Identification and functional characterization of the human ether-a-go-go-related gene Q738X mutant associated with hereditary long QT syndrome type 2.

QT interval prolongation, a risk factor for arrhythmias, may be associated with genetic variants in genes governing cardiac repolarization. Long QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go­go-related gene (hERG). This gene encodes a voltage-gated potassium channel comprised of 4 subunits, and the formation of functional channels requires the proper assembly of these 4 subunits. In the present study, we investigated the role of the LQT2 mutation, Q738X, which causes truncation of the C-terminus of hERG channels, in the assembly and function of hERG channels. When expressed in HEK293 cells, Q738X did not generate an hERG current. The co-expression of Q738X with wild-type (WT)-hERG did not cause the dominant-negative suppression of the WT-hERG current. Western blot analysis and confocal microscopy revealed that the Q738X mutation caused defective trafficking of hERG channel proteins. Co-immunoprecipitation demonstrated that Q738X did not exhibit dominant-negative effects due to the failure of the mutant and WT subunits to co-assemble. In conclusion, the functional loss caused by the Q738X mutation in hERG K+ channels may be attributed to the disruption of tetrameric assembly.

Blockage of hERG current and the disruption of trafficking as induced by roxithromycin.

Roxithromycin is an oral macrolide antibiotic agent that has been repeatedly reported to provoke excessive prolongation of the Q-T interval and torsades de pointes in clinical settings. To investigate the mechanisms underlying the arrhythmogenic side effects of roxithromycin, we studied the molecular mechanisms of roxithromycin on human ether-à-go-go-related gene (hERG) K(+) channels expressed in human embryonic kidney (HEK293) cells. Roxithromycin was found to inhibit wild-type (WT) hERG currents in a concentration-dependent manner with a half-maximum block concentration (IC50) of 55.8 ± 9.1 µmol/L. S6 residue hERG mutants (Y652A and F656C) showed reduced levels of hERG current blockage attributable to roxithromycin. Roxithromycin also inhibited the trafficking of hERG protein to the cell membrane, as confirmed by Western blot analysis and confocal microscopy. These findings indicate that roxithromycin may cause acquired long-QT syndrome via direct inhibition of hERG current and by disruption of hERG protein trafficking. Mutations in drug-binding sites (Y652A or F656C) of the hERG channel were found to attenuate hERG current blockage by roxithromycin, but did not significantly alter the disruption of trafficking.

Lack of association between SLCO1B1 polymorphism and the lipid-lowering effects of atorvastatin and simvastatin in Chinese individuals.

PURPOSE: There is significant inter-individual variability in the lipid-lowering effects of atorvastatin and simvastatin. Our goal was to investigate the impact of SLCO1B1 genetic polymorphism on the lipid-lowering effects of atorvastatin and simvastatin. METHODS: We recruited 363 unrelated hyperlipidemic patients with the CYP3A4 1/1, CYP3A5 1/1, and CYP3AP1 1/1 genotypes: 189 of these were treated with atorvastatin and 174 were treated with simvastatin as a single-agent therapy (20 mg day(-1) orally) for 4 weeks. The genotyping of SLCO1B1 c.521T > C (p.V174A, OATP-C5) was performed with allele-specific polymerase chain reaction (AS-PCR), and PCR restriction fragment length polymorphism (RFLP) was performed to detect the carriers of SLCO1B1 c.388A > G (p.N130D, OATP-C1b). Serum triglyceride (TGs), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels were determined before and after treatment. RESULTS: The frequencies of the SLCO1B1 521T > C and 388A > G variant alleles in Chinese hyperlipidemic patients were found to be 16.2% and 72.1% respectively. After treatment with 20 mg simvastatin or atorvastatin daily for 4 weeks, TC, TG, and LDL-C concentrations were lower than at baseline, on average, by 18.1 ± 3.7%, 25.8 ± 9.7%, 27.7 ± 5.4% in the simvastatin-treated group, and 17.5 ± 3.7%, 22.6 ± 8.6%, 27.5 ± 5.5% in the atorvastatin-treated group respectively, and the mean relative reduction in serum HDL cholesterol did not reach statistical significance (-1.0 ± 10.9%, 0.5 ± 9.3%). However, no significant differences were observed in the lipid-lowering effects of atorvastatin and simvastatin between subjects with different SLCO1B1 genotypes. CONCLUSION: The SLCO1B1 521T > C and 388A > G variants were found to be relatively common in Chinese patients with essential hyperlipidemia. These frequencies were found to be similar to those observed in healthy Chinese and Japanese individuals, but significantly different from Caucasians and blacks. SLCO1B1 521T > C and 388A > G polymorphisms may not be associated with the lipid-lowering effects of atorvastatin and simvastatin.

