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