Demethylation Of Dickkopf1(Dkk-1) Gene In Acute Leukemia Cell Lines By Arsenic Trioxide

Dickkopf (DKKs) is a kind of secretion type glycoprotein, as the key inhibitory factor of WNT/β-catenin signaling transduction pathway, play an important role in the directional differentiation of embryonic cells, it is the hot spot in the tumor molecular biology research[1]. The abnormal activation of WNT/β-catenin signaling transduction pathway is closely related to the generation development and prognosis of various tumors. Dickkopfl (DKK-1) is one newly discovered member of the DKKs family. Studies show that DNA abnormal methylation is one of the important ways to inactivate DKK-1gene. It was reported, about86%of DNA hypermethylation is detected in acute myelogenous leukemia(AML). Thus, demethylation therapy aimed at DKK-1gene becomes a new therapeutic strategy for acute leukemia (AL).Arsenic trioxide(As203) has been reported that it can inhibit the growth of some tumor cells invitro by inducing apoptosis and differentiation, down-regulating of telomerase activity and inhibiting of angiogenesis. Since AS2O3is detoxified via methylation and it consumes methyl groups, the lack of methyl groups would result in inability to properly maintain methylate cytosine in DNA. Accordingly, in our study AL cell lines were treated by AS2O3in which DKK-1gene was suppressed due to DNA hypermethylation. We tried to explode the DKK-1gene methylation status in AL cells to verify the role of methylation as a gene-silencing mechanism involved in the pathogenesis of AL, and to elucidate the antitumor mechanisms of AS2O3in AL cells in order to expend the train of thought of the clinical application of AS2O3in AL and to some extent, to provide a theoretical and experimental basis for designing new antitumor strategies.The subjects of the thesis were focused on the following three aspects:1.Expression of β-catenin in acute leukemia cell lines:Reverse Transcription Polymerase Chain Reaction (RT-PCR) and flow cytometry were performed to detect the expressions of β-catenin in4human AL cell lines(NB4、Jurkat、 Molt-4、CEM). As results showed, β-catenin was widely expressed in AL cell lines in various degree. β-catenin positive cells percent and fluorescence intensity were elevated in normal (P<0.05).2.Analysis of methylation of Dickkopfl(DKK-1) gene in acute leukemia: RT-PCR was used to examine the DKK-1mRNA expressions in4AL cell lines and normal controls. Methylation-Specific Polymerase Chain Reaction (MSP) was used to examine the methylation status of DKK-1gene in AL cell lines. As results showed, compared with normal controls, the expressions of DKK-1gene is significantly reduced in AL cell lines. None of the normal mononuclear cells showed methylation of DKK-1gene. The methylation status of DKK-1gene was visible in the detected cell lines.3.The impacts on WNT signaling transduction pathway inhibitor—DKK-1in AL cells by arsenic trioxide:4AL cell lines were incubated with8umol/1arsenic trioxide for24hours. Then MSP was used to examine the methylation of DKK-1gene. The expressions of mRNA and protein were determined by RT-PCR and Westen blot analyses. Flow cytometry was used to investigate the expression changes of β-catenin. The major results indicated that after24hours treatment, the methylation of DKK-1gene decreased markedly to disappear. Compared with the untreated group, mRNA and protein expressions in arsenic trioxide-treated were recovered partly or completely, And P-catenin protein positive ratios and intensities reduced obviously.Conclusions:1. The overexpression of β-catenin in AL cell lines, as a key mediator of WNT signaling transduction pathway, indicates that the WNT signaling transduction pathway may be aberrantly activated in AL.2. Methylation and aberrant expression of DKK-1gene are common in AL and also play a role in pathogenesis of AL.3.Arsenic trioxide is able to effectively wipe off the methylation of DKK-1gene, recover the mRNA and protein expressions of DKK-1gene, and restrain the abnormal activation of the WNT/β-catenin pathway.