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NUCLEAR RECEPTORS, ROS & CANCER BIOLOGY

Dr. Balakrishnan Ramanathan, BSc., MSc., MPhil., Ph.D. Research Fellow Department of Biochemistry # 01-05A, MD7, 10 Kent Ridge Crescent, E-Mail: bchbr@nus.edu.sg; ramanatha8@yahoo.com Supervisor: Assoc. Prof. Marie V. Clement Collaborator: Dr. Alan Prem Kumar

To investigate the mechanism by which an intracellular pro-oxidant state induces survival in tumor cells

Introduction: Tumor cell resistance to apoptosis due to over-expression of Bcl-2 through intracellular production of O2·- (Pervaiz et al. 2001). Tumor cell resistance to apoptosis is due to activation of small GTPase Rac1 (Clement et al. 2003).   Increase in intracellular O2·- inhibits tumor cells sensitivity to apoptotic triggers (Clement et al. 2004).   Low dose of resveratrol inhibits apoptosis due to generation of intracellular O2·- (Ahmed et al. 2004).

The exact mechanism involved in O2·- mediated cell survival is not yet clearly understood. Background: Increase in intracellular O2·- activates the promoter activity of the NHE-1 gene (Akram S, et al. 2005). NHE-1 recruits Ezrin/Radixin/Moesin (ERM) proteins to regulate Akt dependent cell survival (Wu et al. 2004). Objectives: 1)Akt kinase phosphorylation status and Akt kinase activity upon O2·- mediated over-expression of NHE-1.  2) Recruitment of ERM proteins and PI3K activity upon O2·- mediated over-expression of NHE-1. We seek to 3) investigate superoxide-mediated activation Akt through NHE-1 and the recruitment of ERM proteins on survival pathways in tumor cells.

Hypothesis to be tested:

 

To investigate NHE-1 role on pHi changes and TOP2 mediated DNA damage/repair
 Acidic pH acts like a TOP2 poison to induce TOP2-mediated DNA damage:

Acidic pH induces TOP2-dependent DNA damage signals as evidenced by up-regulation of p53 and Ser-139 phosphorylation of H2AX.  Acidic pH-induced cytotoxicity in tumor cells is reduced in TOP2-deficient cells. Acidic pH increases the mutation frequency of the hypoxanthine phosphoribosyl transferase (HPRT) gene in a TOP2-dependent manner. Acidic pH induces reversible TOP2-mediated DNA strand breaks in vitro (Xiao, H. et al. 2003. PNAS. 100; 5205-10). 

We propose to investigate NHE-1 role on intracellular pH changes that either facilitate DNA damage or impair the DNA repair pathways. We wish to use the TOP2 mediated DNA damage as a model to test the hypothesis.