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Journal Club May09
Journal Club May09
Journal Club May09
Journal Club May09
Journal Club May09
Journal Club May09
Journal Club May09
Journal Club May09
Journal Club May09
Journal Club May09
Journal Club May09
Journal Club May09
Journal Club May09
Journal Club May09
Journal Club May09
Journal Club May09
Journal Club May09
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Journal Club May09

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  • 1.
  • 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. 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. 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. 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. Apicidin induced P-gp promoter activity is SP1 dependant<br />Mithramycin, inhibitor of SP1 binding abolished Apicidin induced expression<br />
  • 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. Mutation analysis of promoter regions<br />Confirms importance of Y-box and GC box<br />
  • 9. ChIP analysis of TF binding<br />No Change<br />Increase<br />Dissociation<br />Accumulation<br />Active transcription<br />
  • 10. Kinase signalling and Apicidin induction<br />Inhibition of PI3K, but not other kinase pathways reduces P-gp promoter activity<br />
  • 11. Inhibition of PI3K pathway blocks Apicidin induced P-gp<br />
  • 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. 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. 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. 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. 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 />

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