Abstract
Background: P15 protein induced by TGF-β negatively regulates the cell cycle by inhibiting the activity of Cyclin D/CDK4,6 complexes. The p15 gene can be made inactive by 5’ CpG island methylation in the promoter regions in several hematopoietic malignancies such as Acute Myeloid Leukemia (AML). Inactivation of P15 blocks its negative regulation of the cell cycle, which may play an important role in AML. Demethylation of the p15 gene may therefore provide a new target for AML therapy. The chemotherapy agent 5-Azacytidine is an analog of the pyrimidine nucleotide cytosine. It interfered with the process of DNA methylation, induced cell differentiation, and demonstrated its clinical activity as a second-line anti-leukemia agent. In this study, we treated one AML patient with 5-Azacytidine for chemotherapy to investigate the demethylation of the p15 gene. Methods: First, we evaluated p15 gene methylation by Eco52I enzyme digestion-based PCR. DNA methylation levels were calculated as the percentage of PCR product density with or without Eco52I digestion. Next, we investigated p15 mRNA expression by RT-PCR analysis. p15 gene expression levels were evaluated by calculating the mRNA ratios of p15 to β-actin. Results: the p15 gene promoter was not methylated, and it was completely digested by Eco52I in HL60 cells. However, it was almost 100% methylated, and it was not digested by Eco52I in KG1A. In AML bone marrow, p15 DNA was not digested by Eco52I before the 5-Azacytidine treatment. This indicates the hypermethylation of the p15 gene, and P15 mRNA expression was low. After the 5-Azacytidine treatments, the methylation levels of the p15 gene were markedly decreased, and p15 mRNA expression was greatly increased. Conclusion: These results indicated that in AML patients, DNA hypermethylation silenced p15 gene expression and blocked the negative regulation of the cell cycle, which may trigger the pathogenesis of AML. the 5-Azacytidine treatment demethylated p15 DNA and reactivated p15 gene expression subsequently retarded G1/S transition of the cell cycle by inhibiting Cyclin D/CDK4,6. We conclude that demethylation and reactivation of the p15 gene may play important roles in the therapeutic effects of 5-Azacytidine on AML (Am J Transl Med 2018. 2:47-51).