Construction of a toxic insulin molecule: selection and partial characterization of cells resistant to its killing effects

Abstract

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.