Leckett B, Germinario R J
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis - Jewish General Hospital, Montreal, Quebec, Canada.
Cytotechnology. 1992;10(2):125-36. doi: 10.1007/BF00570889.
We have constructed an insulin-diphtheria hormono-toxin which migrates as a single 29 kd band on 10% SDS polyacrylamide gel electrophoresis. This corresponds to a one to one molar ratio of the diphtheria A-chain (23 kDa) and insulin (6 kDa) molecules. The diphtheria A-chain: insulin (DTaI) hormono-toxin demonstrates cytotoxicity in V-79 Chinese hamster cells exhibiting an LD50 of 1.1 x 10(-8) M, which is 22 x more potent than whole diphtheria toxin. Also, DTaI can competitively displace [125I]-insulin with an ED50 of 1.1 x 10(-8) M, which is identical to the ED50 of insulin (1.1 x 10(-8) M) and showed limited cross-reactivity with the IGF-1 receptor (12% displacement of [125I]-IGF-1 with a DTaI concentration of 1.1 x 10(-8) M). We have used DTaI to select conjugate-resistant clones from the V-79 Chinese hamster fibroblast parental cell line. Conjugate-resistant variants expressed insulin binding levels ranging from 8.0 +/- 2.0 fmoles/mg protein down to 3.6 +/- 0.5 fmoles/mg protein while insulin binding in the V-79 parental cell line was 11.2 +/- 0.2 fmoles/mg protein. Additionally, a number of conjugate resistant clones expressed variable ability to grow in medium containing 5% serum. The altered ability of these clones to grow in a serum-containing medium did not correlate directly with the changes observed for insulin binding. One mutant, IV-A1-j, did not grow in a serum-free defined medium containing insulin as the predominant mitogen. This IV-A1-j mutant had a lower number of insulin receptors, no change in insulin binding affinity, no change in the rate of internalization of [125I]-insulin and no apparent difference in [125I]-IGF-1 binding. Further, insulin-stimulated sugar transport was similar to that observed in the parental cell line. Based on these observations we suggest that 1) DTaI elicits its cytotoxicological effects through the insulin receptor trafficking pathway, 2) DTaI can be used to isolate cells altered at the level of insulin binding and/or action, and 3) signal transduction mechanisms responsible for mediating insulin-dependent cell growth can be pursued using mutants such as IV-A1-j.
我们构建了一种胰岛素 - 白喉激素毒素，在10%十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上迁移为单一的29千道尔顿条带。这对应于白喉A链（23千道尔顿）和胰岛素（6千道尔顿）分子的1:1摩尔比。白喉A链：胰岛素（DTaI）激素毒素在V - 79中国仓鼠细胞中表现出细胞毒性，其半数致死剂量（LD50）为1.1×10⁻⁸ M，比完整的白喉毒素强22倍。此外，DTaI能够以1.1×10⁻⁸ M的半数有效剂量（ED50）竞争性取代[¹²⁵I] - 胰岛素，这与胰岛素的ED50（1.1×10⁻⁸ M）相同，并且与胰岛素样生长因子 - 1（IGF - 1）受体的交叉反应有限（在1.1×10⁻⁸ M的DTaI浓度下，[¹²⁵I] - IGF - 1的取代率为12%）。我们使用DTaI从V - 79中国仓鼠成纤维细胞亲本细胞系中筛选抗缀合物克隆。抗缀合物变体表达的胰岛素结合水平范围从8.0±2.0飞摩尔/毫克蛋白质降至3.6±0.5飞摩尔/毫克蛋白质，而V - 79亲本细胞系中的胰岛素结合水平为11.2±0.2飞摩尔/毫克蛋白质。此外，一些抗缀合物克隆在含有5%血清的培养基中表现出不同的生长能力。这些克隆在含血清培养基中生长能力的改变与观察到的胰岛素结合变化没有直接相关性。一个突变体IV - A1 - j在以胰岛素作为主要促有丝分裂原的无血清限定培养基中不能生长。这个IV - A1 - j突变体的胰岛素受体数量较少，胰岛素结合亲和力没有变化，[¹²⁵I] - 胰岛素的内化速率没有变化，并且[¹²⁵I] - IGF - 1结合没有明显差异。此外，胰岛素刺激的糖转运与亲本细胞系中观察到的相似。基于这些观察结果，我们认为：1）DTaI通过胰岛素受体转运途径引发其细胞毒理学效应；2）DTaI可用于分离在胰岛素结合和/或作用水平发生改变的细胞；3）可以使用诸如IV - A1 - j这样的突变体来研究介导胰岛素依赖性细胞生长的信号转导机制。
