes (ENG and NPPB) differentiating the examination grups. Conclusions Analysis of the expression profile of genes involved in angiogenesis, carried out using data obtained from examined individuals’ samples, suggests that necrosis accompanying myocardial infarction is a significant factor leading to elevated expression levels of genes involved in neoangiogenesis.

The G16319A substitution frequency in a hemorrhagic stroke

Background: The aim of this paper is to trace the nucleotide alterations within the D-loop region of the mitochondrial DNA, affecting both the mtDNA ability to replicate and the transcription activity of the coding genes located in the H and L threads, in Caucasian patients with an ischemic and hemorrhagic brain strokes. Materials and Methods: The DNA from the peripheral blood of 85 patients with recent sustained ischemic and primary hemorrhagic brain stroke was analysed. The control group consisted of 24 volunteers. The genetic studies were conducted by the PCR method, with the application of the primers for the tRNA-treonine. Results: In the blood samples examined, 3-striatal mtDNA patterns were detected. Pattern-1 is characterised by the C16126T substitution, pattern-2 by the G16319A substitution, and pattern-3 by the C16242T substitution. The frequency of occurrence for the particular mtDNA-1, -2, and -3 patterns, established for the group with an ischemic stroke (77.3, 15.2, and 7.6%), the group with a hemorrhagic stroke (0, 73.7, and 26.3%), and the control group (75, 0, and 25%), differs significantly. Discussion: The exchange of the nucleotides within the D-loop region may affect both the mtDNA replication ability and the transcription activity of the coding genes located in the H and L threads. A hypothesis might be made. The G16319A mutation may result in the formation of lesions within the vascular wall. These lesions have a tendency to form microaneurysms or other defects, which, in turn, will decrease the strength of the vascular wall, making it more susceptible to ruptures. Conclusion: The G16319A substitution may be considered a factor that increases the risk of a hemorrhagic brain stroke.