Treatment with Valproic Acid Restores Differentiation in AML1/ETO-Positive Leukaemic Cells.

In t (8, 21) acute myeloid leukaemia (AML), the leukaemogenesis is supposed to be promoted by interference with expression of AML1 target genes. Repressor complex associated with AML1/ETO fusion protein recruits class I histone deacetylases (HDAC). Valproic acid (VPA) was found to have an extensive effect on AML blasts, via inhibition of class I HDAC. It was shown previously that VPA treatment disrupts the AML1/ETO-HDAC1 complex from AML1 promoter thus leading to apoptosis at different cell lines. However, there is still lack of in-depth morphological and immunophenotypical proof of the hypothesized restoration of differentiation after treatment with VPA. We aimed to characterize the differentiation effect of VPA on AML1/ETO-positive leukaemic cells. Kasumi-1 (AML1/ETO-positive) cell line and MV4-11 (MLL/AF4-positive) cells were treated with VPA and Trichostatin-A (TSA) another HDAC inhibitor. The effect of VPA on cell cycle and differentiation was examined by flowcytometry. DNA, RNA and AgNORs staining of nucleus and nucleolus were used for morphological evaluation. VPA treatment of Kasumi-1 cells resulted in decreased expression of early myeloid progenitor antigens [CD33 (p<0.001), CD34 (p=0.003), CD117 (p<0.001)] and increased expression of antigens typical for more differentiated myeloid cells [CD11a (p=0.001), CD11b (p=0.007)]. Morphological evaluation revealed nucleus fragmentation without chromatin condensation, which evoked monocytoid look of the nucleus in VPA-treated Kasumi cells. The nucleolar morphology and cytochemistry indicated that these cells entered the ageing process and decreased proliferation activity. Large nucleoli characterised by a reduced number of silver-binding nucleolar organizer regions (AgNORs) as well as by their translocation to the nucleolar periphery represented a base for such interpretation. VPA-treated Kasumi cells exhibited accumulation in G1/G0 phase and decreased proliferation (VPA 0.5mM - G1/G0 70%, S/G2/M 16%; control cells - G1/G0 62%, S/G2/M 28%). Conversely, MV4-11 cell line (representing myeloid leukaemic cells of non-CBF origin) did not show any statistically significant differences in imunophenotype after VPA treatment (CD15, CD11b, CD117, CD45, HLA DR, CD14). VPA treated MV4-11 cells did not show accumulation in G1/G0 phase (VPA 0.5mM G1/G0 63%, S/G2/M 13%; control G1/G0 70%, S/G2/M 22%). We show that treatment with VPA restores differentiation of AML1/ETO positive cells as presented by both changes in immunophenotype and morphological analysis. This observed differentiation correlates with decrease in cell cycle. We demonstrate that the effect on differentiation is specific for AML1/ETO-positive cells as it is not noticeable in leukaemic cells of non-CBF origin.