Characterization of The BNIP3 Promoter Regulating Gene Expression During The Differentiation of U937 Cells

• Main Authors: Ching-Huei, 林靜慧
• Other Authors: 黃志揚
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/37723353800248684062

碩士 === 中山醫學大學 === 生化暨生物科技研究所 === 94 === Human myelomonocytic leukemia cell line U937 and myeloid leukemia cell line HL-60 have been widely used in experiments as model systems to study the molecular mechanisms of myeloid cell differentiation, because these cells respond differently to a variety of agents. To understand the molecular mechanism of all-trans retinoic acid (ATRA) induced differentiation of U937 cells, in our laboratory, we have used homemade cDNA microarrays to profile the gene expression pattern, and functional analyses led to the identification of a pro-apoptotic protein BNIP3 gene expression level was induced that is the pro-apoptotic member belongs the BH3 domain only protein of Bcl-2 family. The subsequent experiments with RT-PCR, flow cytometry, reducing NBT and Western blotting confirmed the ATRA-induced expression pattern, and anti-BNIP3 antisence phosphorothioate oligonucleotide specifically inhibited the differentiation induction. Moreover, we also confirmed the ATRA and TPA-induced differentiation of the human cell lines HL-60 with morphology and flow cytometry, proceed to confirmed the gene expression pattern in U937 cells and HL-60 cells induced by ATRA and TPA with RT-PCR. In our laboratory, further analysis of the transcription regulatory function of the BNIP3 promoter delimited an important region that bound to the AML-1 and MZF-1 both are inducible, but one is a repressor, and the other is inducer. Furthermore, we also analysis the activity of the AML-1 deletion of BNIP3 promoter and MZF-1 deletion of BNIP3 promoter by transient transfection in U937 cells during the ATRA-induced differentiation, and co-transfection with AML-1b expression vector and MZF-1 expression vector respectively. Collectively, these results of the study are totally in agreement with the biochemical property of AML-1 and MZF-1 that the inducible expression pattern of BNIP3. Most importantly, considering the data from our laboratory and the evidences provided in previous reports, we concluded that BNIP3 is a bi-functional protein that is required for differentiation and also plays an important role in caspase-independent apoptosis.