HDAC inhibitors alter gene expression

Show potent anti-tumour activities Possible use as chemotherapeutic agents

HDAC

HAT

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-gpexpression

Apicidin (1 induces P-gp in a cell type specific manner

• Apicidin induces P-gppromoter activity in a context independent manner

• P-gp induction not Apicidinspecific

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

Accumulation of acetylated histoneH3 in both responsive and non-responsive cell lines in a dose dependant manner

Mithramycin, inhibitor of SP1 binding abolished Apicidin induced expression

requires Y-box (NF-Y)

and GC box (SP1)

Does not require C/EBP or AP-1

Confirms importance of Y-box and GC box

No Change

Increase

Dissociation

Accumulation

Active transcription

Inhibition of PI3K, but not other kinasepathways reduces P-gp promoter activity

Blocking SP1 phosphorylation by PI3K pathway inhibits dissociation of HDAC1 and recruitment of C/EBP

Inactive

SP1

+ Apicidin

Activation of PI3K pathway

HDAC1

C/EBP

pCAF PolII

• Active

Apicidin induces PI3K pathway,

PI3K results in phosphorylation of SP1

SP1 phosphorylation signals chromatin remodelling and activation of P-gp

This is independent of changes in methylation

Effect of prolonged treatment /different dose

Change in Methylation status

Was methylation effected by Aza treatment

Range of 0.3 to 3 M

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/EBP pCAF to Y-box/each other

Accumulation of HDAC2 in non-responsive cell line