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Gender specific association of ABCA1 gene R219K variant in coronary disease risk through interactions with serum triglyceride elevation in Turkish adults.

OBJECTIVE ATP binding cassette transporter A1 (ABCA1) controls the reverse cholesterol transport. Some ABCA1 variants are correlated with serum high-density lipoprotein cholesterol (HDL-C) and other lipid concentrations. We aimed to explore the relationship of ABCA1 gene with both the lipid profile and coronary heart disease (CHD) risk. METHODS Selected 627 individuals of the Turkish Adult Risk Factor Study were genotyped for ABCA1 R219K polymorphism using PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method. Students t-test, one-way ANOVA, Chi-square test, linear and logistic regression was used for statistical analysis. RESULTS We demonstrated a gender-specific effect of the R219K polymorphism on plasma lipids and CHD. In men, while homozygosity of the K allele was associated with increased plasma low-density lipoprotein cholesterol (LDL-C) (p<0.05) and total cholesterol concentrations (p<0.05), carriage of this allele was associated with higher HDL-C concentrations (p<0.05) after adjustment for associated risk factors, but not with CHD. In women, however, without being related to HDL-C levels, each 219K allele was associated with 10% higher triglycerides (TG) concentrations (p<0.05). R219K heterozygosity in women independently doubled (95% CI 1.00; 3.80) the odds ratio for CHD risk in regression models, after adjustment for several variables. Interaction of TG elevation (>140 mg/dL) with CHD was demonstrated in female 219RK genotype carriers. CONCLUSION R219 allele of the ABCA1 gene independently confers CHD risk in heterozygote Turkish women, not via reduced HDL-C, but interacting with elevated TG expressed by the 219K allele, but not in men.

The variations of BOP gene in hypertrophic cardiomyopathy

OBJECTIVE The observation that Bop null allele mice show underdeveloped right ventricle and excessive development of left ventricle, suggests the possible relationship between human BOP gene and hypertrophic cardiomyopathy (HCMP). In our study, we investigated this possible relationship between BOP gene variations and QT dispersion, a noninvasive arrhythmic risk marker for HCMP. METHODS This cross-sectional study consisted of 50 patients clinically diagnosed with HCMP and 60 healthy subjects. Exonic regions of BOP gene were amplified by polymerase chain reaction and amplified exonic regions were analyzed by Single-Strand Conformation Polymorphisms (SSCP). The samples with different migration patterns were sequenced through an automated sequencing system. Continuous variables were compared by unpaired t-test for independent samples or Mann-Whitney U test. Genotype-disease relationship was tested by Chi-square test. RESULTS The nucleotide substitutions G275<or=A and C965<or=A in exon 2 and 7 were determined only in HCMP group. The G707<or=C, C710<or=T, T761<or=C, T1217<or=C SNPs in exon 6 and 9 are also found in the control group. Significant differences were found between two groups (p=0.002 and p=0.001). It was found that SNPs in exon 6 constitute a haplotype and that QT dispersion and corrected QT dispersion in the rare homozygote (707C/710T/761C) type carriers of HCMP patients for this haplotype were significantly lower than other genotypes (p=0.032 and p=0.030, respectively). CONCLUSION The human BOP gene was analyzed for the first time in HCMP to investigate possible association. The result that homozygosity of 707C/710T/761C haplotype is associated with lower QT dispersion and corrected QT dispersion supports the modifier role of BOP gene in HCMP.

SET oncogene is upregulated in pediatric acute lymphoblastic leukemia

AIMS AND BACKGROUND The SET gene is a target of chromosomal translocations in acute leukemia and encodes a widely expressed multifunctional phosphoprotein. It has been shown that SET is upregulated in BCR-ABL1-positive cell lines, patient-derived chronic myeloid leukemia CD34-positive cells, and some solid tumors. METHODS AND STUDY DESIGN We determined the expression level of SET in 59 pediatric acute lymphoblastic leukemia patients who were BCR-ABL-negative using quantitative real-time reverse-transcriptase-polymerase chain reaction. Results. We showed that SET expression was significantly upregulated in 96.5% of B-acute lymphoblastic leukemia (28 of 29; 16.6 fold) and 93% of T-acute lymphoblastic leukemia (28 of 30; 47.6 fold) patients. This upregulation was not associated with any clinical features or overall and relapse-free survival. CONCLUSIONS Our results showed that SET is significantly overexpressed in pediatric acute lymphoblastic leukemia samples, and an increased level of SET might contribute to leukemic process.

