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Objective • To investigate the effect of mammalian target of rapamycin (mTOR) inhibitor rapamycin on acute myeloid leukemia (AML) with DNA methyltransferase 3A (DNMT3A) R882 mutation in mouse model. Methods • AML cell line OCI-AML3 cells with DNMT3A R882 mutation were cultured with the treatment of rapamycin or DMSO, and then these cells were injected into the tail vein of sublethally irradiated NOD/SCID mice, respectively. The disease progression was monitored by blood routine examination and flow cytometry analysis of CD45+, OCI-AML3 cells, in peripheral blood. Survival time was recorded. Samples from bone marrow, spleen and liver were harvested for flow cytometry analysis and pathological examination. Results • The increasing trend of peripheral leukocytes in the rapamycin treated group was obviously slower than that in the DMSO treated group. The proportion of peripheral blood CD45+ cells in the rapamycin treated group was (4.44±2.58)% (1 week after transplantation) and (34.42±13.64)% (2 weeks after transplantation), which were lower than (16.71±8.96)% and (51.55±5.36)% in the DMSO treated group in the same period, respectively. The median survival time of the rapamycin treated group (27 d) was significantly longer than that of the DMSO group (23 d). The ratios of CD45+ cell infiltration in bone marrow, spleen and liver of the rapamycin treated group were all less than 5%, which were significantly lower than those [(51.32±27.81)% in spleen and (50.03±28.74)% in liver] of the DMSO treated group. Compared with the DMSO treated group, the spleen size of the mice was significantly smaller, and the spleen infiltration and structural damage were significantly alleviated in the rapamycin treated group. Conclusion • Rapamycin shows effective inhibition on the progression of AML with DNMT3A R882 mutation in NOD/SCID mouse model.
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<p><b>OBJECTIVE</b>To explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm' tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells.</p><p><b>METHODS</b>The HEL cells were treated with bufalin at various concentrations to observe cellular morphology, proliferation assay and cell cycle. The mRNA and protein expression levels of WT1 were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry, DNA methylation of WT1 and protein expression levels of DNA methyltransferase 3a (DNMT3a) and DNMT3b were analyzed by methylation-specific PCR, and Western blot respectively.</p><p><b>RESULTS</b>The bufalin was effective to inhibit proliferation of HEL cells in a dose-dependent manner, their suppression rates were from 23.4%±2.1% to 87.2%±5.4% with an half maximal inhibit concentration (IC) of 0.046 μmol/L. Typical apoptosis morphology was observed in bufalin-treated HEL cells. The proliferation index of cell cycle decreased from 76.4%±1.9% to 49.7%±1.3%. The expression levels of WT1 mRNA and its protein reduced gradually with increasing doses of bufalin, meanwhile, the methylation status of WT1 gene changed from unmethylated into partially or totally methylated. While, the expression levels of DNMT3a and DNMT3b protein gradually increased by bufalin treatment in a dose-dependent manner.</p><p><b>CONCLUSIONS</b>Bufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G/Gphase, but also induce cellular apoptosis and down-regulate the expression level of WT1. Our results provide the evidence of bufalin for anti-leukemia, its mechanism may involve in increasing WT1 methylation status which is related to the up-regulation of DNMT3a and DNMT3b proteins in erythroid leukemic HEL cells.</p>
Asunto(s)
Humanos , Apoptosis , Genética , Bufanólidos , Farmacología , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Forma de la Célula , ADN (Citosina-5-)-Metiltransferasas , Metabolismo , Metilación de ADN , Genética , Regulación Leucémica de la Expresión Génica , Leucemia Eritroblástica Aguda , Genética , Patología , ARN Mensajero , Genética , Metabolismo , Regulación hacia Arriba , Genética , Proteínas WT1 , Genética , MetabolismoRESUMEN
DNA methyltransferase 3A (DNMT3A) is one of the critical epigenetic modifiers responsible for de novo DNA methylation. DNMT3A mutations are identified in some kinds of hematological malignancies, especially in acute myeloid leukemia (AML) with high frequency, indicating poor prognosis. Recent researches have shown that abnormalities of epigenetic related genes such as DNMT3A play important roles in the development and progression of hematological malignancies, which was named as class Ⅲ mutation associated with the pathogenesis of AML. In this review, the progresses of clinical and basic researches about DNMT3A mutation in hematological malignancies were summarized.
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DNA methyltransferase 3A (DNMT3A) is one of the critical epigenetic modifiers responsible for de novo DNA methylation. DNMT3A mutations are identified in some kinds of hematological malignancies, especially in acute myeloid leukemia (AML) with high frequency, indicating poor prognosis. Recent researches have shown that abnormalities of epigenetic related genes such as DNMT3A play important roles in the development and progression of hematological malignancies, which was named as class Ⅲ mutation associated with the pathogenesis of AML. In this review, the progresses of clinical and basic researches about DNMT3A mutation in hematological malignancies were summarized.
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Objective To study the relationship between DNA methylation and pathogenesis of childhood immune thrombocytopenic purpura (ITP) by examining the expression of DNA methyltransferase 1(Dnmt1) and DNA methyltransferase 3a (Dnmt3a) mRNA in peripheral blood lymphocytes of the children with ITP. Methods Expression of Dnmt 1 and Dnmt3a mRNA in the peripheral blood lymphocytes in 36 children with newly diagnosed ITP and 26 healthy children were detected using RT-PCR. Results Dnmt1 mRNA expression in peripheral blood lymphocytes in children diagnosed with ITP was 3.02±0.49, significantly lower than 4.58±0.52 in the control group (t=11.95, P<0.001). Dnmt3a mRNA expression in peripheral blood lymphocytes in children diagnosed with ITP was 1.49±0.44, signiifcantly lower than 2.41±0.32 in the control group (t=9.12, P<0.001). Conclusions Children with newly diagnosed ITP have lower DNA methylation status in peripheral blood lymphocytes as compared to that in healthy children. The DNA methylation may play an important role in the etiology of acute ITP in children.