研究生： 林秀盆 指導教授 : 劉興璟 博士 林建煌 教授

1. Characterization of the combinational effects of DNA hypomethylating agents and histone deacetylase inhibitors in human acute myeloid leukemia 研究生： 林秀盆 指導教授: 劉興璟 博士 林建煌 教授

2. Introduction

5. HDACIs

6. HDAC(Histone Deacetylase ), HAT(Histone Acetyltransferase)

7. (Cancer cell:July 2003)

9. A).Compare the effects of sodium butyrate, phenylbutyrate & suberoylanilide hydroxamic acid(SAHA) on apoptosis, cell cycle arrest, cell proliferation and differentiation in HL-60 .

10. MATERIALS AND METHODS

12. HL-60 AML FAB M2

13. Histone Deacetylase Inhibitors -Sodium Butyrate,Phenylbutyrate & SAHA Induce Death of HL-60 Cells.

14. Fig.1)Sodium Butyrate,Phenylbutyrate, & SAHA Induce Death of HL-60 Cells in a dose- and time-dependent manner.

15. HDACIs apicidin and CBHA induced cell death via activation of the death receptor pathway (Bernhard et al., 1999) • SAHA and sodium butyrate, show no requirement for the death receptor pathway(Ruefli,A.A.,2001)

16. Table.1 Phenylbutyrate induced cell death may be via activation of the death receptor pathway , SAHA and sodium butyrate may be not.

17. HDACIs effect Cell Cycle Arrest (Richon et al.,2000)

18. 2)CanSodium Butyrate,Phenylbutyrate & SAHA effect Cell Cycle Arrest ?

19. Fig.2) Sodium Butyrate,Phenylbutyrate, & SAHA effect Cell Cycle Arrest.

20. HDACIs can Antiproliferative activity in cancer cells. (Jaboin et al., 2002)

21. 3)Can Sodium butyrate, Phenylbutyrate & SAHA effect cells proliferation?

22. Fig.3) CFSE stain -proliferation Table 1. SB=2mM Day0 untreated

23. Sodium Butyrate,Phenylbutyrate & SAHA can Antiproliferative activity in a dose- and time-dependent manner.

24. HDACIs Induce Cancer cells Differentiation. (Ferrara et al.,2002;Gottlicher et al.,2001)

25. 4) )CanSodium Butyrate,Phenylbutyrate & SAHA Induce HL-60 Differentiation ?

26. Fig.4a)NBT(nitroblue tetrazolium) reduction test untreated SAHA=2uM SB=2mM PB=2mM

27. Fig.4b) α-Naphthyl Acetate Esterase stain untreated macrophage vitD3=0.1uM SAHA=2uM SB=2mM PB=2mM

29. HL-60 CD11b CD13 CD14 CD95 no drug 0.4±0.1% 0.5±0.1% 0.2±0.1% 0.19±0.01% PB=1mM 2.4±0.32% 1.65±0.7% 2.3±0.1% 4.2±0.21% SB=1mM 2.5±0.05% 4.2±0.5% 2.3±0.1% 0.2±0.1% SAHA=1uM 5.1±0.3% 3.1±0.3% 1.2±0.1% 0.21±0.02% SAHA=2uM 14±0.5% 8.0±0.8% 2.3±0.1% 0.3±0.02%

30. Sodium Butyrate,Phenylbutyrate & SAHA Can Induce HL-60 Monocytes

32. 5-azacytidine and histone deacetylase inhibitors are synergistic with demethylation for reexpressing the silenced genes (Jones and Baylin, 2002)

33. 5AZA in combination with HDAC inhibitors produces a synergistic antineoplastic effect against leukemia cells. (Shaker et al., 2003).

34. B).Hypothesis: Synergy of DNA methylation inhibitor(Zebularine) and HDACI (SAHA) in the re-expression of genes silenced in Leukemia.

36. DNAMethyltransferases and inhibitors (Cheng JC, et al.,2003)

37. E-cadherin gene is a common target for hypermethylation in hematologic malignancies. (Melki et al.,Blood. 2000)

38. Zeb SAHA

39. (Maralice Conacci-Sorrell., J. Clin. Invest.2002)

46. HDAC & suberoylanilidehydroxamic acid(SAHA)

47. The HDACIs SAHA, sodium butyrate, and trichostatin A have allbeen shown to induce apoptosisof CEM cells (Ruefli,A.A.,2001)

49. untreated SB=1mM PB=1mM SAHA=1uM

50. Hypermethylation of E-cadherin in leukemia • (Melki,John R., BLOOD, 15 MAY 2000)