Monocytic Differentiation of Myeloid Leukemia Cell Lines Induced by ATRA and 5-Aza-2'-Deoxycitidine.

The CCAAT/enhancer binding protein (C/EBP)αa is a transcriptional factor of hematopoietic system and plays a key role in monocytic differentiation. Recently, several studies have reported that C/EBPαa expression is down-regulated in acute myeloid leukemia (AML), leading to block of granulocytic and monocytic differentiation. Differentiation therapy of ATRA is highly effective for acute promyelocytic leukemia (APL).

The mechanism of induction of differentiation in the treatment of APL is induction of a set of transcriptional factors which are responsible for myeloid differentiation. However, ATRA alone is not sufficient to treat another type of AML. Thus, it is worth to explore the agents which intensify the efficacy of ATRA. To assess the possibility of differentiation therapy in AML, except for APL, we evaluated the efficacy of demethylation agent combined with ATRA for various AML cell lines.

The five AML cell lines (K562, U-937, HL-60, THP-1, and KOCL48 expressing MLL-AF4) were treated with 50nM 5-Aza-2'-deoxycytidine (5-Aza) for two days and 1 mM ATRA for additional five days. Then, we analyzed cell growth with counting nuclei using Coulter counter. The cell cycle analysis was also performed by flow cytometry (FCM). In addition, Annexin V assay was performed to determine whether apoptosis occurred or not. To assess whether monocytic differentiation was induced or not, the expression of CD11b was evaluated by FCM. In addition, the expression of transcriptional factors, such as C/EBPαa, PU.1 and c-myc ) were analyzed by real time PCR analysis. Methylation specific PCR was also performed to evaluate the methylation status of promoter region of C/EBPαa.

HL-60 was highly sensitive to ATRA (growth inhibition rate: 80%). THP-1 and KOCL-48 were moderately sensitive to ATRA (growth inhibition rate: 50-60%). Addition of 5-Aza induced suppression of the growth in these two cell lines efficiently (growth inhibition rate: 80%). K562 and U937 were resistant to ATRA (growth inhibition rate: 10-20%). Addition of 5-Aza induced suppression of cell-growth in U937 (growth inhibition rate: 70%). However, 5-Aza did not show the effect in K562. Morphological studies revealed the characteristic features, such as extended cytoplasm with vacuoles, fine granules and irregular shaped nucleus, were evident in four cell lines which was sensitive to 5-Aza. FCM analysis revealed intensification of CD11b expression. In addition, real time PCR determined the increased expression of C/EBPαa and PU.1 (Fold change: 2-3.0) in the four cell lines except for K562. On the other hand, the expression level of c-myc was decreased under treatment with ATRA and 5-Aza (Fold change: 0.2). Cell cycle analysis revealed G1 arrest was occurred. Annexin V assay also revealed that combination therapy of ATRA and 5-Aza induced apoptosis in theses cell lines except for K562. Methylation specific PCR did not identified hypermetylation of pormorter region of C/EBPαa in these cell lines except for K562.

Addition of 5-Aza to ATRA induced further expression of C/EBPαa and PU.1 efficiently, leading to monocytic differentiation in AML cell lines. Monocytic differentiation was accompanied with G1 arrest through down-regulation of c-myc, and apoptosis was induced finally. Combination of ATRA and 5-Aza might be effective therapeutic option even for AML which is resistant to differentiation therapy with ATRA.

No relevant conflicts of interest to declare.