Effect of methylation inhibitor on EphB4 gene expression, proliferation and apoptosis in CEM cells

LI Yu-Hua; WEN Fei-Qiu; CHEN Yi-Xin; LI Chang-Gang; ZHANG Zhao-Xia; CHEN Xiao-Wen; LI Bo

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Chinese Journal of Contemporary Pediatrics ›› 2012, Vol. 14 ›› Issue (3) : 205-209.

EXPERIMENTAL RESEARCH

Effect of methylation inhibitor on EphB4 gene expression, proliferation and apoptosis in CEM cells

LI Yu-Hua, WEN Fei-Qiu, CHEN Yi-Xin, LI Chang-Gang, ZHANG Zhao-Xia, CHEN Xiao-Wen, LI Bo

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Abstract

OBJECTIVE: To study the regulation of methylation inhibitor 5-aza-2'-deoxycytidine on transcription of EphB4 gene and effects on the proliferation and apoptosis of human acute lymphocyte leukemia cell line CEM.MethodsBisulfite sequencing PCR was used to detect CpG island methylation density in EphB4 promoter. The expression of EphB4 mRNA and protein was determined by Q-PCR and Western blot. MTS assay and flow cytometry were used to detect the apoptosis of CEM cells after treatment with different concentrations of 5-aza-2'-deoxycytidine (1.0, 2.5 and 5 μmol/L).ResultsMethylation of EphB4 gene promoter was detected in CEM cells (31.4%). The methylation level of EphB4 gene was down-regulated after treatment with various concentrations of 5-aza-2'-deoxycytidine. The EphB4 mRNA and protein expression in CEM cells increased after 5-aza-2'-deoxycytidine treatment. 5-Aza-2'-deoxycytidine significantly inhibited the cell growth in dose and time dependent manners. Early apoptosis rates of CEM cells increased from 4.1% to 24.8% 96 hrs after 5-aza-2'-deoxycytidine treatment. CEM cells in G1 phase decreased from 62.4% to 46.8%, cells in G2 phase increased from 2.1% to 16.2%, and CEM cells were arrested in G2 phase after treatment with 5 μmol/L 5-aza-2'-deoxycytidine for 96 hrs. CONCLUSIONS: 5-Aza-2'-deoxycytidine, an inhibitor of specific methylation transferase, can induce expression of the silent EphB4 gene in CEM cells, inhibit the proliferation of leukemia cells and induce cell apoptosis.

Key words

5-Aza-2'-deoxycytidine / EphB4 gene / Proliferation / Apotosis / CEM cell

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LI Yu-Hua, WEN Fei-Qiu, CHEN Yi-Xin, LI Chang-Gang, ZHANG Zhao-Xia, CHEN Xiao-Wen, LI Bo. Effect of methylation inhibitor on EphB4 gene expression, proliferation and apoptosis in CEM cells[J]. Chinese Journal of Contemporary Pediatrics. 2012, 14(3): 205-209

References

［1］李玉华，文飞球. DNA甲基化与儿童白血病的临床研究进展［J］. 中国当代儿科杂志，2011，13(2): 174-176.

［2］Batlle E, Bacani J, Begthel H, Jonkeer S, Gregorieff A, van de Born M, et al. EphB receptor activity suppresses colorectal cancer progression［J］. Nature, 2005, 435(7045): 1126-1130.

［3］Noren NK, Foos G, Hauser CA, Pasquale EB. The EphB4 receptor suppresses breast cancer cell tumorigenicity through an Abl-Crk pathway［J］. Nat Cell Biol, 2006, 8(8): 815-825.

［4］Alazzouzi H, Davalos V, Kokko A, Domingo E, Woerner SM, Wilson AJ, et al. Mechanisms of inactivation of the receptor tyrosine kinase EPHB2 in colorectal tumors［J］. Cancer Res, 2005, 65(22): 10170-10173.

［5］Davalos V, Dopeso H, Casta~no J, Wilson AJ, Vilardell F, Romero-Gimenez J, et al. EPHB4 and survival of colorectal cancer patients［J］. Cancer Res, 2006, 66(18): 8943-8948.

［6］Fox BP, Kandpal RP. Transcriptional silencing of EphB6 receptor tyrosine kinase in invasive breast carcinoma cells and detection of methylated promoter by methylation specific PCR［J］. Biochem Biophys Res Commun, 2006, 340(1): 268-276.

［7］Guo H, Miao H, Gerber L, Singh J, Denning MF, Gilliam AC, et al. Disruption of EphA2 receptor tyrosine kinase leads to increased susceptibility to carcinogenesis in mouse skin［J］. Cancer Res, 2006, 66(14): 7050-7058.

［8］Huusko P, Ponciano-Jackson D, Wolf M, Kiefer JA, Azorsa DO, Tuzmen S, et al. Nonsense-mediated decay microarray analysis identifies mutations of EPHB2 in human prostate cancer［J］. Nat Genet, 2004, 36(9): 979-983.

［9］Kuang SQ, Tong WG, Yang H, Lin W, Lee MK, Fang ZH, et al. Genome-wide identification of aberrantly methylated promoter associated CpG islands in acute lymphocytic leukemia［J］. Leukemia, 2008, 22(8): 1529-1538.

［10］Egger G, Liang G, Aparicio A, Jones PA. Epigenetics in human disease and prospects for epigenetic therapy［J］. Nature, 2004, 429(6990): 457-463.

［11］Plimack ER, Kantarjian HM, Issa JP. Decitabine and its role in the treatment of hematopoietic malignancies［J］. Leuk Lymphoma, 2007, 48(8): 1472-1481.

［12］Kuang SQ, Bai H, Fang ZH, Lopez G, Yang H, Tong W, et al. Aberrant DNA methylation and epigenetic inactivation of Eph receptor tyrosine kinases and ephrin ligands in acute lymphoblastic leukemia［J］. Blood, 2010, 115(12): 2412-2419.