CYP19A1, MIF and ABCA1 genes are targets of the RORα in monocyte and endothelial cells†

RORα is a member of nuclear receptor superfamily of transcription factors, which has a vital role in the regulation of various physiological processes. Cholesterol is a known ligand of RORα and is one of the key components that take part in cardiovascular diseases such as atherosclerosis. Therefore, it is possible that RORα might have a role in the development of atherosclerosis. To test this hypothesis, we investigated the presence of novel RORα response elements (ROREs) located in the promoter of CYP19A1, MIF and ABCA1 genes. Briefly, the occupancy of RORα in the promoter regions of these genes was demonstrated in THP‐1 and HUVEC cell lines by ChIP analysis. In order to modulate RORα activity, THP‐1 and HUVEC cells were treated with specific RORα ligands (CPG 52608 and SR1001) and then the expression levels of target genes were analysed. In the next step, we tested whether RORα activity in THP‐1 macrophages was influenced by the presence of simvastatin, a cholesterol lowering drug. We found that in the presence of simvastatin the expression of the investigated target genes were down regulated and that this regulation was partially prevented by CPG 52608 and SR1001. Results of this study suggest that CYP19A1, MIF and ABCA1 are the direct target genes of RORα. In conclusion, it is important to demonstrate that certain genes involved in the development of atherosclerosis could be modulated by an inducible transcription factor. Therefore, these results offer a potential therapeutic approach for the treatment of atherosclerosis.

Role of simvastatin and RORα activity in the macrophage apoptotic pathway

Objective: Atherosclerosis is a chronic inflammatory condition and is one of the main causes of death worldwide. Macrophages play important roles in the formation of atherosclerotic plaques. Apoptosis is progressively observed while plaques develop, although the precise mechanisms and outcomes of apoptosis in atherosclerosis development and progression are still contradictory. This study was conducted to explore the effects of simvastatin and retinoic acid receptor-related orphan receptor alpha (RORa) ligands on apoptosis in human acute monocytic leukemia (THP-1) macrophage cells. Methods: Briefly, the occupancy of RORa in the promoter regions of apoptotic pathway genes was demonstrated in THP-1 cell lines using chromatin immunoprecipitation (ChIP) analysis. In order to modulate RORa activity, THP-1 macrophage cells were treated with specific ligands (CPG52608 and SR1001) and then viability as well as count of THP-1 macrophage cells were analyzed. Results: We observed that simvastatin and both RORa ligands had a tendency to decrease THP-1 macrophage cell viability in culture. When compared with non-treated controls, simvastatin significantly decreased cell viability (p=0.04) and cell count (p=0.03). However, this negative effect of simvastatin seemed to be partly prevented by RORa ligands. In addition, bioinformatics analysis of ChIP-on-chip data demonstrated that several genes that are involved in the apoptotic pathway were likely RORa target genes. These genes were involved in the regulation of apoptosis through various pathways. Conclusion: In summary, our study suggest that simvastatin-mediated macrophage apoptosis might be modulated by SR1001 administration. However, involvement of RORa in this modulation through potential apoptotic target genes remains elusive.OBJECTIVE Atherosclerosis is a chronic inflammatory condition and is one of the main causes of death worldwide. Macrophages play important roles in the formation of atherosclerotic plaques. Apoptosis is progressively observed while plaques develop, although the precise mechanisms and outcomes of apoptosis in atherosclerosis development and progression are still contradictory. This study was conducted to explore the effects of simvastatin and retinoic acid receptor-related orphan receptor alpha (RORα) ligands on apoptosis in human acute monocytic leukemia (THP-1) macrophage cells. METHODS Briefly, the occupancy of RORα in the promoter regions of apoptotic pathway genes was demonstrated in THP-1 cell lines using chromatin immunoprecipitation (ChIP) analysis. In order to modulate RORα activity, THP-1 macrophage cells were treated with specific ligands (CPG52608 and SR1001) and then viability as well as count of THP-1 macrophage cells were analyzed. RESULTS We observed that simvastatin and both RORα ligands had a tendency to decrease THP-1 macrophage cell viability in culture. When compared with non-treated controls, simvastatin significantly decreased cell viability (p=0.04) and cell count (p=0.03). However, this negative effect of simvastatin seemed to be partly prevented by RORα ligands. In addition, bioinformatics analysis of ChIP-on-chip data demonstrated that several genes that are involved in the apoptotic pathway were likely RORα target genes. These genes were involved in the regulation of apoptosis through various pathways. CONCLUSION In summary, our study suggest that simvastatin-mediated macrophage apoptosis might be modulated by SR1001 administration. However, involvement of RORα in this modulation through potential apoptotic target genes remains elusive.

