Demethylation Of TMS1Gene In Chronic Leukemia Cell Line K562Cells By Arsenic Trioxide

Masumoto[1] discovered TMS1gene (target of methylation-induced silencing1) in his research of cell morphological changes associated with apoptosis. TMS1is a bipartite protein comprising two protein-protein interaction domains, a pyrin domain (PYD) and a caspase recruitment domain (CARD). Proteins containing these domains play pivotal roles in regulating apoptosis and immune response pathways[2] There is a growing body of evidence suggests that loss of TMS1experssion with concomitant over expression of DNMT possibly facilitate tumor cells escape from apoptosis so as to promote tumorigenesis. Methylation-mediated silencing of TMS1confers a survival advantage by allowing cells to escape from apoptosis, playing a role for carcinogenesis, adversely affecting prognosis for cancer patients.Arsenic trioxide(As203) has been founded that it can inhibit the growth of tumor cells invitro by inducing apoptosis and differentiation, down-regulating telomerase activity and inhibiting angiogenesis. Although As2O3is a pivotal anti-cancer drug, the potential and mechanism of its demethylating effect remains elusive. We conducted a research over K562cells, testing the methylation state of TMS1gene in K562cells treated with As2O3, using Decitabine(DCA) as control in order to provide a theoretical and experimental basis for designing new antitumor strategies.The subjects of the thesis were focused on the following three parts:1、Expression of TMS1mRNA in K562cells and patients without blood diseases:We selected samples of6cases of bone marrow of two normal, four non hematologic malignancies patients as the case-control. Reverse Transcription Polymerase Chain Reaction (RT-PCR) were performed to detect the TMS1expression in K562cells and case-control; Methylation-Specific Polymerase Chain Reaction (MSP) was used to examine the methylation status of TMS1gene in K562cells. Levels of TMS1mRNA expression in K562cells and normal bone marrow specimens were detected using RT-PCR. TMS1mRNA was found in patients with non-hematological disease, but was absent in K562cells which indicates loss expression of TMS1may play a role in pathogenesis of lekemia。2、Analysis of methylation of TMS1gene in K562cells and demethylation of As2O3on it:K562cells were treated with different concentrations of As2O3for48hours. MSP was used to determine the methylation degree of TMS1. RT-PCR and Western Blot were used to detect TMS1expression; Western Blot was used to detect TMS1associated apoptosis protein Bcl-2/Bax expression.Apoptosis rates were measured by flow cytometry using annexin V/propium iodide (PI) double staining method. Results showed TMS1was completely methylated in K562cells.Both of TMS1mRNA and protein showed a low expression (0.01±0.01,0.09±0.02);2μmol/LAs2O3could significantly restore the expression of TMS1gene both on mRNA and protein level (0.72±0.04,1.3±0.06)(P<0.01) by fully reversing DNA methylation level; Flow cytometry showed that the experiment group (As2O3,2μmol/L) significantly increased cell apoptosis rate compared with the control group (As2O3,0μmol/L)(12.24±1.06vs.2.05±0.16, P<0.05). In experiment group, Western Blot showed the expression of anti-apoptotic protein Bcl-2significantly decreased, however, pro-apoptotic protein Bax clearly increased and the ratio of Bcl-2/Bax was obviously reduced (0.56±0.12vs.1.94±0.14, P<0.01).3、Expression changes of NF-κB after promoter demethylation of TMS1in K562cells:K562cells were cultured and treated with decitabine (DCA) at different concentrations and different time. Set up the blank control group and experiment group(0.5μmol/L DCA、1μmol/L DCA) separately. Methylated degree of TMS1was detected by MSP, mRNA and protein of TMS1and NF-κB in K562cells by RT-PCR and Westernblot,the growth of cells by CCK-8and morphologic changes by Light microscope. Results showed the TMS1gene presents exhaustive methylation in blank control group.After promoter demethylation of TMS1, the expression of NF-κB significantly decreased(P<0.05).The inhibition on K562cells proliferation was observerd and showed dual realtion which include quantity and time.Conclusion:1、TMS1gene presents exhaustive methylation in K562cells.2、As2O3could restore the expression of TMS1by reversing the hypermethylation and induced apoptosis of K562cell by down-regulation of Bcl-2/Bax expression.3、 Silent expression of TMS1gene in K562cells may abnormally activate NF-κB signaling pathway and plays a role in the pathogenesis of some leukemia.