Kiess W, Koepf G, Christiansen H, Blum W F
Children's Hospital, Justus Liebig University of Giessen, Germany.
Regul Pept. 1997 Sep 26;72(1):19-29. doi: 10.1016/s0167-0115(97)01026-4.
Neuroblastoma cells are thought to depend upon autocrine stimulation by IGF-II but not by IGF-I. We have studied the expression of IGF, IGFBP and IGF receptor mRNA in two human neuroblastoma cell lines, SK-N-MC and CHP, and asked whether or not the expression of the IGF system in these malignant cells determines their growth pattern. SK-N-MC cells grow with a cell doubling time of 36 hours in medium supplemented with 10% fetal calf serum whereas CHP cells only grow with a doubling time of 72 h. In addition, the SK-N-MC cell line has a plating efficiency ten times greater than the CHP cell line. RNase protection assays were performed using (32)P-labelled riboprobes and RNA that had been purified from SK-N-MC and CHP cells respectively. A 520 bases human IGF-I, a 556 bases human IGF-II, a 480 bases human IGF-I receptor and a 250 human IGF-II/mannose-6-phosphate (M6P) receptor probe were radiolabelled as were human IGFBP-1, -2, -3, -4, -5 and -6 probes. While both SKNMC and CHP neuroblastoma cells expressed mRNAs for IGFBP-2, -4, and -6 no signal was detected for IGFBP-1, and -3 and only SK-N-MC cells expressed IGFBP-5 mRNA. In addition, a 400 bases protected band was seen with the IGF-I receptor probe and a 260 bases protected band with the IGF-IIM6P receptor probe in either cell line. Interestingly, a 300 bases protected species was detected with the IGF-II probe in CHP cell RNA whereas SK-N-MC cells did not express IGF-II transcripts. Conversely, SK-N-MC cells expressed a 520 bases IGF-I transcript while CHP cells did not show IGF-I mRNA expression. As determined by specific radioimmunoassays SK-N-MC cells secreted 0.75+/-0.02 ng/ml IGF-I, 1.2+/-0.04 ng/ml IGF-II and 149+/-2.1 ng/ml IGFBP-2 within 24 h, whereas CHP cells secreted 0.1+/-0.01 ng/ml IGF-I, but 6.2+/-0.1ng/ml IGF-II and 254.8+/-5.5 ng/ml IGFBP-2 (N=5). IGFBP-2 secretion correlated positively with IGF-II secretion in CHP cells (r=0.85, P=0.05) and negatively with IGF-I (r= -0.9, P<0.01) in SK-N-MC cells. In conclusion, SK-N-MC cells which grow rapidly and have a high plating efficiency, express IGF-I, while CHP cells that grow more slowly express IGF-II. We hypothesize that neuroblastoma cells depend upon autocrine stimulation by either IGF-I or IGF-II. Variable sensitivity to growth inhibitors or apoptotic processes may be related to the differential expression of the IGF system.
人们认为神经母细胞瘤细胞依赖胰岛素样生长因子-II（IGF-II）的自分泌刺激，而非胰岛素样生长因子-I（IGF-I）。我们研究了两种人神经母细胞瘤细胞系SK-N-MC和CHP中IGF、IGF结合蛋白（IGFBP）和IGF受体mRNA的表达，并探讨了这些恶性细胞中IGF系统的表达是否决定其生长模式。在添加10%胎牛血清的培养基中，SK-N-MC细胞的倍增时间为36小时，而CHP细胞的倍增时间仅为72小时。此外，SK-N-MC细胞系的接种效率比CHP细胞系高10倍。使用分别从SK-N-MC和CHP细胞中纯化的RNA，与（32）P标记的核糖探针进行核糖核酸酶保护分析。对一条520个碱基的人IGF-I、一条556个碱基的人IGF-II、一条480个碱基的人IGF-I受体和一条250个碱基的人IGF-II/甘露糖-6-磷酸（M6P）受体探针进行放射性标记，人IGFBP-1、-2、-3、-4、-5和-6探针也进行了放射性标记。虽然SKNMC和CHP神经母细胞瘤细胞均表达IGFBP-2、-4和-6的mRNA，但未检测到IGFBP-1和-3的信号，只有SK-N-MC细胞表达IGFBP-5 mRNA。此外，在任一细胞系中，用IGF-I受体探针可观察到一条400个碱基的受保护条带，用IGF-IIM6P受体探针可观察到一条260个碱基的受保护条带。有趣的是，在CHP细胞RNA中用IGF-II探针检测到一条300个碱基的受保护条带，而SK-N-MC细胞不表达IGF-II转录本。相反，SK-N-MC细胞表达一条520个碱基的IGF-I转录本，而CHP细胞未显示IGF-I mRNA表达。通过特异性放射免疫分析测定，SK-N-MC细胞在24小时内分泌0.75±0.02 ng/ml IGF-I、1.2±0.04 ng/ml IGF-II和149±2.1 ng/ml IGFBP-2，而CHP细胞分泌0.1±0.01 ng/ml IGF-I、6.2±0.1 ng/ml IGF-II和254.8±5.5 ng/ml IGFBP-2（N = 5）。在CHP细胞中，IGFBP-2分泌与IGF-II分泌呈正相关（r = 0.85，P = 0.05），在SK-N-MC细胞中与IGF-I分泌呈负相关（r = -0.9，P < 0.01）。总之，生长迅速且接种效率高的SK-N-MC细胞表达IGF-I，而生长较慢的CHP细胞表达IGF-II。我们推测神经母细胞瘤细胞依赖IGF-I或IGF-II的自分泌刺激。对生长抑制剂或凋亡过程的不同敏感性可能与IGF系统的差异表达有关。
