Apoptosis-related gene expression in glioblastoma (LN-18) and medulloblastoma (Daoy) cell lines.

The expression of apoptosis genes in a commercial pre-designed low-density array from Applied Biosystems was evaluated in two human brain cancer cell models, LN-18 and Daoy (HTB-186™) in comparison to the reference human primary endothelial cells under basic conditions. Analysis of the gene expression in the cancer cell lines compared to the normal control revealed features reflecting anti-apoptotic and inflammatory characteristics of the former. There was an overall downregulation of apoptosis-stimulating genes in both cancer cell lines, along with an upregulation of certain apoptosis inhibitors. A number of genes demonstrated statistically significant changes in their expressions, including BAX (BCL2-associated X protein); the CARD4/NLR family, CARD domain containing 4; CASP10 (caspase 10, apoptosis-related cysteine peptidase); DAP1 (death-associated protein kinase 1), and BIRC5 (baculoviral IAP repeat-containing 5). Anti-apoptotic potential in both cell lines was demonstrated by changes in the Bax:Bcl-2 ratio and downregulation of the APAF1 gene in LN18 cells. There was also significant downregulation of extrinsic signals and the TNF/FADD/inflammatory cascade, and upregulation of caspase inhibitors (IAPs). These results provided a novel molecular characterization of important human cancer cell lines, which might provide a useful research tool for investigating the experimental model of the CNS cell.

The CpG island methylation regulated expression of endothelial proangiogenic genes in response to ß-carotene and arachidonic acid.

ß-carotene (BC) and arachidonic acid (AA) were demonstrated to modulate carcinogenesis by influencing angiogenesis. DNA methylation is an epigenetic mechanism regulating gene expression. The aim of this study was to investigate whether BC and AA change DNA methylation and expression of the proangiogenic genes, which might help explain their impact on carcinogenesis. Human umbilical vein endothelial cells (HUVECs) and endothelial progenitors (EPCs) were incubated with BC or AA for 24 h. Based on microarray results, we selected 18 genes for DNA methylation analysis. CpG island methylation was quantified using the combined bisulphite restriction analysis method and methylation sensitive restriction enzymes. Relative gene expression was quantified using a quantitative real-time PCR (qRT-PCR) method. Incubation with AA significantly decreased methylation of the promoters of both KDR (P = 0.048) and Notch4 (P = 0.027) genes in HUVECs. In EPCs, BC increased methylation of the connexin 43 gene (P = 0.036). qRT-PCR showed that AA (P = 0.059) and BC (P = 0.044) upregulated the KDR gene expression in HUVECs. Connexin 43 gene expression was induced in the presence of 1 µM (P = 0.039) and 3 µM (P = 0.043) BC in EPCs. No significant changes in the Notch4 gene expression were found. The impact of BC and AA on carcinogenesis may be due, at least in part, to changes in expression of angiogenic genes and these transcriptional effects may be mediated by changes in methylation of CpG islands in the gene promoters. However more research is necessary to confirm this hypothesis.

Phospholamban gene mutations are not associated with hypertrophic cardiomyopathy in patients from southern Poland.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a genetic disease. The role of phospholamban (PLN) gene mutations is the development of HCM has not been established. AIM: To screen for PLN gene mutations in a group of HCM patients from the southern Poland. METHODS: We included 50 consecutive patients (31 males, mean age 42 ± 14 years) diagnosed with HCM on the basis of typical clinical, echocardiographic, and haemodynamic features. The control group consisted of 50 (sex-, age-matched) healthy subjects with normal echocardiograms. RESULTS: The genetic analysis was focused on R9C mutation with the ability to block PLN phosphorylation leading to chronic inhibition of SERCA2a activity. Another analysed mutation causing the alteration of PLN level in cells was related to the substitution of a leucine residue at position 39 with a premature stop codon (L39X). The sequence analysis of selected coding regions of the PLN gene did not show the presence of mutations in either the patients or the control subpopulations. CONCLUSIONS: Systematic mutation screening did not reveal any mutation in the selected regions of the PLN gene. Additionally, no polymorphisms were detected in any patients. Therefore, PLN gene mutations were not found to be associated with HCM in the study group.

