Experimental Study On Triptolid Reversing Hypermethylation Of APC And Activating Its Transcription In Acute Lymphoblastic Leukemia Cell Line Jurkat

Objective To measure methylation level of gene APC(ademomatous Poylposis coli,APC) in the cell lines of ten malignant hematonosis by nested Methylation Specific PCR(nMSP) and DNA sequencing, and further to explore the relationship between patterns of methylation or and the development of adult acute lymphoblastic leukemia; and to clarify the possible mechanism in the development of adult acute lymphoblastic leukemia. To investigate the mechanism of Triptolid(TPL)induced APC gene demethylation and proteinum expression in the acute lymphoblastic leukemia cell line--Jurkat.Method nested Methylation Specific Polymerase chain reaction (n-MSP) and DNA sequencing were adopted to analyze APC methylation or deletion patterns in the methylation level of cell lines. The MTT assay, growth curve as well as clone formation inhibition ratio methods were used to evaluate the effect that Triptolid had on the ability of cell reduplication and cell vitality.Both methylation levels of Jurkat before and after TPL disposal were analyzed by n-MSP; The expression of APC gene, DNA methyltransferase 1 (DNMT1), DNMT3A and DNMT3B were analyzed by RT-PCR; DNA ploid analytical method was used to analyze the effect that TPL would bring on the cell life cycle of the target cell line. The expression of APC protein was detected by western blot analysis.Results 1. Among the cell lines of ten malignant hematonosis ,APC gene only in Jurkat was found hypermethylated.2. Compared with the untreated group, differentiation was induced and cell growth was arrested by treatment with TPL, the cell cycle test showed that most of the cells were arrested at G0-G1 phrase. 3. The methylation level of APC gene was apparently attenuated after 48h disposal of TPL, and hypermethylation of APC had been successfully reversed. 4. APC gene and APC 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 48h disposal of TPL, the expression of DNMT3A and DNMT3B was obviously down-regulated in a concentration-dependent manner, while expression of DNMT1 stood nearly unchanged.Conclusions 1. With a higher sensitivity and specificity, nMSP can be used in analyzing methylation level in a variety of cell lines.2. TPL could reverse the hypermethylation of APC gene and active the expression of APC gene and APC protein by its direct and/or indirect inhibition of the DNMTs, and finally blocking the cell at G0 or G1 phase.