Wang L, Adamo M L
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78229-3900, USA.
Endocrinology. 2000 Jul;141(7):2481-9. doi: 10.1210/endo.141.7.7577.
The effect of cellular density on insulin-like growth factor I (IGF-I) gene expression was characterized in several tumor-derived cell lines. IGF-I messenger RNA (mRNA) transcripts increased more than 200-fold when C6 glioma cells grew to postconfluence. IGF-I receptor and beta-actin mRNAs were induced by 6- and 2-fold, respectively, as a function of confluence. IGF-I mRNA transcripts in GH3 and SK-N-MC cells increased about 4- to 5-fold in confluent cultures compared with sparse cultures. In OVCAR-3 cells, the IGF-I mRNA level remained constant as the cell density increased. Transient transfection experiments were performed with IGF-I exon 1 promoter/luciferase fusion constructs in C6 cells. The luciferase activity of a construct containing exon 1 sequence between +75 and +282 (the most 5' transcription initiation site was designated +1) was stimulated by 2.5-fold in dense cultures compared with that in sparse cultures of C6 cells. Luciferase activities of other constructs containing at least 282 bp of exon 1 sequence were also stimulated about 2- to 4-fold by cell density. However, 3' deletion to +192 led to loss of the cell density stimulatory effect. In contrast, luciferase activities of IGF-I promoter constructs were not altered by cell density in SK-N-MC cells. When the conditioned medium of low density C6 cultures was exchanged with that of high density cultures, the IGF-I mRNA level remained the same. In summary, cell density has a cell type- and gene type-specific effect on IGF-I gene expression. A cell density response element(s) may be located between +192 and +282 of the exon 1 promoter region in C6 cells.
在几种肿瘤来源的细胞系中研究了细胞密度对胰岛素样生长因子I（IGF-I）基因表达的影响。当C6胶质瘤细胞生长至汇合后，IGF-I信使核糖核酸（mRNA）转录物增加了200多倍。IGF-I受体和β-肌动蛋白mRNA分别随着汇合度的增加而被诱导增加6倍和2倍。与稀疏培养相比，GH3和SK-N-MC细胞在汇合培养时IGF-I mRNA转录物增加了约4至5倍。在OVCAR-3细胞中，随着细胞密度增加，IGF-I mRNA水平保持恒定。在C6细胞中用IGF-I外显子1启动子/荧光素酶融合构建体进行了瞬时转染实验。与C6细胞的稀疏培养相比，含有+75至+282之间外显子1序列的构建体（最上游的转录起始位点指定为+1）的荧光素酶活性在高密度培养中被刺激了2.5倍。其他含有至少282 bp外显子1序列的构建体的荧光素酶活性也因细胞密度而被刺激了约2至4倍。然而，3'端缺失至+192导致细胞密度刺激效应丧失。相反，SK-N-MC细胞中IGF-I启动子构建体的荧光素酶活性不受细胞密度影响。当将低密度C6培养物的条件培养基换成高密度培养物的条件培养基时，IGF-I mRNA水平保持不变。总之，细胞密度对IGF-I基因表达具有细胞类型和基因类型特异性效应。细胞密度反应元件可能位于C6细胞中外显子1启动子区域的+192至+282之间。
