The Study Of T(8;21) Acute Myeloid Leukemia Cells Growth Inhibiton Induced By Dexamethasone

Objective:Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by the abnormal clonal expansion of myeloid blasts in the bone marrow, peripheral blood, and/or extra-medullary tissues. About 55% of adult AML patients harbor chromosome aberrations. The t(8;21) translocation generates the AML1-ETO fusion protein and is the most common chromosomal abnormality, which identified in approximately 12% of adult AML. Dexamethasone-induced apoptosis in t(8;21) AML cell lines has been reported previously by other research groups. This study aims to investigate the mechanisms of the inhibitory effect of dexamethasone on the growth of t(8;21) AML cells.Methods:The cell count kit-8 (CCK-8) and flow cytometry were carried out to test the influence of dexamethasone on the proliferation, apoptosis and cell cycle of AML1-ETO positive cells. To examine the effects of dexamethasone on mRNA and protein expression of AML1-ETO gene, RT-PCR and Western blot were performed. The expression of C-KIT, cyclin D1 and caspase proteins were also analysed by Western blot. We compared the mRNA expression level of the NR3C1 gene which encodes the glucocorticoid receptor between AML1-ETO positive and negative cell lines by RT-PCR. We also used AML1-ETO negative cell lines U937, Si-AE-SKNO-1 and AML1-ETO positive cell lines U937-AE, SKNO-1 as the object of research to examine the influence of AML1-ETO fusion protein on the mRNA and protein expression of NR3C1 gene. Furthermore, in order to explore the relationship between AML1-ETO and NR3C1 in blast cells from AML patients, data from gene chip GSE1159, GSE28317 and GSE18700 which deposited in the gene chip databases GEO were analysed using bioinformatics methods. Finally, the inhibitory effect of dexamethasone on t(8;21) AML cells was checked in vivo.Results:Dexamethason inhibited proliferation, induced apoptosis and caused G1-phase cell cycle arrest in AML1-ETO positive cell lines. Dexamethason decreased the mRNA and protein expression of AML1-ETO gene. Similarly, the level of C-KIT protein was also reduced by dexamethason. The AML1-ETO positive cell lines expressed significantly higher levels of NR3C1 mRNA and AML1-ETO fusion protein than the AML1-ETO negative cell lines. AML1-ETO fusion protein induced the expression of NR3C1 gene at both mRNA and protein levels. Data from gene chip analysis demonstrated that blast cells from t(8;21) AML patients expressed higher levels of NR3C1 mRNA as compared to blast cells from other type of AML. Methylation level in the promoter region of NR3C1 in blast cells from t(8;21) AML patients was lower than that from AML-M2 patients with normal karotype. After transfection with AML1-ETO, the human CD34+ hematopoietic cells isolated from cord blood expressed higher levels of NR3C1 mRNA as compared to control. In vivo study, dexamethason also inhibited the growth of t(8;21) AML cells.Conclusion:AML1-ETO fusion protein upregulates the expression levels of NR3C mRNA and protein, while the activation of glucocorticoid receptor by dexamethasone leading to proliferation inhibition, apoptosis induction and cell cycle arrest in AML-ETO positive cells. Thus, the clarification of the interactions between AML1-ETO fusion protein and glucocorticoid receptor may shed light on a new potential therapeutic target in t(8;21)AML.