Epigenetic inactivation of Wnt inhibitory factor-1 in human esophageal squamous cell carcinoma.

Wnt inhibitory factor-1 (WIF1), as one of most important Wnt antagonists, has been detected frequently silenced by promoter hypermethylation in various types of cancer. In this study, we aimed to investigate the promoter methylation profiles of WIF1 in human esophageal squamous cell carcinoma (ESCC) tissues and cell lines, as well as the functional roles of WIF1 in the human ESCC metastatic behavior. WIF1 mRNA levels and promoter methylation status in ESCC tissues and cell lines were detected using RT-PCR and methylation-specific PCR (MS-PCR), respectively. WIF1 protein levels were assessed by Western blot. Stable ESCC cell line with restoration of WIF1 was generated in EC109 cells, which naturally do not express detectable WIF1 mRNA. The effects of reexpressed WIF1 on EC109 cell proliferation and migration were investigated using crystal violet and wound healing assay, respectively. Also the effects of WIF1 reexpression on the beta-catenin/T-cell factor-dependent transcription activity was measured by luciferase assay. WIF1 promoter methylation was frequently observed in ESCC tissues (46%, 23/50) and cell lines (50%, 2/4). Treatment with demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), increased or restored WIF1 expression in these ESCC cell lines. Restoration of the WIF1 in EC109 cells resulted in a significant inhibition on both cell proliferation and migration. Moreover, reexpression of WIF1 caused significant decrease of beta-catenin/T-cell factor-dependent transcription activity. These findings demonstrated that WIF1 silencing due to promoter hypermethylation is a major mechanism during carcinogenesis of ESCC. This would be an opportunity to prevent the development and progression of HCC through modulation of WIF1.

[Construction of a siRNA vector targeting human MTA1 gene and the gene-silencing effect].

OBJECTIVE: To construct an expression vector of siRNA targeting human MTA1 gene and observe its gene-silencing effect in esophageal carcinoma cells. METHODS: The siRNA sequences targeting MTA1 gene were designed and synthesized with two complementary oligonucleotide strands. The oligonucleotide strands were annealed and recombined into pRNAT-U6.2 vector, which was identified by sequencing following transformation and amplification. The siRNA expression vector pRNAT-U6.2-MTA1 was transfected into human esophageal carcinoma EC9706 cells via liposome. RT-PCR and Western blotting were used to detect expression levels of MTA1 mRNA and protein in the transfected EC9706 cells, respectively. RESULTS: The double-stranded oligonucleotide fragments of the siRNA targeting MTA1 gene were cloned into pRNAT-U6.2 vector, which was validated by sequence analysis. RT-PCR and Western blotting indicated that MTA1 mRNA and protein expressions were significantly decreased in the transfected cells, especially in those transfected with the siRNA targeting the sequence of GACCCTGCTGGCAGATAAA (481-499), which induced almost complete silencing of MTA1 protein expression. CONCLUSION: The siRNA expression vector pRNAT-U6.2-MTA1 for silencing MTA1 gene expression in the esophageal carcinoma cells has been successfully constructed, which may facilitate further study for decreasing the invasive and metastatic potentials of malignant tumors by MTA1 gene silencing.

Endoreduplication of human smooth muscle cells induced by 2-methoxyestradiol: a role for cyclin-dependent kinase 2.

Endoreduplication has been suggested to contribute to the development of hypertrophy of smooth muscle cells (SMCs) in hypertension. However, endoreduplication in vascular SMCs and the underlying molecular mechanisms are not clear. Treatment of human SMCs with 10 microM 2-methoxyestradiol (2-ME) for 24 h induces accumulation of cells with > or =4N DNA content, and some polyploid/aneuploid cells actively synthesize their DNA, suggesting the occurrence of endoreduplication. In addition, 2-ME treatment upregulates the expression of cyclin-dependent kinase 2 (Cdk2). The present study was designed to characterize endoreduplication of human SMCs and explore the potential roles of Cdk2 in endoreduplication induced by 2-ME. Treatment with 2-ME (10 microM) for 2-4 days not only caused increases in >4N cells and their reentry into S phase but also induced overduplication of chromosomes. Furthermore, 2-ME increased the kinase activity of Cdk2 and its interaction with cyclin E. Inducible overexpression of dominant-negative Cdk2 in human SMCs inhibited both DNA synthesis of >4N cells and the accumulation of >4N cells induced by 2-ME. We conclude that 2-ME induces endoreduplication of human SMCs and Cdk2 plays an important role in endoreduplication in response to 2-ME.