Bradshaw S L, D'Ercole A J, Han V K
Department of Biochemistry, University of Western Ontario, The Lawson Research Institute, London, Canada.
Endocrinology. 1999 Feb;140(2):575-84. doi: 10.1210/endo.140.2.6498.
To examine the relationship between the expression of insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) and cell growth in a cell type with a defined IGF/IGFBP system, an ovine IGFBP-2 complementary DNA was overexpressed in C6 glioma cells. C6 cells produce IGFBP-3, IGFBP-4, a negligible amount of IGFBP-2, and IGF-I. An ovine IGFBP-2 complementary DNA was transfected into C6 cells, and nine colonies that stably expressed variable levels of IGFBP-2 messenger RNA were selected. Synthesis of corresponding levels of IGFBP-2 was confirmed by ligand blot and immunoblot analyses of conditioned media. Three clones exhibited significantly reduced growth rates, and the remainder showed growth rates similar to those of the wild-type C6 cells. The clones, which overexpressed high levels of IGFBP-2 and IGF-I, had growth rates similar to the wild-type cells, whereas the three clones that overexpressed IGFBP-2 without a concomitant increase in IGF-I had reduced growth rates. In addition, a cell-associated IGFBP was identified in the slow growing clones, but not in the wild-type or the fast growing clones. This cell-associated IGFBP was deduced to be IGFBP-5 based on its molecular size, detection of IGFBP-5 messenger RNA only in slow growing clones, and competition of its binding by heparin. Growth of the slow growing clone, C6BP2-1, could not be overcome by the addition of exogenous IGF-I, suggesting that the cell-associated IGFBP-5 was the dominant regulator of IGF action. These observations suggested that 1) in C6 glioma cells cellular growth is altered by a disturbance in the equilibrium between IGF-I and IGFBPs and/or the functional properties of the IGFBPs; and 2) C6 cells may have a limited capacity to modulate IGF/IGFBP expression in response to changes in endogenous expression of IGFBPs. Endogenous regulation of the balance between IGFs and IGFBPs may be a model of regulation of cellular growth in tumor cells.
为了在具有明确胰岛素样生长因子（IGF）/IGF结合蛋白（IGFBP）系统的细胞类型中研究IGF结合蛋白-2（IGFBP-2）表达与细胞生长之间的关系，将绵羊IGFBP-2互补DNA在C6胶质瘤细胞中过表达。C6细胞产生IGFBP-3、IGFBP-4、少量的IGFBP-2以及IGF-I。将绵羊IGFBP-2互补DNA转染到C6细胞中，选择了9个稳定表达不同水平IGFBP-2信使RNA的克隆。通过对条件培养基进行配体印迹和免疫印迹分析，证实了相应水平IGFBP-2的合成。3个克隆的生长速率显著降低，其余克隆的生长速率与野生型C6细胞相似。那些过表达高水平IGFBP-2和IGF-I的克隆，其生长速率与野生型细胞相似，而3个过表达IGFBP-2但IGF-I没有相应增加的克隆，其生长速率降低。此外，在生长缓慢的克隆中鉴定出一种细胞相关的IGFBP，但在野生型或生长快速的克隆中未发现。基于其分子大小、仅在生长缓慢的克隆中检测到IGFBP-5信使RNA以及其结合被肝素竞争，推断这种细胞相关的IGFBP为IGFBP-5。添加外源性IGF-I无法克服生长缓慢的克隆C6BP2-1的生长抑制，这表明细胞相关的IGFBP-5是IGF作用的主要调节因子。这些观察结果表明：1）在C6胶质瘤细胞中，细胞生长因IGF-I与IGFBPs之间平衡的紊乱和/或IGFBPs的功能特性而改变；2）C6细胞响应IGFBPs内源性表达变化调节IGF/IGFBP表达的能力可能有限。IGFs与IGFBPs之间平衡的内源性调节可能是肿瘤细胞中细胞生长调节的一种模式。
