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Journal Club May09

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Journal Club May09

  1. 1.
  2. 2. Background<br />HDAC inhibitors alter gene expression<br />Show potent anti-tumour activities<br />Possible use as chemotherapeutic agents<br />HAT<br />HDAC<br />
  3. 3. However...<br />HDI Apicidin shown to induce P-glycoprotein<br />P-gp = Integral membrane protein involved in drug efflux and potentially multidrug resistance<br />Aim<br />Elucidate mechanism of Apicidin induced P-gp expression<br />
  4. 4. Induction of P-gp in cancer cell lines by Apicidin<br />Apicidin (1mM) induces P-gp in a cell type specific manner<br /><ul><li>Apicidin induces P-gp promoter activity in a context independent manner
  5. 5. P-gp induction notApicidin specific</li></li></ul><li>P-gp induction and CpG methylation<br />Apicidin treatment does not affect methylation status of P-gp in HeLa or KB cell lines <br /><ul><li>No significant effect of AzadCpretreatment on P-gp activity</li></li></ul><li>Effect of apicidin on histone H3 acetylation<br />Accumulation of acetylated histone H3 in both responsive and non-responsive cell lines in a dose dependant manner<br />
  6. 6. Apicidin induced P-gp promoter activity is SP1 dependant<br />Mithramycin, inhibitor of SP1 binding abolished Apicidin induced expression<br />
  7. 7. Activation P-gp reporter deletion mutants by Apicidin<br />requires <br />Y-box (NF-Y) and GC box (SP1)<br />Does not require<br />C/EBPbor AP-1<br />
  8. 8. Mutation analysis of promoter regions<br />Confirms importance of Y-box and GC box<br />
  9. 9. ChIP analysis of TF binding<br />No Change<br />Increase<br />Dissociation<br />Accumulation<br />Active transcription<br />
  10. 10. Kinase signalling and Apicidin induction<br />Inhibition of PI3K, but not other kinase pathways reduces P-gp promoter activity<br />
  11. 11. Inhibition of PI3K pathway blocks Apicidin induced P-gp<br />
  12. 12. Effect of PI3K pathway inhibition on TF binding and SP1 phosphorylation<br />Blocking SP1 phosphorylation by PI3K pathway inhibits dissociation of HDAC1 and recruitment of C/EBPb<br />
  13. 13. Inactive<br /><ul><li>Active</li></ul>pCAF<br />PolII<br />HDAC1<br />SP1<br />C/EBPb<br />Activation of PI3K pathway<br />+ Apicidin<br />
  14. 14. Summary...<br />Apicidin induces PI3K pathway, <br />PI3K results in phosphorylationof SP1 <br />SP1 phosphorylation signals chromatin remodelling and activation of P-gp<br />This is independent of changes in methylation<br />
  15. 15. Further work<br />Effect of prolonged treatment /different dose<br />Change in Methylation status<br />Was methylation effected by Aza treatment<br />Range of 0.3 to 3mM<br />
  16. 16. Further work<br />Cell recovery after <br />Short term treatment<br />Long term treatment<br />Effect of apicidin MDR sensitive cell lines/induction of MDR by Apicidin<br />Polymophisms/Translocations?<br />Proof of elimination of TF binding in mutants?<br />Binding of C/EBPb/pCAFto Y-box/each other<br />Accumulation of HDAC2 in non-responsive cell line<br />