Detection Of P16 Gene Methylation Status In Adult Acute Leukemia By Using N-MSP And Experimental Study On EGCG Reversing Hypermethylation Of P16 Gene In Acute Leukemia Cell U937

Objective To detect the methylation or deletion status in the adult acute leukemia using n-MSP and further To explore the relationship between patterns of methylation or deletion and the developmet of adult acute leukemia; and to clarify the possible mechanism in the development of adult acute leukemia. To investigate the mechanism of Epigallocatechin-3-galate(EGCG)induced P16 gene demethylation and proteinum expression in the acute leukemia cell line--U937. Method Nested methylation specific polymerase chain reaction (n-MSP) were adopted to analyze P16 methylation or deletion patterns in 82 adult acute leukemia patients with different subtypes and stages. The growth curve and cell viability were obtained by the SRB assay. Both methylation levels of U937 before and after EGCG disposal were analyzed by n-MSP; The expression of P16 gene, DNA methyltransferase 1 (DNMT1), DNMT3A and DNMT3B were analyzed by RT-PCR; DNA ploid analytical method was used to analyze the effect that EGCG would bring on the cell life cycle of the target cell line. The expression of P16 protein was detected by western blot analysis.Results 1. Rate of P16 gene hypermethylation was 39.0% in 82 adult acute leukemia patients, respectively, acute myelogenous leukemia(AML) 41.4% and 33.3% in acute lymphoblastic leukemia(ALL).It were found that 36.6%of initial treatment AL and 54.5% of relapsed AL developed the hypermethylation of P16 gene. Among the 82 patients,6 seemed to have deletion of P16 gene,including 1 AML(1.7%) and 5 ALL(20.8%).There were no hypermethylation or deletion in the 16 controls. 2. Compared with the untreated group, differentiation was induced and cell growth was arrested by treatment with EGCG, the cell cycle test showed that most of the cells were arrested at G0-G1 phrase. 3. The methylation level of P16 gene was apparently attenuated after 72h disposal of EGCG, and hypermethylation of P16 had been successfully reversed. 4. P16 gene and P16 protein in untreated group was fail to express while in the treated groups they had been greatly strengthened. 5. Compared with the untreated group, after 72h disposal of EGCG, the expression of DNMT3A and DNMT3B was obviously down-regulated in a concentration-dependent manner, while expression of DNMT1 stood nearly unchanged. Conclusions 1. The rate of methylated P16 gene detection was high in adult acute leukemia .Methylation of P16 gene may play a important role than in the leukemogenesis and progression of adult acute leukemia.2. EGCG could reverse the hypermethylation of P16 gene and active the expression of P16 gene and P16 protein by its direct and/or indirect inhibition of the DNMTs, and finally blocking the cell at G0 or G1 phase.