Gianní M, Terao M, Zanotta S, Barbui T, Rambaldi A, Garattini E
Unità di Biologia Molecolare, Centro Catullo e Daniela Borgomainerio, Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy.
Blood. 1994 Apr 1;83(7):1909-21.
In this report we show a strong synergistic interaction between granulocyte colony-stimulating factor (G-CSF) and all-trans retinoic acid (ATRA) on the expression of leukocyte alkaline phosphatase (LAP) in freshly isolated acute promyelocytic leukemia (APL) blasts as well as in NB40 and HL-60 cell lines. The strong synergism observed in these cell types was not evident in two acute leukemia cell lines (K562 and GF-D8), in normal granulocytes, and in monocytes. In freshly isolated leukocytes derived from chronic myelogenous leukemia (CML), in the stable phase of the disease, a weaker interaction between ATRA and G-CSF was documented. The cross-talk between the cytokine and the retinoid was studied in detail in NB4, an immortalized APL leukemia cell line, retaining the 15-17 chromosomal translocation involving the retinoic acid receptor type alpha. The treatment of NB4 cells with G-CSF alone or ATRA alone leads to no increase and to minor induction in LAP activity, respectively. If the cells are treated with the two compounds simultaneously, a dramatic elevation of LAP is observed after 4 days. The synergism between G-CSF and ATRA is evident at concentrations of the retinoid between 10(-7) and 10(-5) mol/L and at concentrations of the cytokine between 1 and 10 ng/mL. The simultaneous presence of the two compounds is necessary to obtain maximal increase of LAP activity and the effect is cell density-dependent. Synergism is specific for G-CSF, and it is not observed with other cytokines and functional inducers of the granulocyte. The augmentation of LAP activity is the consequence of an increased transcriptional rate of the liver/bone/kidney-type (L/B/K-type) alkaline phosphatase gene, as determined by Northern blotting and nuclear run-on analysis using specific cDNA probes. Only one of the two possible alternatively spliced forms of L/B/K-type alkaline phosphatase transcript is detected in NB4 cells after stimulation with G-CSF and ATRA. This mRNA form, which is the one observed in normal polymorphonuclear leukocytes, contains the most upstream leader exon. In NB4 cells, ATRA induces G-CSF, alpha, and beta retinoic acid receptor transcripts, whereas G-CSF has minor effects on the expression of these mRNAs.
在本报告中，我们展示了粒细胞集落刺激因子（G-CSF）和全反式维甲酸（ATRA）在新鲜分离的急性早幼粒细胞白血病（APL）原始细胞以及NB40和HL-60细胞系中对白细胞碱性磷酸酶（LAP）表达具有强烈的协同相互作用。在这些细胞类型中观察到的强烈协同作用在两种急性白血病细胞系（K562和GF-D8）、正常粒细胞和单核细胞中并不明显。在疾病稳定期的慢性粒细胞白血病（CML）新鲜分离的白细胞中，记录到ATRA和G-CSF之间存在较弱的相互作用。在NB4（一种永生化的APL白血病细胞系，保留涉及α型维甲酸受体的15 - 17号染色体易位）中详细研究了细胞因子与类视黄醇之间的相互作用。单独用G-CSF或单独用ATRA处理NB4细胞分别不会导致LAP活性增加或仅有轻微诱导。如果同时用这两种化合物处理细胞，4天后会观察到LAP显著升高。G-CSF和ATRA之间的协同作用在类视黄醇浓度为10(-７)至10(-５)mol/L以及细胞因子浓度为1至10 ng/mL时明显。两种化合物同时存在对于获得LAP活性的最大增加是必要的，并且该效应是细胞密度依赖性的。协同作用对G-CSF具有特异性，在其他细胞因子和粒细胞功能诱导剂中未观察到。通过使用特异性cDNA探针的Northern印迹和核转录分析确定，LAP活性的增强是肝/骨/肾型（L/B/K型）碱性磷酸酶基因转录速率增加的结果。在用G-CSF和ATRA刺激后的NB4细胞中，仅检测到L/B/K型碱性磷酸酶转录本两种可能的可变剪接形式之一。这种mRNA形式是在正常多形核白细胞中观察到的形式，包含最上游的前导外显子。在NB4细胞中，ATRA诱导G-CSF、α和β维甲酸受体转录本，而G-CSF对这些mRNA的表达影响较小。