[Role of the M235T (c.704c>T) polymorphism of angiotensynogen gene as well as A724A (c.2171G>A) polymorphism of SERCA2a gene in ethiopathogenesis of left ventricular hypertrophy in essential hypertension]. / Rola polimorfizmu M235T (c.704c>T) genu dla angiotensynogenu oraz A724A (c.2171G>A) genu dla SERCA2a w etiopatogenezie przerostu miesnia lewej komory serca w pierwotnym nadcisnieniu tetniczym.

Left ventricle hypertrophy (LVH) is main organ complication developing in the course of primary hypertension. Among 'candidates genes' related with development of hypertension as well as LVH; the promoting, but not crucial, influence of (c.704C>T) angiotensinogen (AGT) gene was found. The elevated calcium ions concentration in the cytosol of muscle cells, might be one of the element in the ethiopatomechanism of essential hypertension development. The ATP-related ions pomp--SERCA2A regulates the intracellular calcium concentration. Mutations in the ATP2A2 gene coding the SERCA2A protein, has been associated with elevated calcium level in cardiomyocytes. The aim of the study was to analyze frequency of the M235T (c.704C>T) AGT gene polymorphism. The new mutations in the ATP2A2 gene was searched for in hypertensive patients, independently to the LVH presence in compare to the control group. 157 people participated in the study. Based on the echo-cardiographic examination participants were divided into subgroups: patients with essential hypertension (NT) and patients with NT and LVH. 50 healthy volunteers served as the control group. The frequency of the CC homozygotes, of the M235T (c.704C>T) AGT gene polymorphism, was the highest in the patients with essential hypertension and LVH in compare with patients without LVH (p = 0.67) and control group (p = 0.64). The value of the LVMI was the highest in CC carriers in compare to homozygotes TT (p = 0.33) and CT group (p = 0.66). In homozygotes TT as well as in the carriers of allel T, the elevated blood pressures value was detected. In the exon 15 of ATPA2A gene the new polymorphism A724A (C.2171G>A) was found. only the presence of GG and heterozygotes GA was detected in analyzed group. The frequency of GA genotype was significantly higher in control group vs patients with essential hypertension with (p = 0.05)/or without LVH (p = 0.04). The GA carriers had lower blood pressures values measured in doctor office as well as using ABPM method. The LVM as well as LVMI values were lower in group with mutated genotype GA in compare to GG group (p = 0.107 for LVM; p = 0.154 for LVMI). Results suggest a protective role of the c.2171G>A polymorphism of the ATP2A2 gene against the hypertension as well as LVH development. It seems also that c.704C>T polymorphism of AGT gene does not play crucial role in the essential hypertension development.

beta-Carotene stimulates chemotaxis of human endothelial progenitor cells.

Angiogenesis is a crucial process in tissue remodeling during growth, both in the embryo and the adult. In our study we concentrated on the direct effect of beta-carotene on human umbilical cord originating from endothelial progenitor cells (EPCs). beta-Carotene uptake by EPCs was measured using a HPLC method. The determination of cell surface antigens was performed by flow cytometry. The effect on cell proliferation was estimated by measuring bromo-deoxyuridine incorporation. The influence on the formation of a tubular-like structure was investigated in a 3D assay in matrigel. Quantitative gene expression was estimated using real-time PCR. We demonstrated that beta-carotene in the physiological range of concentrations found in human blood is a potent activator of EPC chemotaxis, which is accompanied by a change in the expression of genes mediating cell adhesion and homing, but does not activate the final markers of endothelial differentiation. This study points to the prochemotactic and homing activity of beta-carotene in undifferentiated endothelial cell progenitors for the first time, which may suggest a potential role of this carotenoid in progenitor cell therapy aimed at angiogenesis and tissue repair.