| Objective: Lymphoma is a kind of tumor of lymphoid stem cell prosoplasia and clonal proliferation. It is one of the common cancers in pediatrics with an increasing incidence, whose etiopathogenisis is unknown. At present, traditional chemotherapy is still the main method of therapy, but the drug resistance and side effect of chemotherapy is one of the important factors to influence long term survival. Therefore, it is significant that searching for etiological factor and exploring a more effective prevention and cure measure to improve the patient's prognosis and quality of life. Inhibitor of DNA binding 4 (Id4) is a kind of suppressor of DNA binding. With a series of researches in a mouse model of cancer, Id4 gene has been identified as a new putative tumor-suppressor by Professor Yu Li. It was rarely reported that methylation of Id4 gene appeared in malignant lymphoma. DNA methylation inhibitor can reverse methylation effect. Arsenic Trioxide was first discovered and succeeded in inducing differentiation therapy of many kinds of malignancy hemopathy recently at home, such as acute promyelocytic leukemia and et al, significant curative effect gained. It was found that Arsenic Trioxide can be deoxidized to generate methylation in vivo, and further interferes with demic methylation mode; therefore, demethylation of Arsenic Trioxide may treat malignancy hemopathy. At present, little has been elucidated that Arsenic Trioxide has been treated malignancy lymphoma by demethylation at home and abroad. In this study, in order to investigate the effect of Arsenic Trioxide treat malignancy lymphoma and the possible antitumor mechanism of Arsenic Trioxide cure malignancy lymphoma, we detected methylated degree of Id4 gene and observed the effect of Arsenic Trioxide on proliferation, apoptosis, cell cycle distribution and methylation degree of Id4 gene and expression of Id4 gene in Raji human malignant lymphoma cell line. Thus, theory evidence and cure strategy for pathogenesy of malignant lymphoma and laboratory data for therapy malignant lymphoma with demethylated medicine will be provided.Methods: Human Burkitt's lymphoma cells Raji were cultivated in vitro, and detected methylated degree of Id4 gene by Methylation Specificity Polymerase Chain Reaction (MS-PCR), then treated with various concentrations of Arsenic Trioxide. Cell inhibitive rate was investigated by MTT assay. Apoptosis rate and cell cycle distribution were detected by Flow Cytometry (FCM). Effect of methylated degree of Id4 gene in Raji cell treated with Arsenic Trioxide was detected by MS-PCR. Expression of Id4 mRNA in Raji cell treated with Arsenic Trioxide was detected by Reverse Transcription Polymerase Chain Reaction (RT-PCR). Results1 MS-PCR showed: The Id4 gene presented exhaustive methylation.2 MTT showed: Cell inhibitive rate of Raji after treated with various concentrations of Arsenic Trioxide for 24 hours were 12.15%, 33.72%, 38.99%, 62.06% respectively; Cell inhibitive rate of Raji after treated with various concentrations of Arsenic Trioxide for 48 hours were 21.86%, 45.38%, 52.10%, 75.79% respectively; Cell inhibitive rate of Raji after treated with various concentrations of Arsenic Trioxide for 72 hours were 40.03%, 66.64%, 74.09%, 92.17% respectively. Cell inhibitive rate in experiment groups after treated Raji cells for 24h,48h,72h were significantly different compared with control group (P<0.05), at the same time, there was a significant difference between concentration groups and time groups (P<0.05).3 FCM showed: Apoptosis peak emerged significantly after Raji cells treated with Arsenic Trioxide for 48 hours. Apoptosis percentage (AP) were (8.93±0.24)%, (24.21±0.97)%, (33.54±0.83)%, (44.76±1.21)% respectively; there was no or only low apoptosis peak in control group, AP in control was (1.97±0.73)%. There was remarkable difference between experiment groups and control group by least significant difference test, AP were significant higher in experiment groups than that in control group. By one-way ANOVA, AP were significantly different among concentration groups (P<0.05).4 FCM showed: After Raji cells treated with various concentrations of Arsenic Trioxide for 48 hours, the cell percentage in G0/G1 phase of test groups were (50.41±0.70)%, (61.87±0.89)%, (68.31±0.12)%, (74.97±0.31)% respectively; the cell percentage in S phase were (39.13±0.63)%, (28.83±1.84)%, (23.62±0.11)%, (17.57±0.18)% respectively; the cell percentage in G2/M phase were (10.46±0.07)%, (9.30±0.30)%, (8.07±0.06)%, (7.46±0.06)% respectively; the cell percentage in G0/G1, S, G2/M phase of control group were (44.85±0.73)%, (43.78±0.27)%, (11.38±0.16)% respectively. Compared with control group, cell cycle distribution in test groups of Arsenic Trioxide existed a significant difference (P<0.05). By one-way ANOVA, cell cycle distribution were significantly different among concentration groups (P<0.05).5 MS-PCR showed: Id4 gene presented exhaustive methylation in 3μmol/L group. It presented meri- methylation in 6μmol/L and 9μmol/L group. It presented exhaustive unmethylation in 12μmol/L group. It hinted that hypermethylated state of Id4 gene can be reversed by Arsenic Trioxide, wich was dose-dependent.6 RT-PCR showed: As compared with theβ-actin, expression of Id4 mRNA in Raji cells after treated with Arsenic Trioxide for 48 hours at the concentrations of 6μmol/L,9μmol/L,12μmol/L, except 3μmol/L, increased gradually with Arsenic Trioxide concentrations increased, Which was also dose-dependent.Conclusions1 Id4 gene presented exhaustive methylation in Raji Human Burkitt's lymphoma cells.2 Arsenic Trioxide can reverse the hypermethylated state of Id4 gene and recover the expression of Id4 mRNA with a dose and time dependent manners from 6~12μmol/L. The demethylation may be another possible anti-neoplastic hematologic disorder mechanism of Arsenic Trioxide.3 The survival of Raji cells was inhibited by Arsenic Trioxide in vitro with a dose and time dependent manners from 3~12μmol/L.4 Arsenic Trioxide can induce the apoptosis in Raji cells, with the increase of Raji cells in G0/G1 phase, and decrease of Raji cells in S and G2/M phase.5 Hypermethylated state of Id4 gene was one reason of malignant proliferations in Raji cells.
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