title: Isolation and characterization of mutant cell lines defective in transforming growth factor beta signaling.
abstract: To isolate and characterize effector molecules of the transforming growth factor beta (TGFbeta) signaling pathway we have used a genetic approach involving the generation of stable recessive mutants, defective in their TGFbeta signaling, which can subsequently be functionally complemented to clone the affected genes. We have generated a cell line derived from a hypoxanthine-guanine phosphoribosyltransferase negative (HPRT-) HT1080 clone that contains the selectable marker Escherichia coli guanine phosphoribosyltransferase (gpt) linked to a TGFbeta-responsive promoter. This cell line proliferates or dies in the appropriate selection medium in response to TGFbeta. We have isolated three distinct TGFbeta-unresponsive mutants following chemical mutagenesis. Somatic cell hybrids between pairs of individual TGFbeta-unresponsive clones reveal that each is in a distinct complementation group. Each mutant clone retains all three TGFbeta receptors yet fails to induce a TGFbeta-inducible luciferase reporter construct or TGFbeta-mediated plasminogen activator inhibitor-1 (PAI-1) expression. Two of the three have an attenuated TGFbeta-induced fibronectin response, whereas in the other mutant the fibronectin response is intact. These TGFbeta-unresponsive cells should allow selection and identification of signaling molecules through functional complementation.
authors: Department of Cell Biology, Cleveland Clinic Research Institute, OH 44195, USA.
authors: B A
authors: BA
authors: Hocevar
authors: P H
authors: PH
authors: Howe
mesh_terms: D002460:Cell Line; D002999:Clone Cells; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005353:Fibronectins / Q000494:pharmacology; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics; D008156:Luciferases / Q000096:biosynthesis; D016296:Mutagenesis; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000096:biosynthesis
chemical_list: D005353:Fibronectins; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D008156:Luciferases; D007041:Hypoxanthine Phosphoribosyltransferase; D001616:beta-Galactosidase