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NUCLEAR RECEPTORS, ROS & CANCER BIOLOGY

CHARIS CHUA SUI HUAY Dip.(Biotech), BSc.(Hon) NGS Postgraduate student Department of Biochemistry MD11, 10 Medical Drive, #B1-07 Email: g0601791@nus.edu.sg Major Supervisor: Assoc. Prof. Marie V. Clement Co-supervisor: Dr. Alan Prem Kumar Tel: (65) 6516-4445 Fax: (65) 6773-5461

Evidences are emerging that at non-toxic doses, hydrogen peroxide (H2O2) plays an important role in cellular signaling and gene regulation. Our lab has showed that H2O2 could repress Na+/H+ exchanger 1 (NHE1) gene expression through an iron dependent but OH∙ independent pathway as iron chelators, deferioxamine and O-phenanthroline, but not OH∙ chelator, sodium formate could attenuate H2O2’s actions. The down-regulation of NHE1 promoter is abrogated by pre-treating cells with reducing agents, dithiothreitol and beta mecaptoethanol; this indicates that H2O2 induces reversible oxidation of NHE1’s transcription factor(s). Sequential deletion of mouse NHE1 promoter revealed that the stretch between 120 and 150 base-pair of the proximal end is important for repressive actions of H2O2. Using bioinformatics analysis we found a potential AP2 binding site within the 30 base-pair region and over-expressing dominant negative AP2 represses NHE1 promoter activity, mRNA and protein expression. Conversely, over-expression of active AP2 protein increases NHE1 expression in L6 rat myoblast and human breast epithelial MCF10A. The delta isoform of AP2 was found highly expressed in L6 cells and could bind to the 30 base-pair region of NHE1 promoter. In addition to undergoing reversible oxidation, AP2 delta protein level decreases upon H2O2 treatment. Taken together these findings suggest that in L6 cells AP2 delta maybe the transcription factor involved in the inhibition of NHE1 expression by non-toxic dose of H2O2.

It has been shown blocking NF-kB activation leads to exaggerated accumulation of H2O2 following stimulation of TNF-Rs and that suppression of this accumulation virtually abrogates TNFα-induced toxicity in NF-KB-deficient cells. We showed that H2O2 down-regulates NHE1 expression, induces intracellular acidification and increases cells’ sensitivity to apoptosis. Hence, this proposal is to study the role NHE1 gene expression in TNFα-mediated cell death and to determine the role NF-KB partakes in this regulation. Preliminary results showed that TNFα has cytostatic effects on NF-KB p65KO but not wild type NIH3T3 cells. NHE1 was found to be downregulated both at the protein and mRNA level following TNFα treatment. Silencing of NHE1 expression resulted in increasing cells’ sensitivity to TNFα mediated growth inhibition. In the present study, we will first 1) Confirm the effects of TNFα on NHE-1 gene and protein expression in wild type and p65KO NIH3T3. An ongoing research demonstrated H2O2 mediated down-regulation of NHE1 to be caspasedependent. Therefore, here we will 2) Assess the role of ROS and caspases in TNFα-mediated down-regulation of NHE-1 expression. Since we have previously shown a dependence on iron for H2O2 mediated repression of NHE1, we will 3) Assess if H2O2-dependent NHE1 repression in p65KO NIH3T3 cells is dependent on an iron-dependent activation of caspase 3 or/and caspase 6. In addition, we will also 4) Assess the effect of NHE-1 down-regulation on intracellular pH upon exposure of p65KO NIH3T3 cells to TNFα and 5) Determine the role of NHE-1 protein down-regulation in TNFα regulation of p65KO NIH3T3 cell growth and induction of apoptotic cell death. Finally, in view of reports linking up-regulation of NHE-1 gene expression in a variety of tumor cells, we will 6) Determine if silencing p65 in tumor cells increase cells’ sensitivity to TNFα and if this is a result of NHE-1 protein down-regulation due to increased ROS. More importantly, these results will provide novel insight into intracellular pathways that could be used as targets for enhancing the sensitivity of cancer cells to TNFα therapy and overcoming the problem of severe toxicities from having to use high pharmacologic doses of TNFα, which is a problem today.

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