Combined Effect Of Phenylbutyrate And 5-aza-2'-deoxycytidine In T(8;21) AML Leukemia Cells In Vitro And In Vivo

Dysregulation of histone acetylation has been demonstrated in several hematological neoplasias. It has been reported that a positive interaction between DNA methylation and histone deacetylation takes place to inhibit transcription. In t(8;21) AML, a stable association of AML1/ETO fusion protein with the nuclear histone deacetylase complex (HDAC) is crucial to repressing transcription of AMLl target genes and blocking differentiation of hematopoietic precursors. Phenylbutyrate (PB) is a competitive inhibitor of HDAC1. 5-aza-2'-deoxycytidine (5-Aza-CdR) is a potent inhibitor of DNA methylation. We investigated whether PB alone and combination with 5-Aza-CdR could reverse transcription repression and induce AML1/ETO cells to differentiate and undergo apoptosis.Cells from the AML1/ETO cell line, Kasumi-1, were treated with PB in suspension culture for 3 days. Incubation of Kasumi-1 cells with PB caused a dose-dependent inhibition of proliferation, with an IC50 of 2.64mM. The proliferation data were also confirmed by colony formation assays. PB also induced a time- and dose-dependent increase in expression of myeloid cell surface proteins CD11b and CD13. By flow cytometric analysis, PB treatment led to a progressive decline in the fraction of S-phase cells and an increase in G0-G1 cells. The induction of apoptosis by PB was demonstrated by annexin V binding coupled with propidium staining, with a dose-dependent increase in early apopotosis as confirmed by DNA ladder assays. Morphological changes of monocytic differentiation and apoptosis were seen by Wright-Giemsa staining. We also evaluated the biological response to PB of primary t(8;21) AML cells. Treatment with PB for 2 days resulted in a dose-dependent reduction in primary AML cell growth as measured by MTT assays, with an IC50 of 3.1mM. After exposure to PB, expression of myeloid cell surface proteins CD11b and CD13 was increased and apoptosis was induced. The combination of PB with 5-Aza-CdR produced a greater inhibition of growth than PB alone, with an IC50...