[Effect of AML1-ETO Fusion Protein on Transcriptional Regulation of p14^ARF]

To investigate the effect of transcriptional regulation of aberrant transcription factor AML1-ETO on p14^ARF.

P14^ARF expression both in AML1-ETO-expressing cells or U937 nonexpressing cells and in leukemia cells of AML patients with or without t(8;21) was assessed by quantitative PCR. Methylation-specific polymerase chain reaction (MSP) was used to analyze the methylation status of p14^ARF promoter. The chromatin immunoprecipitation (ChIP)-based PCR was used to investigate the direct interaction between the AML1-ETO and p14^ARF promoter in AML1-ETO positive leukemia cell line. And the p14^ARF mRNA expression level was detected by qRT-PCR after treatment with 5-Aza.

AML1-ETO-expressing cell subclone displayed low level of p14^ARF mRNA in comparison with the non-transfected U937. In primary bone marrow cells of acute myeloid leukemia containing AML1-ETO, level of p14^ARF mRNA was markedly lower when compared with other acute myeloid leukemias lacking this translocation. P14^ARF gene promoter was non-methylated in control group and primary leukemia cells of AML patients without t(8;21) and was hyper-methylated in U937-A/E1-4 and primary leukemia cells of AML patients with t(8;21). The enriched regions in transfected cells were located within p14^ARF promoter. 5-Aza could increase the expression of p14^ARF.

P14^ARF is a possible target gene of AML1-ETO. The p14^ARF silencing induced by hyper-methlylation may be an important factor for occurrence and development of the M_2b subtype of acute myeloid leukemia.