Identification of potential target genes of ROR-alpha in THP1 and HUVEC cell lines

Abstract ROR‐alpha is a nuclear receptor, activity of which can be modulated by natural or synthetic ligands. Due to its possible involvement in, and potential therapeutic target for atherosclerosis, we aimed to identify ROR‐alpha target genes in monocytic and endothelial cell lines. We performed chromatin immunoprecipitation (ChIP) followed by tiling array (ChIP‐on‐chip) for ROR‐alpha in monocytic cell line THP1 and endothelial cell line HUVEC. Following bioinformatic analysis of the array data, we tested four candidate genes in terms of dependence of their expression level on ligand‐mediated ROR‐alpha activity, and two of them in terms of promoter occupancy by ROR‐alpha. Bioinformatic analyses of ChIP‐on‐chip data suggested that ROR‐alpha binds to genomic regions near the transcription start site (TSS) of more than 3000 genes in THP1 and HUVEC. Potential ROR‐alpha target genes in both cell types seem to be involved mainly in membrane receptor activity, signal transduction and ion transport. While SPP1 and IKBKA were shown to be direct target genes of ROR‐alpha in THP1 monocytes, inflammation related gene HMOX1 and heat shock protein gene HSPA8 were shown to be potential target genes of ROR‐alpha. Our results suggest that ROR‐alpha may regulate signaling receptor activity, and transmembrane transport activity through its potential target genes. ROR‐alpha seems also to play role in cellular sensitivity to environmental substances like arsenite and chloroprene. Although, the expression analyses have shown that synthetic ROR‐alpha ligands can modulate some of potential ROR‐alpha target genes, functional significance of ligand‐dependent modulation of gene expression needs to be confirmed with further analyses.

High MN1 expression increases the in vitro clonogenic activity of primary mouse B-cells

The MN1 (Meningioma 1) gene is overexpressed in certain subtypes of acute myeloid leukemia (AML) and high levels of MN1 expression in mouse bone marrow cells results in myeloid leukemia. We showed that compared with control bone marrow (BM) MN1 expression was increased (2-fold or more) in 29 out of 73 (40%) pediatric B-cell acute lymphoblastic leukemia (B-ALL) patient BM. Additional analysis of MN1 expression in sub-groups within our cohort carrying different chromosome translocations showed that carriers of the good prognostic marker t(12;21)(TEL-AML1) (n=27) expressed significantly more MN1 than both healthy controls (n=9) (P=0.02) and the group carrying the t(9;22)(BCR-ABL) (n=9) (P=0.001). In addition, AML1 expression was also upregulated in 31 out of 45 (68%) B-ALL patient BM compared with control and there was a significant correlation between MN1 and AML1 expression (r=0.3552, P=0.0167). Retroviral MN1 overexpression increased the colony forming activity of mouse Pro-B/Pre-B cells in vitro. Our results suggest that deregulated MN1 expression contributes to the pathogenesis of pediatric B-ALL. Further investigation into the clinical and biological significance of elevated MN1 expression in TEL-AML1(positive) leukemia might provide insight into additional molecular mechanisms contributing to B-ALL and may lead to improved treatment options for patients.

