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NUCLEAR RECEPTORS, ROS & CANCER BIOLOGY

Michelle Chang Ker Xing, BSc (Hon) Research Assistant/Postgraduate student Department of Biochemistry MD7, 10 Kent Ridge Crescent, #01-05A Email: bchckx@nus.edu.sg Major Supervisor: Assoc. Prof. Marie V. Clement Mentor: Dr. Alan Prem Kumar Tel: 6516-3461 Fax: 6779-1453

Down- regulation of Na+/H+ Exchanger 1 gene expression by hydrogen peroxide via activation of caspases: A new pathway involved in the redox inhibition of gene transcription.

Oxidative stress has been implicated in various pathological conditions including cancer. Persistently elevated reactive oxygen species (ROS) has been described to activate other redox sensitive transcription factors, such as NF-kB and AP-1, which act as molecular switches to turn normal cells into premalignant cells. However, very little is known about oxidative repression on the transcription machinery of a survival gene. Our research has provided an insight for such a mechanism through the study of a pH regulator, Na+/H+ exchanger 1 (NHE1) gene expression by non-toxic doses of hydrogen peroxide (H2O2). We have demonstrated that the down-regulation of NHE1 promoter activity and protein expression can be achieved by exposing the cells to 50µM H2O2. This down-regulation was abrogated by the presence of reducing agents: beta- mercaptoethanol and dithiothreitol; as well as pan-caspase inhibitor zVAD-fmk in two different phases, simply an early oxidation phase and a late phase. Unlike beta-mercaptoethanol, the inhibition of caspases was ineffective in rescuing the early phase of NHE1 repression. But interestingly, caspase inhibition was observed after 9 hours of exposure to H2O2 and can completely restored NHE1 promoter activity by 18-24 hours. Taken together, these results provide evidence for the activation of caspases 3 and 6 in the absence of cell death as novel mechanism involved in the redox mediated repression of gene transcription such as NHE1. The next challenge is to investigate how iron aid in the activation of caspases 3 and 6 without the activation of initiator caspases 8 and 9. At the same time, we would like to find out where is caspases 3 and 6 activated in the cell.