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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 etfier-a-go-go-related gene (hERG)[K.sup.+] 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 ([IC.sub.50]) 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. Key words: roxithromycin, Q-T interval, hERG channel, HEK293 cells, long-QT syndrome, hERG protein trafficking, hERG mutants. La roxithromycine est antibiotique de la classe des macrolides qui, selon de nombreux rapports, provoque une prolongation excessive de l'intervalle Q-T et des torsades de pointe en contexte clinique. Afin d'examiner les mecanismes sous-jacents aux effets secondaires arythmogenes de la roxithromycine, nous avons etudie les mecanismes moleculaires de la roxithromycine sur le gene codant le canal potassique hERG (ether-a-go-go- related gene) exprime chez les cellules de rein embyronnaire HEK293. La roxithromycine inhibait les courants du hERG sauvage en fonction de la concentration, avec une [IC.sub.50] de 55,8 ± 9,1 µmol/L. L'inhibition du courant par la roxithromycine etait reduite chez les mutants du segment S6 du hERG (Y652A et F656C). La roxithromycine inhibait aussi le trafic de hERG a la membrane cellulaire, comme l'ont confirme le buvardage Western et la microscopie confocale. Ces donnees indiquent que la roxithromycine peut provoquer un syndrome du QT long au moyen de l'inhibition directe du courant du hERG et par la perturbation du trafic de hERG. Des mutations des sites de liaison du medicament (Y652A ou F656C) du canal hERG attenuent le blocage du courant de hERG par la roxithromycine mais n'affectent pas la perturbation du trafic. [Traduit par la Redaction] Mots-cles: roxithromycine, intervalle Q-T, canal hERG, cellules HEK293, syndrome du QT long, trafic de hERG, mutants du hERG., Introduction Acquired long-QT syndrome (LQTS) is associated with many clinically available drugs and developmental compounds (Mitcheson et al. 2000). Most drugs that can cause acquired LQTS do so through direct [...]

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 [micro]M 2-methoxyestradiol (2-ME) for 24 h induces accumulation of cells with [greater than or equal to] 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 [micro]M) 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. estrogen metabolite; polyploidization; cell cycle; laser scanning; cytometry; microtubule-interfering agents

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.