Sequence variations of NKX2-5 and HAND1 genes in patients with atrial isomerism.

OBJECTIVE Atrial isomerism is a congenital disorder, which is characterized by lateralization defects in normally asymmetrical developing organs like the heart. Atrial isomerism is supposed to be caused by molecular defects during early development. The NKX2-5 is a cardiac specific transcription factor, which initiates and regulates downstream transcriptional cascades of cardiogenesis. The HAND1 is another transcription factor expressed in the heart, and it is characterized by an asymmetrical pattern of expression. In this study, we aimed to test whether mutations in NKX2-5 and HAND1 genes play a role in the etiology of atrial isomerism. METHODS This case-control study consisted of 70 patients who underwent surgical treatment for congenital heart defects including atrial isomerism, 80 healthy subjects (HAND1 gene) and 40 healthy subjects (NKX2-5 gene). All exons and exon-intron boundaries of NKX2-5 and HAND1 genes were analyzed by SSCP, and suspected samples were sequenced for mutation analysis. Digestion with appropriate restriction enzymes was performed for analysis of known mutations and polymorphisms. The frequencies of the alleles and the genotypes were compared among patient and control groups using the Chi-square and the Fisher tests when appropriate. RESULTS In intronic region of HAND1 gene, we identified a C>G substitution both in patients and controls. Frequency of mutant allele (11, 42%) was found higher (p=0.046) in patient group than that of the control group (2.5%). Association between atrial isomerism and genotypes with mutant allele was found borderline significant (p=0.054). In NKX2-5 gene, we identified heterozygous Q170X (Gln170ter) mutation in one patient. We did not found any correlation between defined sequence variations and clinical properties of the patients. CONCLUSION Our results suggest that mutations or sequence variations in HAND1 or NKX2-5 genes may play role in etiology or pathogenesis of atrial isomerism.

Hypoxia Induces Erythropoietin Receptor Expression on K562 Cell Line

ABSTRACT The erythropoietin receptor (EpoR) and erythropoietin (Epo) mediate erythropoietin-induced erythroblast proliferation, differentiation and survival. This study examined the effects of the expression of EpoR on K562 (erythroleukemia) cells upon normoxia and hypoxia. In addition, the impact of the combined effect of recombinant human Epo and hypoxia was investigated. K562 (erythroleukemia) cells and as control group HL60 (promyeloblast) cells were cultured. Hypoxic incubation was performed with 5% O2, 5% CO2 and balance Nitrogen for 24 hours. After 24 hours, K562 and HL60 cells were transferred to normoxic and 5% hypoxic conditions. Recombinant Erythropoietin-alpha was added (10 U/ml) to the cells at the beginning of the experiment. Cultured cells were subjected to viability analysis, total RNA isolation, and protein isolation at three timepoints: 3 h, 6 h, and 24 h. Viability was analysed with a trypan blue exclusion using the Vi-Cell automated cell viability system. RT-PCR and western blot results of normoxic and hypoxic K562 and HL60 cell lines with/without rhEPO were compared. We showed that hypoxia upregulates the expression of EpoR on K562 erythroleukemia cells, and longer exposition to hypoxia turns the upregulated EpoR expression to basal levels. Recombinant human Epo (rhEpo) did not produce further impact in hypoxia-induced upregulation of EpoR neither in normoxia nor in hypoxia. Although addition of the rhEpo caused change in expression of cell-cycle regulators, it seems not to effect cell proliferation or cell viability. In the HL60 cell line, however, we detected EpoR mRNA, but not Epo mRNA by RT-PCR. Hypoxia did not alter the level of EpoR mRNA expression. The results of this study suggest that the expression of EpoR might be regulated by hypoxia, but not by Epo, and the expression of cell-cycle regulators might be regulated by both hypoxia and Epo.