在人體正常的免疫系統中，血液中的單核細胞在進駐到組織時會分化成巨噬細胞。然而在急性前骨髓細胞白血病 (Acute promyelocytic leukemia，簡稱APL) 的病人體內則存在大量未分化的骨髓細胞且會伴隨凝血因子缺陷的臨床症狀。由於此疾病是因15號染色體上的pml及17號染色體上的rarα 這兩個gene重組在一起所造成，因此目前此疾病的控制方式是以All-trans retinoic acid (ATRA)將PML-RARα fusion protein釋放出來並且活化與retinoic acid有關下游gene的轉錄作用，促使未成熟myeloid細胞的分化達到治療效果，然而有關分子的調控機制目前仍不清楚。本篇即利用U937這株人類骨髓單核血球細胞株以1µM ATRA誘導分化，探討參與細胞分化的基因。U937細胞以ATRA處理不同時間0、1、4、12及24小時，收集細胞純化mRNA，隨後利用cDNA晶片做microarray hybridization，分析分化過程中基因表達的差異性。我們發現有46個基因表達有變化，其中14-3-3 eta、c-myc、Egr-1、FRAP2、Hsp90及Uracil-DNA glycosylase等為表達下降的基因，而CD14、HCK、BNip3、PI3Kγ及STC等為表達上升的基因。實驗發現加入BNip3 antisense oligonucleotide可使ATRA誘導U937細胞分化有減少的現象，同時，CD14 (monocyte/macrophage分化的標記) 的mRNA表達也下降。這些結果可推測BNip3在U937細胞的分化過程中，佔有重要功能。BNip3與Bcl-2會形成heterodimer，誘導細胞死亡，故在ATRA刺激U937細胞分化的結果BNip3基因表達上升也許代表著細胞即將進入死亡。在ATRA誘導U937細胞分化的過程中其他因細胞分化而基因表達有差異的基因，是否也佔有重要的地位，則有待進一步實驗來證明之。 The acute promelocytic leukemia (APL) is characterized by a genetic rearrangement between the promelocytic leukemia (PML) gene on chromosome 15 and the retinoic acid receptor α gene (RARα) on chromosome 17 t (15:17). In differentiation therapy, this has been highlighted by the use of all-trans retinoic acid (ATRA) in the treatment of APL. ATRA is known to stimulate the growth inhibition and the differentiation of human myelomonocytic U937 cells to macrophage-like phenotype. To understand the molecular mechanisms controlling the differentiation and proliferation of the cells, we have applied cDNA microarray technology to profile the gene expression patterns of U937 cell differentiation induced with ATRA. For this, the cells were treated with ATRA (1.0µM) for 0, 1, and 4, 12, 24 hours, followed by cellular mRNA purification and hybridization individually to the cDNA-chips (Millennia-Chip version 2). We thus identified 46 cellular genes whose expressions were induced or repressed during U937 cell differentiation and their expression patterns were later confirmed by RT-PCR. Among the identified genes, the expression levels for the Egr-1, FRAP2, Hsp90, BNip3, and Stanniocalcin genes were substantially altered. Anti-BNip3 oligonucleotide demonstrated the inhibition of ATRA-induced U937 cell differentiation. RT-PCR also confirmed the gene expression of BNip3 and CD14 (monocyte/macrophage differentiation marker) were downregulated, these results suggest BNip3 may play an important role in differentiation of U937 cell. BNip3 is a pro-apoptotic, mitochondrial protein, BNip3 formed heterodimer with Bcl-2 and induces cell death. The up-regulated of BNip3 expression during ATRA-induced U937 cell differentiation may also represent the program of cell death. The physiological roles of the other genes in mediating the growth inhibition and differentiation processes warrant further investigation.
