Detection Of P15 Methylation Status In Adult Acute Leukemia Using Hn-MSP And Experimental Study On Reversing Hypermethylation Of P15 Gene

Objective To detect the methylation or deletion status in the adult acute leukemia using Hn-MSP and further to explore the relationship between patterns of methylation or deletion and the developmet of adult acute leukemia.To understand the mono and synergic effect of histone deacetylase inhibitor Sodium Valproate (VPA) alone and in combination with traditional Chinese medicine Arsenic Trioxide(AS2O3)on ce11 growth and to explore the possible mechanisms of inducing hypomethylation and re-expression of the hypermethylated and silenced p15 gene in the acute T cell leukemia ce11 line.Methods Hemi-nested methylation specific polymerase chain reaction (Hn-MSP) and Bisulfit-sequencing PCR (BSP) were adopted to analyze p15 methylation or deletion patterns in 85 adult acute leukemia patients with different subtypes and stages, cell lines of malignant hematopathy and normal lymphocytes.It also analized Hn-MSP's sensitivity and specificity.The acute T cell leukemia cell line cells were cultured in RPMI 1640 with the exposure to varied doses of VPA and AS2O3, and the growth curve and cell viability were obtained by the MTT assay. The synergistic activity in combination was determined by the Q format. The cell cycle was anaslyzed by flow cytometry and the expression level of p15, DNA methyltransferase 1(DNMT-1), DNMT3A and 3B mRNA were detected by RT-PCR. The methylation level was detected by Hn-MSP.Results (1) The sensitivity of detection of p15 methylation was up to 1.0×10-5, and it had great specificity. Rate of p15 gene hypermethylation was 72.9% in 85 adult acute leukemia patients, respectively, acute myeloid leukemia (AML) 75.0% and 68.0% in acute lymphoblastic leukemia (ALL). It was found that 71.2% of primary AL and 81.8% of relapsed AL developed the hypermethylation of p15 gene. Among the 85 patients, 6 seemed to have deletion of p15 gene exon 1, including 4 AML (6.67%) and 3 ALL patients (12%). There were no hypermethylation or deletion in the 22 controls. (2) Differentiation was induced and cell growth was arrested by treatment with VPA alone or in combination with AS2O3. The cell cycle test showed that most of the cells were arrested at G0-G1 phrase. It seemed that Molt-4 cells were not so sensitive as to lower dose of AS2O3 than VPA. The G0-G1 phrase was not varied after the exposure to 2μM AS2O3. The MTT assay showed that VPA and AS2O3 alone could inhibit the viability in a concentration-dependent manner. The inhibition level of cell viability was increased significantly by synergic application of VPA and AS2O3, the values of Q were all>0.85. (3) In Molt-4 cell line, p15 gene fails to express for its hypermethylation. The level of expression of cell p15 gene mRNA was increased significantly after exposure to VPA in combination with AS2O3 for 48h, though VPA alone could only induced its low-level expression. Compared to control group, the expression of DNMT-1 was decreased in a dose-dependent manner, whereas DNMT3A had no significant differences from the control. The level of expression of DNMT3B seemed to decrease at 10mM concentration. There were significant differences between the combination of the two drugs and the control group. As was detected by Hn-MSP, p15 gene were hypermethylated in Molt-4 cell line, the grey scale of methylated bands were decreased after the treatment of VPA alone and in combination with AS2O3 in a dose-dependent manner.Conclusions 1) Hn-MSP was highly sensitive and specific in analyzing p15 methylation status, deserving in clinical application. 2) The detection rate of p15 methylation in many tumors, especially in adult acute leukemia, is frequent as the literatures had reported, correlating with patients'progression and prognosis. 3)VPA could induce hypomethylation of p15 gene by lowering the DNMT-1 and DNMT3B genes'activities directly, and the expression of p15 gene was activated after the treatment of VPA alone or in combination with AS2O3. 4) VPA in combination with AS2O3 would have the synergetic additional effect on the inhibition to cells'viabilities, so that VPA can reduce the side effect to liver function of AS2O3, which would be verificated in the clinical pratice.