Brizzi M F, Rossi P R, Rosso A, Avanzi G C, Pegoraro L
Dipartimento di Scienze Biomediche e Oncologia Umana, Università di Torino, Italy.
Br J Haematol. 1995 Jun;90(2):258-65. doi: 10.1111/j.1365-2141.1995.tb05145.x.
To elucidate the regulatory mechanisms of granulocyte-macrophage colony-stimulating factor (GM-CSF) production in human myeloid leukaemic cells we studied GM-CSF gene transcription, mRNA expression and GM-CSF secretion in human growth factor dependent M-07e cells. GM-CSF transcript was detected in cells cultured in the presence of interleukin-3 (IL-3). GM-CSF or mast cell growth factor (MGF), whereas it was undetectable in growth factor deprived cells. Growth factor re-addition induced, within 2 h, the appearance of GM-CSF mRNA. Nuclear run-on experiments demonstrated that the increase of GM-CSF mRNA levels depends on GM-CSF gene transcription. The simultaneous addition, to deprived cells, of the growth factor, and of cycloheximide (CHX) for 2 h inhibited GM-CSF mRNA expression, suggesting the requirement for newly made proteins for GM-CSF gene transcription. By means of the M-07e bioassay, which allows the detection of GM-CSF, IL-3 and MGF activities, and neutralizing antibodies to each of these factors, GM-CSF activity was detected in the cell-free extract of both IL-3- and MGF-sustained cells and of cells deprived for 24 h. This finding demonstrates that M-07e cells produce and store biologically active GM-CSF in response to both IL-3 and MGF. In contrast, analysis of the growth stimulatory activity present in the culture supernatants revealed that MGF, unlike IL-3, is able to induce the secretion of consistent amounts of GM-CSF. Taken together, our results suggest that, in M-07e cells, GM-CSF gene transcription and GM-CSF production are mediated, unlike its secretion, by mechanisms shared by IL-3 and MGF.
为阐明人髓系白血病细胞中粒细胞巨噬细胞集落刺激因子（GM-CSF）产生的调控机制，我们研究了人生长因子依赖性M-07e细胞中GM-CSF基因转录、mRNA表达及GM-CSF分泌情况。在白细胞介素-3（IL-3）、GM-CSF或肥大细胞生长因子（MGF）存在的情况下培养的细胞中可检测到GM-CSF转录本，而在缺乏生长因子的细胞中未检测到。重新添加生长因子后2小时内，GM-CSF mRNA出现。细胞核连续转录实验表明，GM-CSF mRNA水平的增加取决于GM-CSF基因转录。向缺乏生长因子的细胞同时添加生长因子和环己酰亚胺（CHX）2小时可抑制GM-CSF mRNA表达，这表明GM-CSF基因转录需要新合成的蛋白质。通过M-07e生物测定法（可检测GM-CSF、IL-3和MGF活性）及针对每种因子的中和抗体，在IL-3和MGF维持培养的细胞以及缺乏生长因子24小时的细胞的无细胞提取物中均检测到了GM-CSF活性。这一发现表明，M-07e细胞在IL-3和MGF刺激下可产生并储存具有生物活性的GM-CSF。相比之下，对培养上清液中生长刺激活性的分析显示，与IL-3不同，MGF能够诱导分泌相当数量的GM-CSF。综上所述，我们的结果表明，在M-07e细胞中，与GM-CSF分泌不同，GM-CSF基因转录和GM-CSF产生是由IL-3和MGF共有的机制介导的。
